EP1758640A2 - Inhibition of hiv-1 replication by disruption of the processing of the viral capsid-spacer peptide 1 protein - Google Patents

Inhibition of hiv-1 replication by disruption of the processing of the viral capsid-spacer peptide 1 protein

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Publication number
EP1758640A2
EP1758640A2 EP05779995A EP05779995A EP1758640A2 EP 1758640 A2 EP1758640 A2 EP 1758640A2 EP 05779995 A EP05779995 A EP 05779995A EP 05779995 A EP05779995 A EP 05779995A EP 1758640 A2 EP1758640 A2 EP 1758640A2
Authority
EP
European Patent Office
Prior art keywords
seq
nucleotides
spl
compound
gag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05779995A
Other languages
German (de)
French (fr)
Other versions
EP1758640A4 (en
Inventor
Karl Salzwedel
Feng Li
Carl T. Wild
Graham P. Allaway
Eric O. Freed
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Panacos Pharmaceuticals Inc
Original Assignee
US Department of Health and Human Services
Panacos Pharmaceuticals Inc
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Priority claimed from US10/851,637 external-priority patent/US7537765B2/en
Application filed by US Department of Health and Human Services, Panacos Pharmaceuticals Inc filed Critical US Department of Health and Human Services
Publication of EP1758640A2 publication Critical patent/EP1758640A2/en
Publication of EP1758640A4 publication Critical patent/EP1758640A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the invention includes methods of inhibiting, inhibitors and methods of discovery of inhibitors of HTV infection.
  • HIV Human Immunodeficiency Virus
  • lentivimses a subfamily of retro vimses.
  • the viral genome contains many regulatory elements which allow the vims to control its rate of replication in both resting and dividing cells.
  • HIN infects and invades cells of the immune system; it breaks down the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms.
  • the immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
  • HIN-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4, also known as OKT4, T4 and leu3.
  • the viral tropism is due to the interactions between the viral envelope glycoprotein, gpl20, and the cell-surface CD4 molecules (Dalgleish et al, Nature 312:763-767 (1984)). These interactions not only mediate the infection of susceptible cells by HIN, but are also responsible for the vims-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in HIN-infected patients. These events result in HIN-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
  • the host range of HIN includes cells of the mononuclear phagocytic lineage (Dalgleish et al, supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes.
  • HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage and monocytes are major reservoirs of HIV. They can interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
  • Therapeutic agents for HIN can include, but not are not limited to, at least one of AZT, 3TC, ddC, d4T, ddl, tenofovir, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfmavir, lopinavir, amprenavir, atazanavir and fosamprenavir, or any other antiretroviral drugs or antibodies in combination with each other, or associated with a biologically based therapeutic, such as, for example, gp41- derived peptides enfuvirtide (Fuzeon; Timeris-Roche) and T-1249 (Trimeris), or soluble CD4, antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionally presented herein. Combinations of these
  • HIN virion assembly takes place at the surface membrane of the infected cell where the viral Gag polyprotein accumulates, leading to the assembly of immature virions that bud from the cell surface.
  • Gag is cleaved by the viral proteinase (PR) into the matrix (MA), capsid (CA), nucleocapsid ( ⁇ C), and C-terminal p6 structural proteins (Wiegers K. et al, J. Virol. 72:2846-2854 (1998)).
  • Gag processing induces a reorganization of the internal virion structure, a process termed "maturation.”
  • MA lines the inner surface of the membrane, while CA forms the conical core which encases the genomic R ⁇ A that is complexed with ⁇ C. Cleavage and maturation are not required for particle formation but are essential for infectivity (Kohl, ⁇ . et al, Proc. Nat/. Acad. Sci. USA 55:4686-4690, (1998)).
  • CA and ⁇ C as well as ⁇ C and p6 are separated on the Gag polyprotein by short spacer peptides of 14 and 10 amino acids (p2), respectively (spacer peptide 1 (SP1) and SP2, respectively) (Wiegers K. et al, J. Virol 72:2846- 2854 (1998), Pettit, S.C. et al, J. Virol. 6°: 8017-8027 (1994), Liang et al. J Virol. 76:11729-11737 (2002)). These spacer peptides are released by PR- mediated cleavages at their ⁇ and C termini during particle maturation.
  • the individual cleavage sites on the HIN Gag and Gag-Pol polyproteins are processed at different rates and this sequential processing results in Gag intermediates appearing transiently before the final products. Such intermediates may be important for virion morphogenesis or maturation but do not contribute to the structure of the mature viral particle (Weigers et al. and Pettit, et al, supra).
  • the initial Gag cleavage event occurs at the C terminus of SP1 and separates an ⁇ -terminal MA-CA-SP1 intermediate from a C- terminal ⁇ C-SP2-p6 intermediate. Subsequent cleavages separating MA from CA-SP1 and NC-SP2 from p6 occur at an approximately 10- fold-lower rate.
  • Cleavage of SP1 from the C terminus of CA is a late event and occurs at a 400-fo Id-lower rate than cleavage at the SP1-NC site (Weigers et al. and Pettit, et al, supra).
  • the uncleaved CA-SP1 intermediate protein is alternatively termed "p25,” whereas the cleaved CA protein is alternatively termed “p24” and the cleaved SP1 peptide is alternatively termed "p2".
  • Cleavage of SP1 from the C terminus of CA appears to be one of the last events in the Gag processing cascade and is required for final capsid condensation and formation of mature, infectious viral particles. Electron micrographs of mature virions reveal particles having electron dense conical cores. On the other hand, electron microscopy studies of viral particles defective for CA-SP1 cleavage show particles having a spherical electron- dense ribonucleoprotein core and a crescent-shaped, electron-dense layer . located just inside the viral membrane (Weigers et al, supra).
  • Betulinic acid and platanic acid were isolated from Syzigium claviflorum and were determined to have anti-HIV activity.
  • Betulinic acid and platanic acid exhibited inhibitory activity against HIN-1 replication in H9 lymphocyte cells with EC 50 values of 1.4 ⁇ M and 6.5 ⁇ M, respectively, and therapeutic index (T.I.) values of 9.3 and 14, respectively.
  • Hydrogenation of betulinic acid yielded dihydrobetulinic acid, which showed slightly more potent anti- HIV activity with an EC 50 value of 0.9 and a T.I. value of 14 (Fujioka, T., et al, J. Nat. Prod. 57:243-247 (1994)).
  • Japanese Patent Application No. JP 01 143,832 discloses that betulin and 3,28-diesters thereof are useful in the anti-cancer field.
  • U.S. Patent No. 6,172,110 discloses betulinic acid and dihydrobetulin derivatives which have the following formulae or pharmaceutically acceptable salts thereof, Betulin and Dihydrobetulin Derivatives
  • Ri is a C 2 -C 20 substituted or unsubstituted carboxyacyl
  • R 2 is a C 2 -C 20 substituted or unsubstituted carboxyacyl
  • R 3 is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or ⁇ OR 4 , where t is hydrogen, alkanoyl, benzoyl, or C 2 -C 20 substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29.
  • U.S. Patent Application No. 60/413,451 discloses 3,3-dimethylsuccinyl betulin and is herein incorporated by reference.
  • the strategy of this invention is to provide therapeutic methods and compounds that inhibit the vims in different ways from approved therapies.
  • the compound and methods of the present invention have a novel mechanism of action and therefore are active against HIN strains that are resistant to current reverse transcriptase and protease inhibitors. As such, this invention offers a completely new approach for treating HIN/ ADDS.
  • the invention provides methods of inhibiting, inhibitory compounds and methods of identifying inhibitory compounds that target proteolytic processing of the HIN-1 Gag protein.
  • such compounds may directly or indirectly inhibit the interaction of a protease enzyme with HIV-l Gag protein.
  • such inhibition of interaction occurs via the binding of a compound to Gag.
  • the inhibition of protease cleavage of the CA-SP1 protein of HIN-1 Gag by 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB) is one example, but other proteolytic cleavage sites can be targeted by a similar approach using inhibitory compounds that interact with the substrate in a manner similar to that in which DSB interacts with Gag.
  • Another aspect of the invention is directed to a method of inhibiting the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but having no effect on other Gag processing steps.
  • a further aspect of the invention is directed to a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but have no effect on other Gag processing steps.
  • the invention is drawn to a compound or pharmaceutical composition identified by the method for identifying compounds that inhibit HIV-l replication disclosed herein.
  • the present invention is directed to a polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • This aspect of the invention is also directed to a vector, virus and host cell comprising said polynucleotide, and a method of making said protein.
  • a further aspect of the present invention is directed to an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'- dimethylsuccinyl) betulinic acid.
  • An additional aspect of the invention is directed to an antibody which selectively binds an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid. Also included in this aspect of the invention are a method of making said antibody, a hybridoma producing said antibody and a method of making said hybridoma.
  • the invention is directed to a kit comprising a polynucleotide, polypeptide or antibody disclosed herein.
  • the invention further relates to a method of inhibiting HIV-l infection in cells of an animal by contacting said cells with a compound that blocks the maturation of vims particles released from treated infected cells.
  • the released vims particles exhibit non-condensed cores and a distinctive thin electron-dense layer near the viral membrane and have reduced infectivity.
  • a method is included of contacting animal cells with a compound that both inhibits processing of the viral Gag p25 protein and that disrupts the maturation of virus particles.
  • This invention further includes a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but have no significant effect on other Gag processing steps.
  • the method involves contacting HIN-1 infected cells with a test compound, and thereafter analyzing virus particles that are released to detect the presence of p25.
  • Methods to detect p25 include western blotting of viral proteins and detecting using an antibody to p25, gel electrophoresis, and imaging of metabolically labeled proteins.
  • Methods to detect p25 also include rmmunoassays using an antibody to p25 or SP1 ( ⁇ 2) or to an epitope tag inserted into the SP1 sequence.
  • the invention is further directed to a method for identifying compounds involving contacting HIV-l infected cells with a compound, and - thereafter analyzing vims particles released by the contacted cells, by thin- sectioning and transmission electron microscopy, and determining whether viral particles with non-condensed cores and a distinctive thin electron-dense layer near the viral membrane are present.
  • the invention is also directed to compounds identified by the aforementioned screening methods.
  • the invention is drawn to a method of treating HIV-l infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps.
  • CA-SPl viral Gag p25 protein
  • such inhibition may be accompanied by a different observable phenotypes.
  • inhibition may not necessarily significantly reduce the quantity of virions released from treated infected cells; and/or said inhibition may have little or no significant effect on the amount of R ⁇ A incorporation into the released virions; and/or said inhibition disrupts the maturation of virions released from infected cells treated with said compound.
  • the virion stracture may be affected, and a majority of virions released from treated infected cells exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and/or and have reduced or no infectivity.
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits the interaction of HIN protease with CA-SPl, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps.
  • Such inhibition may be direct or, alternatively, indirect; and/or may involve said compound binding to the viral Gag protein such that interaction of HIN protease with CA-SPl is inhibited.
  • the invention is also drawn to a method of treating HIN in a patient with a compound that binds at or near the site of cleavage of the viral Gag p25 protein (CA-SPl) to p24 (CA), thereby inhibiting the interaction of HIN ' protease with the CA-SPl cleavage site and resulting in the inhibition of processing of p25 to p24.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • the invention is drawn to a method of treating HIN-1 -infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (A) K ⁇ WMTETFLVQ ⁇ A ⁇ PDCKTILKALGPAATLEEMMTAC QGVGGPSHKAR ⁇ LAEAMSQVT ⁇ SAT ⁇ M (SEQ ⁇ D NO: 21); (B) K ⁇ WMTETLLVQ ⁇ A ⁇ PDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKHKARILAEAMSQVTNSA
  • the invention is drawn to a method of treating HIN-1 -infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18 (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828- 1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) by administration of a compound.
  • a compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%), 90%, 95%o or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLNQNANPDCKTILKALGPAATLEEMMTAC QGVGGPSHKARILAEAMSQVTNSAT ⁇ M (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (a) KNWMTETFLNQNA
  • the invention is drawns to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) by administration of a compound wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%) or 99% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828- 1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372- 1419 of
  • the invention may be useful in the treatment of HIN in patients who are not adequately treated by other HIN-1 therapies. Accordingly, the invention is also drawn to a method of treating a patient in need of therapy, wherein the HIV-l infecting said cells does not respond to other HIV-l therapies. In another embodiment, methods of the invention are practiced on a subject infected with an HIV that is resistant to a drug used to treat HIN infection. In one application, the HIV is resistant to a protease inhibitor, a polymerase inhibitor, a nucleoside analog, a vaccine, a binding inhibitor, an immunomodulator, or any other inhibitor.
  • methods of the invention are practiced on a subject infected with an HIN that is resistant to a drag used to treat HIN infection is selected from the group consisting of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate
  • the invention is drawn to a method of treating HIN in a patient, wherein said patient is administered said compound in combination with at least one anti-viral agent.
  • Anti-viral agents suitable include, but are not limited to: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir,
  • the invention is also directed to compounds. Such compounds are useful in a method of treating patients infected with HIN; in a method for inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), or in a method for treating human blood and human blood products.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • Such compounds useful in the present invention include, but are not limited to derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, or is selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'-dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-0-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'- dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid and combinations thereof.
  • Compounds of the invention may be used alone, or administered with additional compounds, including zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusi
  • the invention is directed to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA).
  • said compound does not significantly affect other Gag processing steps.
  • said inhibition does not significantly reduce the quantity of virions released from treated infected cell; and or has little or no significant effect on the amount of RNA incorporation into the released virions; and/or inhibits the maturation of virions released from infected cells treated with said compound; and or affects viral morphology.
  • Such effects on viral morphology include, but are not limited to: the virions released from treated infected cells to exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and/or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and or and have reduced or no infectivity.
  • the method involves the administration of the compound which inhibits the interaction of HIV protease with CA-SPl, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) but has no significant effect on other Gag processing steps.
  • This may be via direct, or indirect inhibition of the interaction of HIV protease with CA- SPl; and/or may involve said compound binds to the viral Gag protein such that interaction of HIN protease with CA-SPl is inhibited; and/or said compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SPl) to p24 (CA), thereby inhibiting the interaction of HIN protease with the CA-SPl cleavage site and resulting in the inhibition of processing of p25 to p24.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTAC
  • the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%o or 99%) identity, or which is identical a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ED NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ED NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO:
  • the invention also embodies methods for identifying compounds that inhibit HIV-l replication. Accordingly, the invention also includes a method of identifying compounds that inhibit HIV-l replication in cells of an animal, comprising: contacting a Gag protein comprising a CA-SPl cleavage site with a test compound; adding a labeled substance that selectively binds near the CA-SPl cleavage site; and measuring competition between the binding of the test compound and the labeled substance to the CA-SPl cleavage site. In further embodiments of this method, the compounds inhibits the interaction of HIV-l protease with a target site by binding to said target site.
  • these methods also include embodiments wherein the CA-SPl cleavage site region is contained within a polypeptide fragment or recombinant peptide; and/or wherein the labeled substance is a labeled antibody specific for CA-SPl, and measuring the change in the amount of labeled antibody bound to the protein in the presence of test compound compared with a control.
  • Labels include, but are not limited to, an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope and a combination thereof.
  • the method of identifying compounds that inhibit HIN-1 replication in cells of an animal also comprises, in one embodiment, measuring the change in the amount of labeled 3-0-(3',3'-dimethylsuccinyl) betulinic acid bound to the protein in the presence of test compound, compared with a control, and wherein the labeled substance is 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • the invention comprises a method for identifying compounds that inhibit HIN-1 replication in the cells of an animal which comprises: contacting a polypeptide comprising a CA-SPl cleavage site, with a protease in the presence of a test compound.
  • a protease is related to HIN-1 protease, or is HIN protease.
  • the method comprises ; contacting a polypeptide comprising a wild type CA- SPl cleavage site, with a protease in the presence of a test compound and also contacting a polypeptide comprising a mutant CA-SPl cleavage site or a protein comprising an alternative protease cleavage site with HIN-1 protease in the presence of the test compound, detecting the cleavage, and comparing the amount of cleavage of the native wild-type polypeptide to the amount of cleavage of the mutant polypeptide or to amount of cleavage of the protein comprising an alternative protease cleavage site.
  • the wild-type CA-SPl or mutant CA-SPl or alternative protease cleavage site region is contained within a polypeptide fragment or recombinant peptide.
  • the polypeptide is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety.
  • the polypeptide is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound.
  • the effect of the test compound on cleavage of the polypeptide is detected by measuring the amount of a labeled antibody that is bound to SP1 or p24 (CA).
  • the labeled antibody that binds CA, or the antibody that binds SP1 is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope, and combinations thereof.
  • the invention is also directed to a method for identifying compounds that inhibit HIN-1 replication in cells of an animal.
  • the method comprises: contacting a test compound with cells infected with wild- type virus isolates and with cells infected with virus isolates having significantly reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl) betulinic acid; and selecting test compounds that are more active against the wild-type virus isolate compared with virus isolates that have reduced sensitivity to 3-0-(3',3'- dimethylsuccinyl) betulinic acid.
  • the method comprises contacting HIN-1 infected cells with a test compound; lysing the infected cells or the released viral particles to form a lysate, and analyzing the lysate to determine whether cleavage of the CA-SPl protein has occurred.
  • said analyzing may comprise measuring the presence or absence of p25; and or performing a western blot of viral proteins and detecting p25 using an antibody to p25; and/or performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins; and or performing an immunoassay.
  • an immunoassay may be performed by any methods known in the art, including, but not limited to: (a) capturing p25 and p24 on a substrate using an antibody that selectively binds p24; and (b) detecting the presence or absence of p25 on the substrate by using an antibody that selectively binds p25.
  • the invention also includes such modifications of the above assay as would be obvious to one of ordinary skill in the art.
  • the method of identifying a compound according to the invention comprises the use of an epitope tag sequence inserted into SP1 and the selective detection of p25 is performed using an antibody to the epitope tag.
  • the invention is also directed to a method for identifying compounds that inhibit HIN-1 replication in the cells of an animal comprising: contacting HIN-1 infected cells with a test compound and thereafter analyzing the virus particles using transmission electron microscopy.
  • Such analysis includes for example, looking for the presence of spherical cores that are acentric with respect to the viral particle; and/or having crescent-shaped, electron-dense layers lying just inside the viral membrane.
  • the invention is drawn to an isolated polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in an HIV Gag p25 protein (CA SP1), said mutation resulting in a decrease in inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-0-(3',3'-dimethylsuccinyl) betulinic acid (DSB).
  • CA SP1 HIV Gag p25 protein
  • DSB 3-0-(3',3'-dimethylsuccinyl) betulinic acid
  • This inhibition of processing of p25 may be due to a decrease in inhibition of the interaction of HIN-1 protease with Gag; and/or a decrease in the binding of 3-O-(3',3'- dimethylsuccinyl) betulinic acid to Gag; and/or a decrease in the binding of DSB at or near the CA-SPl cleavage site of Gag.
  • Suitable polynucleotides also include those encoding a mutation at or near the CA-SPl cleavage site or in the SP1 domain of CA-SPl; and or those encoding a mutation at or near the amino acid sequence G/SHKARV/TLAEAMSQV (SEQ ID NO: 1); and/or those encoding the amino acid sequences GHKARVLVEAMSQV (SEQ ED NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and/or isolated polynucleotide which is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and/or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ED NO: 4, and SEQ ID NO: 6; and/or having at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ED NO: 8 and SEQ ID NO: 9
  • the invention is also drawn to vectors comprising such polynucleotides as described above; to a host cell composing such a vector; and to a method of producing a polypeptide comprising incubating the host cell containing such a vector in a medium and recovering the polypeptide from said medium.
  • the invention is directed to an antibody.
  • Such an antibody may bind to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTAC QGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (e) SHKARE AEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO:
  • the invention is drawn to an antibody which binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide with a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729- 1920 of SEQ ED NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (f) about nucleotides 1370-1413 of SEQ ID NO: 18; (g) about nucleotides 1857-1899 of SEQ ID NO: 19 (h) about nucleotides 1372-1419 of SEQ ID NO: 18; (i) about nucleotides 1858-1905 of SEQ ID NO: 19;
  • the antibody binds to amino acids of the CA-SPl region of the HIN-1 Gag polypeptide, wherein said amino acids comprise: SHKARILAEAMSQN (SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26).
  • the invention is drawn to an antibody that inhibits the binding of 3-O-(3',3'-dimethylsuccinyl) betulinic acid to the CA-SPl region of the Gag polypeptide.
  • the invention is also drawn to mutant HIV-l viruses.
  • the invention is an isolated mutant recombinant HIV-l viras, wherein the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in said viras is not significantly inhibited by 3-O-(3',3 , -dimethylsuccinyl) betulinic acid.
  • this viras is not inhibited by 3-0- (3',3'-dimethylsuccinyl) betulinic acid.
  • 3-0-(3',3'- dimethylsuccinyl) betulinic acid does not inhibit the interaction of protease with the Gag polypeptide in this viras.
  • the virus does not bind to 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • the invention is drawn to viruses wherein the amino acids of the CA-SPl region are replaced with alternative amino acids, or amino acids are added to the CA- SP1 region, or where amino acids are deleted.
  • one or more amino acids are deleted from the AEAMSQV (amino acid no. 8-14 of SEQ DD NO:26) amino acid sequence in the CA-SPl region.
  • a mutant viruses may be used in the methods of the invention described elsewhere herein.
  • such viruses are useful in a method of identifying a compound which inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method comprising comparing the ability of said compound to inhibit HIV-l replication compared with the replication of a the mutant virus outlined above. Such inhibition may be examined in a cell, or in an animal, or in vitro.
  • the invention is also drawn to non-HIV-1 retroviruses that are sensitive to 3-0-(3',3'-dimethylsuccinyl) betulinic acid.
  • said retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence AEAMSQV (amino acid no. 8-14 of SEQ ED NO: 26) at or near the CA-SPl cleavage site.
  • the retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence VLAEAMSQV (amino acid no. 6-14 of SEQ ID NO: 26) at or near the CA-SPl cleavage site.
  • the retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near the CA-SPl cleavage site; in another the retroviras composes the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence enocoded by the polynucleotide of SEQ ID NO:26, SEQ ID NO: 90; SEQ ED NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98; in another embodiment the retroviras comprises the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence of SEQ ID NO: 91; SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97; or SEQ ID NO: 99.
  • the retroviras comprises the nucleic acid sequence having at least 70%, 80%, 90% or which is identical to the sequence of SEQ ED NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98.
  • Retroviruses of this embodiment of the invention include, but are not limited to HIN-2, HTLV-I, HTLV-II, SIN, avian leukosis viras (ALN), endogenous avian retroviras (EAN), mouse mammary tumor viras (MMTN), feline immunodeficiency virus (FIN), Bovine immunodeficiency virus (BIN), caprine arthritis encephalitis virus (CAEN), Nisna-maedi viras, or feline leukemia viras (FeLV).
  • APN avian leukosis viras
  • EAN endogenous avian retroviras
  • MMTN mouse mammary tumor viras
  • FIN feline immunodeficiency virus
  • BIN Bovine immunodeficiency virus
  • CAEN caprine arthritis encephalitis virus
  • Nisna-maedi viras or feline leukemia viras (FeLV).
  • the invention is drawn to a method of making a recombinant non-HIV-1 lentivirus sensitive to DSB.
  • This method comprises: deleting from the genome of said lentivirus the nucleotides which correspond to nucleotides 1370-1413 from SEQ ID NO: 18, in HIV-l; and inserting nucleotides 1370-1413 from SEQ ID NO: 18 or nucleotides 1857- 1899 of SEQ ID NO: 19 into said region of said non-HIN-1 lentivirus.
  • Examples of chimeric lentivimses that were, are or may be constructed by this method are described in Figure 10.
  • Such viruses may be used in the methods of the invention described elsewhere herein.
  • such recombinant non-HIN-1 lentivimses may be used in a method of identifying a compound which inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method consisting of comparing of the ability of said compound to inhibit replication of a wild-type non-HIN-1 lentivirus with the DSB-sensitive recombinant variant thereof .
  • Such inhibition may occur in a cell; in an animal; or in vitro.
  • the invention is also drawn to an animal model of lentivirus infection comprising a suitable non-human animal host infected with a lentivirus sensitive to 3-0-(3',3'-dimethylsuccinyl) betulinic acid.
  • the lentivirus may include, but is not limited to SIN; FrV; EIAV; BIV; CAEN; and Nisna-Maedi virus.
  • the invention is also drawn to isolated polypeptides.
  • the invention is drawn to a polypeptide containing a mutation in an HIV CA-SPl protein, said mutation which results in a decrease in inhibition of processing of p25 by 3-O-(3',3'-dimethylsucciny ⁇ ) betulinic acid.
  • this polypeptide is encoded by a polynucleotide that contains a mutation located at or near the CA-SPl cleavage site or in the SP1 domain encoded by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10 and/or is encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ED NO: 8 and SEQ ID NO: 9; and/or comprises a sequence that is selected from the group consisting of GHKARNLNEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ED NO: 3); and/or is encoded by an isolated polynucleotide which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and 10; and/or is part of a chimeric or fusion protein.
  • a polynucleotide that contains a mutation located at or
  • the invention is also drawn to antibodies which selectively bind to an amino acid sequence containing a mutation in an HIV CA-SPl protein which results in a decrease in the inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-O-(3 '3 '-dimethylsuccinyl) betulinic acid.
  • the antibody selectively binds to a mutation located at or near the CA-SPl cleavage site or in the SP1 domain of CA-SPl; in another, the antibody selectively binds to a mutation comprising a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment, the antibody selectively binds an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
  • the invention is drawn to an antibody that selectively binds SP1 but not CA-SPl; another that selectively binds CA-SPl but not CA; another that selectively binds CA but not CA-SPl; and a further antibody that selectively binds at or near the CA-SPl cleavage site.
  • the invention is also directed to a compound identified by any of the methods elucidated herein.
  • the compounds is not a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-O-(3',3'-dimethylsuccinyl) betulin, 3-O-(3',3'- dimethylglutaryl) betulin, 3-O-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl- dihydrobetulinic acid, and combinations thereof.
  • the pharmaceutical composition comprises derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin; in another, the pharmaceutical composition comprises a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3 , -dimethylglutaryl) betulin, 3-0-(3',3'- dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof.
  • the pharmaceutical composition comprises one or more compounds identified according to the methods of the invention which are not otherwise listed; or any pharmaceutically acceptable salt, ester or prodrag thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprising an anti-viral agent which may include any one of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclo
  • the invention is also drawn to a method of determining if an individual is infected with HIV-l that is susceptible to treatment by a compound that inhibits p25 processing.
  • the method involves taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the sequence encoding the region of the CA- SPl cleavage site.
  • the invention is also drawn to a method of treating a disease in a patient in need thereof comprising: identifying a compound which inhibits the processing of viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps; obtaining regulatory approval for the sale and use of said compound; packaging the compound for sale and treatment of a disease in a patient in need thereof.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • CA p24
  • Figure 1 DSB does not disrapt the activity of HIN-1 protease at a concentration of 50 ⁇ g/mL. In DSB-containing samples recombinant Gag is processed correctly. In contrast, indinavir blocks protease activity at 5 ⁇ g/mL as evidenced by the absence of bands corresponding to p24 and the MA-CA precursor.
  • Figure 2. Western blots of virion-associated Gag derived from chronically infected H9/HIN-l ⁇ 1IB , H9/HIV-2 ROD , and H9/SIVmac251 in the presence of DSB (1 ⁇ g/mL), indinavir (1 ⁇ g/mL) or control (DMSO). Gag proteins were visualized using HIN-Ig (HIV-l) or monkey anti-SINmac251 serum (HIN-2 and SIV; ⁇ IH AIDS Research and Reference Reagent Program).
  • FIG. 3 EM analysis of DSB-treated HIV-l infected cells.
  • the EM data show two primary differences between DSB-treated and untreated samples. Virions generated in the presence of DSB are characteozed by an absence of conical, mature cores. In these samples the cores are uniformly spherical and often acentric. Secondly, many virions display an electron dense layer inside the lipid bilayer but outside the core (indicated with arrows in the DSB-treated sample panels). In the DSB-treated samples no mature viral particles were observed.
  • Figure 4 depicts amino acid sequences in the region of the CA-SPl cleavage site from DSB-sensitive HIV-l isolates ⁇ L4-3 and RF (#1; SEQ ID NO: 1) and DSB-resistant HIV-l isolates (#2; SEQ ID NO: 2 (NL4-3), and #3; SEQ ID NO: 3 (RF)).
  • the differences between the native and DSB-resistant sequences involve an alanine to valine change at the first downstream residue (#2) and an alanine to valine change in the third downstream residue (#3) from the CA-SPl cleavage site (-
  • Figure 5 depicts the + sense consensus sequence for the A364V DSB- resistant NL4-3 mutant (SEQ ED NO: 4) beginning with the start of gag and continuing into pol, including the entire protease coding region. Missense mutations not found in the wild-type NL4-3 GENBANK Ml 9921 sequence are in bold and gray shadowing.
  • the coding sequence for the consensus CA- SPl cleavage site region is underlined.
  • the shaded area including the cleavage site denotes the SP1 sequence.
  • the first mutation is the A364V mutation.
  • protease The second amino acid change (in protease) was also found in the parental clone and has been confirmed to cooespond to a sequencing error in the original GENBANK entry. Therefore, no mutations actually occurred in protease.
  • Figure 6 depicts the + sense consensus sequence for the DSB-sensitive NL4-3 parental isolate (SEQ ID NO: 5) that was passaged in the absence of drag in parallel with the A364V mutant isolate.
  • Figure 7 depicts the + sense consensus sequence for the A366V DSB- resistant HIV-l RF mutant (SEQ ED NO: 6) beginning with the start of the gag and continuing into pol, including the entire protease coding region. Missense mutations not found in the wild-type HIV-l RF GENBANK M17451 sequence are shadowed in gray. The region of the CA-SPl cleavage site is underlined. The only missense mutation not also found in the identically passaged DSB-sensitive isolate is the A366V mutation in the CA-SPl cleavage site.
  • Figure 8 depicts the + sense consensus sequence for the DSB-sensitive HIV-l RF parental isolate (SEQ ED NO: 7), that was passaged in the absence of drag in parallel with the A366V mutant isolate.
  • Figure 9 depicts the polynucleotide sequences, SEQ ID NO: 8 and SEQ ID NO: 9, which encode the polypeptides designated herein as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • SEQ ID NO: 10 and 12 depict the nucleotide sequences that encode the parental polypeptide sequences designated as SEQ ED NO: 1.
  • SEQ ID NO: 1 is a consensus sequence based on the sequences of the region from NL4-3 and RF
  • Figure 10 10 A. Amino acid sequences in the CA-SPl region of lentivirases. (SEQ ID NO: 13; SEQ ED NO: 11; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 20; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 30; respectively)
  • 10B Amino acid sequences of the CA-SPl region in HIV-l strains RF (SEQ ID NO: 11) and NL4-3 (SEQ ID NO: 13).
  • 10C-10D Nucleotide sequences of gag gene chimeric SIVs.
  • the 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is underlined and in bold.
  • the 42 nucleotide sequences encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage sites are underlined and in bold (nucleotide sequence: SEQ ID NO: 16; amino acid sequence SEQ ED NO: 17). 10F.
  • EIAV Equine Infectious Anemia Virus
  • Nucleotide SEQ ID NO: 96 encoding amino acids SEQ DD NO: 97 10H.
  • Figure 12 Sequential SP1 point deletions in the context of NL4-3 used to identify residues necessary for DSB activity.
  • the amino acid sequence of SP1 domain in NL4-3 is shown. " ⁇ ” indicates the deletion and "— " means identical residues between point deletion mutants and NL4-3 (SEQ ID NO: 13; SEQ DD NO: 33; SEQ DD NO: 34; SEQ ED NO: 35; SEQ DD NO: 36; SEQ DD NO: 85; SEQ DD NO: 86; SEQ DD NO: 87; SEQ ID NO: 88; SEQ DD NO: 89; SEQ DD NO: 100; SEQ DD NO: 101; SEQ DD NO: 102; SEQ DD NO: 103; respectively).
  • Figure 13 Summary of particle production and infectivity of point deletions mutants.
  • Figure 14 Western blots for viruses containing point deletions in SP1, in the presence (+) and absence (-) of DSB.
  • Figure 15. Substitution of HIN-1 CA-SPl residues VL-AEAMSQV (SEQ DD ⁇ O:32) into SIVmac239 backbone renders SINmac239 sensitive to DSB (SEQ ID NO: 14; SEQ DD NO: 15; SEQ DD NO: 20; SEQ DD NO: 27; SEQ DD NO: 28; SEQ DD NO: 13; respectively).
  • Figure 16 Sequence conservation in the CA-SPl region of Lentivimses. Cloning Strategy: Substituting HIV-l specific CA-SPl residues into the cooesponding Gag region of FIN, ELAN or BIN.
  • FIG. HIN-1 ⁇ L4-3 SPl tagged with an epitope. Sequences of SPl peptides with peptide tags inserted are shown. “ ⁇ ” indicates deleted residue and "— " indicates that the residue is identical to that in NL4-3 SPl.
  • Figure 18A-C HIV-l strain RF polynucleotide sequence.
  • the nucleotide sequence of the Gag polyprotein is underlined and in bold.
  • the 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is highlighted in green.
  • An additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (seven amino acids residues) in SPl are highlighted.
  • Figure 19A-E HIN-1 strain ⁇ L4-3 polynucleotide sequence.
  • the nucleotide sequence of the Gag polyprotein is underlined and in bold.
  • the 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is highlighted in green.
  • An additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (seven amino acids residues) in SPl are highlighted.
  • Figure 20 A schematic representation of the Gag protein and the CA- SPl sequences of the SHINs used in this study. The sequences flanking the CA-SPl cleavage site for SIN Mac239 and HIN-1 ⁇ L4-3 are shown at the top and bottom of the list of sequences, respectively.
  • a dashed line (-) represents residues that are identical to the parent SIN MAC239, a delta ( ⁇ ) represents SIV residues that are deleted in the SHINs.
  • Figure 21 Western blot analysis of the Gag processing profiles for panel 1-3 SHIVs.
  • Figure 21 A shows Gag processing from cell-associated viras while
  • Fig. 21B shows the Gag processing profile for cell-free virions. Normal Gag processing is indicated by a plus sign (+), while a defective processing profile is indicated by a minus sign (-).
  • Figure 22 Western blot analysis of the effect of DSB at l ⁇ g/ml on the conversion of the capsid precursor, CA-SPl to mature capsid protein.
  • panel A the viras for Western blotting was obtained using a constant volume of cell culture supernatant. This resulted in variability in the intensity of the viral protein bands due to differences among the SHIVs in the level of viras production.
  • Panel B shows the Gag processing profiles obtained when increased amounts of viral protein are used for Western blot analysis. Only SHTVs that exhibit normal Gag processing are included here.
  • the asterix(*) in panel A indicates the faint CA-SPl band for SHIN GI observed in the autoradiograph cannot be seen, however, the DSB sensitivity for this viras is scored a +/- based on results observed when the analysis is performed using increased amounts of protein (panel B).
  • FIG. 23A-H Alignment of the CA-SPl region in HIV-l clinical isolates, obtained from "HIV Sequence Compendium 2002, " Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, NM. (www.hiv.lanl.gov). Detailed Description of the Invention
  • the present invention is directed to methods of inhibiting HIN-1 replication in the cells of an animal. More specifically, the invention involves methods of inhibiting HIV-l replication in the cells of a mammal by contacting infected cells with a compound that inhibits the processing of the viral Gag p25 protein (CA-SPl) to the p24 protein (CA). More specifically, such compounds inhibit the processing of the viral Gag p25 protein (CA-SPl) to the p24 protein (CA) without significantly affecting other Gag processing steps.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p24 protein
  • a compound that does not significantly affect other Gag processing steps means that the compound in question predominantly inhibits processing of p25 to p24, but does not necessarily preclude the possibility of having additional minor effects on other Gag processing steps.
  • “Significant” or “Significantly,” where not otherwise defined herein, means an observable or measurable change compared to the process in the absence of a compound. However, not all observable or measureable changes may necessarily be significant.
  • a number of viral phenotypes may also be observed in practicing the method of the invention.
  • One result of contacting an infected cell with the compounds of the invention may be the formation of noninfectious viral particles.
  • contacting infected cells with a compound that inhibits p25 to p24 processing results in the formation of non- infectious viral particles, but where there is no significant effect on other Gag processing steps. This may not significantly reduce the quantity of viras released from treated cells and or has no little or no significant effect on the amount of R ⁇ A incorporation into the released virions.
  • the invention is also drawn to a method of inhibiting HIN infection in cells of an animal comprising contacting said cells with a compound that inhibits p25 processing and also affects other viral phenotypes, discribed above.
  • DSB 3-0-(3',3'- dimethylsuccinyl) betulinic acid
  • Mutant forms of HIV-l have been generated in which the amino acid sequence in the region of the CA-SPl cleavage site is modified, decreasing the sensitivity of these strains to compounds that disrapt CA-SPl processing. Data on these mutant viruses have been used to identify the amino acid residues in wild-type Gag that are implicated in the antiviral activity of these compounds.
  • compounds that disrapt CA-SPl processing directly or indirectly inhibit the interaction of HIN-1 protease with the region of the Gag protein containing these amino acid residues.
  • compounds that disrapt CA-SPl processing bind to the region containing these amino acid residues.
  • binding refers to binding or attachment including, e.g., ionic interactions, electrostatic hydrophobic interactions, hydrogen bonds, etc; and also includes associations that may be covalent, e.g., by chemically coupling. Covalent bonds can be, for example, ester, ether, phosphoester, thioester, thioether, urethane, amide, amine, peptide, imide, hydrazone, hydrazide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like.
  • bound is broader than and includes terms such as “coupled,” “conjugated” and “attached.”
  • compounds that disrapt CA-SPl processing bind to another region of Gag and thereby inhibit the interaction of HIN-1 protease with the region of the CA-SPl cleavage site.
  • viruses or recombinant proteins that contain mutations in the region of the CA- SPl cleavage site can be used in screening assays to identify compounds that disrupt CA-SPl processing.
  • amino acid residues in HIN-1 Gag that are involved in the disruption of CA-SPl processing by 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB) were identified by sequencing the gag- pol gene of virus isolates that had been selected for resistance to DSB.
  • the amino acid sequences from these resistant viruses were compared with the gag-pol gene sequences from DSB-sensitive HIV-l isolates. Two single amino acid changes were identified in the DSB-resistant viruses, an alanine (Ala) to valine (Val) substitution at residue 364 (SEQ DD NO: 4) and in a second isolate, at residue 366 (SEQ DD NO: 6), in the Gag polyprotein (see Figure 4). These residues are located immediately downstream of the CA-SPl cleavage site (at the N-terminus of SPl). Alanine is highly conserved at these positions throughout all HIV-l subtypes listed in the Los Alamos National Laboratory database.
  • the five amino acid residues upstream and downstream of the CA-SPl cleavage site are also highly conserved among the various subtypes. However, isoleucine replaces valine at the position two residues upstream of the cleavage site in a number of clades (c.f, Figure 4, SEQ DD NO. 1).
  • HAV Sequence Compendium 2002 " Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, NM.)
  • an A366V mutation was identified in the SPl region of NL4-3 viras cultured in the presence of DSB (note: numbering is relative to the Gag polyprotein).
  • a double mutant was identified that contained a G357S mutation in CA as well as the A366V mutation in SPl.
  • the A366V mutation was identified previously in experiments selecting for resistant variants of the RF isolate.
  • the wild-type RF sequence also contains a serine residue at position 357 in CA ( Figure 4). Since serine is present at this position in isolates (such as RF) that are sensitive to DSB, the CA G357S mutation alone is not sufficient to confer resistance to DSB.
  • the A366V mutation and the A366V/G357S double mutation were re-engineered into the wild-type NL4-3 backbone by site- directed mutagenesis.
  • the resulting constructs were transfected into Jurkat T cells and characterized in a viras replication assay as described above for the selection of resistance.
  • SDS-PAGE analysis of transfected cell lysates and virus released into the media demonstrated that the A366V mutant Gag was processed and released from cells inefficiently (data not shown) and thus replicated very poorly even in the absence of drag (Figure 11)
  • the A366V/G357S double mutant replicated efficiently in the absence or presence of DSB.
  • the resistant mutant, A366V requires a serine at the 357 position in the CA region of Gag to compensate for a deleterious effect on viras replication (Figure 11).
  • HIN-1 point-deletion mutagenesis and SIN insertion studies were undertaken to identify the specific amino acid residues associated with compound activity. The study was carried out as follows. Single residue deletions starting with residue E365 and continuing through residue M377 were engineered into the SPl domain of the infectious HIN-1 molecular clone ⁇ L4-3 ( Figure 12). The effect of these point deletions on viral particle production, infectivity, Gag processing and sensitivity to DSB was determined.
  • ⁇ E365 was generated using NL4-3 as the template with Vent DNA polymerase (NEB) by using deletion-specific downstream primer (Primer 1) with universal upstream primer (Primer 2) (Table 1). The fragment derived from this was termed as a first flanking PCR fragment. A second flanking fragment was amplified using deletion-specific upstream primer (Primer. 3) and universal downstream primer (Primer 4) (Table 1). To generate other deletion constructs ( ⁇ A366, ⁇ M367, ⁇ S368, ⁇ Q369, ⁇ V370, ⁇ T371, ⁇ N372, ⁇ P373, ⁇ A374, ⁇ T375, ⁇ I376, and ⁇ M377).
  • PCR procedures were similarly performed by varying deletion-specific downstream and upstream primers cooesponding to each specific point deletion (Table 1). [0113] Each of these parallel two adjacent PCR fragments was gel purified, phosphorylated using T4 polynucelotide kinase (NEB), and ligated by using T4 DNA ligase (NEB). After inactivation at 65°C for 15 minutes, the ligation reaction was used for a subsequent amplification with universal upstream primer (Primer. 2) and downstream primer (Primer. 4). This product was gel purified, digested with Spel and Apal, and then ligated into the Spel and Apal sites of NL4-3 pro viral DNA clone.
  • NEB T4 polynucelotide kinase
  • NEB T4 DNA ligase
  • Standard PCR conditions were used for the above-described reactions. These included, one cycle of denaturation at 95 °C for 1 minutes 30 seconds, followed by 30 cycles of denaturation at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds.
  • the PCR reactions were set up using the following components: 5 ⁇ L 10 x NEB Thermophilic buffer 2 ⁇ L 10 mM dNTPs l ⁇ L 100nM MgSO 2 1 ⁇ L 50 pmol upstream primer 1 ⁇ L 50 pmol downstream primer 1 ⁇ L 50 ng/ ⁇ L template DNA 0.5 ⁇ L Vent DNA polymerase 38.5 ⁇ L ddH 2 O
  • the phosphorylation reaction was set up as follows: 2 ⁇ L 10X T4 polynucleotide kinase buffer 2 ⁇ L lOmM ATP 1 ⁇ L T4 polynucleotide kinase [0116] 15 ⁇ L gel purified DNA of each of these two adjacent PCR fragments The reaction was incubated at 37°C for 1 hour. Following the inactivation at 65°C for 10 minutes, the adjacent phosphorylated PCR fragments were then ligated together by using T4 DNA ligase (NEB) under following conditions: 3 ⁇ L 10X T4 DNA ligase buffer 13 ⁇ L of each of two adjacent PCR fragments 1 ⁇ L T4 DNA ligase
  • the ligation reaction product was used in a second round PCR reaction to amplify the full-length PCR fragment spanning these two adjacent PCR products.
  • the second round PCR reaction was performed as described above with the exception that only universal upstream primer (Primer. 2) and downstream primer (Primer. 4) were used. Again, a 10 ⁇ L aliquot was run on a agarose gel to make sure the cooect product was amplified. The full-length PCR fragments were then gel isolated and purified using a Qiaex II kit.
  • the digested vector NL4-3 and full length PCR fragment were ligated using T4 DNA ligase under the following procedure: 1 ⁇ L 10X T4 DNA ligase buffer 1 ⁇ L (25-50 ng) digested NL4-3 vector 7 ⁇ L digested (200 ng-400 ng) digested PCR fragment (700 bp) 1 ⁇ L T4 DNA ligase [0119]
  • the ligation reaction was incubated at 16°C overnight and the ligated products were transformed into Escherichila coli Max Efficiency Stbl2 (Invitrogen) by heat shock according to instruction (Invitrogen).
  • proviral DNA clones were then screened by automatically sequencing using a Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems) individually using internal primers (Primer 29 and 30) Following the verification the mutations the proviral DNA clones were used for various future studies.
  • a panel of SIN chimeric constructs harboring various residues of ⁇ L4- 3 CA-SPl boundary region was generated using the SIVmac239 molecular clone by employing PCR and cloning procedures described above. These constructs and their amino acid sequences in the CA-SPl boundary region are shown in Figure 15.
  • SIN mac239 was used to generate the SIN DD and DE constructs.
  • the SIN DD construct was used to generate SIN DM.
  • Different SIN chimeric constructs were produced in the PCR by varying respective mutagenic upstream and downstream primers corresponding to each chimera (Table 1).
  • HeLa cells were maintained in DMEM (Invitrogen) (10% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml Streptomycin) and passaged upon confluence.
  • DMEM Invitrogen
  • Jurkat cells were cultured in RPMI 1640 (Invitrogen) (10% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml Streptomycin) and passaged every two or three days.
  • wild-type HIN-1 ⁇ L4-3 or SF mac239 and respective mutant proviral DNAs were transfected into HeLa cells by employing FuGENE 6 transfection reagent (Roche). Briefly, cells were seeded into a 6-well plate (Coming) at a concentration of 0.5 x 105 per well the day before transfection to reach 60 to 80% confluence on the day of transfection. For each transfection, 3 ⁇ l of FuGENE 6 was diluted into 100 ⁇ l of serum- free DMEM followed by the addition of 1 ⁇ g of DNA.
  • DNA-lipid complexes was gently added drop wise into the cells containing 2 ml of complete DMEM medium. Twenty-four hours post-transfection, medium containing DNA-FuGENE 6 complexes was removed, 2 ml of fresh DMEM was added into the transfected cells. At 48 h post-transfection, medium containing viral particles was collected and clarified by centrifugation at 2,000 rpm at 4°C for 20 min in a Sorvall RT 6000B centrifuge.
  • Viras particle-containing supematants were then concentrated through a 20% sucrose cushion in a microcentrifuge at 13,000 rpm at 4°C for 120 min and pellets were resuspended in a lysis buffer (150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholate, pH 8.0).
  • the level of viral particle production for wild type NL4-3 and point deletion mutants was determined by p24 antigen capture ELISA (ZeptoMetrix, Buffalo, NY). To examine the effect of deletion or substitution on Gag polyprotein processing (in the absence of DSB), SDS-PAGE and Westem-Blot was performed.
  • viral proteins were separated on a 12% NuPAGE Bis-Tris Gel (Invitrogen) and transfeoed to a nitrocellulose membrane (Invitrogen) followed by blocking in a PBS buffer containing 0.5% Tween and 5% dry milk.
  • the membrane was incubated with immunoglobulin from HIV-1- infected patients (HlV-Ig) (NIH ADDS research and reference reagent program) and hybridized with goat anti-human horseradish peroxidase (Sigma).
  • the membrane containing SIV proteins was incubated with a reference polyclonal immune serum from a SIN-infected monkey (N H ADDS Research and Reference Reagent Program) and hybridized with goat-anti-monkey horseradish peroxidase (Sigma). The immune complex was visualized with an ECL system (Amersham Pharmacis Biotech) according to the instructions provided by the manufacturer.
  • ECL system Aminogen-activated Cell Sorting System
  • HeLa cells were transfected with wild-type HIV-l NL4-3 or SINmac239 and respective mutant proviral D ⁇ As by employing the procedure described above.
  • DSB at a concentration of 1 ⁇ g/ml and DMSO control were maintained throughout the entire culture and SDS- PAGE/Western-Blot for analyzing viral proteins derived from these transfections were performed as described in the previous paragraph.
  • TCED 5 0 50% -Tissue Culture Infectious Dose per ml was used as a measure of the infectivity of each deletion mutant.
  • Mutant viruses derived from transfections in HeLa cells were used to infect U87 CD4.CXCR4 cells. Each virus stock was tested in triplicate at a starting dilution of 1 : 10, followed by four-fold serial dilutions. Cells were plated the day before infection at a density of 3xl0 3 cells/well. On the day of infection, culture media was removed from the cell plate and 90 ⁇ l of diluted viras was added. On days 1, 3, and 6 post infection, virus was removed from plate and 200 ⁇ l of culture media was added.
  • TCDD 50 The viras dilution that caused 50% of the culture to be infected was determined according to the method of Reed and Muench (Aldovini A. and B. Walker 1990; Dulbecco R. 1988).
  • Sensitivity to DSB was determined in experiments that characterized the effect of DSB on a late step in Gag processing, CA-SPl cleavage. Specifically, these assays measured the ability of DSB to disrupt CA-SPl processing. As seen, e.g. Example 8, the DSB-induced defect in Gag processing cooelates with the ability of the compound to inhibit virus replication. Results from these experiments indicate that deletion of a single residue at any of the six positions E365 through V370 significantly reduces the affect of DSB on CA-SPl processing ( Figure 14). In contrast, starting with residue T372 and extending away from the CA-SPl cleavage site, all of the characterized point deletions are fully sensitive to DSB-induced disraption of CA-SPl processing ( Figure 14).
  • the SPl residues associated with DSB activity consist of the contiguous residues E365 through V370.
  • HIV-l maturation inhibitors disrapt Gag CA-SPl processing, which results in the formation and release of non-infectious viral particles exhibiting aberrant core morphology. See e.g. Li et al. Proc Natl Acad Sci U S A. 100:13555-60 (2003).
  • the betulinic acid derivative DSB is an example of this class of inhibitors.
  • the viral genetic determinants critical that are associated with the activity of maturation inhibitors map to amino acid residues flanking the HIN-1 CA-SPl cleavage site. When this determinant is introduced into the CA-SPl cleavage sites of DSB-resistant non-HIV-1 viruses, maturation inhibitor sensitive chimeras result.
  • CA-SPl chimeric viruses serve as the basis for an animal efficacy model for HIN-1 maturation inhibitors.
  • the region of HIN-1 CA-SPl necessary for maturation inhibitor sensitivity is introduced into selected lentiviruses. Amino acid residues from HIN-1 CA-SPl junction that are determinants of DSB sensitivity were used to replace the corresponding CA-SPl amino acids in the genome of Simian Immunodeficiency (SIN).
  • HIN-1 CA- SPl junction determinants of DSB
  • FV Feline Immunodeficiency virus
  • BIV Bovine Immunodeficiency Virus
  • EIAV Equine Infectious Anemia Viras
  • CAEV Caprine Arthritis Encephalitis virus
  • Table 2 depicts the Gag polypeptide sequence for HIN-1, SIN, FIN, EIAV and BIN in the region of the CA-SPl cleavage site.
  • the HIV-l CA-SPl sequence used for replacement is as follows: CA SPl GHKARVL AEAMSQV ( SEQ ID NO : 80 ) [0136] The method described above for generating the SHIV CA-SPl chimeric provirus DNA clone is used to generate FIV, EIAV and BIV proviras clones containing selected residues or extended region from CA-SPl region of HIN-1 replacing the corresponding wild-type sequence ( Figure 16). [0137] The SHIN CA-SPl chimeric-provirus D ⁇ A clone was generated by site-directed mutagenesis employing standard molecular biology techniques.
  • the unique restriction enzyme sites in the SIV Gag that suoounding the CA-SPl region were identified i.e., BamHI (in matrix) and Sbf-I (in ⁇ C).
  • BamHI in matrix
  • Sbf-I in ⁇ C.
  • a forward and a reverse primer incorporating the mutated sequence i.e., HTV CA-SPl at their 5' ends were synthesized.
  • the fragments, Bam HI-CA-SPl and CA- SPl -Sbf-I were annealed at their common HTV-CA-SP1 sequence and amplified with a forward SIN Bam HI primer and a reverse SIN Sbf-I primer to generate a full-length chimeric SHIN CA-SPl gag fragment.
  • the chimeric SHIN CA-SPl PCR fragment was cloned into BamHI - Sbf-I window of SIV provirus clone replacing the SIN-Gag wild-type sequence to yield the SHIN CA-SPl provirus cD ⁇ A clone.
  • the chimeric HIN-1 CA-SPl fragment is digested with the appropriate restriction enzyme and cloned into SacI-EcoRI window of FTV provirus; or (ii) KasI-EcoRN window of EIAV provirus; or (iii) BsrGI-Apal window of BTV provirus replacing the cooesponding wild type sequence ( Figure 16).
  • the chimeric FIN/EIAN/BIN-HIN-1 CA-SPl provirus D ⁇ A clones are sequenced to confirm the presence of intended mutations. Based on observed results that indicate the transfer of DSB sensitivity, additional constructs are generated employing the above strategy in order to optimize the results.
  • a chimeric viras was generated in which the CA-SPl determinant of HTV-1 maturation inhibitor sensitivity has replaced the analogous region of Gag in the maturation inhibitor-resistant simian immunodeficiency virus (SIV). Transfer of this region of HTV-1 into the genome of STV results in a maturation inhibitor-sensitive phenotype. Infection of a non-human primate with this HIN-1 /SIN chimeric viras should result in an animal efficacy model for therapeutic development of maturation inhibitors.
  • SIV maturation inhibitor-resistant simian immunodeficiency virus
  • mutant and chimeric viruses of the present invention are useful in a variety of cell based as well as animal based assays.
  • the invention includes a method of identifying a compound that inhibits cleavage of p25 to p24 in wild type HTV-1, but does not inhibit CA-SPl processing in HIN-1 containing a deletion in the CA-SPl region. Compounds obtained by such a method are also included in the present invention.
  • Chimeras of SIN and other lentiviruses that do not readily infect humans have additional advantages. Firstly, these viruses pose a lesser safety hazard to laboratory workers. As a result, cell based assays to identify novel compounds that inhibit CA-SPl processing, for example, can be conducted with less risk. The lower risk may allow assays to be performed that cannot be performed readily or safely with HIN, and may also lower the cost of such assays. [0144] Furthermore, such chimeric vimses are useful in animal models.
  • chimeric SIN that is sensitive to DSB may be used to identify novel compounds that inhibit CA-SPl processing, for example; to identify pharmaceutical compositions, routes of administration and dosage regimes for treatment of disease; and for studying the effect of combination therapies, such as DSB with protease inhibitors.
  • animal models may be used to identify appropriate pharmaceutical compositions for the treatment of animal diseases, of interest in the treatment of companion animals and other high value animals, such as agricultural breeding stock and race horses.
  • Chimeric vimses may be derived from any retroviras.
  • derived HIN-2, HTLV-I, HTLN-II, SIN avian leukosis virus (ALV), endogenous avian retroviras (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency viras (FIN), Bovine immunodeficiency virus (BIV), caprine arthritis encephalitis viras (CAEV), Equine infectious anemia viras (EIAV), Visna-maedi virus, or feline leukemia virus (FeLV).
  • AMV avian leukosis virus
  • EAV endogenous avian retroviras
  • MMTV mouse mammary tumor virus
  • BIV Bovine immunodeficiency virus
  • CAEV caprine arthritis encephalitis viras
  • EIAV Equine infectious anemia viras
  • Visna-maedi virus Visna-maedi virus
  • Such chimeric viruses may be used in the methods of the invention described elsewhere herein.
  • such recombinant non-HIV-1 lentiviruses may be used in a method of identifying a compound which inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method consisting of comparing of the ability of said compound to inhibit replication of a wild-type non-HIN-1 lentivirus with the DSB-sensitive recombinant variant thereof .
  • Such inhibition may occur in a cell; in an animal; or in vitro.
  • the present invention is also drawn to recombinant retroviruses with epitope tags in the CA-SPl region of Gag.
  • Epitope tags may be inserted in the CA domain and/or in the SPl domain.
  • Suitable tags are well known to those of ordinary skill in the art, and include haemagglutinin epitope HA (YPYDVPDYA) (SEQ ED NO: 81), bluetongue viras epitope VP7 (QYPALT) (SEQ ED NO: 82), ⁇ -tubulin epitope (EEF), Flag (DYKDDDDK) (SEQ ED NO: 83), and VSV-G (YTDIEMNRLGK) (SEQ ED NO: 84).
  • Such epitope tagged viruses and fragments thereof are useful in identifying novel compounds that inhibit CA-SPl processing in vitro, in cell based assays, and in vivo, including in animal models. Additional uses of such epitope tagged viruses and fragments thereof are described elsewhere herein.
  • the invention also includes isolated polypeptides and polynucleotides.
  • the invention includes polypeptides at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQ GVGGPSHKARILAEAMSQVTNSATDVI (SEQ ED NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQ GVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (e) SHKARILAEAMSQV (SEQ ED NO: 25); (f) GHKARVLAEAMSQV (a) KNWM
  • the invention includes polynucleotides encoding the above polypeptides.
  • Polynucleotides of the invention include degenerate variants, such as those that differ in the third base of the codon but nevertheless encodes the same amino acid due to coding "degeneracy”.
  • polypeptides and polynucleotides of the invention are useful in the methods of the invention.
  • they may be used in an in vitro assay to identify compounds that bind to the CA-SPl region of Gag.
  • they may be used in the production of antibodies useful in other methods described elsewhere herein.
  • a polynucleotide may be inserted into a vector and thereupon into a host cell for production of polypeptide.
  • the above embodiments are exemplary and are not intended to be limiting.
  • the present invention comprises a polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in the HTV Gag p25 protein (CA-SPl), said mutation resulting in a decrease in the inhibition of processing of ⁇ 25 (CA-SPl) to p24 (CA) by DSB.
  • the polynucleotide of the invention includes a mutation which is optionally located at or near the CA-SPl cleavage site or located in the SPl domain of CA-SPl. Said mutation can be present in an amino acid sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ED NO: 2) and SHKARILAEVMSQV (SEQ DD NO: 3).
  • the polynucleotide of this invention is also drawn to sequences designated as SEQ ED NO: 4, SEQ ED NO: 6, SEQ DD NO: 8 and SEQ ED NO: 9.
  • the invention also includes a vector comprising said polynucleotide, a host cell comprising said vector and a method of producing said polypeptides comprising incubating said host cell in a medium and recovering the polypeptide from the medium.
  • the invention further includes a polynucleotide that hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
  • the invention also includes a polynucleotide which hybridizes to SEQ NO: 5, SEQ ED NO: 7 or SEQ ED NO: 10 or 12, which contains a mutation which results in the decrease in the inhibition of processing of p25 to p24 by 3-0-(3',3'- dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl.
  • the invention is also directed to a vector comprising said polynucleotides, a host cell comprising said vector and a method of producing said polypeptides, comprising incubating said host cell in a medium and recovering said polypeptide from the medium.
  • Near or “adjacent,” as used herein in reference to polypeptides is meant to include about 50, about 25, about 20, or about 15 residues from the point of reference.
  • near may encompass about 50, about 25, about 20 or about 15 residues on either side of the HTV-1 Gag CA-SPl cleavage site; more preferably about ten residues on either side of the HTV-1 Gag CA-SPl cleavage site; and most preferably about seven residues on either side of the HTV-1 Gag CA-SPl cleavage site.
  • the terms “near” or “adjacent refer to about 150, about 75, about 60, about 45, or about 30 nucleotides from the point of reference.
  • isolated means altered “by the hand of man” from the natural state. If an composition or substance occurs in nature, it has been changed or removed from its original environment, or both, when found in its "isolated” form.
  • isolated nucleic acid molecule(s) of the invention is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often refeoed to as oligonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly refeoed to as peptides, oligopeptides or oligomers, and to longer chains, generally refeoed to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, foonation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
  • “Mutant” as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively.
  • a typical mutant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the mutant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical mutant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a mutant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a mutant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a mutant that is not known to occur naturally. Non-naturally occurring mutants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • the mutant, (or fragments, derivatives or analogs) of a polypeptide encoded by any one of the polynucleotides described herein may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (a conserved amino acid residue(s), or at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG:Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or
  • mutants are deemed to be within the scope of those skilled in the art from the teachings herein. Polynucleotides encoding these mutants are also encompassed by the invention. "Mutant” as used herein is equivalent to the term “variant.”
  • Substitutions of charged amino acids with another charged amino acids and with neutral or negatively charged amino acids are included.
  • one or more of the amino acid residues of the polypeptides of the invention e.g., arginine and lysine residues
  • proteases such as, for example, furins or kexins.
  • the prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al, Clin Exp. Immunol.
  • polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
  • nonconservative substitutions of amino acids is used to render a DSB sensitive viras resistant to DSB.
  • the polynucleotides encompassed by this invention may have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with a reference sequence, providing the reference polynucleotide encodes an amino acid sequence containing a mutation in the CA-SPl protein, said mutation which results in the decrease in the inhibition of processing of p25 to p24 by a 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • the polynucleotides also encompassed by this invention include those mutations which are "silent," in which different codons encode the same amino acid (wobble).
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences.
  • identity is used interchangeably with the word “homology” herein. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans.
  • Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Baxevanis and OuUette, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Second Edition, Wiley-Interscience, New York, (2001). Methods to determine identity and similarity are codified in computer programs. Prefeoed computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J. et al, Nucleic Acids Research i2(l):387, (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al, J. Molec. Biol 215:403, (1990)).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid.
  • the polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • the reference sequence may be the entire nucleotide sequence of any one of the nucleotide sequences of the invention or any polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of the invention and or C terminal deletion).
  • nucleotide sequences of the invention can be determined conventionally using known computer programs such as the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, (Advances in Applied Mathematics 2:482-489 (1981)), to find the best segment of homology between two sequences.
  • the parameters are set, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the identity between a sequence of the present invention and a subject sequence is deteonined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is cooected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This cooected score is what is used for the purposes of this embodiment.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence, which are not matched aligned with the query. In this case the percent identity calculated by FASTDB is not manually cooected. Only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually cooected. No other manual cooections are made for the purposes of this embodiment.
  • the present application is directed to nucleic acid molecules having at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%) or 99% identity or which is identical to the nucleic acid sequence disclosed herein, or fragments thereof, ioespective of whether they encode a polypeptide having the disclosed functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having the disclosed functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having the disclosed functional activity include, inter alia: (1) isolating the variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to determine cellular location or presence of the disclosed sequences, and (3) Northern Blot analysis for detecting mRNA expression in specific tissues.
  • FISH in situ hybridization
  • PCR refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis et al, as well as improvements now known in the art.
  • conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • stringent conditions refers to homology in hybridization, is based upon combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions, and well known in the art (Sambrook, et al. supra).
  • the invention includes an isolated nucleic acid molecule comprising, a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the sequence complementary to the coding and/or noncoding (i.e., transcribed, untranslated) sequence of any polynucleotide or a polynucleotide fragment as described herein.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising, or alternatively consisting of: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing in O.lx SSC at about 65°C.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention also includes a viras comprising the polynucleotides of the invention, and wherein the viras includes a retroviras comprising said polynucleotides, and wherein the retroviras may be a member of the group consisting of HTV-1, HIN-2, HTLV-I, HTLV-II, STV, avian leukosis viras (ALV), endogenous avian retroviras (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIN), or feline leukemia viras (FeLN).
  • AAV avian leukosis viras
  • EAV endogenous avian retroviras
  • MMTV mouse mammary tumor virus
  • FIN feline immunodeficiency virus
  • FeLN feline leukemia viras
  • the invention further includes a polypeptide containing a mutation in the CA-SPl protein, said mutation which results in the decrease in inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or located in the SPl domain of SEQ ED NO: 5 or SEQ ED NO: 7 (parental polynucleotide sequences) encoding the CA-SPl protein.
  • Said polypeptide may be encoded by a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ DD NO: 9, or may compose a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ DD NO: 2) and SHKARD AEVMSQV (SEQ ED NO: 3).
  • the polypeptide of this invention may further be encoded by a polynucleotide which hybridizes to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
  • the invention also includes a polypeptide encoded by a polynucleotide which hybridizes to SEQ NO: 5, SEQ DD NO: 7 or SEQ DD NO: 10 or 12, which contains a mutation that results in decrease in inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl.
  • the polypeptide of this invention further includes polypeptides that are part of a chimeric or fusion protein.
  • Said chimeric proteins may be derived from species which include, but are not limited to: primates, including simian and human; rodentia, including rat and mouse; feline; bovine; ovine; including goat and sheep; canine; or porcine. Fusion proteins may include synthetic peptide sequences, bifunctional antibodies, peptides linked with proteins from the above species, or with linker peptides. Polypeptides of the invention may be further linked with detectable labels; metal compounds; cofactors; chromatography separation tags, such as, but not limited to: histidine, protein A, or the like, or linkers; blood stabilization moieties such as, but not limited to: transferrin, or the like; therapeutic agents, and so forth.
  • the invention also includes an antibody which selectively binds an amino acid sequence containing a mutation in the CA-SPl protein that results in a decrease in the inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-0-(3',3'-dimethylsuccinyl) betulinic acid and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl.
  • the invention also includes an antibody which selectively binds the polypeptide having a mutation which comprises a sequence that is one of GHKARVLVEAMSQV (SEQ ED NO: 2), SHKARILAEVMSQV (SEQ ED NO: 3).
  • Said antibody can selectively bind the polypeptide encoded by a polynucleotide sequence selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
  • Said antibody can also selectively bind the polypeptide encoded by a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
  • the invention also includes an antibody that selectively binds SPl, which would enable one to distinguish SPl from CA-SPl (p25).
  • the invention also includes an antibody that selectively binds CA (p24), which would enable one to distinguish CA from CA-SPl.
  • the invention also includes an antibody that selectively binds CA-SPl, which would enable one to distinguish CA from CA-SPl.
  • the invention additionally includes an antibody that selectively binds at or near the CA-SPl cleavage site.
  • the antibody of this invention may be a polyclonal antibody, a monoclonal antibody or said antibody may be chimeric or bifunctional, or part of a fusion protein.
  • the invention further includes a portion of any antibody of this invention, including single chain, light chain, heavy chain, CDR, F(ab') 2 , Fab, Fab', Fv, sFv, or dsFv, or any combinations thereof.
  • an antibody "selectively binds" a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins.
  • the term “selectively binds” also comprises determining whether the antibody selectively binds to the target mutant sequence relative to a native target sequence.
  • An antibody which "selectively binds" a target peptide is equivalent to an antibody which is "specific” to a target peptide, as used herein.
  • An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody.
  • the determination whether the antibody selectively binds to the mutant target sequence composes: (a) deteonining the binding affinity of the antibody for the mutant target sequence and for the native target sequences; and (b) comparing the binding affinities so determined, the presence of a higher binding affinity for the mutant target sequence than for the native indicating that the antibody selectively binds to the mutant target sequence.
  • the invention is further drawn to an antibody immobilized on an insoluble carrier comprising any of the antibodies disclosed herein.
  • the antibody immobilized on an insoluble carrier includes multiple well plates, culture plates, culture tubes, test tubes, beads, spheres, filters, electrophoresis material, microscope slides, membranes, or affinity chromatography medium.
  • the invention also includes labeled antibodies, comprising a detectable signal.
  • the labeled antibodies of this invention are labeled with a detectable molecule, which includes an enzyme, a fluorescent substance, a chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, an electron dense substance, and a radioisotope, or any combination thereof.
  • the invention further includes a method of producing a hybridoma comprising fusing a mammalian myeloma cell with a mammalian B cell that produces a monoclonal antibody which selectively binds an amino acid sequence containing a mutation in the CA-SPl protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'- dimethylsuccinyl) betulinic acid and a hybridoma producing any of the monoclonal antibodies disclosed herein.
  • the invention further includes a method of producing an antibody comprising growing a hybridoma producing the monoclonal antibodies disclosed herein in an appropriate medium and isolating the antibodies from the medium, as is well known in the art.
  • the invention also includes the production of polyclonal antibodies comprising the injection, either one injection or multiple injections of any of the polypeptides of the inventions into any animal known in the art to be useful for the production of polyclonal antibodies, including, but not limited to mouse, rat, hamster, rabbit, goat, sheep, deer, guinea pig, or primate, and recovering the antibodies in sera produced therein.
  • the invention includes high avidity or high affinity antibodies produced therein.
  • the invention also includes B cells produced from the listed species to be further used in cell fusion procedures for the manufacture of monoclonal antibody-producing hybridomas as disclosed herein.
  • the invention is further drawn to a kit comprising the antibody or a portion thereof as disclosed herein, a container comprising said antibody and instructions for use, a kit comprising the polypeptides of this invention and instructions for use and a kit comprising the polynucleotide of the invention, a container comprising said polynucleotide and instructions for use, or any combinations thereof.
  • kits would include, but not be limited to nucleic acid detection kits, which may, or may not, utilize PCR and immunoassay kits. Such kits are useful for clinical diagnostic use and provide standardized reagents as required in current clinical practice. These kits could either provide information as to the presence or absence of mutations prior to treatment or monitor the progress of the patient during therapy.
  • the kits of the invention may also be used to provide standardized reagents for use in research laboratory studies.
  • the invention is also directed to a compound, a method of using a compound, a method of identifying a compound and the like.
  • Compounds useful in the methods of the present invention include derivatives of betulinic acid and betulin that are presented in U.S. Patent Nos. 5,679,828 and 6,172,110 respectively, and in U.S. application Nos. 60/443,180 and 10/670,797, which are herein incorporated by reference. Additional useful compounds include oleanolic acid derivatives disclosed by Zhu et al. (Bioorg. Chem Lett. 77:3115-3118 (2001)); oleanolic acid and promolic acid derivatives disclosed by Kashiwada et al. (J. Nat. Prod.
  • compounds useful in the present invention include, but are not limited to those betulinic acid derivatives having the general Formula /and dihydrobetulinic acid derivatives of Formula//:
  • R is a C 2 -C 20 substituted or unsubstituted carboxyacyl
  • R' is hydrogen, C 2 -C ⁇ o substituted or unsubstituted alkyl, or aryl group.
  • Prefeoed compounds are those wherein R is one of the substituents in Table 4, below, and R' is hydrogen.
  • useful compounds include derivatives of betulin and dihydrobetulin of Formula HI:
  • Ri is a C 2 -C 20 substituted or unsubstituted carboxyacyl, or an ester thereof;
  • R 2 is hydrogen, C(C 6 H 5 ) 3 , or a C 2 -C 2 o substituted or unsubstituted carboxyacyl;
  • R 3 is hydrogen, halogen, amino, optionally substituted mono- or di- alkylamino, or alkanoyl, benzoyl, or C 2 -C 20 substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29.
  • Prefeoed compounds useful in the present invention are those where R ⁇ is one of the substituents in Table 4, R 2 is hydrogen or one of the substituents in Table 4 and R 3 is hydrogen.
  • Table 4 Preferred Substituents for R, R', R R 2 :
  • More prefeoed compounds are 3-0-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, and 3-O-(3',3'-dimethylsuccinyl or glutaryl) dihydrobetulin.
  • a particularly prefeoed compound is 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • R ⁇ -OR ⁇ 4 or -NHR 15 ;
  • R 12 COOR ⁇ , COO " A + , or CH 2 OR 17
  • R 13 -H, halogen, amino, optionally substituted mono-or di-alkylamino, or -OR 16 ;
  • R 14 -H, C 2 - C 2 o substituted or unsubstituted carboxyacyl;
  • Ri 5 -H, C 2 - C 2 o substituted or unsubstituted carboxyacyl;
  • R 16 -H, C - C 7 alkanoyl, benzyloyl, or C 2 - C 20 substituted or unsubstituted carboxyacyl;
  • R 38 moieties other than hydrogen are attached to R 33 oxygen by a covalent bond to the carbonyl carbon.
  • Prefeoed compounds are those where R 38 is not hydrogen.
  • any of R 38 , R 40 and/or R 41 are methyl.
  • compounds useful in the methods of the invention also include those described in U.S. Provisional Application No. 60/559,358, which is entirely incorporated by reference. In one aspect, these compounds are described by reference to the following compounds VIII to XI: [0196] In some embodiments, compounds useful in the present invention have the general Formula VHP.
  • R 5 1 is a carboxyalkanoyl, where the alkanoyl chain can be optionally substituted by one or more hydroxy or halo, or can be interrupted by a nitrogen, sulfur or oxygen atom, or combinations thereof;
  • R 5 2, R 53 and RM are independently hydrogen, methyl, halogen, or hydroxy, carbonyl or -COOR ⁇ , wherein R ⁇ is alkyl or carboxylalkyl, where the alkyl chain can be optionally substituted by one or more nydroxyl or halo, or can be interrupted by nitrogen, sulfur or oxygen atom, or combinations thereof;
  • R 55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyaUcanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which
  • R 51 cannot be glutaryl or succinyl when a double bond exists between C12 and C13; when A is (ii) and is methyl, then R 51 cannot be succinyl; when A is (iii) and R 52 , R 53 and R « are each hydrogen, then R ⁇ cannot be succinyl; and with the proviso that A (i) cannot be
  • R 51 is a carboxy(C 2-6 )alkylcarbonyl group or a carboxy(C 2-6 )alkoxy(C ⁇ - 6 )alkylcarbonyl group.
  • Suitable groups are selected from the group consisting of:
  • the compounds have Formula IX:
  • R 51 , R 54 , R 55 , R 56 , R 57 , R 58 and R 64 are as defined above for Formula VIII.
  • R 56 is ⁇ -methyl
  • R 58 is hydrogen
  • R 55 is hydroxymethyl
  • R 5 ⁇ is 3',3'-dimethylglutaryl, 3',3'-dimethylsuccinyl, glutaryl or succinyl.
  • R 56 is hydrogen
  • R 57 and R 58 are both methyl
  • R 55 is carboxyl
  • R 51 is 3',3'-dimethylglutaryl, 3',3'- dimethylsuccinyl, glutaryl or succinyl.
  • R 55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo, or R 5 is a carboxyl or hydroxymethyl.
  • R55 is selected from a group consisting of carboxyl, hydroxymethyl, -CO 2 (CH 2 ) n COOH, -CO 2 (CH 2 ) n CH 3 , -CH 2 OC(O)(CH 2 ) n CH 3> -CH 2 OC(O)(CH 2 ) n COOH, -CO(CH 2 ) n CH 3 and -CO(CH 2 ) n COOH.
  • R 55 is selected from a group consisting of
  • R 5 5 is hydroxymethyl. In some embodiments, R 55 is carboxyl. In some embodiments, n is from 0 to 20, and preferably 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is from 1 to 6. In some embodiments, n
  • compounds useful in the present invention have the Formula X:
  • R 5 t is 3',3'-dimethylglutaryl, 3 ',3 '-dimethylsuccinyl, glutaryl or succinyl.
  • R ⁇ is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo.
  • R ⁇ is selected from the group consisting of methyl, -CO 2 (CH 2 ) n COOH, -COC(O)(CH 2 ) n CH 3 , -CO(CH 2 ) n CH 3 and -CO(CH 2 ) n COOH.
  • n is from 0 to 20, or preferably 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is from 1 to 6. In some embodiments, n is from 2 to 6.
  • R ⁇ i is methyl. In some embodiments, R ⁇ ⁇ is methoxycarbonyl. In some embodiments, R 6 i is selected from the group consisting of methoxymethyl and ethoxymethyl. In some embodiments, methyl groups found in R ⁇ can be substituted with a halogen or a hydroxy.
  • the compounds useful in the present invention have Formula XI:
  • R 51 , R 5 2, R 53 , R 54 , and R ⁇ 3 are as defined above for Formula VIII.
  • R 51 is 3',3'-dimethylglutaryl, 3',3'-dimethylsuccinyl, glutaryl or succinyl.
  • both R 52 and R 53 are methyl.
  • the compounds of Formula VIII are selected from the group consisting of derivatives of uvaol, ursolic acid, erythrodiol, echinocystic acid, oleanolic acid, sumaresinolic acid, lupeol, dihydrolupeol, betulinic acid methylester, dihydrobetulinic acid methylester, 17- ⁇ -methyl-androstanediol, androstanediol, and 4,4-dimethyl- androstanediol.
  • the compounds of the present invention are defined as in Formula VIII, wherein R 52 and R 3 are both methyl.
  • the compounds of the present invention are defined as in Formula VIII, wherein R 51 is 3',3'-dimethylsuccinyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein R 51 is succinyl, i.e.,
  • the stereochemistry of the sidechain substituents is important.
  • the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R 55 is in the ⁇ position.
  • the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R 56 is in the ⁇ position.
  • the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R 64 is in the ⁇ position.
  • the compounds of the present invention are defined as in Formula VIII, wherein A is (i), R 57 is ⁇ -methyl, and R 58 is hydrogen.
  • the compounds of the present invention are defined as in Formula VIII, wherein A is (i), R 58 is ⁇ -methyl, and R 57 is hydrogen. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and both R 57 and R 58 are methyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (ii) and Re t is in the ⁇ position.
  • 3',3'-dimethylsuccinyl is at the C3 position.
  • the compounds of Formula DC are 3-O-(3',3'- dimethylsuccinyl)uvaol; 3-O-(3',3'-dimethylsuccinyl)erythrodiol; 3- O-(3',3'- dimethylsuccinyl)echinocystic acid or 3-O-(3',3'-dimethylsuccinyl) sumaresinolic acid.
  • the compounds of Formula X are 3-O-(3',3'-dimethylsuccinyl) lupeol; 3-O-(3',3'-dimethylsuccinyl) dihydrolupeol; 3-0-(3',3'-dimethylsuccinyl)17 ⁇ -methylester-betulinic acid; or 3-O-(3',3'-dimethylsuccinyl)17 ⁇ -methylester-dihydrobetulinic acid.
  • the compounds of Formula XI are 3-O-(3',3'-dimethylsuccinyl) 4,4-dimethylandrostanediol; 3-0-(3',3'-dimethylsuccinyl)l 7 ⁇ - methylandrostanediol; 3-O-(3',3'-dimethylsuccinyl) androstanediol.
  • the invention includes compounds and methods that use compounds of Formula ⁇ //:
  • R 72 is one of:
  • compounds useful in the present invention are betulin derivative compounds of Formula AT /: or a pharmaceutically acceptable salt or prodrag thereof, wherein: R 51 is C 3 -C 20 alkanoyl, carboxyalkanoyl, carboxyalkenoyl, alkoxycarbonylalkanoyl, alkenyloxycarbonylalkanoyl, cyanoalkanoyl, hydroxyalkanoyl, aminocarbonylalkanoyl, hydroxyaminocarbonylalkanoyl, monoalkylaminocarbonylalkanoyl, dialkylaminocarbonylalkanoyl, heteroarylalkanoyl, heterocyclylaUcanoyl, heterocyclylcarbonylalkanoyl, heteroarylaminocarbonylalkanoyl, heterocyclylaUcanoyl, heterocyclylcarbonylalkanoyl, heteroarylaminocarbonylalkanoyl, heterocyclylaUcanoyl,
  • R 83 is hydroxyl, isopropenyl, isopropyl, l'-hydroxyisopropyl, l'-haloisopropyl, l'-thioisopropyl, l'-trifluoromethylisopropyl, 2'-hydroxyisopropyl, 2'-haloisopropyl, 2'-thioisopropyl, 2'-trifluoromethylisopropyl, l'-hydroxyethyl, l'-(alkoxy)ethyl,
  • Y is -SRmor -NR ] 13 Rn 4 ;
  • R ⁇ i is methyl;
  • R 112 is hydrogen or hydroxyl;
  • R 113 and R 114 are independently hydrogen, alkyl, alkanoyl, arylalkyl, heteroarylalkyl, arylsulfonyl or arylaminocarbonyl; or R 113 and R 114 can be taken together with the nitrogen to which they are attached to form a heterocycle, wherein the heterocycle can optionally include one or more additional nitrogen, sulfur or oxygen atoms;
  • m is zero to three;
  • R 84 is hydrogen; or
  • R 83 and R 84 can be taken together to form oxo, alkylimino, alkoxyimino or benzyloxyimino;
  • Rg5 is C 2 -C 20 alkyl, alkenyl, C 2 -C 20 carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl,
  • Alkyl groups and alkyl containing groups of the compounds of the present invention can be straight chain or branched alkyl groups, preferably having one to ten carbon atoms.
  • the alkyl groups or alkyl containing groups of the present invention can be substituted with a C 3- cycloalkyl group.
  • the cycloalkyl group may include, but is not limited to, a cyclobutyl, cyclopentyl or cyclohexyl group.
  • the non- toxic pharmaceutically acceptable salts of the compounds of the present invention are included within the scope of the present invention.
  • These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free acid form with a suitable organic or inorganic base and isolating the salt thus formed.
  • These can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, N-methyl glucamine and the like.
  • esters of the compounds of the present invention are included within the scope of the present invention.
  • Ester groups are preferably of the type which are relatively readily hydrolyzed under physiological conditions.
  • examples of pharmaceutically acceptable esters of the compounds of the invention include C 1-6 alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C 5-7 cycloalkyl esters as well as arylalkyl esters, such as, but not limited to benzyl. C 1-4 alkyl esters are prefeoed.
  • esters are selected from the group consisting of alkylcarboxylic acid esters, such as acetic acid esters, and mono- or dialkylphosphate esters, such as methylphoshate ester or dimethylphosphate ester.
  • esters of the compounds of the present invention can be prepared according to conventional methods.
  • prodrugs Certain compounds are listed above derivatives refeoed to as "prodrugs". This includes compounds within the scope of Formula VIII to XI, for example.
  • the expression “prodrag” refers to compounds that are rapidly transformed in vivo by an enzymatic or chemical process, to yield the parent compound of the above formulas, for example, by hydrolysis in blood.
  • prodrugs can be esters, for example, of the compounds of Formulae VIII, DC, X, and XI.
  • a lower alkyl group is substituted with one or more hydroxy or halo groups by a suitable acid.
  • suitable acids include, e.g., carboxylic acids, sulfonic acids, phosphoric acid or lower alkyl esters thereof, and phosphonic acid or lower alkyl esters thereof.
  • suitable carboxylic acids include alkylcarboxylic acids, such as acetic acid, arylcarboxylic acids and arylalkylcarboxylic acids.
  • Suitable sulfonic acids include alkylsulfonic acids, arylsulfonic acids and arylalkylsulfonic acids.
  • Suitable phosphoric and phosphonic acid esters are methyl or ethyl esters.
  • the C3 acyl groups having dimethyl groups or oxygen at the C3' position can be the most active compounds. This observation suggests that these types of acyl groups might be important to the enhanced anti-HIN activity.
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formula / through XIII
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formula /through XIII, with the exception of DSB.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII, or is other than a compound of Formula / through XIII.
  • CA-SPl viral Gag p25 protein
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through XI; or in other embodiments is other than / through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formula Groups /through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII; or in other embodiments is other than a compound of Formula / through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through XI; or in other embodiments is other than a compound of Formula / through XIII.
  • the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formula / through XIII.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII; or in other embodiments is other than a compound of Formula / through XIII.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the derivatives of betulinic acid and dihydrobetulinic acid of the present invention were all synthesized by refluxing a solution of betulinic acid or dihydrobetulinic acid, dimethylaminopyridine (1 equivalent mol), and an appropriate anhydride (2.5-10 equivalent mol) in anhydrous pyridine (5-10 mL). The reaction mixture was then diluted with ice water and extracted with CHC1 3 . The organic layer was washed with water, dried over MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed using silica gel column or semi-preparative-scale HPLC to yield the product.
  • Methods of "inhibiting HTV” or “inhibition of HTV” as used herein means any interference in, inhibition of, and/or prevention of HTV using the methods of the invention.
  • methods of inhibition are useful in inhibiting with the infectivity of HTV, inhibition of p25 processing, inhibition of viral maturation, formation of virions that exhibit altered phenotypes, and the like.
  • methods of the invention act upon p25 processing in the cells of an animal, but are not limited by that method of action.
  • a method of inhibiting HTV with a compound may be relevant to a method of treating HTV infection in a patient. Therefore, a method of inhibiting HIN with a compound may similarly be used to treat a patient.
  • the methods of inhibiting HIN-1 replication in cells of an animal includes contacting infected cells with a compound of Formula /through XIII, above.
  • Related embodiments include a method of treating a HIN-1 infectionin a patient comprising administration of a compound of Formula / through XIII; a method of inhibiting p25 processing either in a cell, in vivo, and/or in vitro, by administration of a compound that inhibits said p25 processing; and a method of treating human blood or blood products by administering a compound of Formula / through XIII. Also included are a method of identifying a compound that inhibits any one of p25 processing, HIN maturation, HIN infectivity, HTV virion phenotypes and the like.
  • the compound is a derivative of betulinic acid, betulin, or dihydrobetulinic acid or dihydrobetulin and which includes the prefeoed substituents of Table 4.
  • Prefeoed compounds include but are not limited to 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-O-(3',3'-di methylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-0-diglycolyl-betulinic acid, and 3-O-diglycolyl-dihydrobetulinic acid.
  • the invention is drawn to a method inhibiting HIV-l replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formulas /through XIII above.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • the invention is drawn to a method of inhibiting HIV-l replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is compound of Formulas / through XIII, with the exception of DSB.
  • CA-SPl viral Gag p25 protein
  • CA viral Gag p25 protein
  • CA viral Gag p24
  • the invention is drawn to a method of inhibiting HIN-1 replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is one that is excluded from those of Formulas / through VI.
  • the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is one that is other than those of Formulas / through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound of Formulas /through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas /through VI.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas /through XIII.
  • the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas / through XI.
  • the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formulas / through XIII.
  • the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound other than those of Formulas /through VI.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound other than those of Formulas / through XIII.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the method disclosed herein further comprises contacting said cells with one or more drags selected from the group consisting of anti-viral agents, anti-fungal agents, anti-bacterial agents, anti-cancer agents, immunostimulating agents, and combinations thereof.
  • the method may include the treatment of human blood products.
  • the invention may also be used in conjunction with a method of treating cancer comprising the administration to an animal of one or more anti- neoplastic agents, exposing an animal to a cancer cell-killing amount of radiation, or a combination of both.
  • the invention further includes a method for identifying compounds that inhibit HTV-1 replication in cells of an animal disclosed herein.
  • said method comprises: (a) contacting a Gag polypeptide comprising a CA-SPl cleavage site with a test compound; (b) adding a labeled substance that selectively binds at or near the CA-SPl cleavage site; and (c) measuring the binding of the test compound at or near the CA- SP1 cleavage site.
  • Labeled substances or molecules include labeled antibodies or labeled DSB and the label includes an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, such as gold, osmium tetroxide, lead or uranyl acetate, and radioisotope, antibodies labeled with such substances of molecules or a combination thereof.
  • the assays could include, but are not limited to ELISA, single and double sandwich techniques, immunodiffusion or immunoprecipitation techniques, as known in the art ( ⁇ Immunoassay Handbook, T d ed.," D. Wild, Nature Publishing Group, (2001)).
  • Said methods of identifying also could include, but are not limited to Western blot assays, colorimetric assays, light and electron microscopic techniques, confocal microscopy, or other techniques known in the art.
  • a method of identifying compounds that inhibit HIV replication in cells of an animal further comprises: (a) contacting a Gag protein comprising a wild-type CA-SPl cleavage site, with HIN-1 protease in the presence of a test compound; (b) separately, contacting a Gag protein comprising a mutant CA- SPl cleavage site or a protein comprising an alternative protease cleavage site with HIN-1 protease in the presence of the test compound; and (c) comparing the cleavage of the native wild-type Gag protein to the amount of cleavage of the mutant Gag protein or to the amount of cleavage of the peptide comprising an alternative protease cleavage site.
  • Step (b) above is performed as a control in order to eliminate compounds that might bind directly to, and therefore inhibit, the protease enzyme.
  • the above method also includes the method wherein the wild-type CA-SPl, mutant CA-SPl or alternative protease cleavage site is contained within a polypeptide fragment or recombinant peptide.
  • the method for identifying compounds that inhibit HTV-1 disclosed herein also includes a method wherein said peptide or protein is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety, or wherein said peptide or protein is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound and HIN-1 protease and comparing said transfer of fluorescent energy to that observed when the same procedure is applied to a peptide that comprises a sequence containing a mutation in the CA-SPl cleavage site or that comprises a sequence containing another cleavage site.
  • fluorescence-based assays of protease activity are well known in the art.
  • a protease substrate is labeled with green fluorescent dye molecules, which fluoresce when the substrate is cleaved by the protease enzyme (Molecular Probes, Protease Assay Kit).
  • the method of comparing the cleavage also includes using a labeled antibody that selectively binds CA or SPl or CA-SPl in order to measure the extent to which the test compound inhibits CA-SPl cleavage.
  • the antibody can be labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, and radioisotope, or combinations thereof.
  • the method also includes the use of an antibody to a specific epitope tag sequence to selectively detect CA-SPl (p25) or SPl, into which the amino acid sequence for that epitope tag has been engineered according to standard methods in the art.
  • Suitable tags are well known to those of ordinary skill in the art, and include haemagglutinin epitope HA (YPYDNPDYA) (SEQ ED NO: 81), bluetongue viras epitope VP7 (QYPALT) (SEQ ED NO: 82), ⁇ -tubulin epitope (EEF), Flag (DYKDDDDK) (SEQ ED NO: 83), and VSV-G (YTDOEMNRLGK) (SEQ ED NO: 84). Examples of SPl containing epitope tags are illustrated in Figure 17.
  • the sequence of the FLAG epitope tag (Sigma- Aldrich) is inserted into the p2 (SPl) region of Gag by oligonucleotide-directed mutagenesis of a Gag expression plasmid.
  • SPl p2
  • the presence of the SPl domain in the cell-expressed protein is then be detected using commercially available anti-FLAG monoclonal antibodies (Sigma-Aldrich). (Hopp, T.P. Biotechnology 6: 1204-1210 (1988)).
  • the method of identifying compounds that disrapt CA-SPl cleavage also includes the addition of a compound to cells infected with HIN-1 and the detection of CA-SPl cleavage products by lysing and analyzing the cells or the released virions.
  • the method included in the invention can be performed using a western blot analysis of viral proteins and detecting p25 using an antibody to p25 or wherein said mixture is analyzed by performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins, or wherein the mixture is analyzed using immunoassays that use an antibody that selectively binds p25 or an antibody that selectively binds in order to distinguish p25 from p24.
  • the invention includes the use of an antibody to a specific epitope tag sequence inserted into the C-terminal domain of SPl to selectively detect p25 or SPl.
  • a sandwich ELISA assay can be performed where p25 and p24 in detergent-solubilized viras are captured using an antibody that selectively binds to the CA region of Gag, which antibody is bound to a multiple well plate.
  • bound p25 is detected using an antibody to an epitope tag inserted in SPl, which is conjugated to an appropriate detection reagent (e.g. alkaline phosphatase for an enzyme-linked immunosorbent assay).
  • an appropriate detection reagent e.g. alkaline phosphatase for an enzyme-linked immunosorbent assay.
  • the disclosed method is drawn to an antibody that selectively binds p25, or an antibody that selectively binds SPl, or an antibody to an epitope tag sequence inserted into SPl, which is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, and radioisotope, or combinations thereof.
  • Infected cells includes cells infected naturally by membrane fusion and subsequent insertion of the viral genome into the cells, or transfection of the cells with viral genetic material through artificial means. These methods include, but are not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, lipid-mediated transfection, electroporation or infection.
  • the invention may be practiced by infecting target cells in vitro with an infectious strain of HIN and in the presence of test compound, under appropriate culture conditions and for varying periods of time. Infected cells or supernatant fluid can be processed and loaded onto a polyacrylamide gel for the detection of viras levels, by methods that are well known in the art. ⁇ on- infected and non-treated cells can be used as negative and positive infection controls, respectively. Alternatively, the invention may be practiced by culturing the target cells in the presence of test compound prior to infecting the cells with an HIN strain.
  • the invention also includes a method for identifying compounds that inhibit HIV-l replication in the cells of an animal, comprising: (a) contacting a test compound with wild-type viras isolates and separately with virus isolates having redued sensitivity to 3-O-(3',3'- dimethylsuccinyl) betulinic acid; and (b) selecting test compounds that are more active against the wild- type viras isolate compared with vims isolates that have reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
  • This invention further includes a method for identifying compounds that act by any of the abovementioned mechanism, involving treating HTV-1 infected or transfected cells with a compound then analyzing the viras particles released by compound-treated cells by thin-sectioning and transmission electron microscopy, by standard methods well known in the art.
  • a compound acts by the abovementioned mechanism if particles are detected that exhibit spherical condensed cores that are acentric with respect to the viral particle and/or a crescent-shaped electron-dense layer just inside the viral membrane.
  • infected cells or centrifuged viras pellets obtained from the supernatant fluid can be contacted with a fixative, such as glutaraldehyde or freshly-made paraformaldehyde, and/or osmium tetroxide or other electron microscopy compatible fixative that is known in the art.
  • a fixative such as glutaraldehyde or freshly-made paraformaldehyde, and/or osmium tetroxide or other electron microscopy compatible fixative that is known in the art.
  • the vims from the supernatant fluid or the cells is dehydrated and embedded in an electron-lucent polymer such as an epoxy resin or methacrylate, thin sectioned using an ultramicrotome, stained using electron dense stains such as uranyl acetate, and/or lead citrate, and viewed in a transmission electron microscope.
  • Non-infected and non-treated cells can be used as negative and positive infection controls, respectively.
  • the invention may be practiced by culturing the target cells in the presence of test compound prior to infecting the cells with an HTV strain. Maturation defects caused by the compounds of the present invention are determined by the presence of morphologically abeoant viral particles, compared with controls, as described herein.
  • the virus-infected cells may be observed for the formation of syncytia, or the supernatant may be tested for the presence of HIN particles. Virus present in the supernatant may be harvested to infect other naive cultures to determine infectivity.
  • Also included in the invention is a method of determining if an individual is infected with HTV-1, is susceptible to treatment by a compound that inhibits p25 processing, the method involves taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the CA-SPl cleavage site.
  • the invention also includes a method for identifying compounds that act by the abovementioned mechanisms, involving testing by a combination of the methods disclosed herein.
  • HTV Gag protein and fragments thereof for use in the aforementioned assays may be expressed or synthesized using a variety of methods familiar to those skilled in the art.
  • Gag can be produced in an in vitro transcription and translation system using a rabbit reticulocyte lysate. Gag expressed in this system has been shown to be processed sequentially in a pattern similar to that observed in infected cells (Pettit, S.C. et al. J. Virol. 76:10226-10233 (2002)).
  • Gag expressed by this method is capable of assembling into immature viral particles when fused to a heterologous type D retroviral cytoplasmic self-assembly domain (Sakalian, M. et al, J.
  • the plasmid pDAB72 available from the NTH AIDS Reagent Program can be used for this purpose (Erickson-Viitanen, S. et al, AIDS Res. Hum. Retroviruses. 5:577-91 (1989); Sidhu M.K. et al, Biotechniques, 18:20, 22, 24 (1995)).
  • Other in vitro transcription/translation systems based on wheat germ or bacterial lysates can also be used for this purpose.
  • HTV Gag may also be expressed in transfected cells using a variety of commercially available expression vectors.
  • the plasmid p55-GAG/GFP may be used to express an HTV Gag-green fluorescent protein fusion protein in mammalian cells for drag interaction studies (Sandefur, S. et al, J. Virol. 72:2723-2732 (1998)). This construct would permit the capture and purification of Gag fusion protein using GFP- specific monoclonal antibodies.
  • Gag may be expressed in cells using recombinant viral vectors, such as those used in the vaccinia viras, adenoviras, or baculoviras systems. Gag can also be expressed by infecting cells with HTV or by transfecting cells with proviral DNA.
  • Gag may be expressed in yeast or bacterial cells transformed with the appropriate expression vectors.
  • peptides cooesponding to various regions of Gag may be commercially synthesized from using standard peptide synthesis techniques.
  • the invention further encompasses compounds identified by the method of this invention and/or a compound which inhibits HIN-1 replication according to the methods of this invention and pharmaceutical compositions comprising one or more compounds as disclosed herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers.
  • compositions according to the present invention have been found to possess anti-retroviral, particularly anti-HIN, activity.
  • the salts and other formulations of the present invention are expected to have improved water solubility, and enhanced oral bioavailability. Also, due to the improved water solubility, it will be easier to formulate the salts of the present invention into pharmaceutical preparations. Further, compounds according to the present invention are expected to have improved biodistribution properties.
  • the compounds are those of Formula / through XIII, in another they are compounds other than the compounds of Formula / through XIII.
  • This invention also includes a pharmaceutical composition comprising a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps, or that inhibits the maturation of viras particles released from treated infected cells, such as the compounds of Formula / through XIII.
  • CA-SPl viral Gag p25 protein
  • CA p24
  • the invention includes a pharmaceutical composition comprising one or more compounds disclosed herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers, wherein said compound is of Formulae / through XIII above, or preferably, wherein said compound is selected from the group consisting of 3-O-(3',3 , -dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-0-(3',3'- dimethylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-0-diglycolyl-betulinic acid, and 3-O-diglycolyl-dihydrobetulinic acid.
  • compositions of the present invention can compose at least one of the compounds of Formulae / through XIII disclosed herein.
  • Pharmaceutical compositions according to the present invention can also further comprise other anti-viral agents such as, but not limited to, AZT (zidovudine, RETROVEt®, GlaxoSmithKline), 3TC (lamivudine, EPTVTR®, GlaxoSmithKline), AZT+3TC, (COMBTVTR®, GlaxoSmithKline), AZT+3TC+abacavir (TRIZTVTR®, GlaxoSmithKline), ddl (didanosine, VIDEX®, Bristol-Myers Squibb), ddC (zalcitabine, HINED®, Hoffmann-La Roche), D4T (stavudine, ZERIT®, Bristol-Myers Squibb), abacavir (ZIAGE ⁇ ®, GlaxoSmithKline), tenofovir (VIREAD®, Gilead Sciences),
  • Additional suitable antiviral agents for optimal use with one of the compounds of Formulae / through XIII of the present invention can include, but are not limited to,amphotericin B (FUNGIZONE®); Ampligen (mismatched RNA) developed by Hemispherx Biopharma; ; BETASERON® ( ⁇ -interferon, Chiron); butylated hydroxytoluene; Caoosyn (polymannoacetate); Castanospermine; Contracan (stearic acid derivative); Creme Pharmatex (containing benzalkonium chloride); 5-unsubstituted derivative of zidovudine; penciclovir (DENAVIR® Novartis); famciclovir (FAMVT ® Novartis); acyclovir (ZOVIRAX® GlaxoSmithKline ); cytofovir (VISTEDE® Gilead); ganciclovir (CYTOVENE®, Hoffman LaRoche); dextran sulfate; D-penici
  • compositions of the present invention can also further comprise immunomodulators.
  • Suitable immunomodulators for optional use with a betulinic acid or betulin derivative of the present invention in accordance with the present invention can include, but are not limited to: ABPP (Bropririmine); Ampligen (mismatched RNA) Hemispherx Biopharma; anti-human interferon- ⁇ -antibody; ; ascorbic acid and derivatives thereof; interferon- ⁇ ; Ciamexon; cyclosporin; cimetidine; CL-246,738; colony stimulating factors, including GM-CSF; dinitrochlorobenzene; HE2000 (Hollis-Eden Pharmaceuticals); inteferon- ⁇ ; glucan; hyperimmune gamma- globulin (Bayer); immuthiol (sodium diethylthiocarbamate); interleukin-1 (Hoffmann-LaRoche; Amgen), interleukin-2 (IL-2) (Chiron); isoprinosine
  • compositions of the present invention can also further comprise anti-cancer therapeutic agents.
  • Suitable anti-cancer therapeutic agents for optional use include an anti-cancer composition effective to inhibit neoplasia comprising a compound, or a pharmaceutically acceptable salt or prodrag of said anti-cancer agent, which can be used for combination therapy include, but are not limited to alkylating agents, such as busulfan, cis-platin, mitomycin C, and carboplatin antimitotic agents, such as colchicine, vinblastine, taxols, such as paclitaxel (TAXOL®, Bristol-Meyers Squibb) docetaxel (TAXOTERE®, Aventis), topo I inhibitors, such as camptothecin, irinotecan and topotecan (HYCAMTIN®, GlaxoSmithKline), topo II inhibitors, such as doxorubicin, daunorabicin and etoposides such as VP16; RNA/DNA antimetabol
  • the invention further provides methods for providing anti-bacterial therapeutics, anti-parasitic therapeutics, and anti-fungal therapeutics, for use in combination with the compounds of the invention and pharmaceutically- acceptable salts thereof.
  • anti-bacterial therapeutics include compounds such as penicillins, ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin, cepharadine, cefadoxil, cefaclor, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, imipenem, clavulanate, timentin, sulbactam, erythromycin, neomycin, gentamycin, streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin
  • anti-parasitic therapeutics include bithionol, diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine, niridazole, oxamniquine and other quinine derivatives, piperazine citrate, praziquantel, pyrantel pamoate and thiabendazole, as well as derivatives and altered forms of each of these compounds.
  • anti-fungal therapeutics include amphotericin B, clotrimazole, econazole nitrate, flucytosine, griseofulvin, ketoconazole and miconazole, as well as derivatives and altered forms of each of these compounds.
  • Anti-fungal compounds also include aculeacin A and papulocandin B.
  • the prefeoed animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human patients.
  • treating means the administering to subjects a compound of Formulae / through XIII or a compound identified by one or more assays within the present invention, for purposes which can include prevention, amelioration, or cure of a retroviral-related pathology.
  • Said compounds for treating a subject that are identified by one or more assays within the present inventions are identified as compounds which have the ability to disrapt Gag processing, described herein.
  • inhibitors the interaction means preventing, or reducing the rate of, direct or indirect association of one or more molecules, peptides, proteins, enzymes, or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes or receptors.
  • Medicaments are considered to be provided "in combination" with one another if they are provided to the patient concurrently or if the time between the admimsfration of each medicament is such as to permit an overlap of biological activity.
  • at least one compound of Formulae / through XIII above comprises a single pharmaceutical composition.
  • compositions for administration according to the present invention can comprise at least one compound of Formulae / through XIII above or compounds identified by one or more assays within the present invention.
  • Said compounds for treating a subject that are identified by one or more assays within the present inventions are identified as compounds which have the ability to disrupt Gag processing, described herein.
  • the compounds according to the present invention are further included in a pharmaceutically acceptable form optionally combined with a pharmaceutically acceptable carrier.
  • These compositions can be administered by any means that achieve their intended purposes. Amounts and regimens for the administration of a compound of Formulae / through XIII according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating a retroviral pathology.
  • administration can be by parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, transmucosal, ocular, rectal, intravaginal, or buccal routes.
  • parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, transmucosal, ocular, rectal, intravaginal, or buccal routes.
  • administration can be by the oral route.
  • the administration may be as an oral or nasal spray, or topically, such as powders, ointments, drops or a patch.
  • the dosage administered depends upon the age, health and weight of the recipient, type of previous or concuoent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • Compounds and methods of the invention are useful in additional ways. For example, such compounds may be used prophylatically, to minimize the risk of infection. In another embodiment, a compound may be used to minimize spread of the disease from an infected person.
  • the invention is also directed to novel methods of treating HIN in an infected individual.
  • the invention is particularly useful in stimulating an immune response in a person infected with HIN. For example, by allowing noninfectious virus to be released from infected cells, such infected cells continue to expose antigens and may be effectively targeted by the immune system or other therapies directed against such antigens. In another example, by continuing to permit the release of noninfectious viras, an infected individual continues to develop an immune response to said virus without suffering the deleterious effects of such a virus.
  • the invention is also useful in expanding the scope of treatment, and offers novel means of treating disease in patients in need thereof.
  • the invention may be practiced in a patient who does not respond to other therapy for reasons other than viral resistance.
  • conventional methods of treating HTV as known in the art, are associated with deleterious side effects.
  • the methods and compositions of the invention are useful in treating a patient without a reduction in one or more deleterious side effects.
  • the invention includes a method of treating a patient with a compound that does not have a particular side effect or has less of a particular side effect.
  • the bioavailability of drags is also relevant in treatment.
  • the invention may be practiced such that compounds are more effectively absorbed into infected cells.
  • the invention encompasses improved methods of delivering a drag to a cell infected with HTV.
  • compositions within the scope of this invention include all compositions comprising at least one compound of Formulae / through XIII above according to the present invention in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • a dose may comprise O.OOOlmg to lOg/kg of body weight.
  • Typical dosages comprise about 0.1 to about 100 mg/kg body weight.
  • the prefeoed dosages comprise about 1 to about 100 mg/kg body weight of the active ingredient. More prefeoed dosages comprise about 5 to about 50 mg/kg body weight.
  • Administration of a compound of the jpresent invention can also optionally include previous, concuoent, subsequent or adjunctive therapy using immune system boosters or immunomodulators.
  • a pharmaceutical composition of the present invention can also contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the prefeoed type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient.
  • compositions of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, e.g., fillers such as saccharide, for example, lactose or sucrose, mannitol or sorbitol; cellulose preparations and or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and or polyvinyl pyoolidone.
  • fillers such as saccharide, for example, lactose or sucrose, mannitol or sorbitol
  • cellulose preparations and or calcium phosphates such as tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, cellulose
  • disintegrating agents can be added such as the above-mentioned starches and also carboxymethyl starch, cross- linked polyvinyl pyoolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyoolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl cellulose phthalate are used.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which an be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the active compounds in the form of granules which can be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin.
  • stabilizers can be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories which consist of a combination of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water- soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions can be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides or glycol-400.
  • Aqueous injection suspensions that can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension can also contain stabilizers.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils such as cottonseed, groundnut, com, germ, olive, castor, and sesame oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, cellulose, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and combinations thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, cellulose, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and combinations thereof.
  • compositions for topical admimsfration include formulations appropriate for administration to the skin, mucosa, surfaces of the lung or eye.
  • Compositions may be prepared as a pressurized or non- pressurized dry powder, liquid or suspension.
  • the active ingredients in non- pressurized powdered formulations may be admixed in a finely divided form in a phaonaceutically-acceptable inert carrier, including but not limited to mannitol, fructose, dextrose, sucrose, lactose, saccharin or other sugars or sweeteners.
  • the pressurized composition may contain a compressed gas, such as nitrogen, or a liquefied gas propellant.
  • the propellant may also contain a surface-active ingredient, which may be a liquid or solid non-ionic or anionic agent.
  • the anionic agent may be in the form of a sodium salt.
  • a formulation for use in the eye would comprise a pharmaceutically acceptable ophthalmic carrier, such as an ointment, oils, such as vegetable oils, or an encapsulating material.
  • a pharmaceutically acceptable ophthalmic carrier such as an ointment, oils, such as vegetable oils, or an encapsulating material.
  • the regions of the eye to be treated include the comeal region, or internal regions such as the iris, lens, ciliary body, anterior chamber, posterior chamber, aqueous humor, vitreous humor, choroid or retina.
  • Compositions for rectal administration may be in the form of suppositories.
  • Compositions for use in the vagina may be in the form of suppositories, creams, foams, or in-dwelling vaginal inserts.
  • compositions may be administered in the form of liposomes.
  • Liposomes may be made from phospholipids, phosphatidyl cholines (lecithins) or other lipoidal compounds, natural or synthetic, as known in the art. Any non-toxic, pharmacologically acceptable lipid capable of forming liposomes may be used.
  • the liposomes may be multilamellar or mono-lamellar.
  • a pharmaceutical formulation for systemic admimsfration according to the invention can be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation can be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, dragees, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds described above or compounds identified by one or more assays within the present invention and have the ability to disrapt Gag processing, can also be administered in the form of an implant when compounded with a biodegradable slow-release carrier.
  • the compounds of the present invention can be formulated as a transdermal patch for continuous release of the active ingredient.
  • a robust vims inhibition assay was used to evaluate the anti-viral activity of DSB against primary HIN-1 isolates propagated in PMBC. Briefly, serial dilutions of DSB were made in medium into 96-well tissue culture plates. 25 - 250 TCED 50 of viras and 5 x 10 5 PHA-stimulated PBMCs were added to each well. On days 1, 3 and 5 post-infection, media was removed from each well and replaced with fresh media containing DSB at the appropoate concentration. On day 7 post-infection, culture supernatant was removed from each well for p24 detection of vims replication and 50% inhibitory concentrations (IC 50 ) were calculated by standard methods.
  • Table 5 shows the potent anti-viral activity of DSB against a panel of primary HIN-1 isolates. DSB exhibits levels of activity similar to approved drags that were tested in parallel. Importantly, the activity of DSB was not restricted by co-receptor usage.Table 5 IC 50 (nM)
  • Table 5 Inhibitory activity (IC 50 ) of DSB and two approved drugs against a panel of primary Clade B HIV-l isolates. Clinical HIV-l isolates denoted by * were isolated at Panacos. All other virus isolates were obtained from the NTH AIDS Reference Repository. Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4 respectively.
  • Toxicity of DSB was analyzed by incubating with PHA-stimulated PBMC for 7 days at a range of concentrations, then determining cell viability using the XTT method. The 50%) cytotoxic concentration was >30 ⁇ M, cooesponding to an in vitro therapeutic index of approximately 5000.
  • DSB The activity of DSB was tested against a panel of HIN-1 isolates resistant to approved drags. These viruses were obtained from the NTH AIDS Research and Reference Reagent Program. Assays were performed using vims propagated in PBMCs with a p24 endpoint (above), or using cell line targets (MT-2 cells) and a cell killing endpoint.
  • the MT-2 assay format was as follows. Serial dilutions of DSB, or each approved drug, were prepared in 96 well plates. To each sample well was added media containing MT-2 cells at 3 x 10 5 cells/mL and viras inoculum at a concentration necessary to result in 80% killing of the cell targets at 5 days post-infection (PI). On day 5 post- infection, virus-induced cell killing was determined by the XTT method and the inhibitory activity of the compound was determined.
  • Table 6 shows the potent anti-viral activity of DSB against a panel of drag-resistant HTV-1 isolates. The results were not significantly different from those obtained with the panel of wild-type isolates (Table 5), demonstrating that DSB retains its activity against virus strains resistant to all of the major classes of approved drags.
  • Table 6 Inhibitory activity (nM IC 50 ) of DSB against a panel of drug resistant HIV-l isolates. Assays were done in fresh PBMC with a p24 endpoint except for the NNRTI- resistant isolates that were performed in MT-2 cells with a cell viability (XTT) endpoint. *Fold Resistance. Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4 respectively.
  • DSB Inhibits HIV-l Replication at a Late Step in the Virus Life Cycle
  • a multinuclear activation of a galactosidase indicator (MAGI) assay was used.
  • the targets are HeLa cells stably expressing CD4, CXCR4, CCR5 and a reporter construct consisting of the - galactosidase gene (modified to localize to the nucleus) driven by a truncated HTV-1 LTR. Infection of these cells results in expression of Tat that drives activation of the ⁇ -galactosidase reporter gene.
  • ⁇ -galactosidase in infected cells is detected using the chromogenic substrate X-gal.
  • the entry inhibitor T-20, the NRTI AZT and the NNRTI nevirapine caused significant reductions in ⁇ -galactosidase gene expression in HTV-1 infected MAGI cells due to their ability to disrupt early steps in viral replication that affect Tat protein expression.
  • the protease inhibitor indinavir targets a late step in viras replication (following Tat expression) and does not prevent ⁇ -galactosidase gene expression in this system. Similar results were obtained with DSB as with indinavir, indicating that DSB blocks virus replication at a time point following the completion of proviral DNA integration and synthesis of the viral transactivating protein (Table 7).
  • Table 7 Effect of DSB and inhibitors of entry (the gp41 peptide T-20), RT (AZT and Nevirapine) and protease (indinavir) on expression of b-galactosidase in HIV-l infected MAGI cells.
  • the DMSO control contained no drug.
  • Kanamoto et al Antimicrob. Agents Chemother., April; 45(4): 1225- 30, (2002) have also reported that DSB acts at a late step in HIN replication. However, they reported that the compound inhibits release of virus from chronically-infected cells. In contrast, our data using a variety of experimental systems indicate that DSB does not have a significant effect on virus release (e.g. Example 6).
  • DSB causes a defect in the final step of Gag processing (CA-SPl cleavage) that has been associated with viral maturation defects
  • HeLa cells were transfected with HIN-1 infectious molecular clone p ⁇ L4-3 and treated as described previously with DSB. Following treatment, DSB-treated infected cells were fixed in glutaraldehyde and analyzed by EM. The results of this analysis are shown in Figure 3.
  • 4-0-(3',3'-dimethylsuccinyl) betulinic acid is an example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-l replication.
  • this compound does not inhibit PR activity, and its action is specific for the p25 to p24 processing step, not other steps in Gag processing.
  • DSB treatment results in the abeoant HTV particle morphology described above.
  • DSB 3-O-(3',3'-dimethylsuccinyl) betulinic acid
  • NL4-3 or RF vims isolate was used to infect two cell cultures. Following infection, one culture was maintained in growth medium containing DSB, while the other culture was maintained in parallel in growth medium lacking DSB.
  • H9 cells that had been infected with RF virus were maintained in the presence or absence of increasing concentrations of DSB (0.05-1.6 ⁇ g/ml). The cells were passaged every 2-3 days with the addition of fresh drag. Virus replication was monitored by p24 ELISA every 7 days. At that time, DSB-treated cultures with high levels of p24 were passaged by co- cultivation with fresh uninfected H9 cells at a 1:1 ratio of cells in the presence of lx or 2x the original concentration of DSB. After 8 weeks of co- cultivation, cell-free viras was collected from the culture containing DSB at a concentration of 1.6 ⁇ g/ml and used to infect fresh H9 cells.
  • viras from cultures containing high levels of p24 was passaged by cell-free infection in the presence of lx or 2x the original concentration of DSB.
  • viras from the culture containing 3.2 ⁇ g/ml DSB was collected and used to infect MT-2 cells.
  • Viras replication in the MT-2 cells was monitored by observing syncytia formation microscopically. Every 1-3 days, the cells were washed to remove input viras, and fresh drag was added to the culture under selection.
  • a stock of viras derived from the molecular clone pNL4-3 (5.7 x 104 TCED50) was used to infect MT-2 cells (6 x 106 cells) and cultures were maintained in the presence or absence of DSB at a concentration of 1.6 ⁇ g/ml. Every 1-3 days, the cells were washed to remove input vims, and fresh drag was added to the culture under selection. Virus replication was monitored by observing syncytia formation microscopically. Every 3-7 days, following the emergence of extensive syncytia in the culture under selection, supernatant from each culture was collected and passed through a 0.45 ⁇ m filter to remove cell debris.
  • This filtered virus supernatant was then used to infect fresh MT-2 cells in the presence or absence of fresh drag. After 5 rounds of cell-free infection, and every other round thereafter, the concentration of drag was doubled. After 10 rounds of cell-free infection (approximately 7 weeks in culture), when the concentration of drag reached 12.8 ⁇ g/ml, viras stocks were collected and frozen for further analysis.
  • HTV-1 isolates selected for resistance to compounds that disrapt the processing of the viral Gag capsid (CA) protein from the C A-spacer peptide 1 protein precursor.
  • CA viral Gag capsid
  • Virus stocks derived as described above were further analyzed both phenotypically and genotypically to characterize the nature of their drag- resistance.
  • the resistance of the viruses to 3-O-(3',3'-dimethylsuccinyl)- betulinic acid (DSB) was determined in viras replication assays. Briefly, the viras stocks were first titered in H9 cells by quantitating the levels of p24 (by ELISA) in cultures 8 days after infection with serial 4-fold dilutions of virus. Virus input was then normalized for a second assay in which each viras is cultured for 8 days in the presence of serial 4-fold dilutions of drug.
  • the IC50 for each viras was determined as the dilution of drug that reduced the p24 endpoint level by 50% as compared to the no-drag control.
  • the two independently derived viras stocks resulted in IC 50 values greater than 2 ⁇ M for DSB, as compared to an IC 50 of 0.02 ⁇ M for viras that had been cultured in parallel in the absence of drug.
  • the A364V mutation was engineered into the HTV-1 NL4-3 proviral DNA, which was subsequently transfected into HeLa cells. Resulting viras was collected and used to test the activity of DSB in a viral replication assay, as described above. In these assays, the DSB-resistant viras resulted in an IC 50 value of 0.1 ⁇ M whereas wild-type NL4-3 gave an IC 50 value of 0.01 ⁇ M.
  • HeLa cells were maintained in DMEM (Invitrogen) (supplemented with 10% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml Streptomycin) and passaged upon confluence. All plasmid DNAs were prepared using the midiprep kit (Qiagen). HeLa cells were transfected with wild-type STVmac239, HIV-l pNL4-3 or SHTV proviral DNAs by employing the FuGENE 6 transfection reagent (Roche). Briefly, cells were seeded into a 6- well plate (Coming) at a concentration of 1.5 x 10 5 cells per well the day prior to use and allowed to reach 60 to 80% confluence on the day of transfection.
  • Particle-containing supematants were then concentrated through a 20%) sucrose cushion in a microcentrifuge at 13,000 rpm at 4°C for 120 min and pellets were resuspended in lysis buffer (150 mM Tris-HCl, 5%> Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], pH 8.0).
  • lysis buffer 150 mM Tris-HCl, 5%> Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], pH 8.0.
  • SDS sodium dodecyl sulfate
  • Viral pellets and cell lysates were separated on a 12% ⁇ uPAGE Bis- Tris Gel (Invitrogen) and transfeoed to a nitrocellulose membrane (Invitrogen) followed by blocking in a PBS buffer containing 0.5% Tween and 5% dry milk powder.
  • the membrane was incubated with anti-S ⁇ Nmac251 p27 McAb (NOT ADDS Research and Reference Reagent Program) and hybridized with goat anti-mouse horseradish peroxidase (Sigma).
  • HTV-1 immunoglobulin from HTV-1 -infected patients
  • HTV-Ig immunoglobulin from HTV-1 -infected patients
  • goat anti-human horseradish peroxidase goat anti-human horseradish peroxidase
  • HTV-1/STV Gag chimeras Three panels of HTV-1/STV Gag chimeras were prepared (Fig. 20).
  • Panel 1 consisted of virases containing the STV backbone into which residues from the HIN-1 SPl domain had been inserted.
  • the HIN-1 inserts in these chimeras ranged in size from a single point substitution (SHIN DA) to the complete replacement of the SIN SPl domain with the SPl sequence from HTV-1 (SHTV D ⁇ ).
  • Panel 2 consisted of virases containing the same SPl substitutions plus the inclusion of the two C-terminal CA resides from HIN-1 (LM to VL).
  • Panel 3 SHTVs were identical to those in panel 2 except that, in addition to the substitutions in the SPl domain and the two C-terminal CA resides from HTV-1 (LM to VL), each of these chimeras also incorporated a Q (STV) to H (HTV-1) change at the 6 th position upstream (P6) from the CA-SPl cleavage site.
  • STV Q to H
  • P6 6 th position upstream
  • Results from SHIN panel 2 comprised of virases containing HIN-1 residues in both the SPl and CA domains, are shown in Figure 21.
  • this panel of SHIVs is identical to panel 1 except that in addition to the substitutions in the SPl domain, each of these chimeras also incorporates the two HIN-1 CA C-terminal residues (VL from HTV-1 replaces LM from SIN).
  • the chimeras in panel 2 were characterized by normal Gag processing profiles (Fig. 21) and, while the cellular expression of SHINs FC and 11 was somewhat reduced (Fig. 21 A), the proportion of virus found in the supernatant of cells transfected with D ⁇ A encoding these SHINs was proportional to the level of viral release observed for all panel 2 virases.
  • Each of the SHTVs in panels 1 , 2 and 3 were characterized for their sensitivity to DSB.
  • DSB disrupts HIN-1 CA-SPl cleavage leading to the release of non-infectious viral particles that exhibit abeoant core morphology (Li, F., R. Goila-Gaur, K. Salzwedel, ⁇ . R. Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, D. E. Martin, J. M. Orenstein, G. P. Allaway, E. O. Freed, and C. T. Wild. (2003) PA-457: a potent HIN inhibitor that disrupts core condensation by targeting a late step.
  • DSB does disrupt CA-SPl processing for a subset of the panel 3 SHIVs.
  • SHINs 23 and GI exhibited a level of DSB-sensitivity comparable to the prototypic HIN-1 isolate ⁇ L4-3 (Fig. 22A).
  • SHIN GH exhibited some level of DSB sensitivity, however, on a qualitative level, the activity observed against this viras was reduced compared to that observed with SHINs 23 and GI.
  • FIG. 20 Three panels of SIN/HIN-1 chimeras were prepared (Fig. 20) and characterized. All panel 1 and 2 SHINs behaved similarly with respect to the effect of SPl or CA/SP1 substitutions on Gag protein processing, viral particle release and sensitivity to DSB (Figs. 21 and 22). Although some of the virases in panel 2 (i.e. FD and 11) were characterized by a reduction in the level of viral particle production, this effect was most likely due to an overall reduction in the amount of viras generated by the transfected cells (Fig 21 A).
  • sequence polymorphisms in HIN have been demonstrated to cooespond with the ability of a viras to replicate in the presence of DSB.
  • Most sequence polymorphisms are clustered in gag, especially in the region encoding CA-SPl. Accordingly, genotyping of a viral isolate may be used to readily determine whether the replication of such a vims is likely to be inhibited by DSB, or any other compound that intereferes with p25 processing.
  • results of such genotyping are useful in, for example, determining whether a viral infection in a patient may be treated with DSB, or any other compound that intereferes with p25 processing in a similar manner, or in determining the emergence of resistant variants during a course of treatment with DSB.
  • Genotyping may be performed by a number of methods. In some embodiments, genotyping is performed by sequencing.
  • a single frozen aliquot (approximate volume 1.2 ml) of plasma is obtained from each patient.
  • the plasma sample is stored at -70°C until ready for processing. Each sample is identified using the three digit patient ED number.
  • each plasma sample On the day of processing, each plasma sample is thawed rapidly in a 37°C water bath and then placed on ice. A 140 ⁇ l aliquot of plasma is removed to a separate tube for nucleic acid purification using the QIAamp Mini Viral RNA Purification Kit (Qiagen). The remainder of the plasma sample is transferred to a separate tube for brief, low speed centrifugation (3 min at 8,000 rpm) to clarify the plasma.
  • viral RNA is eluted in a final volume of approximately 60 ⁇ l. Only 7 ⁇ l of this stock is used initially as a template for reverse transcription using the StrataScript First Strand Synthesis System (Stratagene). The remainder of the RNA stock is stored at -70°C as a backup.
  • the primer for reverse transcription (R+625) anneals approximately 625 bp downstream of the CA-SPl cleavage site. All of the primers to be used for RT-PCR and sequencing in this project have been designed to anneal to regions of ag that are highly conserved among clade B HIN isolates and have been validated using plasma samples from 42 different patients.
  • the reverse transcription reaction is performed in a total volume of 50 ⁇ l. Only 5 ⁇ l of this reaction is used initially as a template for PCR amplification of the CA-SPl region using the PicoMaxx High Fidelity PCR Master Mix (Stratagene). The remainder of the reaction is stored at -20°C as a backup. A two-step "nested" PCR strategy is used which has been found to provide a high yield of very clean D ⁇ A product.
  • the forward and reverse primers for the first-round PCR amplification (F-625 and R+525) anneal approximately 625 bp upstream and 525 bp downstream of the CA-SPl cleavage site, respectively. No product is typically visible by agarose gel analysis following this first PCR reaction.
  • the initial PCR reaction is performed in a total volume of 50 ⁇ l. After cycling, 5 ⁇ l of this reaction is removed and used as a template in a second- round "nested" PCR reaction using primers F-575 and R-450, which anneal to regions of gag that are internal to the regions to which the initial primer pair anneals. The remainder of the first-round PCR reaction is stored at -20°C as a backup. The forward and reverse primers for the second-round PCR reaction anneal approximately 575 bp upstream and 450 bp downstream of the CA-SPl cleavage site, respectively. Five ⁇ l of the final "nested" PCR reaction is removed for analysis of DNA products by agarose gel electrophoresis.
  • the reaction contains only one prominent band of the predicted size for the desired product ( ⁇ 1.1 kb), and the yield is estimated to be sufficient to permit sequencing (i.e. -200 ng total)
  • 40 ⁇ l of the reaction is removed for purification of the DNA product using the MinElute PCR Purification Kit (Qiagen). The remaining ⁇ 5 ⁇ l of the reaction is stored at -20°C as a backup.
  • an appropoate volume (cooesponding to at least 40 ng of DNA) is transfeoed to two tubes, each containing a different sequencing primer (one for each strand of the DNA).
  • the "+” strand sequencing primer (F-300) anneals approximately 300 bp upstream of the CA-SPl cleavage site.
  • the "-" strand sequencing primer (R+275) anneals approximately 275 bp downstream of the CA-SPl cleavage site.
  • the template/primer mixture is shipped for sequencing and analysis.
  • the remainder of the purified DNA product is stored at -20°C as a backup.
  • the resulting sequence analysis provides overlapping reads for each DNA strand to help resolve any ambiguities in any single sequencing reaction.
  • the desired DNA product is purified by running the entire PCR reaction (re- amplified from a backup sample if necessary) on an agarose gel and excising the desired band. The DNA is then purified from the agarose using the QIAEX II Gel Extraction Kit (Qiagen) and eluted product is prepared for sequencing as described above.
  • PCR reaction fails to yield sufficient product for sequencing, then additional RT-PCR or PCR reactions could be run, if necessary, using any of the backup samples outlined above and additional validated primer sets, including four forward primers that anneal approximately 550, 375, 300, and 125 bp upstream (F-550, F-375, F-300 and F-125) and three reverse primers that anneal approximately 400, 275, and 100 bp downstream (R+ 400, R+275 and R+100) of the CA-SPl cleavage site. For example, excellent results have been obtained using primer R+525 for reverse transcription and primers F-575 and R+450 for single-round PCR amplification. Primers F-550 and R+400 also work well for PCR amplification.
  • the resulting genotype is then matched with the genotype of virases identified elsewhere herein to determine whether the viras is inhibited or is not inhibited by DSB. Further confirmation of the genotyping results may be obtained by follow-up experiments such as by direct experiments on viral isolates.
  • genotype of HIN-1 during a course of treatment with DSB the genotype of the virus population in each patient prior to dosing and at the end of the study (day 28) is obtained to determine if any mutations have occurred during the course of treatment. If any mutations are identified in the end of study samples that were not present prior to dosing, then intermediate samples drawn on days 7 and 10 after dosing are also be genotyped to determine when the mutation occuoed.
  • the mutations that may occur in the total viras population during the course of the study are determined.
  • a mutation will be identified as a greater than 25% variation in the amino acid designation for a given codon.
  • a chromatogram of the raw data is reviewed to determine the identities of the amino acids at that position in the minor virus populations. If none of the resistance mutations listed above are identified using these criteria, then the chromatograms from each reaction are reviewed to determine if any minor species (less than 25% of the total population) are present at any of the relevant positions.

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Abstract

Inhibition of HIV-1 replication by disrupting the processing of the viral Gag capsid (CA) protein (p24) from the CA-spacer peptide 1 (SP1) protein precursor (p25) is disclosed. Amino acid sequences containing a mutation in the Gag p25 protein, with the mutation resulting in a decrease in the inhibition of processing of p25 to p24 by dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, polynucleotides encoding such mutated sequences and antibodies that selectively bind such mutated sequences are also included. Methods of inhibiting, inhibitory compounds and methods of discovering inhibitory compounds that target proteolytic processing of the HIV Gag protein are included. In one embodiment, such compounds inhibit the interaction of the HIV protease enzyme with Gag by binding to Gag rather than to the protease enzyme. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the Gag proteolytic cleavage site can be used in screening assays to identify compounds that target proteolytic processing.

Description

INHIBITION OF HIV-l REPLICATION BY DISRUPTION OF THE PROCESSING OF THE VIRAL CAPSID-SPACER PEPTIDE 1 PROTEIN
Statement Regarding Federally-Sponsored Research and Development
[0001] The U.S. Government has a paid-up license in this invention and may have the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. 2R44AI051047-02 awarded by NIH/NIAID.
Background of the Invention
Field of the Invention
[0002] The invention includes methods of inhibiting, inhibitors and methods of discovery of inhibitors of HTV infection.
Background
[0003] Human Immunodeficiency Virus (HIV) is a member of the lentivimses, a subfamily of retro vimses. The viral genome contains many regulatory elements which allow the vims to control its rate of replication in both resting and dividing cells. Most importantly, HIN infects and invades cells of the immune system; it breaks down the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
[0004] HIN-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4, also known as OKT4, T4 and leu3. The viral tropism is due to the interactions between the viral envelope glycoprotein, gpl20, and the cell-surface CD4 molecules (Dalgleish et al, Nature 312:763-767 (1984)). These interactions not only mediate the infection of susceptible cells by HIN, but are also responsible for the vims-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in HIN-infected patients. These events result in HIN-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
[0005] In addition to CD4+ T cells, the host range of HIN includes cells of the mononuclear phagocytic lineage (Dalgleish et al, supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage and monocytes are major reservoirs of HIV. They can interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
[0006] Considerable progress has been made in the development of drugs for HIN-1 therapy. Therapeutic agents for HIN can include, but not are not limited to, at least one of AZT, 3TC, ddC, d4T, ddl, tenofovir, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfmavir, lopinavir, amprenavir, atazanavir and fosamprenavir, or any other antiretroviral drugs or antibodies in combination with each other, or associated with a biologically based therapeutic, such as, for example, gp41- derived peptides enfuvirtide (Fuzeon; Timeris-Roche) and T-1249 (Trimeris), or soluble CD4, antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionally presented herein. Combinations of these drugs are particularly effective and can reduce levels of viral RΝA to undetectable levels in the plasma and slow the development of viral resistance, with resulting improvements in patient health and life span.
[0007] Despite these advances, there are still problems with the currently available drug regimens. Many of the drugs exhibit severe toxicities, have other side-effects (e.g., fat redistribution) or require complicated dosing schedules that reduce compliance and thereby limit efficacy. Resistant strains of HIN often appear over extended periods of time even on combination therapy. The high cost of these drugs is also a limitation to their widespread use, especially outside of developed countries. [0008] There is still a major need for the development of additional drugs to circumvent these issues. Ideally these would target different stages in the viral life cycle, adding to the armamentarium for combination therapy, and exhibit minimal toxicity, yet have lower manufacturing costs.
[0009] HIN virion assembly takes place at the surface membrane of the infected cell where the viral Gag polyprotein accumulates, leading to the assembly of immature virions that bud from the cell surface. Within the virion, Gag is cleaved by the viral proteinase (PR) into the matrix (MA), capsid (CA), nucleocapsid (ΝC), and C-terminal p6 structural proteins (Wiegers K. et al, J. Virol. 72:2846-2854 (1998)). Gag processing induces a reorganization of the internal virion structure, a process termed "maturation." In mature HIN particles, MA lines the inner surface of the membrane, while CA forms the conical core which encases the genomic RΝA that is complexed with ΝC. Cleavage and maturation are not required for particle formation but are essential for infectivity (Kohl, Ν. et al, Proc. Nat/. Acad. Sci. USA 55:4686-4690, (1998)).
[0010] CA and ΝC as well as ΝC and p6 are separated on the Gag polyprotein by short spacer peptides of 14 and 10 amino acids (p2), respectively (spacer peptide 1 (SP1) and SP2, respectively) (Wiegers K. et al, J. Virol 72:2846- 2854 (1998), Pettit, S.C. et al, J. Virol. 6°: 8017-8027 (1994), Liang et al. J Virol. 76:11729-11737 (2002)). These spacer peptides are released by PR- mediated cleavages at their Ν and C termini during particle maturation. The individual cleavage sites on the HIN Gag and Gag-Pol polyproteins are processed at different rates and this sequential processing results in Gag intermediates appearing transiently before the final products. Such intermediates may be important for virion morphogenesis or maturation but do not contribute to the structure of the mature viral particle (Weigers et al. and Pettit, et al, supra). The initial Gag cleavage event occurs at the C terminus of SP1 and separates an Ν-terminal MA-CA-SP1 intermediate from a C- terminal ΝC-SP2-p6 intermediate. Subsequent cleavages separating MA from CA-SP1 and NC-SP2 from p6 occur at an approximately 10- fold-lower rate. Cleavage of SP1 from the C terminus of CA is a late event and occurs at a 400-fo Id-lower rate than cleavage at the SP1-NC site (Weigers et al. and Pettit, et al, supra). The uncleaved CA-SP1 intermediate protein is alternatively termed "p25," whereas the cleaved CA protein is alternatively termed "p24" and the cleaved SP1 peptide is alternatively termed "p2".
[0011] Cleavage of SP1 from the C terminus of CA appears to be one of the last events in the Gag processing cascade and is required for final capsid condensation and formation of mature, infectious viral particles. Electron micrographs of mature virions reveal particles having electron dense conical cores. On the other hand, electron microscopy studies of viral particles defective for CA-SP1 cleavage show particles having a spherical electron- dense ribonucleoprotein core and a crescent-shaped, electron-dense layer . located just inside the viral membrane (Weigers et al, supra). Mutations at or near the CA-SP1 cleavage site have been shown to inhibit Gag processing and disrapt the normal maturation process, thereby resulting in the production of non-infectious viral particles (Weigers et al, supra). Phenotypically, these particles exhibit a defect in Gag processing (which manifests itself in the presence of a p25 (CA-SP1) band in Western blot analysis) and the aberrant particle morphology described above which results from defective capsid condensation.
[0012] Previously, betulinic acid and platanic acid were isolated from Syzigium claviflorum and were determined to have anti-HIV activity. Betulinic acid and platanic acid exhibited inhibitory activity against HIN-1 replication in H9 lymphocyte cells with EC50 values of 1.4 μM and 6.5 μM, respectively, and therapeutic index (T.I.) values of 9.3 and 14, respectively. Hydrogenation of betulinic acid yielded dihydrobetulinic acid, which showed slightly more potent anti- HIV activity with an EC50 value of 0.9 and a T.I. value of 14 (Fujioka, T., et al, J. Nat. Prod. 57:243-247 (1994)). Esterification of betulinic acid with certain substituted acyl groups, such as 3',3'-dimethylglutaryl and 3',3'-dimethylsuccinyl groups produced derivatives having enhanced activity (Kashiwada, Y., et al, J. Med. Chem. 5P:1016-1017 (1996)). Acylated betulinic acid and dihydrobetulinic acid derivatives that are potent anti-HIN agents are also described in U.S. Patent No. 5,679,828. Anti- HIN assays indicated that 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) and the dihydrobetulinic acid analog both demonstrated extremely potent anti- HIV activity in acutely infected H9 lymphocytes with EC50 values of less than 1.7 x 10"5 μM, respectively. These compounds exhibited remarkable T.I. values of more than 970,000 and more than 400,000, respectively. [0013] U.S. Patent No. 5,468,888 discloses 28-amido derivatives of lupanes that are described as having a cytoprotecting effect for HIN-infected cells.
■ R = H (Betulinic acid)
[0015] Japanese Patent Application No. JP 01 143,832 discloses that betulin and 3,28-diesters thereof are useful in the anti-cancer field. [0016] U.S. Patent No. 6,172,110 discloses betulinic acid and dihydrobetulin derivatives which have the following formulae or pharmaceutically acceptable salts thereof, Betulin and Dihydrobetulin Derivatives
wherein Ri is a C2-C20 substituted or unsubstituted carboxyacyl, R2 is a C2-C20 substituted or unsubstituted carboxyacyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or ~OR4, where t is hydrogen, alkanoyl, benzoyl, or C2-C20 substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29.
[0017] U.S. Patent Application No. 60/413,451 discloses 3,3-dimethylsuccinyl betulin and is herein incorporated by reference. Zhu, Y-M. et al, Bioorg. Chem Lett. 11:3115-3118 (2001); Kashiwada Y. et al, J. Nat. Prod. 61:1090- 1095 (1998); Kashiwada Y. et al, J. Nat. Prod. 63:1619-1622 (2000); and Kashiwada Y. et al, Chem. Pharm. Bull. 48: 1387- 1390 (2000) disclose dimethylsuccinyl betulinic acid and dimethylsuccinyl oleanolic acid. Esterification of the 3' carbon of betulin with succinic acid produced a compound capable of inhibiting HIV-l activity (Pokrovskii, A.G. et al, Gos. Nauchnyi Tsentr Virusol. Biotekhnol "Vector, " 9:42,5-491 (2001)).
[0018] Published International Application No. WO 02/26761 discloses the use of betulin and analogs thereof for treating fungal infections.
[0019] There exists a need for new HIN inhibition methods that are effective against drag resistant strains of the virus. The strategy of this invention is to provide therapeutic methods and compounds that inhibit the vims in different ways from approved therapies.
[0020] The compound and methods of the present invention have a novel mechanism of action and therefore are active against HIN strains that are resistant to current reverse transcriptase and protease inhibitors. As such, this invention offers a completely new approach for treating HIN/ ADDS.
Brief Summary of the Invention
[0021] Generally, the invention provides methods of inhibiting, inhibitory compounds and methods of identifying inhibitory compounds that target proteolytic processing of the HIN-1 Gag protein. In one embodiment, such compounds may directly or indirectly inhibit the interaction of a protease enzyme with HIV-l Gag protein. In another embodiment, such inhibition of interaction occurs via the binding of a compound to Gag. The inhibition of protease cleavage of the CA-SP1 protein of HIN-1 Gag by 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB) is one example, but other proteolytic cleavage sites can be targeted by a similar approach using inhibitory compounds that interact with the substrate in a manner similar to that in which DSB interacts with Gag.
[0022] Another aspect of the invention is directed to a method of inhibiting the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but having no effect on other Gag processing steps.
[0023] A further aspect of the invention is directed to a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but have no effect on other Gag processing steps.
[0024] In one aspect, the invention is drawn to a compound or pharmaceutical composition identified by the method for identifying compounds that inhibit HIV-l replication disclosed herein.
[0025] In another aspect, the present invention is directed to a polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid. This aspect of the invention is also directed to a vector, virus and host cell comprising said polynucleotide, and a method of making said protein.
[0026] A further aspect of the present invention is directed to an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'- dimethylsuccinyl) betulinic acid.
[0027] An additional aspect of the invention is directed to an antibody which selectively binds an amino acid sequence containing a mutation in the Gag p25 protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid. Also included in this aspect of the invention are a method of making said antibody, a hybridoma producing said antibody and a method of making said hybridoma.
[0028] In a further embodiment, the invention is directed to a kit comprising a polynucleotide, polypeptide or antibody disclosed herein.
[0029] The invention further relates to a method of inhibiting HIV-l infection in cells of an animal by contacting said cells with a compound that blocks the maturation of vims particles released from treated infected cells. In one embodiment, the released vims particles exhibit non-condensed cores and a distinctive thin electron-dense layer near the viral membrane and have reduced infectivity. A method is included of contacting animal cells with a compound that both inhibits processing of the viral Gag p25 protein and that disrupts the maturation of virus particles. Also, included is a method of treating HIV- infected cells, wherein the HIN infecting said cells does not respond to other HIN therapies.
[0030] This invention further includes a method for identifying compounds that inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but have no significant effect on other Gag processing steps. The method involves contacting HIN-1 infected cells with a test compound, and thereafter analyzing virus particles that are released to detect the presence of p25. Methods to detect p25 include western blotting of viral proteins and detecting using an antibody to p25, gel electrophoresis, and imaging of metabolically labeled proteins. Methods to detect p25 also include rmmunoassays using an antibody to p25 or SP1 (ρ2) or to an epitope tag inserted into the SP1 sequence.
[0031] The invention is further directed to a method for identifying compounds involving contacting HIV-l infected cells with a compound, and - thereafter analyzing vims particles released by the contacted cells, by thin- sectioning and transmission electron microscopy, and determining whether viral particles with non-condensed cores and a distinctive thin electron-dense layer near the viral membrane are present.
[0032] The invention is also directed to compounds identified by the aforementioned screening methods. In additional embodiments, the invention is drawn to a method of treating HIV-l infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps. In related embodiments, such inhibition may be accompanied by a different observable phenotypes. For example, inhibition may not necessarily significantly reduce the quantity of virions released from treated infected cells; and/or said inhibition may have little or no significant effect on the amount of RΝA incorporation into the released virions; and/or said inhibition disrupts the maturation of virions released from infected cells treated with said compound. In related embodiments, the virion stracture may be affected, and a majority of virions released from treated infected cells exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and/or and have reduced or no infectivity.
[0033] In additional embodiments, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits the interaction of HIN protease with CA-SPl, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps. Such inhibition may be direct or, alternatively, indirect; and/or may involve said compound binding to the viral Gag protein such that interaction of HIN protease with CA-SPl is inhibited. The invention is also drawn to a method of treating HIN in a patient with a compound that binds at or near the site of cleavage of the viral Gag p25 protein (CA-SPl) to p24 (CA), thereby inhibiting the interaction of HIN ' protease with the CA-SPl cleavage site and resulting in the inhibition of processing of p25 to p24.
[0034] In other embodiments, the invention is drawn to a method of treating HIN-1 -infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (A) KΝWMTETFLVQΝAΝPDCKTILKALGPAATLEEMMTAC QGVGGPSHKARΓLAEAMSQVTΝSATΓM (SEQ ΓD NO: 21); (B) KΝWMTETLLVQΝAΝPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26); (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNS ATIM (SEQ ID NO: 118); and 0") GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119).
[0035] In other embodiments, the invention is drawn to a method of treating HIN-1 -infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18 (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828- 1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) about nucleotides 1858-1920 of SEQ ID NO: 19.
[0036] In another aspect, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) by administration of a compound. In related embodiments, such a compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%), 90%, 95%o or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLNQNANPDCKTILKALGPAATLEEMMTAC QGVGGPSHKARILAEAMSQVTNSATΓM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26); (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNS ATEvl (SEQ ID NO: 118); and 0) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119).
[0037] In related embodiments; the invention is drawns to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) by administration of a compound wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%) or 99% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828- 1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372- 1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) about nucleotides 1858-1920 of SEQ ID NO: 19.
[0038] The invention may be useful in the treatment of HIN in patients who are not adequately treated by other HIN-1 therapies. Accordingly, the invention is also drawn to a method of treating a patient in need of therapy, wherein the HIV-l infecting said cells does not respond to other HIV-l therapies. In another embodiment, methods of the invention are practiced on a subject infected with an HIV that is resistant to a drug used to treat HIN infection. In one application, the HIV is resistant to a protease inhibitor, a polymerase inhibitor, a nucleoside analog, a vaccine, a binding inhibitor, an immunomodulator, or any other inhibitor. In another embodiment, methods of the invention are practiced on a subject infected with an HIN that is resistant to a drag used to treat HIN infection is selected from the group consisting of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D- penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid,. ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, and combinations thereof.
[0039] Compounds of the invention are also useful as part of combination of therapies. Accordingly, in one aspect the invention is drawn to a method of treating HIN in a patient, wherein said patient is administered said compound in combination with at least one anti-viral agent. Anti-viral agents suitable include, but are not limited to: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, gp41 -derived peptides, antibodies to CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof. In another embodiment, the patient is administered said compound in combination with an immunomodulating agent, anticancer agent, antibacterial agent, antifungal agent, or a combination thereof.
[0040] The invention is also directed to compounds. Such compounds are useful in a method of treating patients infected with HIN; in a method for inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), or in a method for treating human blood and human blood products. Such compounds useful in the present invention include, but are not limited to derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, or is selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'-dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-0-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'- dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid and combinations thereof.
[0041] Compounds of the invention may be used alone, or administered with additional compounds, including zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, gp41 -derived peptides, antibodies to CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof; an antiviral, an immunomodulating agent, anti-cancer agent, antibacterial agent, an anti-fungal agent, or combinations thereof.
[0042] In further embodiments, the invention is directed to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA). In one aspect, said compound does not significantly affect other Gag processing steps. In related embodiments of this method, said inhibition does not significantly reduce the quantity of virions released from treated infected cell; and or has little or no significant effect on the amount of RNA incorporation into the released virions; and/or inhibits the maturation of virions released from infected cells treated with said compound; and or affects viral morphology. Such effects on viral morphology include, but are not limited to: the virions released from treated infected cells to exhibit spherical, electron-dense cores that are acentric with respect to the viral particle; and/or possess crescent-shaped electron-dense layers lying just inside the viral membrane; and or and have reduced or no infectivity. In related embodiments, the method involves the administration of the compound which inhibits the interaction of HIV protease with CA-SPl, which results in the inhibition of the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) but has no significant effect on other Gag processing steps. This may be via direct, or indirect inhibition of the interaction of HIV protease with CA- SPl; and/or may involve said compound binds to the viral Gag protein such that interaction of HIN protease with CA-SPl is inhibited; and/or said compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SPl) to p24 (CA), thereby inhibiting the interaction of HIN protease with the CA-SPl cleavage site and resulting in the inhibition of processing of p25 to p24.
[0043] In a further embodiment, the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTAC
QGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPAT (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26); (g) SHKARTLAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATEVI (SEQ ID NO: 119). In a related embodiment, the invention is drawn to a method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%o or 99%) identity, or which is identical a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ED NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ED NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and 0") about nucleotides 1858-1920 of SEQ ID NO: 19.
[0045] The invention also embodies methods for identifying compounds that inhibit HIV-l replication. Accordingly, the invention also includes a method of identifying compounds that inhibit HIV-l replication in cells of an animal, comprising: contacting a Gag protein comprising a CA-SPl cleavage site with a test compound; adding a labeled substance that selectively binds near the CA-SPl cleavage site; and measuring competition between the binding of the test compound and the labeled substance to the CA-SPl cleavage site. In further embodiments of this method, the compounds inhibits the interaction of HIV-l protease with a target site by binding to said target site.
[0046] These methods also include embodiments wherein the CA-SPl cleavage site region is contained within a polypeptide fragment or recombinant peptide; and/or wherein the labeled substance is a labeled antibody specific for CA-SPl, and measuring the change in the amount of labeled antibody bound to the protein in the presence of test compound compared with a control. Labels include, but are not limited to, an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope and a combination thereof.
[0047] The method of identifying compounds that inhibit HIN-1 replication in cells of an animal also comprises, in one embodiment, measuring the change in the amount of labeled 3-0-(3',3'-dimethylsuccinyl) betulinic acid bound to the protein in the presence of test compound, compared with a control, and wherein the labeled substance is 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
[0048] In an alternative embodiment, the invention comprises a method for identifying compounds that inhibit HIN-1 replication in the cells of an animal which comprises: contacting a polypeptide comprising a CA-SPl cleavage site, with a protease in the presence of a test compound. Preferably the protease is related to HIN-1 protease, or is HIN protease. In one embodiment, the method comprises ; contacting a polypeptide comprising a wild type CA- SPl cleavage site, with a protease in the presence of a test compound and also contacting a polypeptide comprising a mutant CA-SPl cleavage site or a protein comprising an alternative protease cleavage site with HIN-1 protease in the presence of the test compound, detecting the cleavage, and comparing the amount of cleavage of the native wild-type polypeptide to the amount of cleavage of the mutant polypeptide or to amount of cleavage of the protein comprising an alternative protease cleavage site. In a related aspect of this method, the wild-type CA-SPl or mutant CA-SPl or alternative protease cleavage site region is contained within a polypeptide fragment or recombinant peptide. In a further related aspect, the polypeptide is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety. In another related embodiment, the polypeptide is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound. In a further embodiment, the effect of the test compound on cleavage of the polypeptide is detected by measuring the amount of a labeled antibody that is bound to SP1 or p24 (CA). In a related aspect, the labeled antibody that binds CA, or the antibody that binds SP1 is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope, and combinations thereof. The invention is also directed to a method for identifying compounds that inhibit HIN-1 replication in cells of an animal. In one embodiment, the method comprises: contacting a test compound with cells infected with wild- type virus isolates and with cells infected with virus isolates having significantly reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl) betulinic acid; and selecting test compounds that are more active against the wild-type virus isolate compared with virus isolates that have reduced sensitivity to 3-0-(3',3'- dimethylsuccinyl) betulinic acid. In another embodiment, the method comprises contacting HIN-1 infected cells with a test compound; lysing the infected cells or the released viral particles to form a lysate, and analyzing the lysate to determine whether cleavage of the CA-SPl protein has occurred. In this latter embodiment, said analyzing may comprise measuring the presence or absence of p25; and or performing a western blot of viral proteins and detecting p25 using an antibody to p25; and/or performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins; and or performing an immunoassay. Such an immunoassay may be performed by any methods known in the art, including, but not limited to: (a) capturing p25 and p24 on a substrate using an antibody that selectively binds p24; and (b) detecting the presence or absence of p25 on the substrate by using an antibody that selectively binds p25. The invention also includes such modifications of the above assay as would be obvious to one of ordinary skill in the art.
[0050] In a further embodiment, the method of identifying a compound according to the invention comprises the use of an epitope tag sequence inserted into SP1 and the selective detection of p25 is performed using an antibody to the epitope tag.
[0051] The invention is also directed to a method for identifying compounds that inhibit HIN-1 replication in the cells of an animal comprising: contacting HIN-1 infected cells with a test compound and thereafter analyzing the virus particles using transmission electron microscopy. Such analysis includes for example, looking for the presence of spherical cores that are acentric with respect to the viral particle; and/or having crescent-shaped, electron-dense layers lying just inside the viral membrane.
[0052] In additional aspects, the invention is drawn to an isolated polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in an HIV Gag p25 protein (CA SP1), said mutation resulting in a decrease in inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-0-(3',3'-dimethylsuccinyl) betulinic acid (DSB). This inhibition of processing of p25 may be due to a decrease in inhibition of the interaction of HIN-1 protease with Gag; and/or a decrease in the binding of 3-O-(3',3'- dimethylsuccinyl) betulinic acid to Gag; and/or a decrease in the binding of DSB at or near the CA-SPl cleavage site of Gag. Suitable polynucleotides also include those encoding a mutation at or near the CA-SPl cleavage site or in the SP1 domain of CA-SPl; and or those encoding a mutation at or near the amino acid sequence G/SHKARV/TLAEAMSQV (SEQ ID NO: 1); and/or those encoding the amino acid sequences GHKARVLVEAMSQV (SEQ ED NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and/or isolated polynucleotide which is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and/or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ED NO: 4, and SEQ ID NO: 6; and/or having at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ED NO: 8 and SEQ ID NO: 9; and/or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ NO: 5 and SEQ ID NO: 7; and/or having at least about 80% identity to a polynucleotide of SEQ ID NO: 10. In additional embodiments, the polynucleotide having more than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% identity or which is identical to the polynucleotide sequences listed above.
[0053] The invention is also drawn to vectors comprising such polynucleotides as described above; to a host cell composing such a vector; and to a method of producing a polypeptide comprising incubating the host cell containing such a vector in a medium and recovering the polypeptide from said medium.
[0054] In one embodiment, the invention is directed to an antibody. Such an antibody may bind to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTAC QGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (e) SHKARE AEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26); (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117); (i) SHKARD AEAMSQVTNSAT (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPAT (SEQ ID NO: 119). In a further related embodiment, the invention is drawn to an antibody which binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide with a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729- 1920 of SEQ ED NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (f) about nucleotides 1370-1413 of SEQ ID NO: 18; (g) about nucleotides 1857-1899 of SEQ ID NO: 19 (h) about nucleotides 1372-1419 of SEQ ID NO: 18; (i) about nucleotides 1858-1905 of SEQ ID NO: 19; (j) about nucleotides 1372-1434 of SEQ ED NO: 18; and (k) about nucleotides 1858-1920 of SEQ ID NO: 19.
[0056] In one embodiment, the antibody binds to amino acids of the CA-SPl region of the HIN-1 Gag polypeptide, wherein said amino acids comprise: SHKARILAEAMSQN (SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26).
[0057] In one embodiment, the invention is drawn to an antibody that inhibits the binding of 3-O-(3',3'-dimethylsuccinyl) betulinic acid to the CA-SPl region of the Gag polypeptide.
[0058] The invention is also drawn to mutant HIV-l viruses. In one such embodiment, the invention is an isolated mutant recombinant HIV-l viras, wherein the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in said viras is not significantly inhibited by 3-O-(3',3,-dimethylsuccinyl) betulinic acid. In related embodiments, this viras is not inhibited by 3-0- (3',3'-dimethylsuccinyl) betulinic acid. In another embodiment, 3-0-(3',3'- dimethylsuccinyl) betulinic acid does not inhibit the interaction of protease with the Gag polypeptide in this viras. In another, the virus does not bind to 3-O-(3',3'-dimethylsuccinyl) betulinic acid. In further embodiments the invention is drawn to viruses wherein the amino acids of the CA-SPl region are replaced with alternative amino acids, or amino acids are added to the CA- SP1 region, or where amino acids are deleted. In one embodiment, ; one or more amino acids are deleted from the AEAMSQV (amino acid no. 8-14 of SEQ DD NO:26) amino acid sequence in the CA-SPl region.
[0059] A mutant viruses may be used in the methods of the invention described elsewhere herein. For example, such viruses are useful in a method of identifying a compound which inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method comprising comparing the ability of said compound to inhibit HIV-l replication compared with the replication of a the mutant virus outlined above. Such inhibition may be examined in a cell, or in an animal, or in vitro. [0060] The invention is also drawn to non-HIV-1 retroviruses that are sensitive to 3-0-(3',3'-dimethylsuccinyl) betulinic acid. In some embodiment, said retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence AEAMSQV (amino acid no. 8-14 of SEQ ED NO: 26) at or near the CA-SPl cleavage site. In another embodiment, the retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence VLAEAMSQV (amino acid no. 6-14 of SEQ ID NO: 26) at or near the CA-SPl cleavage site. In another embodiment, the retroviras encodes a CA-SPl polypeptide with an amino acid sequence comprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near the CA-SPl cleavage site; in another the retroviras composes the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence enocoded by the polynucleotide of SEQ ID NO:26, SEQ ID NO: 90; SEQ ED NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98; in another embodiment the retroviras comprises the amino acid sequence having at least 60%, 70%, 80%, 90% identity or which is identical to the sequence of SEQ ID NO: 91; SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97; or SEQ ID NO: 99. In another embodiment, the retroviras comprises the nucleic acid sequence having at least 70%, 80%, 90% or which is identical to the sequence of SEQ ED NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98.
[0061] Retroviruses of this embodiment of the invention include, but are not limited to HIN-2, HTLV-I, HTLV-II, SIN, avian leukosis viras (ALN), endogenous avian retroviras (EAN), mouse mammary tumor viras (MMTN), feline immunodeficiency virus (FIN), Bovine immunodeficiency virus (BIN), caprine arthritis encephalitis virus (CAEN), Nisna-maedi viras, or feline leukemia viras (FeLV).
[0062] In a related embodiment, the invention is drawn to a method of making a recombinant non-HIV-1 lentivirus sensitive to DSB. This method comprises: deleting from the genome of said lentivirus the nucleotides which correspond to nucleotides 1370-1413 from SEQ ID NO: 18, in HIV-l; and inserting nucleotides 1370-1413 from SEQ ID NO: 18 or nucleotides 1857- 1899 of SEQ ID NO: 19 into said region of said non-HIN-1 lentivirus. [0063] Examples of chimeric lentivimses that were, are or may be constructed by this method are described in Figure 10.
[0064] Such viruses may be used in the methods of the invention described elsewhere herein. For example, such recombinant non-HIN-1 lentivimses may be used in a method of identifying a compound which inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method consisting of comparing of the ability of said compound to inhibit replication of a wild-type non-HIN-1 lentivirus with the DSB-sensitive recombinant variant thereof . Such inhibition may occur in a cell; in an animal; or in vitro.
[0065] The invention is also drawn to an animal model of lentivirus infection comprising a suitable non-human animal host infected with a lentivirus sensitive to 3-0-(3',3'-dimethylsuccinyl) betulinic acid. In such an embodiment, the lentivirus may include, but is not limited to SIN; FrV; EIAV; BIV; CAEN; and Nisna-Maedi virus.
[0066] The invention is also drawn to isolated polypeptides. In one embodiment, the invention is drawn to a polypeptide containing a mutation in an HIV CA-SPl protein, said mutation which results in a decrease in inhibition of processing of p25 by 3-O-(3',3'-dimethylsuccinyι) betulinic acid. In a related embodiment, this polypeptide is encoded by a polynucleotide that contains a mutation located at or near the CA-SPl cleavage site or in the SP1 domain encoded by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10 and/or is encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ED NO: 8 and SEQ ID NO: 9; and/or comprises a sequence that is selected from the group consisting of GHKARNLNEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ED NO: 3); and/or is encoded by an isolated polynucleotide which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and 10; and/or is part of a chimeric or fusion protein.
[0067] The invention is also drawn to antibodies which selectively bind to an amino acid sequence containing a mutation in an HIV CA-SPl protein which results in a decrease in the inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-O-(3 '3 '-dimethylsuccinyl) betulinic acid. In one such embodiment, the antibody selectively binds to a mutation located at or near the CA-SPl cleavage site or in the SP1 domain of CA-SPl; in another, the antibody selectively binds to a mutation comprising a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment, the antibody selectively binds an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
[0068] In another embodiment, the invention is drawn to an antibody that selectively binds SP1 but not CA-SPl; another that selectively binds CA-SPl but not CA; another that selectively binds CA but not CA-SPl; and a further antibody that selectively binds at or near the CA-SPl cleavage site.
[0069] The invention is also directed to a compound identified by any of the methods elucidated herein. In one embodiment, the compounds is not a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-O-(3',3'-dimethylsuccinyl) betulin, 3-O-(3',3'- dimethylglutaryl) betulin, 3-O-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl- dihydrobetulinic acid, and combinations thereof.
[0070] The invention is also drawn to a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin; in another, the pharmaceutical composition comprises a compound selected from the group consisting of 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3,-dimethylglutaryl) betulin, 3-0-(3',3'- dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof. In another embodiment, the pharmaceutical composition comprises one or more compounds identified according to the methods of the invention which are not otherwise listed; or any pharmaceutically acceptable salt, ester or prodrag thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprising an anti-viral agent which may include any one of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid,. ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, combinations thereof, any other antiviral, immunomodulating agent, anti-cancer agent, anti-fungal agent, anti-bacterial agent, or combinations thereof. [0071] The invention is also drawn to a method of determining if an individual is infected with HIV-l that is susceptible to treatment by a compound that inhibits p25 processing. In one embodiment, the method involves taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the sequence encoding the region of the CA- SPl cleavage site. [0072] The invention is also drawn to a method of treating a disease in a patient in need thereof comprising: identifying a compound which inhibits the processing of viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps; obtaining regulatory approval for the sale and use of said compound; packaging the compound for sale and treatment of a disease in a patient in need thereof.
Brief Description of the Drawings
[0073] Figure 1. DSB does not disrapt the activity of HIN-1 protease at a concentration of 50 μg/mL. In DSB-containing samples recombinant Gag is processed correctly. In contrast, indinavir blocks protease activity at 5 μg/mL as evidenced by the absence of bands corresponding to p24 and the MA-CA precursor. [0074] Figure 2. Western blots of virion-associated Gag derived from chronically infected H9/HIN-lι1IB, H9/HIV-2ROD, and H9/SIVmac251 in the presence of DSB (1 μg/mL), indinavir (1 μg/mL) or control (DMSO). Gag proteins were visualized using HIN-Ig (HIV-l) or monkey anti-SINmac251 serum (HIN-2 and SIV; ΝIH AIDS Research and Reference Reagent Program).
[0075] Figure 3. EM analysis of DSB-treated HIV-l infected cells. The EM data show two primary differences between DSB-treated and untreated samples. Virions generated in the presence of DSB are characteozed by an absence of conical, mature cores. In these samples the cores are uniformly spherical and often acentric. Secondly, many virions display an electron dense layer inside the lipid bilayer but outside the core (indicated with arrows in the DSB-treated sample panels). In the DSB-treated samples no mature viral particles were observed.
[0076] Figure 4 depicts amino acid sequences in the region of the CA-SPl cleavage site from DSB-sensitive HIV-l isolates ΝL4-3 and RF (#1; SEQ ID NO: 1) and DSB-resistant HIV-l isolates (#2; SEQ ID NO: 2 (NL4-3), and #3; SEQ ID NO: 3 (RF)). The differences between the native and DSB-resistant sequences involve an alanine to valine change at the first downstream residue (#2) and an alanine to valine change in the third downstream residue (#3) from the CA-SPl cleavage site (-|-). These residues are underlined and bolded for ease of identification.
[0077] Figure 5 depicts the + sense consensus sequence for the A364V DSB- resistant NL4-3 mutant (SEQ ED NO: 4) beginning with the start of gag and continuing into pol, including the entire protease coding region. Missense mutations not found in the wild-type NL4-3 GENBANK Ml 9921 sequence are in bold and gray shadowing. The coding sequence for the consensus CA- SPl cleavage site region is underlined. The shaded area including the cleavage site denotes the SP1 sequence. The first mutation is the A364V mutation.
[0078] The second amino acid change (in protease) was also found in the parental clone and has been confirmed to cooespond to a sequencing error in the original GENBANK entry. Therefore, no mutations actually occurred in protease.
[0079] Figure 6 depicts the + sense consensus sequence for the DSB-sensitive NL4-3 parental isolate (SEQ ID NO: 5) that was passaged in the absence of drag in parallel with the A364V mutant isolate.
[0080] Figure 7 depicts the + sense consensus sequence for the A366V DSB- resistant HIV-l RF mutant (SEQ ED NO: 6) beginning with the start of the gag and continuing into pol, including the entire protease coding region. Missense mutations not found in the wild-type HIV-l RF GENBANK M17451 sequence are shadowed in gray. The region of the CA-SPl cleavage site is underlined. The only missense mutation not also found in the identically passaged DSB-sensitive isolate is the A366V mutation in the CA-SPl cleavage site.
[0081] Figure 8 depicts the + sense consensus sequence for the DSB-sensitive HIV-l RF parental isolate (SEQ ED NO: 7), that was passaged in the absence of drag in parallel with the A366V mutant isolate.
[0082] Figure 9 depicts the polynucleotide sequences, SEQ ID NO: 8 and SEQ ID NO: 9, which encode the polypeptides designated herein as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. SEQ ID NO: 10 and 12 depict the nucleotide sequences that encode the parental polypeptide sequences designated as SEQ ED NO: 1. SEQ ID NO: 1 is a consensus sequence based on the sequences of the region from NL4-3 and RF
[0083] Figure 10: 10 A. Amino acid sequences in the CA-SPl region of lentivirases. (SEQ ID NO: 13; SEQ ED NO: 11; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 20; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 30; respectively) 10B: Amino acid sequences of the CA-SPl region in HIV-l strains RF (SEQ ID NO: 11) and NL4-3 (SEQ ID NO: 13). 10C-10D: Nucleotide sequences of gag gene chimeric SIVs. The 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is underlined and in bold. 10E-H Nucleotide sequences of gag gene of chimeric FIV, EIAV and BIN to be made according to the invention. The 42 nucleotide sequences encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage sites are underlined and in bold (nucleotide sequence: SEQ ID NO: 16; amino acid sequence SEQ ED NO: 17). 10F. Nucleotide sequence of GAG gene of Chimeric Feline Immunodeficiency Viras (FIN) containing the HIN CA-SPl region: Chimeric FIN-GAG gene nucleotides 1-1353 corresponds to nucleotides 628-1980 in Chimeric FIN genome. Nucleotide sequence SEQ DD NO: 94 encoding amino acids SEQ ID NO: 95. 10G. Nucleotide Sequence of GAG gene of Chimeric Equine Infectious Anemia Virus (EIAV) containing the HIV1 CA-SPl region: Chimeric EIAV-GAG gene nucleotide 1-1587 corresponds to nucleotides 450- 1910 in Chimeric EIAV genome. Nucleotide SEQ ID NO: 96 encoding amino acids SEQ DD NO: 97 10H. Nucleotide Sequence of GAG gene of Chimeric Bovine Immunodeficiency Viras (BIV) containing the HIV1 CA-SPl region: Chimeric BPV-GAG gene nucleotides 1-1471 corresponds to nucleotides 316- 1746 in Chimeric BIN genome. Nucleotide SEQ ID NO: 98 encoding amino acids SEQ ID NO: 99
[0084] Figure 11 : Replication kinetics of PA-457 (DSB)-resistant mutants
[0085] Figure 12: Sequential SP1 point deletions in the context of NL4-3 used to identify residues necessary for DSB activity. The amino acid sequence of SP1 domain in NL4-3 is shown. "Δ" indicates the deletion and "— " means identical residues between point deletion mutants and NL4-3 (SEQ ID NO: 13; SEQ DD NO: 33; SEQ DD NO: 34; SEQ ED NO: 35; SEQ DD NO: 36; SEQ DD NO: 85; SEQ DD NO: 86; SEQ DD NO: 87; SEQ ID NO: 88; SEQ DD NO: 89; SEQ DD NO: 100; SEQ DD NO: 101; SEQ DD NO: 102; SEQ DD NO: 103; respectively).
[0086] Figure 13. Summary of particle production and infectivity of point deletions mutants.
[0087] Figure 14. Western blots for viruses containing point deletions in SP1, in the presence (+) and absence (-) of DSB. [0088] Figure 15. Substitution of HIN-1 CA-SPl residues VL-AEAMSQV (SEQ DD ΝO:32) into SIVmac239 backbone renders SINmac239 sensitive to DSB (SEQ ID NO: 14; SEQ DD NO: 15; SEQ DD NO: 20; SEQ DD NO: 27; SEQ DD NO: 28; SEQ DD NO: 13; respectively). (Top panel) Amino acid sequences near the CA-SPl cleavage site (including entire SPl region) are shown for SINmac239, HIV-l ΝL4-3 and a series of SIV mutants into which various NL4-3 residues (underlined) were inserted. Dashes ("— ") indicates the residues are the same as those in SIVmac239. (Bottom panel) Western blots showing the CA and CA-SPl proteins for this series of vimses in the presence (+) or absence (-) of DSB.
[0089] Figure 16: Sequence conservation in the CA-SPl region of Lentivimses. Cloning Strategy: Substituting HIV-l specific CA-SPl residues into the cooesponding Gag region of FIN, ELAN or BIN.
[0090] Figure 17. HIN-1 ΝL4-3 SPl tagged with an epitope. Sequences of SPl peptides with peptide tags inserted are shown. "Δ" indicates deleted residue and "— " indicates that the residue is identical to that in NL4-3 SPl. (Fig 17 (1); SEQ DD NO: 15; SEQ DD NO: 104; SEQ DD NO: 105; SEQ DD NO: 106; SEQ DD NO: 107; respectively); (Fig 17 (2); SEQ ED NO: 15; SEQ ED NO: 108; SEQ ED NO: 109; respectively); (Fig 17 (3); SEQ DD NO: 15; SEQ DD NO: 110; SEQ ED NO: 111; respectively); (Fig 17 (4); SEQ DD NO: 15; SEQ DD NO: 112; SEQ DD NO: 113; SEQ DD NO: 114; respectively); (Fig 17 (5); SEQ DD NO: 15; SEQ DD NO: 115; respectively).
[0091] Figure 18A-C: HIV-l strain RF polynucleotide sequence. The nucleotide sequence of the Gag polyprotein is underlined and in bold. The 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is highlighted in green. An additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (seven amino acids residues) in SPl are highlighted.
[0092] Figure 19A-E: HIN-1 strain ΝL4-3 polynucleotide sequence. The nucleotide sequence of the Gag polyprotein is underlined and in bold. The 42 nucleotide sequence encoding the seven amino acids upstream and seven amino acids downstream of the CA-SPl cleavage site is highlighted in green. An additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (seven amino acids residues) in SPl are highlighted.
[0093] Figure 20: A schematic representation of the Gag protein and the CA- SPl sequences of the SHINs used in this study. The sequences flanking the CA-SPl cleavage site for SIN Mac239 and HIN-1 ΝL4-3 are shown at the top and bottom of the list of sequences, respectively. A dashed line (-) represents residues that are identical to the parent SIN MAC239, a delta (Δ) represents SIV residues that are deleted in the SHINs.
[0094] Figure 21: Western blot analysis of the Gag processing profiles for panel 1-3 SHIVs. Figure 21 A shows Gag processing from cell-associated viras while Fig. 21B shows the Gag processing profile for cell-free virions. Normal Gag processing is indicated by a plus sign (+), while a defective processing profile is indicated by a minus sign (-).
[0095] Figure 22: Western blot analysis of the effect of DSB at lμg/ml on the conversion of the capsid precursor, CA-SPl to mature capsid protein. In panel A, the viras for Western blotting was obtained using a constant volume of cell culture supernatant. This resulted in variability in the intensity of the viral protein bands due to differences among the SHIVs in the level of viras production. Panel B shows the Gag processing profiles obtained when increased amounts of viral protein are used for Western blot analysis. Only SHTVs that exhibit normal Gag processing are included here. The asterix(*) in panel A indicates the faint CA-SPl band for SHIN GI observed in the autoradiograph cannot be seen, however, the DSB sensitivity for this viras is scored a +/- based on results observed when the analysis is performed using increased amounts of protein (panel B).
[0096] Figure 23A-H. Alignment of the CA-SPl region in HIV-l clinical isolates, obtained from "HIV Sequence Compendium 2002, " Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, NM. (www.hiv.lanl.gov). Detailed Description of the Invention
[0097] The present invention is directed to methods of inhibiting HIN-1 replication in the cells of an animal. More specifically, the invention involves methods of inhibiting HIV-l replication in the cells of a mammal by contacting infected cells with a compound that inhibits the processing of the viral Gag p25 protein (CA-SPl) to the p24 protein (CA). More specifically, such compounds inhibit the processing of the viral Gag p25 protein (CA-SPl) to the p24 protein (CA) without significantly affecting other Gag processing steps.
[0098] "A compound that does not significantly affect other Gag processing steps" means that the compound in question predominantly inhibits processing of p25 to p24, but does not necessarily preclude the possibility of having additional minor effects on other Gag processing steps.
[0099] "Significant" or "Significantly," where not otherwise defined herein, means an observable or measurable change compared to the process in the absence of a compound. However, not all observable or measureable changes may necessarily be significant.
[0100] A number of viral phenotypes may also be observed in practicing the method of the invention. One result of contacting an infected cell with the compounds of the invention may be the formation of noninfectious viral particles. Alternatively, or in addition, contacting infected cells with a compound that inhibits p25 to p24 processing, results in the formation of non- infectious viral particles, but where there is no significant effect on other Gag processing steps. This may not significantly reduce the quantity of viras released from treated cells and or has no little or no significant effect on the amount of RΝA incorporation into the released virions.
[0101] Accordingly, the invention is also drawn to a method of inhibiting HIN infection in cells of an animal comprising contacting said cells with a compound that inhibits p25 processing and also affects other viral phenotypes, discribed above.
[0102] Mutant viruses defective in CA-SPl cleavage have been shown to be non-infectious (Wiegers K. et al, J. Virol. 72:2846-2854 (1998)). 3-0-(3',3'- dimethylsuccinyl) betulinic acid (DSB) is an example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-l replication. This compound's activity is specific for the p25 to p24 processing step, not other steps in Gag processing. Furthermore, DSB treatment results in the aberrant HIN particle morphology as described in Figure 3.
1. Identification of HIN-1 determinants associated with sensitivity to 3- 0-(3',3'-dimethylsuccinyl) betulinic acid
(a) Generation and selection of HIN-1 viruses resistant to DSB.
[0103] Mutant forms of HIV-l have been generated in which the amino acid sequence in the region of the CA-SPl cleavage site is modified, decreasing the sensitivity of these strains to compounds that disrapt CA-SPl processing. Data on these mutant viruses have been used to identify the amino acid residues in wild-type Gag that are implicated in the antiviral activity of these compounds. In one embodiment, compounds that disrapt CA-SPl processing directly or indirectly inhibit the interaction of HIN-1 protease with the region of the Gag protein containing these amino acid residues. In another embodiment, compounds that disrapt CA-SPl processing bind to the region containing these amino acid residues. As used herein, the terms "bind," "bound" or "binding" refers to binding or attachment including, e.g., ionic interactions, electrostatic hydrophobic interactions, hydrogen bonds, etc; and also includes associations that may be covalent, e.g., by chemically coupling. Covalent bonds can be, for example, ester, ether, phosphoester, thioester, thioether, urethane, amide, amine, peptide, imide, hydrazone, hydrazide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. The term "bound" is broader than and includes terms such as "coupled," "conjugated" and "attached."
[0104] In another embodiment, compounds that disrapt CA-SPl processing bind to another region of Gag and thereby inhibit the interaction of HIN-1 protease with the region of the CA-SPl cleavage site. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the CA- SPl cleavage site can be used in screening assays to identify compounds that disrupt CA-SPl processing. [0105] In one set of experiments, amino acid residues in HIN-1 Gag that are involved in the disruption of CA-SPl processing by 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB) were identified by sequencing the gag- pol gene of virus isolates that had been selected for resistance to DSB. The amino acid sequences from these resistant viruses were compared with the gag-pol gene sequences from DSB-sensitive HIV-l isolates. Two single amino acid changes were identified in the DSB-resistant viruses, an alanine (Ala) to valine (Val) substitution at residue 364 (SEQ DD NO: 4) and in a second isolate, at residue 366 (SEQ DD NO: 6), in the Gag polyprotein (see Figure 4). These residues are located immediately downstream of the CA-SPl cleavage site (at the N-terminus of SPl). Alanine is highly conserved at these positions throughout all HIV-l subtypes listed in the Los Alamos National Laboratory database. The five amino acid residues upstream and downstream of the CA-SPl cleavage site are also highly conserved among the various subtypes. However, isoleucine replaces valine at the position two residues upstream of the cleavage site in a number of clades (c.f, Figure 4, SEQ DD NO. 1). ("HIV Sequence Compendium 2002, " Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, NM.)
[0106] In order to more extensively map the viral genetic determinants for DSB resistance, additional experiments were performed to select for viruses in vitro that are drag resistant. Multiple parallel cultures of Jurkat T cells (5 x 105 each) were transfected with the proviral DNA clone pNL4-3 in the presence or absence of 10-50 ng/ml DSB. The cells were passaged every two days, and fresh drag was added at each passage. Virus replication was monitored by measuring reverse transcriptase activity in culture supematants. Vims was isolated from culture supematants harvested at selected timepoints, and genomic DNA was amplified by RT-PCR using primers that spanned the coding region between the N-terminus of CA and the N-terminus of RT. The amplified product was then sequenced using the same set of primers.
[0107] In one experiment, an A366V mutation was identified in the SPl region of NL4-3 viras cultured in the presence of DSB (note: numbering is relative to the Gag polyprotein). Upon further passaging, a double mutant was identified that contained a G357S mutation in CA as well as the A366V mutation in SPl. The A366V mutation was identified previously in experiments selecting for resistant variants of the RF isolate. Interestingly, the wild-type RF sequence also contains a serine residue at position 357 in CA (Figure 4). Since serine is present at this position in isolates (such as RF) that are sensitive to DSB, the CA G357S mutation alone is not sufficient to confer resistance to DSB. To determine the contribution of each of these mutations to drag resistance, the A366V mutation and the A366V/G357S double mutation were re-engineered into the wild-type NL4-3 backbone by site- directed mutagenesis. The resulting constructs were transfected into Jurkat T cells and characterized in a viras replication assay as described above for the selection of resistance. SDS-PAGE analysis of transfected cell lysates and virus released into the media demonstrated that the A366V mutant Gag was processed and released from cells inefficiently (data not shown) and thus replicated very poorly even in the absence of drag (Figure 11) However, the A366V/G357S double mutant replicated efficiently in the absence or presence of DSB. There data indicate that the resistant mutant, A366V, requires a serine at the 357 position in the CA region of Gag to compensate for a deleterious effect on viras replication (Figure 11).
[0108] In a further experiment, ten different resistant isolates were generated. Sequencing of these isolates identified four additional mutations not previously seen in resistance selection experiments. These were H358Y, L363F and L363M in CA, and A402T in the NC region of Gag. None of these mutations are present in the consensus sequences for HIV-l clades A-O, reflecting the breadth of activity of DSB against genetically diverse clades of HIN-1. The L363M substitution in CA was found in the consensus sequence for HIV-2, which may, in part, explain the specificity of DSB for HIV-l.
[0109] These results demonstrate the presence of specific genetic determinants for DSB activity in HIN-1, and that these determinants are centered around the CA-SPl cleavage site.
(a) HIN-1 ΝL4-3 deletion and SIN insertion studies used to identify viral genetic determinants of DSB sensitivity
[0110] Results from in vitro resistance selection experiments indicated that the determinants of DSB HIN-1 inhibitory activity map to the region of Gag flanking the CA-SPl cleavage site. In order to better define the viral genetic determinant for DSB, HIN-1 point-deletion mutagenesis and SIN insertion studies were undertaken to identify the specific amino acid residues associated with compound activity. The study was carried out as follows. Single residue deletions starting with residue E365 and continuing through residue M377 were engineered into the SPl domain of the infectious HIN-1 molecular clone ΝL4-3 (Figure 12). The effect of these point deletions on viral particle production, infectivity, Gag processing and sensitivity to DSB was determined. The results of these experiments were used to identify the Gag residues in the region of the CA-SPl cleavage site that are associated with DSB activity. The residues associated with activity were inserted into the CA-SPl cleavage site region of the DSB-resistant virus SIN (Mac 239 isolate) to generate a HIN-1, SIN chimeric virus (SHIN). Point substitution of HIV-l residues from the Ν-terminus of the CA protein were made into this chimeric viras until the minimal sequence necessary to rescue DSB activity was identified. This minimal sequence necessary to gain DSB activity is considered a primary viral genetic determinant of DSB activity. It may suggest the molecular determinant of DSB activity.
1. Methods:
(a) Constraction of ΝL4-3 single point-deletion mutants.
[0111] Single point-deletion constructs were generated using the PCR- ligation-PCR (PLP) strategy as previously described. HIN-1 ΝL4-3 plasmid DNA was used as the template to perform all PCR reactions for generating point deletions spanning the complete Gag SPl domain with the exception of the first residue of SPl.
[0112] ΔE365 was generated using NL4-3 as the template with Vent DNA polymerase (NEB) by using deletion-specific downstream primer (Primer 1) with universal upstream primer (Primer 2) (Table 1). The fragment derived from this was termed as a first flanking PCR fragment. A second flanking fragment was amplified using deletion-specific upstream primer (Primer. 3) and universal downstream primer (Primer 4) (Table 1). To generate other deletion constructs (ΔA366, ΔM367, ΔS368, ΔQ369, ΔV370, ΔT371, ΔN372, ΔP373, ΔA374, ΔT375, ΔI376, and ΔM377). PCR procedures were similarly performed by varying deletion-specific downstream and upstream primers cooesponding to each specific point deletion (Table 1). [0113] Each of these parallel two adjacent PCR fragments was gel purified, phosphorylated using T4 polynucelotide kinase (NEB), and ligated by using T4 DNA ligase (NEB). After inactivation at 65°C for 15 minutes, the ligation reaction was used for a subsequent amplification with universal upstream primer (Primer. 2) and downstream primer (Primer. 4). This product was gel purified, digested with Spel and Apal, and then ligated into the Spel and Apal sites of NL4-3 pro viral DNA clone. [0114] Standard PCR conditions were used for the above-described reactions. These included, one cycle of denaturation at 95 °C for 1 minutes 30 seconds, followed by 30 cycles of denaturation at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds. The PCR reactions were set up using the following components: 5 μL 10 x NEB Thermophilic buffer 2 μL 10 mM dNTPs l μL 100nM MgSO2 1 μL 50 pmol upstream primer 1 μL 50 pmol downstream primer 1 μL 50 ng/μL template DNA 0.5 μL Vent DNA polymerase 38.5 μL ddH2O
[0115] A 10 μL aliquot was run on a 1.0% agarose gel to make sure the correct size product was amplified. The PCR products were then gel isolated and purified with a Qiaex II gel extraction Kit (Qiagen). The gel-purified two adjacent PCR fragments were individually phosphorylated in the following reaction by using T4 polynucleotide kinase (NEB) prior to ligation. The phosphorylation reaction was set up as follows: 2 μL 10X T4 polynucleotide kinase buffer 2 μL lOmM ATP 1 μL T4 polynucleotide kinase [0116] 15 μL gel purified DNA of each of these two adjacent PCR fragments The reaction was incubated at 37°C for 1 hour. Following the inactivation at 65°C for 10 minutes, the adjacent phosphorylated PCR fragments were then ligated together by using T4 DNA ligase (NEB) under following conditions: 3 μL 10X T4 DNA ligase buffer 13 μL of each of two adjacent PCR fragments 1 μL T4 DNA ligase
[0117] After overnight incubation at 16°C the ligation reaction product was used in a second round PCR reaction to amplify the full-length PCR fragment spanning these two adjacent PCR products. The second round PCR reaction was performed as described above with the exception that only universal upstream primer (Primer. 2) and downstream primer (Primer. 4) were used. Again, a 10 μL aliquot was run on a agarose gel to make sure the cooect product was amplified. The full-length PCR fragments were then gel isolated and purified using a Qiaex II kit. The purified full-length PCR fragment, together with NL4-3, were then cut with Spel and Apal under the following conditions: 2 μL 10X NE buffer 4 (NEB) 1 μL Apal (NEB) 1 μL Spel (NEB) 16 μL full length PCR product (1 μg) or NL4-3 (500 ng) [0118] The above restriction enzyme digestion mixture was incubated at 37°C for 2 hours. Digested DNA fragments for the full-length PCR product and the NL4-3 plasmid were individually gel isolated and purified using a Qiaex H kit. The digested vector NL4-3 and full length PCR fragment were ligated using T4 DNA ligase under the following procedure: 1 μL 10X T4 DNA ligase buffer 1 μL (25-50 ng) digested NL4-3 vector 7 μL digested (200 ng-400 ng) digested PCR fragment (700 bp) 1 μL T4 DNA ligase [0119] The ligation reaction was incubated at 16°C overnight and the ligated products were transformed into Escherichila coli Max Efficiency Stbl2 (Invitrogen) by heat shock according to instruction (Invitrogen). The proviral DNA clones were then screened by automatically sequencing using a Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems) individually using internal primers (Primer 29 and 30) Following the verification the mutations the proviral DNA clones were used for various future studies.
1. Construction of SIV chimeric mutants
[0120] A panel of SIN chimeric constructs harboring various residues of ΝL4- 3 CA-SPl boundary region was generated using the SIVmac239 molecular clone by employing PCR and cloning procedures described above. These constructs and their amino acid sequences in the CA-SPl boundary region are shown in Figure 15. SIN mac239 was used to generate the SIN DD and DE constructs. The SIN DD construct was used to generate SIN DM. Different SIN chimeric constructs were produced in the PCR by varying respective mutagenic upstream and downstream primers corresponding to each chimera (Table 1). Each of these parallel two adjacent PCR fragments was gel purified and directly used without phospohorylation treatment for a subsequent amplification with universal upstream primer (Primer. 31) and downstream .primer (Primer 32). This product was gel purified, digested with BamHI and Sbfl, and then ligated into the BamHI and Sbfl sites of SIVmac239 proviral DΝA clone. The proviral DΝA clones were then screened by automatically sequencing using a Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems) individually using an internal primer (Primer. 39). Following the verification the mutations the proviral DΝA clones were used for various future studies.
2. Cell culture and DΝA transfection
[0121] HeLa cells were maintained in DMEM (Invitrogen) (10% FBS, 100 U/ml penicillin, and 100 μg/ml Streptomycin) and passaged upon confluence. Jurkat cells were cultured in RPMI 1640 (Invitrogen) (10% FBS, 100 U/ml penicillin, and 100 μg/ml Streptomycin) and passaged every two or three days.
[0122] To characterize the effect of deletion or substitution on viral particle production and Gag polyprotein processing, wild-type HIN-1 ΝL4-3 or SF mac239 and respective mutant proviral DNAs were transfected into HeLa cells by employing FuGENE 6 transfection reagent (Roche). Briefly, cells were seeded into a 6-well plate (Coming) at a concentration of 0.5 x 105 per well the day before transfection to reach 60 to 80% confluence on the day of transfection. For each transfection, 3 μl of FuGENE 6 was diluted into 100 μl of serum- free DMEM followed by the addition of 1 μg of DNA. After gently mixing, the mixture of DNA-lipid complexes was gently added drop wise into the cells containing 2 ml of complete DMEM medium. Twenty-four hours post-transfection, medium containing DNA-FuGENE 6 complexes was removed, 2 ml of fresh DMEM was added into the transfected cells. At 48 h post-transfection, medium containing viral particles was collected and clarified by centrifugation at 2,000 rpm at 4°C for 20 min in a Sorvall RT 6000B centrifuge. Viras particle-containing supematants were then concentrated through a 20% sucrose cushion in a microcentrifuge at 13,000 rpm at 4°C for 120 min and pellets were resuspended in a lysis buffer (150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholate, pH 8.0). The level of viral particle production for wild type NL4-3 and point deletion mutants was determined by p24 antigen capture ELISA (ZeptoMetrix, Buffalo, NY). To examine the effect of deletion or substitution on Gag polyprotein processing (in the absence of DSB), SDS-PAGE and Westem-Blot was performed. In brief, viral proteins were separated on a 12% NuPAGE Bis-Tris Gel (Invitrogen) and transfeoed to a nitrocellulose membrane (Invitrogen) followed by blocking in a PBS buffer containing 0.5% Tween and 5% dry milk. The membrane was incubated with immunoglobulin from HIV-1- infected patients (HlV-Ig) (NIH ADDS research and reference reagent program) and hybridized with goat anti-human horseradish peroxidase (Sigma). For the membrane containing SIV proteins, the membrane was incubated with a reference polyclonal immune serum from a SIN-infected monkey (N H ADDS Research and Reference Reagent Program) and hybridized with goat-anti-monkey horseradish peroxidase (Sigma). The immune complex was visualized with an ECL system (Amersham Pharmacis Biotech) according to the instructions provided by the manufacturer. [0124] To address the effect of deletion or substitution on the ability of DSB to inhibit CA-SPl processing, HeLa cells were transfected with wild-type HIV-l NL4-3 or SINmac239 and respective mutant proviral DΝAs by employing the procedure described above. DSB at a concentration of 1 μg/ml and DMSO control were maintained throughout the entire culture and SDS- PAGE/Western-Blot for analyzing viral proteins derived from these transfections were performed as described in the previous paragraph.
[0125] The 50% -Tissue Culture Infectious Dose (TCED50) per ml was used as a measure of the infectivity of each deletion mutant. Mutant viruses derived from transfections in HeLa cells were used to infect U87 CD4.CXCR4 cells. Each virus stock was tested in triplicate at a starting dilution of 1 : 10, followed by four-fold serial dilutions. Cells were plated the day before infection at a density of 3xl03 cells/well. On the day of infection, culture media was removed from the cell plate and 90μl of diluted viras was added. On days 1, 3, and 6 post infection, virus was removed from plate and 200μl of culture media was added. On days 6 and 8 post infection, supernatant was collected for p24 ELISA analysis. The viras dilution that caused 50% of the culture to be infected (TCDD50) was determined according to the method of Reed and Muench (Aldovini A. and B. Walker 1990; Dulbecco R. 1988).
3. Results
[0126] Vimses containing sequential point deletions within the Gag SPl domain (Figure 12) were characterized for particle production, infectivity, Gag processing and sensitivity to DSB. The results from these experiments were used to identify SPl residues associated with DSB activity.
[0127] As expected, the effect of point deletions on viral particle production varied as a function of the proximity of the change from the proteolytic cleavage site. The results from these experiments are summarized in Figure 13. Viruses with deletions at residues E366, A367 and M368 were most affected, generating <25% the number of particles normally observed in wild- type viras infection. In vitro infectivity assays were used to characterize the ability of the deletion mutants to support virus replication. These experiments indicated that deletion of single residues at any of the five positions E365 through Q369 resulted in a viras that was either non-infectious or significantly impaired for replication (Figure 13). In contrast, starting with residue V370 and extending away from the CA-SPl cleavage site, none of the characterized point deletions resulted in a decrease in viras infectivity (Figure 13). With the exception of viruses with deletions at positions 1376 and M377 all mutant viruses exhibited a normal or near normal Gag processing phenotype (Figure 13). The results from these three sets of experiments permitted the design and interpretation of experiments to identify the genetic determinants of DSB activity.
[0128] Sensitivity to DSB was determined in experiments that characterized the effect of DSB on a late step in Gag processing, CA-SPl cleavage. Specifically, these assays measured the ability of DSB to disrupt CA-SPl processing. As seen, e.g. Example 8, the DSB-induced defect in Gag processing cooelates with the ability of the compound to inhibit virus replication. Results from these experiments indicate that deletion of a single residue at any of the six positions E365 through V370 significantly reduces the affect of DSB on CA-SPl processing (Figure 14). In contrast, starting with residue T372 and extending away from the CA-SPl cleavage site, all of the characterized point deletions are fully sensitive to DSB-induced disraption of CA-SPl processing (Figure 14).
[0129] The SPl residues associated with DSB activity consist of the contiguous residues E365 through V370.
[0130] Residues A364 through V370 were inserted into the analogous position of the Gag SPl domain in the DSB-resistant retroviras SIV (Mac 239 isolate). Additionally, the N-terminus of the CA protein of this chimeric viras was modified by cumulative substitution of residues found in SIN with HIN-1- specific residues. This approach is summarized in Figure 15. Next, the effect of DSB on the Gag processing phenotype of each of the chimeric viruses was determined. As shown in Figure 15, the SIV.DM virus displays a Gag processing phenotype indicative of sensitivity to DSB. Thus, the minimum sequence of HIV-l CA-SPl -specific residues that needs to be inserted to rescue DSB activity in the SHIVs extends from V362 to V370 Table 1. PCR Mutagenesis Primers
[0131] The resistance and mutagenesis data presented above suggest that the GHKARNL- AEAMSQV amino acid sequence in the region of the HIN-1 Gag CA-SPl cleavage site serves as a genetic determinant of viral sensitivity to DSB.
Extending The Determinants Of Dsb Sensitivity To Other Lentiviruses: Ca-Spl Chimeras As Animal Efficacy Models For Development Of Maturation Inhibitors.
[0132] The development of anti-HIN therapeutics has been hindered by the lack of an animal efficacy model. This lack of an animal model is primarily due to the inability of most HIV-l strains to replicate and cause disease in non-human primates. In some instances this problem has been overcome through the use of chimeric viruses that incorporate the region(s) of interest from the HIV-l viral target into an SIV viral backbone that will support replication in a non-human primate. The most notable example of this approach involves the HIV-l /SIV (SHIN) chimeric viruses in which the proteins making up the infectious viras are exclusively SIN in origin with the exception of Env (gpl20/gp41) which is derived form HIN-1. These SHIN envelope chimeras have been used extensively in HIN-1 vaccine development.
[0133] HIV-l maturation inhibitors disrapt Gag CA-SPl processing, which results in the formation and release of non-infectious viral particles exhibiting aberrant core morphology. See e.g. Li et al. Proc Natl Acad Sci U S A. 100:13555-60 (2003). The betulinic acid derivative DSB is an example of this class of inhibitors. The viral genetic determinants critical that are associated with the activity of maturation inhibitors map to amino acid residues flanking the HIN-1 CA-SPl cleavage site. When this determinant is introduced into the CA-SPl cleavage sites of DSB-resistant non-HIV-1 viruses, maturation inhibitor sensitive chimeras result. These CA-SPl chimeric viruses serve as the basis for an animal efficacy model for HIN-1 maturation inhibitors. [0134] The region of HIN-1 CA-SPl necessary for maturation inhibitor sensitivity is introduced into selected lentiviruses. Amino acid residues from HIN-1 CA-SPl junction that are determinants of DSB sensitivity were used to replace the corresponding CA-SPl amino acids in the genome of Simian Immunodeficiency (SIN). Similarly, the amino acid residues from HIN-1 CA- SPl junction that are determinants of DSB can be replaced in Feline Immunodeficiency virus (FIV), Bovine Immunodeficiency Virus (BIV), Equine Infectious Anemia Viras (EIAV), Visna-Maedi, and Caprine Arthritis Encephalitis virus (CAEV). Table 2 depicts the Gag polypeptide sequence for HIN-1, SIN, FIN, EIAV and BIN in the region of the CA-SPl cleavage site.
Table 2. Sequence comparison in the region of the CA-SPl cleavage site region of HIN-1 with SIV, FIV, EIAV and BIV CA SPl ΝC HIV-l GHKARVL AEAMSQVTΝPATIM IQKG (SEQ ID NO: 76) FIV GYKMQL AEALTKVQ WQS (SEQ ID NO: 77) EIAV KQKMMLL. AKALQ TGLA (SEQ ID NO: 78) BIV KSKMQF VAAMKEMGIQSPIPAVLPHTPEAYA SQTS (SEQ ID NO: 79)
[0135] The HIV-l CA-SPl sequence used for replacement is as follows: CA SPl GHKARVL AEAMSQV ( SEQ ID NO : 80 ) [0136] The method described above for generating the SHIV CA-SPl chimeric provirus DNA clone is used to generate FIV, EIAV and BIV proviras clones containing selected residues or extended region from CA-SPl region of HIN-1 replacing the corresponding wild-type sequence (Figure 16). [0137] The SHIN CA-SPl chimeric-provirus DΝA clone was generated by site-directed mutagenesis employing standard molecular biology techniques. Briefly, the unique restriction enzyme sites in the SIV Gag that suoounding the CA-SPl region were identified i.e., BamHI (in matrix) and Sbf-I (in ΝC). Starting from the CA-SPl region where the mutagenisis is intended two overlapping primers, a forward and a reverse primer incorporating the mutated sequence i.e., HTV CA-SPl at their 5' ends were synthesized. Using the wild- type STV provirus DΝA as a template, two separate PCR reactions were set up to amplify SIN-Gag fragments in either direction from the site of mutagenisis (CA-SPl region), i.e., yield two amplified fragments that overlapped in the mutated CA-SPl region, a Bam HI-CA-SPl fragment and a CA-SPl-Sbf-I fragment. In a third PCR reaction, the fragments, Bam HI-CA-SPl and CA- SPl -Sbf-I were annealed at their common HTV-CA-SP1 sequence and amplified with a forward SIN Bam HI primer and a reverse SIN Sbf-I primer to generate a full-length chimeric SHIN CA-SPl gag fragment. The chimeric SHIN CA-SPl PCR fragment was cloned into BamHI - Sbf-I window of SIV provirus clone replacing the SIN-Gag wild-type sequence to yield the SHIN CA-SPl provirus cDΝA clone.
[0138] Similarly, unique restriction enzyme cloning sites suoounding the CA- SPl regions in FIN (Genbank Accession # ΝC_001482), EIAV (GA# AF016316), and BTV (GA# M32690) genome have been identified (Fig 16). Specific FrV/ELAV/BTV-HTVlCA-SPl chimeric primers along with genome specific primers of FIN, ELAN or BIN incorporating the specific cloning site sequence are synthesized. These primers along with cooesponding provirus DΝA clone as template (FIN, EIAV or BIAV) in PCR reactions to generate the Chimeric HIN-1 CA-SPl fragment. The chimeric HIN-1 CA-SPl fragment is digested with the appropriate restriction enzyme and cloned into SacI-EcoRI window of FTV provirus; or (ii) KasI-EcoRN window of EIAV provirus; or (iii) BsrGI-Apal window of BTV provirus replacing the cooesponding wild type sequence (Figure 16). The chimeric FIN/EIAN/BIN-HIN-1 CA-SPl provirus DΝA clones are sequenced to confirm the presence of intended mutations. Based on observed results that indicate the transfer of DSB sensitivity, additional constructs are generated employing the above strategy in order to optimize the results.
[0139] In summary, a chimeric viras was generated in which the CA-SPl determinant of HTV-1 maturation inhibitor sensitivity has replaced the analogous region of Gag in the maturation inhibitor-resistant simian immunodeficiency virus (SIV). Transfer of this region of HTV-1 into the genome of STV results in a maturation inhibitor-sensitive phenotype. Infection of a non-human primate with this HIN-1 /SIN chimeric viras should result in an animal efficacy model for therapeutic development of maturation inhibitors.
[0140] Analogous approaches are used to prepare and characterize HTV-1 CA- SPl chimeras with FIN, BIN and EIAV. These additional DSB-sensitive chimeric viruses should enable the development of additional animal efficacy models for the study of HTV-1 maturation inhibitors.
Uses Of Mutant And Chimeric Viruses
[0141] The mutant and chimeric viruses of the present invention, as described above, are useful in a variety of cell based as well as animal based assays.
[0142] By comparing the phenotypes associated with a viras that is resistant to DSB, with a virus that is sensitive DSB, one may identify compounds that act by a mechanism similar to that of DSB. Thus the invention includes a method of identifying a compound that inhibits cleavage of p25 to p24 in wild type HTV-1, but does not inhibit CA-SPl processing in HIN-1 containing a deletion in the CA-SPl region. Compounds obtained by such a method are also included in the present invention.
[0143] Chimeras of SIN and other lentiviruses that do not readily infect humans have additional advantages. Firstly, these viruses pose a lesser safety hazard to laboratory workers. As a result, cell based assays to identify novel compounds that inhibit CA-SPl processing, for example, can be conducted with less risk. The lower risk may allow assays to be performed that cannot be performed readily or safely with HIN, and may also lower the cost of such assays. [0144] Furthermore, such chimeric vimses are useful in animal models. For example chimeric SIN that is sensitive to DSB may be used to identify novel compounds that inhibit CA-SPl processing, for example; to identify pharmaceutical compositions, routes of administration and dosage regimes for treatment of disease; and for studying the effect of combination therapies, such as DSB with protease inhibitors.
[0145] As SIN is generally limited to infection of monkeys, the generation of additional lentiviral chimeras allows animal studies to be performed in animals that are less expensive, easier to handle, have a faster disease progression or otherwise more appropriate for a particular aspect of human disease, for example.
[0146] Furthermore, animal models may be used to identify appropriate pharmaceutical compositions for the treatment of animal diseases, of interest in the treatment of companion animals and other high value animals, such as agricultural breeding stock and race horses.
[0147] Chimeric vimses may be derived from any retroviras. For example, derived HIN-2, HTLV-I, HTLN-II, SIN, avian leukosis virus (ALV), endogenous avian retroviras (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency viras (FIN), Bovine immunodeficiency virus (BIV), caprine arthritis encephalitis viras (CAEV), Equine infectious anemia viras (EIAV), Visna-maedi virus, or feline leukemia virus (FeLV).
[0148] Such chimeric viruses may be used in the methods of the invention described elsewhere herein. For example, such recombinant non-HIV-1 lentiviruses may be used in a method of identifying a compound which inhibit processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), the method consisting of comparing of the ability of said compound to inhibit replication of a wild-type non-HIN-1 lentivirus with the DSB-sensitive recombinant variant thereof . Such inhibition may occur in a cell; in an animal; or in vitro.
Construction And Use Of Viruses Or Polypeptides With Epitope Tags
[0149] The present invention is also drawn to recombinant retroviruses with epitope tags in the CA-SPl region of Gag. Epitope tags may be inserted in the CA domain and/or in the SPl domain. Suitable tags are well known to those of ordinary skill in the art, and include haemagglutinin epitope HA (YPYDVPDYA) (SEQ ED NO: 81), bluetongue viras epitope VP7 (QYPALT) (SEQ ED NO: 82), α-tubulin epitope (EEF), Flag (DYKDDDDK) (SEQ ED NO: 83), and VSV-G (YTDIEMNRLGK) (SEQ ED NO: 84). Examples of SPl containing epitope tags are illustrated in Figure 17. [0150] Such epitope tagged viruses and fragments thereof are useful in identifying novel compounds that inhibit CA-SPl processing in vitro, in cell based assays, and in vivo, including in animal models. Additional uses of such epitope tagged viruses and fragments thereof are described elsewhere herein.
Polynucleotides, Polypeptides and Antibodies of the Invention
[0151] The invention also includes isolated polypeptides and polynucleotides. In one embodiment, the invention includes polypeptides at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQ GVGGPSHKARILAEAMSQVTNSATDVI (SEQ ED NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQ GVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24); (e) SHKARILAEAMSQV (SEQ ED NO: 25); (f) GHKARVLAEAMSQV (SEQ ED NO: 26); (g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117); (i) SHKARTLAEAMSQVTNS ATM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATTM (SEQ ID NO: 119).
[0152] In another embodiment the invention includes polynucleotides encoding the above polypeptides. Polynucleotides of the invention include degenerate variants, such as those that differ in the third base of the codon but nevertheless encodes the same amino acid due to coding "degeneracy".
[0153] The term "about" as used herein refers to a value that is 10% more or less than the stated value, and preferably is 5% more or less.
[0154] The polypeptides and polynucleotides of the invention are useful in the methods of the invention. In one aspect, they may be used in an in vitro assay to identify compounds that bind to the CA-SPl region of Gag. In another, they may be used in the production of antibodies useful in other methods described elsewhere herein. In another, a polynucleotide may be inserted into a vector and thereupon into a host cell for production of polypeptide. The above embodiments are exemplary and are not intended to be limiting.
[0155] The present invention comprises a polynucleotide comprising a sequence which encodes an amino acid sequence containing a mutation in the HTV Gag p25 protein (CA-SPl), said mutation resulting in a decrease in the inhibition of processing of ρ25 (CA-SPl) to p24 (CA) by DSB. The polynucleotide of the invention includes a mutation which is optionally located at or near the CA-SPl cleavage site or located in the SPl domain of CA-SPl. Said mutation can be present in an amino acid sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ED NO: 2) and SHKARILAEVMSQV (SEQ DD NO: 3). The polynucleotide of this invention is also drawn to sequences designated as SEQ ED NO: 4, SEQ ED NO: 6, SEQ DD NO: 8 and SEQ ED NO: 9. The invention also includes a vector comprising said polynucleotide, a host cell comprising said vector and a method of producing said polypeptides comprising incubating said host cell in a medium and recovering the polypeptide from the medium.
[0156] The invention further includes a polynucleotide that hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9. The invention also includes a polynucleotide which hybridizes to SEQ NO: 5, SEQ ED NO: 7 or SEQ ED NO: 10 or 12, which contains a mutation which results in the decrease in the inhibition of processing of p25 to p24 by 3-0-(3',3'- dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl. The invention is also directed to a vector comprising said polynucleotides, a host cell comprising said vector and a method of producing said polypeptides, comprising incubating said host cell in a medium and recovering said polypeptide from the medium.
[0157] "Near" or "adjacent," as used herein in reference to polypeptides is meant to include about 50, about 25, about 20, or about 15 residues from the point of reference. For example, near may encompass about 50, about 25, about 20 or about 15 residues on either side of the HTV-1 Gag CA-SPl cleavage site; more preferably about ten residues on either side of the HTV-1 Gag CA-SPl cleavage site; and most preferably about seven residues on either side of the HTV-1 Gag CA-SPl cleavage site. In reference to polynucleotides, the terms "near" or "adjacent refer to about 150, about 75, about 60, about 45, or about 30 nucleotides from the point of reference.
[0158] "Isolated" means altered "by the hand of man" from the natural state. If an composition or substance occurs in nature, it has been changed or removed from its original environment, or both, when found in its "isolated" form. Also, "isolated" nucleic acid molecule(s) of the invention is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
[0159] "Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often refeoed to as oligonucleotides. "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly refeoed to as peptides, oligopeptides or oligomers, and to longer chains, generally refeoed to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, foonation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
[0161] "Mutant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively. A typical mutant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the mutant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical mutant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A mutant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A mutant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a mutant that is not known to occur naturally. Non-naturally occurring mutants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
[0162] Thus, the mutant, (or fragments, derivatives or analogs) of a polypeptide encoded by any one of the polynucleotides described herein may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (a conserved amino acid residue(s), or at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG:Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such mutants are deemed to be within the scope of those skilled in the art from the teachings herein. Polynucleotides encoding these mutants are also encompassed by the invention. "Mutant" as used herein is equivalent to the term "variant."
[0163] Substitutions of charged amino acids with another charged amino acids and with neutral or negatively charged amino acids are included. Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al, Clin Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 70:307-377 (1993)). Thus, the polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
[0164] As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 3). TABLE 3. Conservative Amino Acid Substitutions
[0165] However, in some embodiments, it is desirable to use nonconservative substitutions of amino acids. For example nonconservative substitution of amino acids is used to render a DSB sensitive viras resistant to DSB.
[0166] The polynucleotides encompassed by this invention may have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with a reference sequence, providing the reference polynucleotide encodes an amino acid sequence containing a mutation in the CA-SPl protein, said mutation which results in the decrease in the inhibition of processing of p25 to p24 by a 3-O-(3',3'-dimethylsuccinyl) betulinic acid. The polynucleotides also encompassed by this invention include those mutations which are "silent," in which different codons encode the same amino acid (wobble).
[0167] "Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. The term "identity" is used interchangeably with the word "homology" herein. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans. Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Baxevanis and OuUette, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Second Edition, Wiley-Interscience, New York, (2001). Methods to determine identity and similarity are codified in computer programs. Prefeoed computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J. et al, Nucleic Acids Research i2(l):387, (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al, J. Molec. Biol 215:403, (1990)).
[0168] A polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
[0169] Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence, is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid. To obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire nucleotide sequence of any one of the nucleotide sequences of the invention or any polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of the invention and or C terminal deletion).
[0170] Whether any particular nucleic acid molecule having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity or which are identical to, for instance, the nucleotide sequences of the invention can be determined conventionally using known computer programs such as the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, (Advances in Applied Mathematics 2:482-489 (1981)), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
[0171] In a specific embodiment, the identity between a sequence of the present invention and a subject sequence, also refeoed to as a global sequence alignment, is deteonined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Prefeoed parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the reference sequence because of 5' or 3' deletions, not because of internal deletions, a manual cooection is made to the results to take into consideration the fact that the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is cooected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This cooected score is what is used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence, which are not matched aligned with the query. In this case the percent identity calculated by FASTDB is not manually cooected. Only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually cooected. No other manual cooections are made for the purposes of this embodiment. The present application is directed to nucleic acid molecules having at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%) or 99% identity or which is identical to the nucleic acid sequence disclosed herein, or fragments thereof, ioespective of whether they encode a polypeptide having the disclosed functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having the disclosed functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having the disclosed functional activity include, inter alia: (1) isolating the variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to determine cellular location or presence of the disclosed sequences, and (3) Northern Blot analysis for detecting mRNA expression in specific tissues.
[0173] As used herein, the term "PCR" refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis et al, as well as improvements now known in the art. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
[0174] The term "stringent conditions," as used herein refers to homology in hybridization, is based upon combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions, and well known in the art (Sambrook, et al. supra). The invention includes an isolated nucleic acid molecule comprising, a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the sequence complementary to the coding and/or noncoding (i.e., transcribed, untranslated) sequence of any polynucleotide or a polynucleotide fragment as described herein. By "stringent hybridization conditions" is intended overnight incubation at 42°C in a solution comprising, or alternatively consisting of: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing in O.lx SSC at about 65°C. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
[0175] The invention also includes a viras comprising the polynucleotides of the invention, and wherein the viras includes a retroviras comprising said polynucleotides, and wherein the retroviras may be a member of the group consisting of HTV-1, HIN-2, HTLV-I, HTLV-II, STV, avian leukosis viras (ALV), endogenous avian retroviras (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIN), or feline leukemia viras (FeLN).
[0176] The invention further includes a polypeptide containing a mutation in the CA-SPl protein, said mutation which results in the decrease in inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or located in the SPl domain of SEQ ED NO: 5 or SEQ ED NO: 7 (parental polynucleotide sequences) encoding the CA-SPl protein. Said polypeptide may be encoded by a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ DD NO: 9, or may compose a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ DD NO: 2) and SHKARD AEVMSQV (SEQ ED NO: 3). The polypeptide of this invention may further be encoded by a polynucleotide which hybridizes to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9. The invention also includes a polypeptide encoded by a polynucleotide which hybridizes to SEQ NO: 5, SEQ DD NO: 7 or SEQ DD NO: 10 or 12, which contains a mutation that results in decrease in inhibition of processing of p25 to p24 by 3-O-(3',3'-dimethylsuccinyl) betulinic acid, and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl. The polypeptide of this invention further includes polypeptides that are part of a chimeric or fusion protein. Said chimeric proteins may be derived from species which include, but are not limited to: primates, including simian and human; rodentia, including rat and mouse; feline; bovine; ovine; including goat and sheep; canine; or porcine. Fusion proteins may include synthetic peptide sequences, bifunctional antibodies, peptides linked with proteins from the above species, or with linker peptides. Polypeptides of the invention may be further linked with detectable labels; metal compounds; cofactors; chromatography separation tags, such as, but not limited to: histidine, protein A, or the like, or linkers; blood stabilization moieties such as, but not limited to: transferrin, or the like; therapeutic agents, and so forth. [0177] The invention also includes an antibody which selectively binds an amino acid sequence containing a mutation in the CA-SPl protein that results in a decrease in the inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-0-(3',3'-dimethylsuccinyl) betulinic acid and also wherein said mutation is optionally located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl. The invention also includes an antibody which selectively binds the polypeptide having a mutation which comprises a sequence that is one of GHKARVLVEAMSQV (SEQ ED NO: 2), SHKARILAEVMSQV (SEQ ED NO: 3)., Said antibody can selectively bind the polypeptide encoded by a polynucleotide sequence selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9. Said antibody can also selectively bind the polypeptide encoded by a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9. The invention also includes an antibody that selectively binds SPl, which would enable one to distinguish SPl from CA-SPl (p25). The invention also includes an antibody that selectively binds CA (p24), which would enable one to distinguish CA from CA-SPl. The invention also includes an antibody that selectively binds CA-SPl, which would enable one to distinguish CA from CA-SPl. The invention additionally includes an antibody that selectively binds at or near the CA-SPl cleavage site. The antibody of this invention may be a polyclonal antibody, a monoclonal antibody or said antibody may be chimeric or bifunctional, or part of a fusion protein. The invention further includes a portion of any antibody of this invention, including single chain, light chain, heavy chain, CDR, F(ab')2, Fab, Fab', Fv, sFv, or dsFv, or any combinations thereof.
[0178] As used herein, an antibody "selectively binds" a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. The term "selectively binds" also comprises determining whether the antibody selectively binds to the target mutant sequence relative to a native target sequence. An antibody which "selectively binds" a target peptide is equivalent to an antibody which is "specific" to a target peptide, as used herein. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross- reactivity. In another embodiment, the determination whether the antibody selectively binds to the mutant target sequence composes: (a) deteonining the binding affinity of the antibody for the mutant target sequence and for the native target sequences; and (b) comparing the binding affinities so determined, the presence of a higher binding affinity for the mutant target sequence than for the native indicating that the antibody selectively binds to the mutant target sequence.
[0179] The invention is further drawn to an antibody immobilized on an insoluble carrier comprising any of the antibodies disclosed herein. The antibody immobilized on an insoluble carrier includes multiple well plates, culture plates, culture tubes, test tubes, beads, spheres, filters, electrophoresis material, microscope slides, membranes, or affinity chromatography medium.
[0180] The invention also includes labeled antibodies, comprising a detectable signal. The labeled antibodies of this invention are labeled with a detectable molecule, which includes an enzyme, a fluorescent substance, a chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, an electron dense substance, and a radioisotope, or any combination thereof.
[0181] The invention further includes a method of producing a hybridoma comprising fusing a mammalian myeloma cell with a mammalian B cell that produces a monoclonal antibody which selectively binds an amino acid sequence containing a mutation in the CA-SPl protein, said mutation resulting in a decrease in the inhibition of processing of p25 to p24 by 3-O-(3',3'- dimethylsuccinyl) betulinic acid and a hybridoma producing any of the monoclonal antibodies disclosed herein. The invention further includes a method of producing an antibody comprising growing a hybridoma producing the monoclonal antibodies disclosed herein in an appropriate medium and isolating the antibodies from the medium, as is well known in the art. The invention also includes the production of polyclonal antibodies comprising the injection, either one injection or multiple injections of any of the polypeptides of the inventions into any animal known in the art to be useful for the production of polyclonal antibodies, including, but not limited to mouse, rat, hamster, rabbit, goat, sheep, deer, guinea pig, or primate, and recovering the antibodies in sera produced therein. The invention includes high avidity or high affinity antibodies produced therein. The invention also includes B cells produced from the listed species to be further used in cell fusion procedures for the manufacture of monoclonal antibody-producing hybridomas as disclosed herein. The invention is further drawn to a kit comprising the antibody or a portion thereof as disclosed herein, a container comprising said antibody and instructions for use, a kit comprising the polypeptides of this invention and instructions for use and a kit comprising the polynucleotide of the invention, a container comprising said polynucleotide and instructions for use, or any combinations thereof. These kits would include, but not be limited to nucleic acid detection kits, which may, or may not, utilize PCR and immunoassay kits. Such kits are useful for clinical diagnostic use and provide standardized reagents as required in current clinical practice. These kits could either provide information as to the presence or absence of mutations prior to treatment or monitor the progress of the patient during therapy. The kits of the invention may also be used to provide standardized reagents for use in research laboratory studies. Compounds of the Invention
[0183] In one aspect, the invention is also directed to a compound, a method of using a compound, a method of identifying a compound and the like.
[0184] The term "a", "an" or "one", as used in the present invention may refer to either the singular or the plural. For example, "a compound" encompasses one or more compounds.
[0185] Compounds useful in the methods of the present invention include derivatives of betulinic acid and betulin that are presented in U.S. Patent Nos. 5,679,828 and 6,172,110 respectively, and in U.S. application Nos. 60/443,180 and 10/670,797, which are herein incorporated by reference. Additional useful compounds include oleanolic acid derivatives disclosed by Zhu et al. (Bioorg. Chem Lett. 77:3115-3118 (2001)); oleanolic acid and promolic acid derivatives disclosed by Kashiwada et al. (J. Nat. Prod. 61 : 1090- 1095 (1998)); 3-O-acyl ursolic acid derivatives described by Kashiwada et al. (J. Nat. Prod. 65:1619-1622 (2000)); and 3-alkylamido-3-deoxy-betulinic acid derivatives, disclosed by Kashiwada et al. (Chem. Pharm. Bull. #:1387-1390 (2000)). (All references incorporated by reference).
[0186] In some embodiments, compounds useful in the present invention include, but are not limited to those betulinic acid derivatives having the general Formula /and dihydrobetulinic acid derivatives of Formula//:
or a pharmaceutically acceptable salt thereof, wherein, R is a C2-C20 substituted or unsubstituted carboxyacyl, R' is hydrogen, C2-Cιo substituted or unsubstituted alkyl, or aryl group. Prefeoed compounds are those wherein R is one of the substituents in Table 4, below, and R' is hydrogen. [0187] In other embodiments, useful compounds include derivatives of betulin and dihydrobetulin of Formula HI:
or a pharmaceutically acceptable salt thereof, wherein, Ri is a C2-C20 substituted or unsubstituted carboxyacyl, or an ester thereof; R2 is hydrogen, C(C6H5)3, or a C2-C2o substituted or unsubstituted carboxyacyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or di- alkylamino, or alkanoyl, benzoyl, or C2-C20 substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29. [0188] Prefeoed compounds useful in the present invention are those where R\ is one of the substituents in Table 4, R2 is hydrogen or one of the substituents in Table 4 and R3 is hydrogen. Table 4: Preferred Substituents for R, R', R R2:
[0189] More prefeoed compounds are 3-0-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, and 3-O-(3',3'-dimethylsuccinyl or glutaryl) dihydrobetulin.
[0190] A particularly prefeoed compound is 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
[0191] In some embodiments, compounds useful in the present invention are described by the Formulas IV, V, VI and VII.
Rπ = -ORι4 or -NHR15; R12 = COORπ, COO"A+, or CH2OR17 R13 = -H, halogen, amino, optionally substituted mono-or di-alkylamino, or -OR16; R14 = -H, C2 - C2o substituted or unsubstituted carboxyacyl; Ri5 = -H, C2 - C2o substituted or unsubstituted carboxyacyl;
R16 = -H, C - C7 alkanoyl, benzyloyl, or C2 - C20 substituted or unsubstituted carboxyacyl;
Rι7 = -H, C(C6H5)3, or C2 - C20 substituted or unsubstituted carboxyacyl; wherein dashed line represents optional bond between C 0 and C2 , and wherein A = Na+, K+, or other cation,
wherein
C5
90
O o t 5
H U α.
o
C5
O o o
[0192] R38 moieties other than hydrogen are attached to R33 oxygen by a covalent bond to the carbonyl carbon. [0193] Prefeoed compounds are those where R38 is not hydrogen.
[0194] In additional embodiments, any of R38, R40 and/or R41 are methyl.
[0195] In some embodiments, compounds useful in the methods of the invention also include those described in U.S. Provisional Application No. 60/559,358, which is entirely incorporated by reference. In one aspect, these compounds are described by reference to the following compounds VIII to XI: [0196] In some embodiments, compounds useful in the present invention have the general Formula VHP.
or a pharmaceutically acceptable salt or ester thereof: wherein A is a fused ring of formula
or (i) (ϋ) (iii) wherein the ring carbons designated x and y in the formulas of A are the same as the ring carbons designated x and y in Formula VIII; R51 is a carboxyalkanoyl, where the alkanoyl chain can be optionally substituted by one or more hydroxy or halo, or can be interrupted by a nitrogen, sulfur or oxygen atom, or combinations thereof; R52, R53 and RM are independently hydrogen, methyl, halogen, or hydroxy, carbonyl or -COOR^, wherein R^ is alkyl or carboxylalkyl, where the alkyl chain can be optionally substituted by one or more nydroxyl or halo, or can be interrupted by nitrogen, sulfur or oxygen atom, or combinations thereof; R55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyaUcanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo, or R55 is a carboxyl or hydroxymethyl; R56 is hydrogen, methyl, hydroxy or halogen; R57 and R58 are independently hydrogen or C1-6 alkyl; R59 is CH2 or CH3; Rβo is hydrogen, hydroxy or methyl; Rόi is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo; Rό2 is hydrogen or methyl; Rβ3 is hydrogen or methyl; R^ is hydrogen or hydroxy; Res is hydrogen if C12 and C13 form a single bond, or R^ is absent if C12 and C13 form a double bond; and wherein the straight dashed line represents an optional double bond between C12 and C13 or C20 and C29; with the proviso that when A is
then R51 cannot be glutaryl or succinyl when a double bond exists between C12 and C13; when A is (ii) and is methyl, then R51 cannot be succinyl; when A is (iii) and R52, R53 and R« are each hydrogen, then R ι cannot be succinyl; and with the proviso that A (i) cannot be
when R52 and R53 are both methyl and a double bond exists between C12 and C13. In some embodiments, R51 is a carboxy(C2-6)alkylcarbonyl group or a carboxy(C2-6)alkoxy(Cι-6)alkylcarbonyl group. Suitable groups are selected from the group consisting of:
[0198] According to the invention, in some embodiments the compounds have Formula IX:
wherein R51, R54, R55, R56, R57, R58 and R64 are as defined above for Formula VIII. In one embodiment, R56 is β-methyl, R58 is hydrogen, R55 is hydroxymethyl and R5ι is 3',3'-dimethylglutaryl, 3',3'-dimethylsuccinyl, glutaryl or succinyl. In another embodiment, R56 is hydrogen, R57 and R58 are both methyl, R55 is carboxyl and R51 is 3',3'-dimethylglutaryl, 3',3'- dimethylsuccinyl, glutaryl or succinyl. [0199] In some embodiments, R55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo, or R 5 is a carboxyl or hydroxymethyl. In some embodiments, R55 is selected from a group consisting of carboxyl, hydroxymethyl, -CO2(CH2)nCOOH, -CO2(CH2)nCH3, -CH2OC(O)(CH2)nCH3> -CH2OC(O)(CH2)nCOOH, -CO(CH2)nCH3 and -CO(CH2)nCOOH. In some embodiments, R55 is selected from a group consisting of
H02C όo
[0200] In some embodiments, R55 is hydroxymethyl. In some embodiments, R55 is carboxyl. In some embodiments, n is from 0 to 20, and preferably 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is from 1 to 6. In some embodiments, n
[0201] In some embodiments, compounds useful in the present invention have the Formula X:
wherein R51, R59, Rβo, and are as defined above for Formula VIII. In one embodiment, R5t is 3',3'-dimethylglutaryl, 3 ',3 '-dimethylsuccinyl, glutaryl or succinyl.
[0202] In some embodiments, R^ is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more hydroxy or halo. In some embodiments, R^ is selected from the group consisting of methyl, -CO2(CH2)nCOOH, -COC(O)(CH2)nCH3, -CO(CH2)nCH3 and -CO(CH2)nCOOH.
[0203] In some embodiments, n is from 0 to 20, or preferably 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is from 1 to 6. In some embodiments, n is from 2 to 6. In some embodiments, Rόi is methyl. In some embodiments, R^\ is methoxycarbonyl. In some embodiments, R6i is selected from the group consisting of methoxymethyl and ethoxymethyl. In some embodiments, methyl groups found in R^ can be substituted with a halogen or a hydroxy.
[0204] In some embodiments, the compounds useful in the present invention have Formula XI:
wherein R51, R52, R53, R54, and R^3 are as defined above for Formula VIII. In one embodiment, R51 is 3',3'-dimethylglutaryl, 3',3'-dimethylsuccinyl, glutaryl or succinyl. In one embodiment, both R52 and R53 are methyl. [0205] Any triterpene which falls within the scope of Formula VIII can be used. According to the invention, in some embodiments the compounds of Formula VIII are selected from the group consisting of derivatives of uvaol, ursolic acid, erythrodiol, echinocystic acid, oleanolic acid, sumaresinolic acid, lupeol, dihydrolupeol, betulinic acid methylester, dihydrobetulinic acid methylester, 17-α-methyl-androstanediol, androstanediol, and 4,4-dimethyl- androstanediol. [0206] In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein R52 and R 3 are both methyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein R51 is 3',3'-dimethylsuccinyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein R51 is succinyl, i.e.,
[0207] According to the invention, in some embodiments the stereochemistry of the sidechain substituents is important. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R55 is in the β position. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R56 is in the β position. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R64 is in the α position. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i), R57 is α-methyl, and R58 is hydrogen. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i), R58 is α-methyl, and R57 is hydrogen. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and both R57 and R58 are methyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (ii) and Ret is in the β position.
[0208] In some embodiments, 3',3'-dimethylsuccinyl is at the C3 position. In some embodiments, the compounds of Formula DC are 3-O-(3',3'- dimethylsuccinyl)uvaol; 3-O-(3',3'-dimethylsuccinyl)erythrodiol; 3- O-(3',3'- dimethylsuccinyl)echinocystic acid or 3-O-(3',3'-dimethylsuccinyl) sumaresinolic acid. In some embodiments, the compounds of Formula X are 3-O-(3',3'-dimethylsuccinyl) lupeol; 3-O-(3',3'-dimethylsuccinyl) dihydrolupeol; 3-0-(3',3'-dimethylsuccinyl)17β-methylester-betulinic acid; or 3-O-(3',3'-dimethylsuccinyl)17β-methylester-dihydrobetulinic acid. In some embodiments, the compounds of Formula XI are 3-O-(3',3'-dimethylsuccinyl) 4,4-dimethylandrostanediol; 3-0-(3',3'-dimethylsuccinyl)l 7α- methylandrostanediol; 3-O-(3',3'-dimethylsuccinyl) androstanediol. [0209] In an additional embodiment, the invention includes compounds and methods that use compounds of Formula^//:
where R72 is one of:
wherein Z is hydroxy or halogen; and R73 is lower alkyl, such as methyl, ethyl or propyl. [0210] In additional embodiments, compounds useful in the present invention are betulin derivative compounds of Formula AT /: or a pharmaceutically acceptable salt or prodrag thereof, wherein: R51 is C3-C20 alkanoyl, carboxyalkanoyl, carboxyalkenoyl, alkoxycarbonylalkanoyl, alkenyloxycarbonylalkanoyl, cyanoalkanoyl, hydroxyalkanoyl, aminocarbonylalkanoyl, hydroxyaminocarbonylalkanoyl, monoalkylaminocarbonylalkanoyl, dialkylaminocarbonylalkanoyl, heteroarylalkanoyl, heterocyclylaUcanoyl, heterocyclylcarbonylalkanoyl, heteroarylaminocarbonylalkanoyl, heterocyclylaminocarbonylalkanoyl, cyanoaminocarbonylalkanoyl, alkylsulfonylaminocarbonylalkanoyl, arylsulfonylaminocarbonylalkanoyl, sulfoaminocarbonylalkanoyl, phosphonoaminocarbonylalkanoyl, tetrazolylalkanoyl, phosphono, sulfo, phosphonoalkanoyl, sulfoalkanoyl, alkylsulfonylalkanoyl, or alkylphosphonoalkanoyl; R82 is formyl, carboxyalkenyl, heterocyclyl, heteroaryl, -CH2SR94,
(i) (") (iii) (IV)
(v) (vi) (vii) (viii) R83 is hydroxyl, isopropenyl, isopropyl, l'-hydroxyisopropyl, l'-haloisopropyl, l'-thioisopropyl, l'-trifluoromethylisopropyl, 2'-hydroxyisopropyl, 2'-haloisopropyl, 2'-thioisopropyl, 2'-trifluoromethylisopropyl, l'-hydroxyethyl, l'-(alkoxy)ethyl,
1 '-(alkoxyalkoxy)ethyl, 1 '-(arylalkoxy)ethyl; 1 '-(arylcarbonyloxy)ethyl, 1 '-(oxo)ethyl, 1 '-(hydroxyl)- 1 '-(hydroxyalkyl)ethyl, 1 '(oxo)oxazolidinyl, l',2'-epoxyisopropyl, 2'-haloisopropenyl, 2'-hydroxyisopropenyl,
2'-aminoisopropenyl, or
wherein Y is -SRmor -NR] 13Rn4; Rπi is methyl; R112 is hydrogen or hydroxyl; R113 and R114 are independently hydrogen, alkyl, alkanoyl, arylalkyl, heteroarylalkyl, arylsulfonyl or arylaminocarbonyl; or R113 and R114 can be taken together with the nitrogen to which they are attached to form a heterocycle, wherein the heterocycle can optionally include one or more additional nitrogen, sulfur or oxygen atoms; m is zero to three; R84 is hydrogen; or R83 and R84 can be taken together to form oxo, alkylimino, alkoxyimino or benzyloxyimino; Rg5 is C2-C20 alkyl, alkenyl, C2-C20 carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl, diaUcylaminoalkyl, alkoxyalkyl, cyano, cyanoalkyl, alkylthioalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkanoylaminoalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, heterocyclylcarbonylalkyl, cycloalkylcarbonylalkyl, heteroarylalkylaminocarbonylalkyl, arylalkylaminocarbonylalkyl, heterocyclylalkylaminocarbonylalkyl, carboxyaUcylaminocarbonylalkyl, arylsulfonylaminocarbonylalkyl, alkylsulfonylaminocarbonylalkyl, arylphosphonoaminocarbonylalkyl, alkylphosphonoaminocarbonylalkyl, or hydroxyimino(amino)aUcyl; R86 is hydrogen, phosphono, sulfo, cyano, alkyl, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyalkyl, alkoxycarbonylalkyl, or cyanoalkyl; Rg or Rs8 are independently hydrogen, alkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxyalkyl, alkoxyaUcoxyalkyl, alkoxycarbonylaminoalkoxyalkyl, alkoxycarbonylaminoalkyl, aminoalkoxyaUcyl, aUcylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, or cycloalkyl, or Rs7 and R«8 can together with the nitrogen atom to which they are attached form a heterocyclyl or heteroaryl group, wherein the heterocyclyl or heteroaryl can optionally include one or more additional nitrogen, sulfur or oxygen atoms; R89 is hydrogen, phosphono, sulfo, cyano, alkyl, alkylsilyl, cycloalkyl, carboxyalkyl, alkoxycarbonyloxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, cyanoalkyl, phosphonoalkyl, sulfoalkyl, alkylsulfonyl, alkylphosphono, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, or dialkoxyalkyl; R 0 and R 1 are independently hydrogen, hydroxyl, cyano, alkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyl, carboxyalkyl, alkanoyloxyalkyl, alkoxyalkyl, alkoxyaUcoxyalkyl, alkoxycarbonylaminoalkoxyalkyl, alkoxycarbonylaminoalkyl, aminoalkoxyalkyl, aUcylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, arylsulfonyl, or cycloalkyl, or alkyl interrupted by one or more oxygen atoms, or R90 and R91 can together with the nitrogen atom to which they are attached form a heterocyclyl group, wherein the heterocyclyl can optionally include one or more additional nitrogen, sulfur or oxygen atoms; R92 and R 3 are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloalkyloxy, heterocyclylaminoalkyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R92 and R93 can together with the nitrogen atom to which they are attached form a heterocyclyl group, wherein the heterocyclyl can optionally include one or more additional nitrogen, sulfur or oxygen atoms, or R92 and R93 can together with the nitrogen atom to which they are attached form an alkylazo group, and b is one to six; R94 is hydrogen, alkyl, alkenyl, arylalkyl, carboxyalkyl, carboxyalkenyl, alkoxycarbonylalkyl, alkenyloxycarbonylalkyl, cyanoalkyl, hydroxyalkyl, carboxybenzyl, aminocarbonylalkyl; R 5 and R 6 are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloalkyloxy, heterocyclylaminoaUcyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R95 and R96 can together with the nitrogen atom to which they are attached form a heterocyclyl group, wherein the heterocyclyl can optionally include one or more additional nitrogen, sulfur or oxygen atoms, or R95 and R96 can together with the nitrogen atom to which they are attached form an alkylazo group; R97 is hydrogen, alkyl, alkenyl, carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, cyanoalkyl, alkylthioalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, alkanoylaminoalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, heterocyclylcarbonylalkyl, cycloalkylcarbonylalkyl, heteroarylalkylaminocarbonylalkyl, arylalkylaminocarbonylalkyl, heterocyclylalkylaminocarbonylalkyl, carboxyalkylaminocarbonylalkyl, arylsulfonylaminocarbonylalkyl, alkylsulfonylaminocarbonylalkyl, or hydroxyimino(amino)alkyl; R98 and R99 are independently hydrogen, methyl or ethyl, preferably hydrogen or methyl; and d is from one to six. Alkyl groups and alkyl containing groups of the compounds of the present invention can be straight chain or branched alkyl groups, preferably having one to ten carbon atoms. In some embodiments, the alkyl groups or alkyl containing groups of the present invention can be substituted with a C3- cycloalkyl group. In some embodiments, the cycloalkyl group may include, but is not limited to, a cyclobutyl, cyclopentyl or cyclohexyl group. [0212] Also, included within the scope of the present invention are the non- toxic pharmaceutically acceptable salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free acid form with a suitable organic or inorganic base and isolating the salt thus formed. These can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, N-methyl glucamine and the like.
[0213] Also, included within the scope of the present invention are the non- toxic pharmaceutically acceptable esters of the compounds of the present invention. Ester groups are preferably of the type which are relatively readily hydrolyzed under physiological conditions. Examples of pharmaceutically acceptable esters of the compounds of the invention include C1-6 alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-7 cycloalkyl esters as well as arylalkyl esters, such as, but not limited to benzyl. C1-4 alkyl esters are prefeoed. In some embodiments, the esters are selected from the group consisting of alkylcarboxylic acid esters, such as acetic acid esters, and mono- or dialkylphosphate esters, such as methylphoshate ester or dimethylphosphate ester. Esters of the compounds of the present invention can be prepared according to conventional methods.
[0214] Certain compounds are listed above derivatives refeoed to as "prodrugs". This includes compounds within the scope of Formula VIII to XI, for example. The expression "prodrag" refers to compounds that are rapidly transformed in vivo by an enzymatic or chemical process, to yield the parent compound of the above formulas, for example, by hydrolysis in blood. A thorough discussion is provided by Higuchi, T. and V. Stella in Pro-drugs as Novel Delivery Systems, Vol. 14, A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association, Pergamon Press, 1987. Useful prodrugs can be esters, for example, of the compounds of Formulae VIII, DC, X, and XI. In some prodrag embodiments, a lower alkyl group is substituted with one or more hydroxy or halo groups by a suitable acid. Suitable acids include, e.g., carboxylic acids, sulfonic acids, phosphoric acid or lower alkyl esters thereof, and phosphonic acid or lower alkyl esters thereof. For example, suitable carboxylic acids include alkylcarboxylic acids, such as acetic acid, arylcarboxylic acids and arylalkylcarboxylic acids. Suitable sulfonic acids include alkylsulfonic acids, arylsulfonic acids and arylalkylsulfonic acids. Suitable phosphoric and phosphonic acid esters are methyl or ethyl esters.
[0215] In some embodiments, the C3 acyl groups having dimethyl groups or oxygen at the C3' position can be the most active compounds. This observation suggests that these types of acyl groups might be important to the enhanced anti-HIN activity.
[0216] In one embodiment, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formula / through XIII
[0217] In one embodiment, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formula /through XIII, with the exception of DSB.
[0218] In one embodiment, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII, or is other than a compound of Formula / through XIII. In one embodiment, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through XI; or in other embodiments is other than / through XIII. [0219] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formula Groups /through XIII.
[0220] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII; or in other embodiments is other than a compound of Formula / through XIII.
[0221] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through XI; or in other embodiments is other than a compound of Formula / through XIII.
[0222] In another embodiment, the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formula / through XIII.
[0223] In another embodiment, the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is other than a compound of Formula / through VII; or in other embodiments is other than a compound of Formula / through XIII.
Synthesis of Dsb and Related Compounds
[0224] Reaction of betulinic acid and dihydrobetulinic acid with dimethylsuccinic anhydride produced a mixture of 3-0-(2',2'- dimethylsuccinyl) and 3-0-(3',3'-dimethylsuccinyl)-betulinic acid and dihydrobetulinic acid, respectively. The mixtures were successfully separated by preparative scale HPLC yielding pure samples. The structures of these isomers were assigned by long-range 1H-13C COSY examinations. [0225] The derivatives of betulinic acid and dihydrobetulinic acid of the present invention were all synthesized by refluxing a solution of betulinic acid or dihydrobetulinic acid, dimethylaminopyridine (1 equivalent mol), and an appropriate anhydride (2.5-10 equivalent mol) in anhydrous pyridine (5-10 mL). The reaction mixture was then diluted with ice water and extracted with CHC13. The organic layer was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was chromatographed using silica gel column or semi-preparative-scale HPLC to yield the product.
[0226] Preparation of 3-O-(3',3*-dimethylsuccinyl) betulinic acid: yield 70% (starting with 542 mg of betulinic acid); crystallization from MeOH gave colorless needles; mp 274°-276°C; [α]D 19+23.5° (c=0.71), CHCl3-MeOH [1:1]); Positive FABMS m z 585 (M+H)+; Negative FABMS m/z 583 (M-HV; HR-FABMS calcd for C36H57O6 585.4155, found m/z 585.4161; 1H NMR (pyridine-d5): 0.73, 0.92, 0.97, 1.01, 1.05 (each 3H, s; 4-(CH3)2, 8-CH3, 10- CH3, 14-CH3), 1.55 (6H, s, 3'-CH3 x 2), 1.80 (3H, s, 20-CH3), 2.89, 2.97 (each 1H, d, J=15.5 Hz, H-2'), 3.53 (1H, m, H-19), 4.76 (1H, dd, J=5.0, 11.5 Hz, H- 3), 4.78, 4.95 (each 1H, br s, H-30).
[0227] 3-O-(3',3'-dimethylsuccinyl) dihydrobetulinic acid: yield 24.5% (starting with 155.9 mg of dihydrobetulinic acid); crystallization from MeOH- H2O gave colorless needles; mp 291°-292°C; [α]D 20-13.4° (c=l.l, CHC13- MeOH [1:1], 1H NMR (pyridine-d5): 0.85, 0.94 (each 3H, d, J=7.0 Hz; 20- (CH3)2), 0.75, 0.93, 0.97, 1.01, 1.03 (each 3H, s; 4-(CH3)2, 8-CH3, 10-CH3, 14- CH3), 1.55 (6H, s; 3'-CH3 x 2), 2.89, 2.97 (each 1H, d, J=15.5 Hz; H-2'), 4.77 (1H, dd, J=5.0, 11.0 Hz, H-3); Anal. Calcd for C36H58O6.5/2H2O: C 68.43, H 10.04; found C 68.64, H 9.78.
[0228] The synthesis of 3-O-(3',3'-dimethylglutaryl) betulinic acid was disclosed U.S. Patent No. 5,679,828, as COMPOUND NO. 4.
[0229] 3-O-(3',3'-dimethylglutaryl) dihydrobetulinic acid: yield 93.3% (starting with 100.5 mg of dihydrobetulinic acid); crystallization from needles MeOH-H2O gave colorless needles; mp 287°-289°C; [α]D 20-17.9° (c=0.5, CHCl3-MeOH[l:l]); 1H-NMR (pyridine-d5): 0.86, 0.93 (each 3H, d, J=6.5 Hz; 20-(CH3)2), 0.78, 0.92, 0.96, 1.02, 1.05 (each 3H, s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3), 1.38, 1.39 (each 3H, s; 3'-CH3 x 2), 2.78 (4H, m, H2 -2' and 4% 4.76 (1H, dd, J=4.5, 11.5 Hz; H-3). Anal. Calcd for C37H60O6 : C 73.96, H 10.06; found C 73.83, H 10.10.
[0230] The synthesis for 3-O-diglycolyl-betulinic acid was disclosed in U.S. Patent No. 5,679,828, as COMPOUND NO. 5.
[0231] 3-O-diglycolyl-dihydrobetulinic acid: yield 79.2% (starting with 103.5 mg of dihydrobetulinic acid); an off-white amorphous powder; [α]D 20-9.8° (c=l.l, CHC13 -MeOH[l:l]); 1H-NMR (pyridine-d5): 0.79, 0.87 (each 3H, d, J=6.5 Hz; 20-(CH3)2), 0.87, 0.88, 0.91, 0.98, 1.01 (each 3H, s; 4-(CH3)2, 8- CH3, 10-CH3, 14-CH3), 4.21, 4.23 (each 2H, s, H2-2' and 4'), 4.57 (1H, dd, J=6.5, 10.0 Hz, H-3); Anal. Calcd for C34H54O7.2H2O: C 66.85, H 9.57; found C 67.21, H 9.33.
[0232] The syntheses of 3-O-(3',3'-dimethylsuccinyl) betulin and 3-0-(3',3'- dimethylglutaryl) betulin were disclosed in U.S. Application 10/670,797.
Methods Of Inhibiting Hiv With A Compound
[0233] Methods of "inhibiting HTV" or "inhibition of HTV" as used herein, means any interference in, inhibition of, and/or prevention of HTV using the methods of the invention. As such, methods of inhibition are useful in inhibiting with the infectivity of HTV, inhibition of p25 processing, inhibition of viral maturation, formation of virions that exhibit altered phenotypes, and the like. Preferably, methods of the invention act upon p25 processing in the cells of an animal, but are not limited by that method of action.
[0234] A method of inhibiting HTV with a compound may be relevant to a method of treating HTV infection in a patient. Therefore, a method of inhibiting HIN with a compound may similarly be used to treat a patient.
[0235] The methods of inhibiting HIN-1 replication in cells of an animal includes contacting infected cells with a compound of Formula /through XIII, above. Related embodiments include a method of treating a HIN-1 infectionin a patient comprising administration of a compound of Formula / through XIII; a method of inhibiting p25 processing either in a cell, in vivo, and/or in vitro, by administration of a compound that inhibits said p25 processing; and a method of treating human blood or blood products by administering a compound of Formula / through XIII. Also included are a method of identifying a compound that inhibits any one of p25 processing, HIN maturation, HIN infectivity, HTV virion phenotypes and the like.
[0236] In one embodiment, the compound is a derivative of betulinic acid, betulin, or dihydrobetulinic acid or dihydrobetulin and which includes the prefeoed substituents of Table 4. Prefeoed compounds include but are not limited to 3-O-(3',3'-dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-O-(3',3'-di methylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-0-diglycolyl-betulinic acid, and 3-O-diglycolyl-dihydrobetulinic acid.
[0237] In one embodiment, the invention is drawn to a method inhibiting HIV-l replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is a compound of Formulas /through XIII above.
[0238] In one embodiment, the invention is drawn to a method of inhibiting HIV-l replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is compound of Formulas / through XIII, with the exception of DSB.
[0239] In one embodiment, the invention is drawn to a method of inhibiting HIN-1 replication in cells of an animal by contacting infected cells with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is one that is excluded from those of Formulas / through VI. In one embodiment, the invention is drawn to a method of treating HIN-1 infection in a patient by administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps, wherein said compound is one that is other than those of Formulas / through XIII.
[0240] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound of Formulas /through XIII.
[0241] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas /through VI.
[0242] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas /through XIII.
[0243] In another embodiment, the invention is drawn to a method of inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affecting other Gag processing steps, wherein said compound is a compound other than those of Formulas / through XI. In another embodiment, the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound of Formulas / through XIII.
[0244] In another embodiment, the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound other than those of Formulas /through VI.
[0245] In another embodiment, the invention is drawn to a method of treating human blood or blood products by inhibiting processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) in a cell, but without significantly affect other Gag processing steps, wherein said compound is a compound other than those of Formulas / through XIII.
[0246] The method disclosed herein, further comprises contacting said cells with one or more drags selected from the group consisting of anti-viral agents, anti-fungal agents, anti-bacterial agents, anti-cancer agents, immunostimulating agents, and combinations thereof. The method may include the treatment of human blood products. [0247] The invention may also be used in conjunction with a method of treating cancer comprising the administration to an animal of one or more anti- neoplastic agents, exposing an animal to a cancer cell-killing amount of radiation, or a combination of both.
Methods of Identifying Compounds
[0248] The invention further includes a method for identifying compounds that inhibit HTV-1 replication in cells of an animal disclosed herein. In one embodiment, said method comprises: (a) contacting a Gag polypeptide comprising a CA-SPl cleavage site with a test compound; (b) adding a labeled substance that selectively binds at or near the CA-SPl cleavage site; and (c) measuring the binding of the test compound at or near the CA- SP1 cleavage site.
[0249] Labeled substances or molecules include labeled antibodies or labeled DSB and the label includes an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, such as gold, osmium tetroxide, lead or uranyl acetate, and radioisotope, antibodies labeled with such substances of molecules or a combination thereof. The assays could include, but are not limited to ELISA, single and double sandwich techniques, immunodiffusion or immunoprecipitation techniques, as known in the art (^Immunoassay Handbook, Td ed.," D. Wild, Nature Publishing Group, (2001)). Said methods of identifying also could include, but are not limited to Western blot assays, colorimetric assays, light and electron microscopic techniques, confocal microscopy, or other techniques known in the art.
[0250] A method of identifying compounds that inhibit HIV replication in cells of an animal further comprises: (a) contacting a Gag protein comprising a wild-type CA-SPl cleavage site, with HIN-1 protease in the presence of a test compound; (b) separately, contacting a Gag protein comprising a mutant CA- SPl cleavage site or a protein comprising an alternative protease cleavage site with HIN-1 protease in the presence of the test compound; and (c) comparing the cleavage of the native wild-type Gag protein to the amount of cleavage of the mutant Gag protein or to the amount of cleavage of the peptide comprising an alternative protease cleavage site.
[0251] Step (b) above is performed as a control in order to eliminate compounds that might bind directly to, and therefore inhibit, the protease enzyme. The above method also includes the method wherein the wild-type CA-SPl, mutant CA-SPl or alternative protease cleavage site is contained within a polypeptide fragment or recombinant peptide.
[0252] The method for identifying compounds that inhibit HTV-1 disclosed herein, also includes a method wherein said peptide or protein is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety, or wherein said peptide or protein is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound and HIN-1 protease and comparing said transfer of fluorescent energy to that observed when the same procedure is applied to a peptide that comprises a sequence containing a mutation in the CA-SPl cleavage site or that comprises a sequence containing another cleavage site. Examples of fluorescence-based assays of protease activity are well known in the art. In one such example, a protease substrate is labeled with green fluorescent dye molecules, which fluoresce when the substrate is cleaved by the protease enzyme (Molecular Probes, Protease Assay Kit).
[0253] The method of comparing the cleavage, above, also includes using a labeled antibody that selectively binds CA or SPl or CA-SPl in order to measure the extent to which the test compound inhibits CA-SPl cleavage. The antibody can be labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, and radioisotope, or combinations thereof. The method also includes the use of an antibody to a specific epitope tag sequence to selectively detect CA-SPl (p25) or SPl, into which the amino acid sequence for that epitope tag has been engineered according to standard methods in the art. Suitable tags are well known to those of ordinary skill in the art, and include haemagglutinin epitope HA (YPYDNPDYA) (SEQ ED NO: 81), bluetongue viras epitope VP7 (QYPALT) (SEQ ED NO: 82), α-tubulin epitope (EEF), Flag (DYKDDDDK) (SEQ ED NO: 83), and VSV-G (YTDOEMNRLGK) (SEQ ED NO: 84). Examples of SPl containing epitope tags are illustrated in Figure 17.
[0254] As an example, the sequence of the FLAG epitope tag (Sigma- Aldrich) is inserted into the p2 (SPl) region of Gag by oligonucleotide-directed mutagenesis of a Gag expression plasmid. The presence of the SPl domain in the cell-expressed protein is then be detected using commercially available anti-FLAG monoclonal antibodies (Sigma-Aldrich). (Hopp, T.P. Biotechnology 6: 1204-1210 (1988)).
[0255] The method of identifying compounds that disrapt CA-SPl cleavage also includes the addition of a compound to cells infected with HIN-1 and the detection of CA-SPl cleavage products by lysing and analyzing the cells or the released virions. The method included in the invention can be performed using a western blot analysis of viral proteins and detecting p25 using an antibody to p25 or wherein said mixture is analyzed by performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins, or wherein the mixture is analyzed using immunoassays that use an antibody that selectively binds p25 or an antibody that selectively binds in order to distinguish p25 from p24. The invention includes the use of an antibody to a specific epitope tag sequence inserted into the C-terminal domain of SPl to selectively detect p25 or SPl. For example, a sandwich ELISA assay can be performed where p25 and p24 in detergent-solubilized viras are captured using an antibody that selectively binds to the CA region of Gag, which antibody is bound to a multiple well plate. Following a washing step, bound p25 is detected using an antibody to an epitope tag inserted in SPl, which is conjugated to an appropriate detection reagent (e.g. alkaline phosphatase for an enzyme-linked immunosorbent assay). Viras released by cells treated with compounds that act via this mechanism will generally have increased levels of p25 compared with untreated virions.
[0256] The disclosed method is drawn to an antibody that selectively binds p25, or an antibody that selectively binds SPl, or an antibody to an epitope tag sequence inserted into SPl, which is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, and radioisotope, or combinations thereof.
[0257] "Infected cells," as used herein, includes cells infected naturally by membrane fusion and subsequent insertion of the viral genome into the cells, or transfection of the cells with viral genetic material through artificial means. These methods include, but are not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, lipid-mediated transfection, electroporation or infection.
[0258] The invention may be practiced by infecting target cells in vitro with an infectious strain of HIN and in the presence of test compound, under appropriate culture conditions and for varying periods of time. Infected cells or supernatant fluid can be processed and loaded onto a polyacrylamide gel for the detection of viras levels, by methods that are well known in the art. Νon- infected and non-treated cells can be used as negative and positive infection controls, respectively. Alternatively, the invention may be practiced by culturing the target cells in the presence of test compound prior to infecting the cells with an HIN strain.
[0259] The invention also includes a method for identifying compounds that inhibit HIV-l replication in the cells of an animal, comprising: (a) contacting a test compound with wild-type viras isolates and separately with virus isolates having redued sensitivity to 3-O-(3',3'- dimethylsuccinyl) betulinic acid; and (b) selecting test compounds that are more active against the wild- type viras isolate compared with vims isolates that have reduced sensitivity to 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
[0260] This invention further includes a method for identifying compounds that act by any of the abovementioned mechanism, involving treating HTV-1 infected or transfected cells with a compound then analyzing the viras particles released by compound-treated cells by thin-sectioning and transmission electron microscopy, by standard methods well known in the art. A compound acts by the abovementioned mechanism if particles are detected that exhibit spherical condensed cores that are acentric with respect to the viral particle and/or a crescent-shaped electron-dense layer just inside the viral membrane.
[0261] For electron microscopic studies, infected cells or centrifuged viras pellets obtained from the supernatant fluid can be contacted with a fixative, such as glutaraldehyde or freshly-made paraformaldehyde, and/or osmium tetroxide or other electron microscopy compatible fixative that is known in the art. The vims from the supernatant fluid or the cells, is dehydrated and embedded in an electron-lucent polymer such as an epoxy resin or methacrylate, thin sectioned using an ultramicrotome, stained using electron dense stains such as uranyl acetate, and/or lead citrate, and viewed in a transmission electron microscope. Non-infected and non-treated cells can be used as negative and positive infection controls, respectively. Alternatively, the invention may be practiced by culturing the target cells in the presence of test compound prior to infecting the cells with an HTV strain. Maturation defects caused by the compounds of the present invention are determined by the presence of morphologically abeoant viral particles, compared with controls, as described herein.
[0262] For cell culture studies, the virus-infected cells may be observed for the formation of syncytia, or the supernatant may be tested for the presence of HIN particles. Virus present in the supernatant may be harvested to infect other naive cultures to determine infectivity. [0263] Also included in the invention, is a method of determining if an individual is infected with HTV-1, is susceptible to treatment by a compound that inhibits p25 processing, the method involves taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the CA-SPl cleavage site.
[0264] The invention also includes a method for identifying compounds that act by the abovementioned mechanisms, involving testing by a combination of the methods disclosed herein.
[0265] HTV Gag protein and fragments thereof for use in the aforementioned assays may be expressed or synthesized using a variety of methods familiar to those skilled in the art. Gag can be produced in an in vitro transcription and translation system using a rabbit reticulocyte lysate. Gag expressed in this system has been shown to be processed sequentially in a pattern similar to that observed in infected cells (Pettit, S.C. et al. J. Virol. 76:10226-10233 (2002)). Moreover, Gag expressed by this method is capable of assembling into immature viral particles when fused to a heterologous type D retroviral cytoplasmic self-assembly domain (Sakalian, M. et al, J. Virol 76:10811- 10820 (2002)). The plasmid pDAB72, available from the NTH AIDS Reagent Program can be used for this purpose (Erickson-Viitanen, S. et al, AIDS Res. Hum. Retroviruses. 5:577-91 (1989); Sidhu M.K. et al, Biotechniques, 18:20, 22, 24 (1995)). Other in vitro transcription/translation systems based on wheat germ or bacterial lysates can also be used for this purpose. HTV Gag may also be expressed in transfected cells using a variety of commercially available expression vectors. The plasmid p55-GAG/GFP, available from the NIH ADDS Reagent Program, may be used to express an HTV Gag-green fluorescent protein fusion protein in mammalian cells for drag interaction studies (Sandefur, S. et al, J. Virol. 72:2723-2732 (1998)). This construct would permit the capture and purification of Gag fusion protein using GFP- specific monoclonal antibodies. In addition, Gag may be expressed in cells using recombinant viral vectors, such as those used in the vaccinia viras, adenoviras, or baculoviras systems. Gag can also be expressed by infecting cells with HTV or by transfecting cells with proviral DNA. Finally, Gag may be expressed in yeast or bacterial cells transformed with the appropriate expression vectors.
[0266] In addition to Gag proteins expressed in cells or in vitro using cell lysates, peptides cooesponding to various regions of Gag may be commercially synthesized from using standard peptide synthesis techniques.
[0267] The invention further encompasses compounds identified by the method of this invention and/or a compound which inhibits HIN-1 replication according to the methods of this invention and pharmaceutical compositions comprising one or more compounds as disclosed herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers.
Pharmaceutical Compositions
[0268] Compounds according to the present invention have been found to possess anti-retroviral, particularly anti-HIN, activity. The salts and other formulations of the present invention are expected to have improved water solubility, and enhanced oral bioavailability. Also, due to the improved water solubility, it will be easier to formulate the salts of the present invention into pharmaceutical preparations. Further, compounds according to the present invention are expected to have improved biodistribution properties.
[0269] In one embodiment, the compounds are those of Formula / through XIII, in another they are compounds other than the compounds of Formula / through XIII.
[0270] This invention also includes a pharmaceutical composition comprising a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps, or that inhibits the maturation of viras particles released from treated infected cells, such as the compounds of Formula / through XIII. The invention includes a pharmaceutical composition comprising one or more compounds disclosed herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers, wherein said compound is of Formulae / through XIII above, or preferably, wherein said compound is selected from the group consisting of 3-O-(3',3,-dimethylsuccinyl) betulinic acid, 3-0-(3',3'- dimethylsuccinyl) betulin, 3-O-(3',3'-dimethylglutaryl) betulin, 3-0-(3',3'- dimethylsuccinyl) dihydrobetulinic acid, 3-O-(3',3'-dimethylglutaryl) betulinic acid, (3',3'-dimethylglutaryl) dihydrobetulinic acid, 3-0-diglycolyl-betulinic acid, and 3-O-diglycolyl-dihydrobetulinic acid. The pharmaceutical compositions according to the invention, further comprise one or more drags selected from an anti-viral agent, anti-fungal agent, anti-cancer agent or an immunostimulating agent.
[0271] Pharmaceutical compositions of the present invention can compose at least one of the compounds of Formulae / through XIII disclosed herein. Pharmaceutical compositions according to the present invention can also further comprise other anti-viral agents such as, but not limited to, AZT (zidovudine, RETROVEt®, GlaxoSmithKline), 3TC (lamivudine, EPTVTR®, GlaxoSmithKline), AZT+3TC, (COMBTVTR®, GlaxoSmithKline), AZT+3TC+abacavir (TRIZTVTR®, GlaxoSmithKline), ddl (didanosine, VIDEX®, Bristol-Myers Squibb), ddC (zalcitabine, HINED®, Hoffmann-La Roche), D4T (stavudine, ZERIT®, Bristol-Myers Squibb), abacavir (ZIAGEΝ®, GlaxoSmithKline), tenofovir (VIREAD®, Gilead Sciences), nevirapine (VIRAMUΝE®, Boehringer Ingelheim), delavirdine (Pfizer), emtricitabine (EMTRIVA®, Gilead Sciences), efavirenz (SUSTTVA®, DuPont Pharmaceuticals), saquinavir (IΝVIRASE®, FORTOVASE®, Hofϊmann-LaRoche), ritonavir (ΝORVIR®, Abbott Laboratories), indinavir (CRDOVAΝ®, Merck and Company), nelfinavir (VIRACEPT®, Pfizer), amprenavir (AGEΝERASE®, GlaxoSmithKline), adefovir (PREVEOΝ®, HEPSERA®, Gilead Sciences), atazanavir (Bristol-Myers Squibb), fosamprenavir (LEXINA®, GlaxoSmithKline) and hydroxyurea (HYDREA®, Bristol-Meyers Squibb), or any other antiretroviral drags or antibodies in combination with each other, or associated with a biologically based therapeutic, such as, for example, gp41 -derived peptides enfuvirtide (FUZEOΝ®, Roche and Trimeris) and T-1249, or soluble CD4, antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionally presented herein.
[0272] Additional suitable antiviral agents for optimal use with one of the compounds of Formulae / through XIII of the present invention can include, but are not limited to,amphotericin B (FUNGIZONE®); Ampligen (mismatched RNA) developed by Hemispherx Biopharma; ; BETASERON® (β-interferon, Chiron); butylated hydroxytoluene; Caoosyn (polymannoacetate); Castanospermine; Contracan (stearic acid derivative); Creme Pharmatex (containing benzalkonium chloride); 5-unsubstituted derivative of zidovudine; penciclovir (DENAVIR® Novartis); famciclovir (FAMVT ® Novartis); acyclovir (ZOVIRAX® GlaxoSmithKline ); cytofovir (VISTEDE® Gilead); ganciclovir (CYTOVENE®, Hoffman LaRoche); dextran sulfate; D-penicillamine (3 -mercapto-D- valine); FOSCARNET® (trisodiimi phosphonoformate; AstraZeneca); fusidic acid; glycyohizin (a constituent of licorice root); HPA-23 (ammonium-21-tungsto-9-antimonate); ORNEDYL® (eflornithine; Aventis); nonoxynol; pentamidine isethionate (PENTAM-300); Peptide T (octapeptide sequence; ,Peninsula Laboratories); Phenytoin (Pfizer); TNH or isoniazid; ribavirin (VIRAZOLE®, Valeant Pharmaceuticals); rifabutin, ansamycin (MYCOBUTTN® Pfizer); CD4-IgG2 (Progenies Pharmaceuticals) or other CD4-containing or CD4-based molecules; Trimetrexate (Medimmune); suramin and analogues thereof (Bayer); and WELLFERON® (α-interferon, GlaxoSmithKline). Pharmaceutical compositions of the present invention can also further comprise immunomodulators. Suitable immunomodulators for optional use with a betulinic acid or betulin derivative of the present invention in accordance with the present invention can include, but are not limited to: ABPP (Bropririmine); Ampligen (mismatched RNA) Hemispherx Biopharma; anti-human interferon-α-antibody; ; ascorbic acid and derivatives thereof; interferon-β; Ciamexon; cyclosporin; cimetidine; CL-246,738; colony stimulating factors, including GM-CSF; dinitrochlorobenzene; HE2000 (Hollis-Eden Pharmaceuticals); inteferon-γ; glucan; hyperimmune gamma- globulin (Bayer); immuthiol (sodium diethylthiocarbamate); interleukin-1 (Hoffmann-LaRoche; Amgen), interleukin-2 (IL-2) (Chiron); isoprinosine (inosine pranobex); Krestin ; LC-9018 (Yakult); lentinan (Yamanouchi); LF- 1695; methionine-enkephalin; Minophagen C; muramyl tripeptide, MTP-PE; naltrexone ( Bao Laboratories); RNA immunomodulator ; REMUNE® (Immune Response Corporation),;RETICULOSE® (Advanced Viral Research Corporation); shosaikoto; ginseng; thymic humoral factor; Thymopentin; thymosin factor 5; thymosin 1 (ZADAXIN®, SciClone), thymostimulin, TNF (tumor necrosis factor, Genentech), and vitamin preparations.
[0274] Pharmaceutical compositions of the present invention can also further comprise anti-cancer therapeutic agents. Suitable anti-cancer therapeutic agents for optional use include an anti-cancer composition effective to inhibit neoplasia comprising a compound, or a pharmaceutically acceptable salt or prodrag of said anti-cancer agent, which can be used for combination therapy include, but are not limited to alkylating agents, such as busulfan, cis-platin, mitomycin C, and carboplatin antimitotic agents, such as colchicine, vinblastine, taxols, such as paclitaxel (TAXOL®, Bristol-Meyers Squibb) docetaxel (TAXOTERE®, Aventis), topo I inhibitors, such as camptothecin, irinotecan and topotecan (HYCAMTIN®, GlaxoSmithKline), topo II inhibitors, such as doxorubicin, daunorabicin and etoposides such as VP16; RNA/DNA antimetabolites, such as 5-azacytidine, 5-fluorouracil and methotrexate, DNA antimetabolites, such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea, thioguanine, and antibodies, such as trastuzumab (HERCEPTIN®, Genentech), and rituximab (RITUXAN®, Genentech and Biogen-Idec), melphalan, chlorambucil, cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarabicin, bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid, tamoxifen, alanosine, and combinations thereof.
[0275] The invention further provides methods for providing anti-bacterial therapeutics, anti-parasitic therapeutics, and anti-fungal therapeutics, for use in combination with the compounds of the invention and pharmaceutically- acceptable salts thereof. Examples of anti-bacterial therapeutics include compounds such as penicillins, ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin, cepharadine, cefadoxil, cefaclor, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, imipenem, clavulanate, timentin, sulbactam, erythromycin, neomycin, gentamycin, streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin, quinolones, rifampin, sulfonamides, bacitracin, polymyxin B, vancomycin, doxycycline, methacycline, minocycline, tetracycline, amphotericin B, cycloserine, ciprofloxacin, norfloxacin, isoniazid, ethambutol, and nalidixic acid, as well as derivatives and altered forms of each of these compounds.
[0276] Examples of anti-parasitic therapeutics include bithionol, diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine, niridazole, oxamniquine and other quinine derivatives, piperazine citrate, praziquantel, pyrantel pamoate and thiabendazole, as well as derivatives and altered forms of each of these compounds.
[0277] Examples of anti-fungal therapeutics include amphotericin B, clotrimazole, econazole nitrate, flucytosine, griseofulvin, ketoconazole and miconazole, as well as derivatives and altered forms of each of these compounds. Anti-fungal compounds also include aculeacin A and papulocandin B.
[0278] The prefeoed animal subject of the present invention is a mammal. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human patients.
[0279] The term "treating" means the administering to subjects a compound of Formulae / through XIII or a compound identified by one or more assays within the present invention, for purposes which can include prevention, amelioration, or cure of a retroviral-related pathology. Said compounds for treating a subject that are identified by one or more assays within the present inventions are identified as compounds which have the ability to disrapt Gag processing, described herein.
[0280] The term "inhibits the interaction" as used herein, means preventing, or reducing the rate of, direct or indirect association of one or more molecules, peptides, proteins, enzymes, or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes or receptors.
[0281] Medicaments are considered to be provided "in combination" with one another if they are provided to the patient concurrently or if the time between the admimsfration of each medicament is such as to permit an overlap of biological activity. [0282] In one prefeoed embodiment, at least one compound of Formulae / through XIII above comprises a single pharmaceutical composition.
[0283] Pharmaceutical compositions for administration according to the present invention can comprise at least one compound of Formulae / through XIII above or compounds identified by one or more assays within the present invention. Said compounds for treating a subject that are identified by one or more assays within the present inventions are identified as compounds which have the ability to disrupt Gag processing, described herein. The compounds according to the present invention are further included in a pharmaceutically acceptable form optionally combined with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purposes. Amounts and regimens for the administration of a compound of Formulae / through XIII according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating a retroviral pathology.
[0284] For example, administration can be by parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, transmucosal, ocular, rectal, intravaginal, or buccal routes. Alternatively, or ■ concuoently, administration can be by the oral route. The administration may be as an oral or nasal spray, or topically, such as powders, ointments, drops or a patch. The dosage administered depends upon the age, health and weight of the recipient, type of previous or concuoent treatment, if any, frequency of treatment, and the nature of the effect desired.
[0285] Compounds and methods of the invention are useful in additional ways. For example, such compounds may be used prophylatically, to minimize the risk of infection. In another embodiment, a compound may be used to minimize spread of the disease from an infected person.
[0286] The invention is also directed to novel methods of treating HIN in an infected individual. In one embodiment, the invention is particularly useful in stimulating an immune response in a person infected with HIN. For example, by allowing noninfectious virus to be released from infected cells, such infected cells continue to expose antigens and may be effectively targeted by the immune system or other therapies directed against such antigens. In another example, by continuing to permit the release of noninfectious viras, an infected individual continues to develop an immune response to said virus without suffering the deleterious effects of such a virus.
[0287] The invention is also useful in expanding the scope of treatment, and offers novel means of treating disease in patients in need thereof. In another embodiment, the invention may be practiced in a patient who does not respond to other therapy for reasons other than viral resistance. For example, conventional methods of treating HTV, as known in the art, are associated with deleterious side effects. In one embodiment, the methods and compositions of the invention are useful in treating a patient without a reduction in one or more deleterious side effects. In one embodiment the invention includes a method of treating a patient with a compound that does not have a particular side effect or has less of a particular side effect.
[0288] The bioavailability of drags is also relevant in treatment. In an embodiment, the invention may be practiced such that compounds are more effectively absorbed into infected cells. In one embodiment the invention encompasses improved methods of delivering a drag to a cell infected with HTV.
[0289] Compositions within the scope of this invention include all compositions comprising at least one compound of Formulae / through XIII above according to the present invention in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. For example, a dose may comprise O.OOOlmg to lOg/kg of body weight. Typical dosages comprise about 0.1 to about 100 mg/kg body weight. The prefeoed dosages comprise about 1 to about 100 mg/kg body weight of the active ingredient. More prefeoed dosages comprise about 5 to about 50 mg/kg body weight.
[0290] Administration of a compound of the jpresent invention can also optionally include previous, concuoent, subsequent or adjunctive therapy using immune system boosters or immunomodulators. In addition to the pharmacologically active compounds, a pharmaceutical composition of the present invention can also contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the prefeoed type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient.
[0291] Pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
[0292] Suitable excipients are, e.g., fillers such as saccharide, for example, lactose or sucrose, mannitol or sorbitol; cellulose preparations and or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and or polyvinyl pyoolidone. If desired, disintegrating agents can be added such as the above-mentioned starches and also carboxymethyl starch, cross- linked polyvinyl pyoolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyoolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl cellulose phthalate are used. Dyestuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
[0293] Other pharmaceutical preparations which an be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which can be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin. In addition, stabilizers can be added.
[0294] Possible pharmaceutical preparations which can be used rectally include, for example, suppositories which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
[0295] Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water- soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides or glycol-400. Aqueous injection suspensions that can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension can also contain stabilizers.
[0296] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils such as cottonseed, groundnut, com, germ, olive, castor, and sesame oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
[0297] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, cellulose, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and combinations thereof.
[0298] Pharmaceutical compositions for topical admimsfration include formulations appropriate for administration to the skin, mucosa, surfaces of the lung or eye. Compositions may be prepared as a pressurized or non- pressurized dry powder, liquid or suspension. The active ingredients in non- pressurized powdered formulations may be admixed in a finely divided form in a phaonaceutically-acceptable inert carrier, including but not limited to mannitol, fructose, dextrose, sucrose, lactose, saccharin or other sugars or sweeteners.
[0299] The pressurized composition may contain a compressed gas, such as nitrogen, or a liquefied gas propellant. The propellant may also contain a surface-active ingredient, which may be a liquid or solid non-ionic or anionic agent. The anionic agent may be in the form of a sodium salt.
[0300] A formulation for use in the eye would comprise a pharmaceutically acceptable ophthalmic carrier, such as an ointment, oils, such as vegetable oils, or an encapsulating material. The regions of the eye to be treated include the comeal region, or internal regions such as the iris, lens, ciliary body, anterior chamber, posterior chamber, aqueous humor, vitreous humor, choroid or retina. [0301] Compositions for rectal administration may be in the form of suppositories. Compositions for use in the vagina may be in the form of suppositories, creams, foams, or in-dwelling vaginal inserts.
[0302] The compositions may be administered in the form of liposomes. Liposomes may be made from phospholipids, phosphatidyl cholines (lecithins) or other lipoidal compounds, natural or synthetic, as known in the art. Any non-toxic, pharmacologically acceptable lipid capable of forming liposomes may be used. The liposomes may be multilamellar or mono-lamellar.
[0303] A pharmaceutical formulation for systemic admimsfration according to the invention can be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation can be used simultaneously to achieve systemic administration of the active ingredient.
[0304] Suitable formulations for oral administration include hard or soft gelatin capsules, dragees, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
[0305] The compounds described above or compounds identified by one or more assays within the present invention and have the ability to disrapt Gag processing, can also be administered in the form of an implant when compounded with a biodegradable slow-release carrier. Alternatively, the compounds of the present invention can be formulated as a transdermal patch for continuous release of the active ingredient.
[0306] The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Examples
Example 1
Anti- Viral Activity Against Primary HIV-l Isolates:
[0307] A robust vims inhibition assay was used to evaluate the anti-viral activity of DSB against primary HIN-1 isolates propagated in PMBC. Briefly, serial dilutions of DSB were made in medium into 96-well tissue culture plates. 25 - 250 TCED50 of viras and 5 x 105 PHA-stimulated PBMCs were added to each well. On days 1, 3 and 5 post-infection, media was removed from each well and replaced with fresh media containing DSB at the appropoate concentration. On day 7 post-infection, culture supernatant was removed from each well for p24 detection of vims replication and 50% inhibitory concentrations (IC50) were calculated by standard methods.
[0308] Table 5 shows the potent anti-viral activity of DSB against a panel of primary HIN-1 isolates. DSB exhibits levels of activity similar to approved drags that were tested in parallel. Importantly, the activity of DSB was not restricted by co-receptor usage.Table 5 IC50 (nM)
Table 5: Inhibitory activity (IC50) of DSB and two approved drugs against a panel of primary Clade B HIV-l isolates. Clinical HIV-l isolates denoted by * were isolated at Panacos. All other virus isolates were obtained from the NTH AIDS Reference Repository. Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4 respectively. [0309] Toxicity of DSB was analyzed by incubating with PHA-stimulated PBMC for 7 days at a range of concentrations, then determining cell viability using the XTT method. The 50%) cytotoxic concentration was >30 μM, cooesponding to an in vitro therapeutic index of approximately 5000.
Example 2
Anti- Viral Activity of DSB against Drug Resistant HIV-l Isolates:
[0310] The activity of DSB was tested against a panel of HIN-1 isolates resistant to approved drags. These viruses were obtained from the NTH AIDS Research and Reference Reagent Program. Assays were performed using vims propagated in PBMCs with a p24 endpoint (above), or using cell line targets (MT-2 cells) and a cell killing endpoint. The MT-2 assay format was as follows. Serial dilutions of DSB, or each approved drug, were prepared in 96 well plates. To each sample well was added media containing MT-2 cells at 3 x 105 cells/mL and viras inoculum at a concentration necessary to result in 80% killing of the cell targets at 5 days post-infection (PI). On day 5 post- infection, virus-induced cell killing was determined by the XTT method and the inhibitory activity of the compound was determined.
[0311] Table 6 shows the potent anti-viral activity of DSB against a panel of drag-resistant HTV-1 isolates. The results were not significantly different from those obtained with the panel of wild-type isolates (Table 5), demonstrating that DSB retains its activity against virus strains resistant to all of the major classes of approved drags.
ill
Table 6
IC50(nM)
Table 6: Inhibitory activity (nM IC50) of DSB against a panel of drug resistant HIV-l isolates. Assays were done in fresh PBMC with a p24 endpoint except for the NNRTI- resistant isolates that were performed in MT-2 cells with a cell viability (XTT) endpoint. *Fold Resistance. Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4 respectively.
Example 3
DSB Inhibits HIV-l Replication at a Late Step in the Virus Life Cycle To distinguish the inhibitory activity of DSB against early and late replication targets, a multinuclear activation of a galactosidase indicator (MAGI) assay was used. In this assay, the targets are HeLa cells stably expressing CD4, CXCR4, CCR5 and a reporter construct consisting of the - galactosidase gene (modified to localize to the nucleus) driven by a truncated HTV-1 LTR. Infection of these cells results in expression of Tat that drives activation of the β-galactosidase reporter gene. Expression of β-galactosidase in infected cells is detected using the chromogenic substrate X-gal. As shown in Table 7, the entry inhibitor T-20, the NRTI AZT and the NNRTI nevirapine caused significant reductions in β-galactosidase gene expression in HTV-1 infected MAGI cells due to their ability to disrupt early steps in viral replication that affect Tat protein expression. In contrast, the protease inhibitor indinavir targets a late step in viras replication (following Tat expression) and does not prevent β-galactosidase gene expression in this system. Similar results were obtained with DSB as with indinavir, indicating that DSB blocks virus replication at a time point following the completion of proviral DNA integration and synthesis of the viral transactivating protein (Table 7).
Table 7
Table 7: Effect of DSB and inhibitors of entry (the gp41 peptide T-20), RT (AZT and Nevirapine) and protease (indinavir) on expression of b-galactosidase in HIV-l infected MAGI cells. The DMSO control contained no drug. [0313] Kanamoto et al (Antimicrob. Agents Chemother., April; 45(4): 1225- 30, (2002)) have also reported that DSB acts at a late step in HIN replication. However, they reported that the compound inhibits release of virus from chronically-infected cells. In contrast, our data using a variety of experimental systems indicate that DSB does not have a significant effect on virus release (e.g. Example 6).
Example 4
DSB does not Inhibit HIV-l Protease Activity
[0314] It was previously determined that DSB had no effect on HIN-1 protease function using a cell-free fluorometric assay that characterized enzyme activity by following the cleavage of a synthetic peptide substrate. The results of these experiments indicated that at concentrations up to 50 μg/mL that DSB had no effect on protease function. As a result of the observation that DSB blocks virus replication at a late step, studies were also performed using a recombinant form of the Gag protein, which is a more relevant system than the synthetic peptide substrate used in the initial assays. The use of the recombinant Gag protein as substrate resulted in a similar experimental outcome. In these experiments DSB did not disrupt protease- mediated Gag protein processing at concentrations as high as 50 μg/mL. In contrast, as expected, the protease inhibitor indinavir blocked Gag protein processing at 5 μg/mL (Figure 1).
Example 5
DSB causes a defect in the final step of Gag processing (CA-SPl cleavage) that has been associated with viral maturation defects
[0315] In order to better define DSB's mechanism of action, a detailed examination was undertaken of the viras produced from HIN-1- infected cell lines treated with DSB. Briefly, H9 cells chronically infected with the HIN- IIHB isolate were treated with DSB at 1 μg/mL for a period of 48 hrs. Indinavir was used as a control. At the 48hr time-point, spent media was removed and fresh media containing compound was added. At 24, 48 and 72 hrs post fresh compound addition, both cells and supernatant were recovered for analysis. The level of viras in the culture supernatant was determined and western blots were used to characterize viral protein production in both cell- associated and cell-free virus. As observed in previous experiments, DSB did not cause a significant reduction in the amount of virus produced by chronically infected H9 cells, however, there was a defect in Gag processing in both cell-associated and cell-free virus. This defect took the form of an additional band in the western blots cooesponding to p25 (Figure 2). This p25 band results from the incomplete processing of the capsid CA-SPl precursor. DSB treatment of HTV-2 and STV chronically infected cell lines exhibited normal Gag processing consistent with the observed lack of antiviral activity against these vimses. The Gag processing defect seen in the presence of DSB is completely distinct from that observed with the protease inhibitor indinavir (Figure 2). As discussed above, mutations at the p25 to p24 cleavage site that prevent processing are associated with defects in viral maturation and infectivity (Wiegers K. et al, J. Virol. 72:2846-54 (1998)).
[0316] As previously discussed (C.T. Wild et al., XIV Int. AIDS Conf. Barcelona, Spain, Abstract MoPeA3030, (July 2002)), abnormal p25 to ρ24 processing is also seen in other maturation budding defects. These include mutations in the Gag late domain (PTAP) or defects in TSG-101 mediated viral assembly that disrupt budding (Garras, J.E et al, Cell, 107:55-65, (2001); Demirov, D. G. et al, J. Virology 76:105-117, (2002)). However, these mutations cause inhibition of viras release, while DSB treatment does not have a significant effect on viras release. The morphology of these maturation/budding mutants is also quite distinct from that following DSB- treatment (see Example 6).
[0317] In addition, mutations that interfere with viral RNA dimerization and lead to the production of immature virus with defective core structures give a similar Gag processing phenotype (Liang, C. et al, J. Virology, 75:6147-6151, (1999)). However, in those cases RNA incorporation is inhibited and the morphology of particles released is distinct from those following DSB treatment (see Example 6).
Example 6
DSB treatment effects HIV-l maturation as determined by electron microscopy (EM)
[0318] It has been demonstrated that mutations in HTV-1 Gag that disrapt p25 to p24 processing give rise to non-infectious viral particles characterized by an internal morphology distinct from normal viras (Wiegers K. et al, J. Virol. 72:2846-54 (1998)). To determine if virus generated in the presence of DSB exhibited this distinct morphology the following experiment was carried out.
[0319] HeLa cells were transfected with HIN-1 infectious molecular clone pΝL4-3 and treated as described previously with DSB. Following treatment, DSB-treated infected cells were fixed in glutaraldehyde and analyzed by EM. The results of this analysis are shown in Figure 3.
[0320] These results are consistent with a compound that disrupts p25 to p24 processing which generates non-infectious morphologically abeoant viral particles.
[0321] 3-0-(3',3'-dimethylsuccinyl) betulinic acid (DSB) is an example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-l replication. However, this compound does not inhibit PR activity, and its action is specific for the p25 to p24 processing step, not other steps in Gag processing. Furthermore, DSB treatment results in the abeoant HTV particle morphology described above.
Example 7
[0322] In vitro selection for HTV-1 isolates resistant to compounds that disrapt the processing of the viral Gag capsid (CA) protein from the CA-spacer peptide 1 protein precursor.
[0323] A series of experiments were performed to select for vimses resistant to inhibition by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), an inhibitor HTV-1 maturation. For each experiment, either NL4-3 or RF vims isolate was used to infect two cell cultures. Following infection, one culture was maintained in growth medium containing DSB, while the other culture was maintained in parallel in growth medium lacking DSB.
[0324] In one experiment, H9 cells that had been infected with RF virus were maintained in the presence or absence of increasing concentrations of DSB (0.05-1.6 μg/ml). The cells were passaged every 2-3 days with the addition of fresh drag. Virus replication was monitored by p24 ELISA every 7 days. At that time, DSB-treated cultures with high levels of p24 were passaged by co- cultivation with fresh uninfected H9 cells at a 1:1 ratio of cells in the presence of lx or 2x the original concentration of DSB. After 8 weeks of co- cultivation, cell-free viras was collected from the culture containing DSB at a concentration of 1.6 μg/ml and used to infect fresh H9 cells. Every 7 days, viras from cultures containing high levels of p24 was passaged by cell-free infection in the presence of lx or 2x the original concentration of DSB. After 5 weeks of cell-free passaging, viras from the culture containing 3.2 μg/ml DSB was collected and used to infect MT-2 cells. Viras replication in the MT-2 cells, was monitored by observing syncytia formation microscopically. Every 1-3 days, the cells were washed to remove input viras, and fresh drag was added to the culture under selection. Every 3-4 days, following the emergence of extensive syncytia in the culture under selection, supernatant from each culture was collected and passed through a 0.45 μm filter to remove cell debris. This filtered viras supernatant was then used to infect fresh MT-2 cells in the presence or absence of fresh drag. After 4 rounds of cell-free infection (approximately 2 weeks in culture), with the concentration of drag at 3.2 μg/ml, virus stocks were collected and frozen for further analysis.
[0325] In a second experiment, a stock of viras derived from the molecular clone pNL4-3 (5.7 x 104 TCED50) was used to infect MT-2 cells (6 x 106 cells) and cultures were maintained in the presence or absence of DSB at a concentration of 1.6 μg/ml. Every 1-3 days, the cells were washed to remove input vims, and fresh drag was added to the culture under selection. Virus replication was monitored by observing syncytia formation microscopically. Every 3-7 days, following the emergence of extensive syncytia in the culture under selection, supernatant from each culture was collected and passed through a 0.45 μm filter to remove cell debris. This filtered virus supernatant was then used to infect fresh MT-2 cells in the presence or absence of fresh drag. After 5 rounds of cell-free infection, and every other round thereafter, the concentration of drag was doubled. After 10 rounds of cell-free infection (approximately 7 weeks in culture), when the concentration of drag reached 12.8 μg/ml, viras stocks were collected and frozen for further analysis.
Example 8
[0326] Characterization of HTV-1 isolates selected for resistance to compounds that disrapt the processing of the viral Gag capsid (CA) protein from the C A-spacer peptide 1 protein precursor.
[0327] Virus stocks derived as described above were further analyzed both phenotypically and genotypically to characterize the nature of their drag- resistance. The resistance of the viruses to 3-O-(3',3'-dimethylsuccinyl)- betulinic acid (DSB) was determined in viras replication assays. Briefly, the viras stocks were first titered in H9 cells by quantitating the levels of p24 (by ELISA) in cultures 8 days after infection with serial 4-fold dilutions of virus. Virus input was then normalized for a second assay in which each viras is cultured for 8 days in the presence of serial 4-fold dilutions of drug. The IC50 for each viras was determined as the dilution of drug that reduced the p24 endpoint level by 50% as compared to the no-drag control. In these assays, the two independently derived viras stocks resulted in IC50 values greater than 2 μM for DSB, as compared to an IC50 of 0.02 μM for viras that had been cultured in parallel in the absence of drug. In a subsequent series of experiments, the A364V mutation was engineered into the HTV-1 NL4-3 proviral DNA, which was subsequently transfected into HeLa cells. Resulting viras was collected and used to test the activity of DSB in a viral replication assay, as described above. In these assays, the DSB-resistant viras resulted in an IC50 value of 0.1 μM whereas wild-type NL4-3 gave an IC50 value of 0.01 μM.
[0328] To determine if the resistant vimses were able to escape the CA-SPl cleavage defect caused by DSB in wild-type viras, stocks of each viras grown in either the presence or absence of drag were analyzed by Western blot. Viras was pelleted through a 20% sucrose cushion from filtered culture supematants that were collected 60 hr post-infection and 18 hr after the cells had been washed and fresh drag added. The virases were lysed, and the amount of each virus was normalized by quantitating p24 levels in each sample. Western blot analysis of the viral proteins in each sample demonstrated that the drag-resistant viruses did not contain the CA-SPl product in the presence of DSB, confirming that these virases were resistant to the effects of the drug on this cleavage event. [0329] Finally, to identify the genetic determinants of DSB resistance, the entire Gag and PR coding regions of the viral genomes were amplified by high-fidelity RT-PCR for sequencing. The viral RNA was purified from each viras lysate prepared as described above and digested with DNase to remove any contaminating DNA. The RT-PCR products were then gel-purified to remove any non-specific PCR products. Finally, both strands of the resulting DNA fragments were sequenced using overlapping a series of primers. Two amino acid mutations were identified that are independently capable of conferring resistance to DSB, an alanine to valine substitution in the Gag polyprotein at residue 364 in the NL4-3 isolate and at residue 366 in the RF isolate. These are the first and the third residues, respectively, downstream of the CA-SPl cleavage site (the N-terminus of SPl). Alanine is highly conserved at each of these positions throughout all HIV-l clades in the database.
Example 9
Determinants of Activity of the HIV-l Maturation Inhibitor DSB Map to the Gag Protein CA-SPl Domain.
[0330] To further define the molecular determinants of DSB activity, a series of chimeric virases were prepared in which residues proximal to the CA-SPl cleavage site from the DSB-sensitive viras, HIN-1, were inserted into the analogous region of the Gag protein of the DSB-resistant retroviras, SIN. Characterization of these STV/HTV chimeras (SHINs) with respect to DSB activity allowed further identification of minimal HIN-1 Gag sequences both necessary and sufficient for DSB activity.
Materials and Methods
Constraction of SHIN DΝA clones
[0331] Three panels of SHINs were generated using the DSB-resistant STVmac239 as the backbone in conjunction with residues from the Gag CA- SPl region of the DSB-sensitive HIV-l pΝL4-3. The CA-SPl sequences for these SHIN constracts are shown in Fig. 20. All DΝA mutagenesis was carried out using the PCR-overlapping-PCR strategy. (Ho, S. Ν., H. D. Hunt, R. M. Horton, J. K. PuUen, and L. R. Pease (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59) and other standard molecular cloning approaches (Sambrook, J., E. F. Fritsch, and T. Maniatis (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, ΝY.).
Cell culture and DNA transfection
[0332] HeLa cells were maintained in DMEM (Invitrogen) (supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml Streptomycin) and passaged upon confluence. All plasmid DNAs were prepared using the midiprep kit (Qiagen). HeLa cells were transfected with wild-type STVmac239, HIV-l pNL4-3 or SHTV proviral DNAs by employing the FuGENE 6 transfection reagent (Roche). Briefly, cells were seeded into a 6- well plate (Coming) at a concentration of 1.5 x 105 cells per well the day prior to use and allowed to reach 60 to 80% confluence on the day of transfection. For each transfection, 3 μl of FuGENE 6 was diluted into 100 μl of serum-free DMEM followed by the addition of 1 μg of DNA. After gently mixing, the mixture of DNA-lipid complexes was gently added drop-wise to the cells in 1.5 ml of complete DMEM medium. Twenty-four hours post-transfection, medium containing the DNA-FuGENE 6 complexes was removed and 1.5 ml of fresh DMEM was added to the transfected cells. At 48 h post-transfection, both cells and culture supernatant were harvested for further analysis. SDS-PAGE and Western blot
[0333] To characterize the effect of incorporation of residues from the CA- SPl domain of HIN-1 into STV on viral particle production and Gag polyprotein processing Western blotting was performed. Briefly, at 48h post- transfection, culture medium containing viral particles was collected and clarified by centrifugation at 2,000 rpm and 4°C for 20 min in a Sorvall RT 6000B centrifuge. Particle-containing supematants were then concentrated through a 20%) sucrose cushion in a microcentrifuge at 13,000 rpm at 4°C for 120 min and pellets were resuspended in lysis buffer (150 mM Tris-HCl, 5%> Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], pH 8.0). For cell lysates, at 48 h post-transfection, cells were washed once with PBS and lysed (150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholate, pH 8.0) followed by centrifugation at 13,000 rpm at 4°C for 5 min to remove nuclear fractions. Viral pellets and cell lysates were separated on a 12% ΝuPAGE Bis- Tris Gel (Invitrogen) and transfeoed to a nitrocellulose membrane (Invitrogen) followed by blocking in a PBS buffer containing 0.5% Tween and 5% dry milk powder. The membrane was incubated with anti-SιNmac251 p27 McAb (NOT ADDS Research and Reference Reagent Program) and hybridized with goat anti-mouse horseradish peroxidase (Sigma). For the membrane containing HTV-1 proteins, the membrane was incubated with immunoglobulin from HTV-1 -infected patients (HTV-Ig) (NIH ADDS Research and Reference Reagent Program) and hybridized with goat anti-human horseradish peroxidase (Sigma). The immune complex was visualized with an ECL system (Amersham Pharmacia Biotech) according to the instructions provided by the manufacturer.
Effect of DSB on SHTV Gag processing
[0334] To address the effect of the Gag substitutions on the ability of DSB to inhibit CA-SPl processing, HeLa cells were transfected with wild-type SιNmac239, HTV-1 pNL4-3 or SHIN proviral DΝAs by employing the procedure described above. DSB at a concentration of 1 μg/ml or DMSO (no drag control) was maintained throughout the entire period of the culture and SDS-PAGE/Western-Blot for analyzing viral proteins derived from these cultures was performed as described above. [0335] Only virases that were fully sensitive to the effects of DSB were scored as sensitive, while those with residual resistance were scored as resistant. As such, the scoring in Experiment 9 differs from previous scoring, in that viruses that were not fully resistant were scored as sensitive. For this reason, SHIN.DM was scored as DSB resistant in Experiment 9, but was previously scored as DSB sensitive.
Results
Characterization of Gag SHINs
[0336] Three panels of HTV-1/STV Gag chimeras were prepared (Fig. 20). Panel 1 consisted of virases containing the STV backbone into which residues from the HIN-1 SPl domain had been inserted. The HIN-1 inserts in these chimeras ranged in size from a single point substitution (SHIN DA) to the complete replacement of the SIN SPl domain with the SPl sequence from HTV-1 (SHTV DΝ). Panel 2 consisted of virases containing the same SPl substitutions plus the inclusion of the two C-terminal CA resides from HIN-1 (LM to VL). Panel 3 SHTVs were identical to those in panel 2 except that, in addition to the substitutions in the SPl domain and the two C-terminal CA resides from HTV-1 (LM to VL), each of these chimeras also incorporated a Q (STV) to H (HTV-1) change at the 6th position upstream (P6) from the CA-SPl cleavage site.
[0337] The level of viral particle release from transfected cells and the Gag processing profile for each of the SHINs were determined (Fig. 21). All panel 1 SHTVs behaved similarly with respect to particle production and Gag processing; these chimeric vimses exhibited near wild-type levels of particle production and a normal Gag processing profile compared with the parental SIV. The majority of chimeras in panel 2 were characterized by normal Gag processing profiles, whereas the cellular expression of SHINs FC and 11 was somewhat reduced (Fig. 21 A). For all panel 2 SHTVs, the amount of vims production relative to cell-associated expression was comparable to the parental STV. In contrast to the panel 1 and 2 SHINs, most of the chimeras in panel 3 exhibited defects in Gag processing. Of these SHTVs, only GH, GI and 23 exhibited a normal Gag processing profile. It is clear from these results that the Q to H change six residues upstream from the CA-SPl cleavage site affects the ability of STV PR to process the chimeric Gag proteins.
[0338] Results from SHIN panel 2, comprised of virases containing HIN-1 residues in both the SPl and CA domains, are shown in Figure 21. As described previously, this panel of SHIVs is identical to panel 1 except that in addition to the substitutions in the SPl domain, each of these chimeras also incorporates the two HIN-1 CA C-terminal residues (VL from HTV-1 replaces LM from SIN). As with the viruses in panel 1, the chimeras in panel 2 were characterized by normal Gag processing profiles (Fig. 21) and, while the cellular expression of SHINs FC and 11 was somewhat reduced (Fig. 21 A), the proportion of virus found in the supernatant of cells transfected with DΝA encoding these SHINs was proportional to the level of viral release observed for all panel 2 virases.
[0339] Results from SHIN panel 3 with virases containing HIN-1 residues in both the Gag SPl and CA domains are shown in Fig. 21. This panel of SHINs is identical to those in panel 1 except that in addition to the substitutions in the SPl domain, each of these chimeras also incorporates the two C-terminal resides from HIN-1 (LM from STV to VL from HIN-1) plus a Q (STV) to H (HTV-1) change at the 6th position upstream from the CA-SPl cleavage site. Unlike the first two panels, most of the chimeras in panel 3 exhibited defects in Gag processing (Fig. 21). Of these SHINs, only GH, GI and 23 exhibited a normal Gag processing profile. It is clear from these results that the Q to H change six residues upstream from the CA-SPl cleavage site has a significant affect on the ability of STV protease to process the resulting chimeric Gag protein.
Sensitivity of Gag SHINs to DSB
[0340] Each of the SHTVs in panels 1 , 2 and 3 were characterized for their sensitivity to DSB. As described above, DSB disrupts HIN-1 CA-SPl cleavage leading to the release of non-infectious viral particles that exhibit abeoant core morphology (Li, F., R. Goila-Gaur, K. Salzwedel, Ν. R. Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, D. E. Martin, J. M. Orenstein, G. P. Allaway, E. O. Freed, and C. T. Wild. (2003) PA-457: a potent HIN inhibitor that disrupts core condensation by targeting a late step. in Gag processing. Proc. Natl Acad. Sci. USA 700:13555-13560.). In the current study, SHIN-expressing cells were cultured in the presence of DSB at a concentration of 1 μg/ml for a period of 48hrs. At the end of that time viras was harvested from the culture supernatant and the Gag processing profile for each chimeric viras was analyzed and compared to Gag protein processing in the absence of compound (Fig. 22).
[0341] As can be seen in Fig. 22, none of the SPl SHINs (panel 1) exhibited sensitivity to DSB. Even SHIN DΝ, which contains the complete HIV-l SPl domain, exhibited a normal Gag processing profile in the presence of compound at concentrations in excess of the tissue culture determined IC50. (Fig. 22) (Li et al). The results from the panel 1 chimeras demonstrate that the determinants of DSB activity include regions of HIN-1 Gag outside of the SPl domain. Results from the panel 2 SHINs are identical to those observed for panel 1. Specifically, none of these CA-SPl chimeras exhibited DSB sensitivity (Fig. 22). Since the panel 2 virases contained the C-terminal NL amino acid residues, in addition to the SPl sequence from HTV-1, these results demonstrate that HTV-1 Gag residues other than those immediately flanking the CA-SPl cleavage site play a role in DSB sensitivity.
[0342] As shown in figure 22 A, DSB does disrupt CA-SPl processing for a subset of the panel 3 SHIVs. Of these chimeric virases, SHINs 23 and GI exhibited a level of DSB-sensitivity comparable to the prototypic HIN-1 isolate ΝL4-3 (Fig. 22A). In repeat experiments the magnitude of the effect of the compound on each of these virases, as determined by the relative ratios of CA-SPl to CA, was nearly identical. Also in panel 3, SHIN GH exhibited some level of DSB sensitivity, however, on a qualitative level, the activity observed against this viras was reduced compared to that observed with SHINs 23 and GI. For SHIN GH the effect of DSB on Gag processing was reduced to the point where the very faint CA-SPl band observed in the immunoblot is not apparent in Fig. 22A. A 5X increase in the amount of viral proteins loaded onto the gel enhanced the sensitivity of the Western blot assay to a level that allowed the DSB-mediated processing defect for SHIN GH to be observed (Fig. 22B). For the remainder of the vimses in panel 3 the effect of the compound could not be determined due to sequence-related defects in Gag processing (Fig. 2 IB). The results from the panel 3 SHINs indicate that the His residue at the 6th position upstream from the CA-SPl cleavage site plays an important role in DSB sensitivity and that significant portions of both CA and SPl beyond the immediate vicinity of the cleavage site are necessary for DSB activity.
Discussion
[0343] Three panels of SIN/HIN-1 chimeras were prepared (Fig. 20) and characterized. All panel 1 and 2 SHINs behaved similarly with respect to the effect of SPl or CA/SP1 substitutions on Gag protein processing, viral particle release and sensitivity to DSB (Figs. 21 and 22). Although some of the virases in panel 2 (i.e. FD and 11) were characterized by a reduction in the level of viral particle production, this effect was most likely due to an overall reduction in the amount of viras generated by the transfected cells (Fig 21 A). The fact that none of these panel 1 and 2 SHINs were sensitive to DSB as determined by the effect of the compound on CA-SPl processing indicates that Gag sequences outside the immediate vicinity of the HIN-1 CA-SPl cleavage site play a critical role in DSB activity.
[0344] In contrast to the viruses in panels 1 and 2, three members of the panel 3 SHINs exhibited some degree of DSB sensitivity. Among these virases, SHINs 23 and GI exhibited ΝL4-3-like sensitivity to DSB while SHTV GH displayed a somewhat reduced level of sensitivity to the compound. Of the additional panel 3 viruses, the effect of the CA-SPl substitutions on Gag processing made it impossible to determine the effect of the compound on the remaining chimeras.
[0345] The effect of DSB on the Gag processing profile of these 3 panels of CA-SPl SHrVs suggests that the determinants of compound activity include a relatively large region of Gag flanking the CA-SPl cleavage site. Comparison of the activity of DSB against panel 2 SHIN 11 (insensitive) with panel 3 SHTV 23 (sensitive) indicates that the His residue located at the 6th CA position upstream from the cleavage site is critical to DSB activity (Fig. 22B). Consistent with this observation, in vitro resistance selection studies have identified a mutation at this position that confers some level of DSB insensitivity. [0346] The results herein provide for the existence of both high and low affinity binding sites in the CA-SPl region. A low-affinity interaction would result in partial DSB activity (i.e., SINm3) while high affinity binding would give full compound sensitivity (i.e., SHIN 23).
Example 10
Genotyping of Viral Isolates
[0347] As shown above, sequence polymorphisms in HIN have been demonstrated to cooespond with the ability of a viras to replicate in the presence of DSB. Most sequence polymorphisms are clustered in gag, especially in the region encoding CA-SPl. Accordingly, genotyping of a viral isolate may be used to readily determine whether the replication of such a vims is likely to be inhibited by DSB, or any other compound that intereferes with p25 processing.
[0348] The results of such genotyping are useful in, for example, determining whether a viral infection in a patient may be treated with DSB, or any other compound that intereferes with p25 processing in a similar manner, or in determining the emergence of resistant variants during a course of treatment with DSB.
[0349] Genotyping may be performed by a number of methods. In some embodiments, genotyping is performed by sequencing.
Methods
[0350] A single frozen aliquot (approximate volume 1.2 ml) of plasma is obtained from each patient. The plasma sample is stored at -70°C until ready for processing. Each sample is identified using the three digit patient ED number. [0351] On the day of processing, each plasma sample is thawed rapidly in a 37°C water bath and then placed on ice. A 140 μl aliquot of plasma is removed to a separate tube for nucleic acid purification using the QIAamp Mini Viral RNA Purification Kit (Qiagen). The remainder of the plasma sample is transferred to a separate tube for brief, low speed centrifugation (3 min at 8,000 rpm) to clarify the plasma. One ml of the clarified supernatant is then transfeoed to a fresh tube and centrifuged for 2 hr at full speed (13,000 rpm) to pellet vims. The supernatant is carefully removed using a pipet and transfeoed to a separate tube for storage at -70°C as a precaution against possible disruption of the viral pellet. The viras pellet is resuspended in 140 μl of PBS and stored at -70°C as a backup sample in case sufficient RT-PCR product is not obtained from the initial aliquot of non-pelleted plasma.
Table 8 HTV-1 Gag primers HIV-l Gag CA/SP1 primers conserved in clade B: Name Sequence Length Tm %GC Forward ("+" strand) primers F-625 cacctagaactttaaatgcatgg 23 51 39 F-575 gcccagaagtaatacccatgttttcagc 28 62 46 F-550 cagaaggagccaccccacaag 21 58 62 F-525 caccatgctaaacacagtggg 21 53 52 F-375 ggaagtgacatagcaggaactactag 26 52 46 F-300 ccacctatcccagtaggag 19 53 58 F-125 ggatgacagaaaccttgttggtcc 24 58 50 Reverse ("-" strand) primers (5 '-3') R+100 cctttccacatttccaacagccc 23 57 52 R+200 cttccctaaaaaattagcctgtc 23 51 39 R+275 ctggtggggctgttggctc 19 64 68 R+400 gggtcgttgccaaagagtg 19 60 58 R+450 ctgtatcatctgctcctgtatctaatag 28 52 39 R+525 caattccccctatcatttttggtttcc 27 62 41 R+625 cttccaattatgttgacaggtgtaggtcc 29 60 45
[0352] Following purification, viral RNA is eluted in a final volume of approximately 60 μl. Only 7 μl of this stock is used initially as a template for reverse transcription using the StrataScript First Strand Synthesis System (Stratagene). The remainder of the RNA stock is stored at -70°C as a backup. The primer for reverse transcription (R+625) anneals approximately 625 bp downstream of the CA-SPl cleavage site. All of the primers to be used for RT-PCR and sequencing in this project have been designed to anneal to regions of ag that are highly conserved among clade B HIN isolates and have been validated using plasma samples from 42 different patients.
[0353] The reverse transcription reaction is performed in a total volume of 50 μl. Only 5 μl of this reaction is used initially as a template for PCR amplification of the CA-SPl region using the PicoMaxx High Fidelity PCR Master Mix (Stratagene). The remainder of the reaction is stored at -20°C as a backup. A two-step "nested" PCR strategy is used which has been found to provide a high yield of very clean DΝA product. The forward and reverse primers for the first-round PCR amplification (F-625 and R+525) anneal approximately 625 bp upstream and 525 bp downstream of the CA-SPl cleavage site, respectively. No product is typically visible by agarose gel analysis following this first PCR reaction.
[0354] The initial PCR reaction is performed in a total volume of 50 μl. After cycling, 5 μl of this reaction is removed and used as a template in a second- round "nested" PCR reaction using primers F-575 and R-450, which anneal to regions of gag that are internal to the regions to which the initial primer pair anneals. The remainder of the first-round PCR reaction is stored at -20°C as a backup. The forward and reverse primers for the second-round PCR reaction anneal approximately 575 bp upstream and 450 bp downstream of the CA-SPl cleavage site, respectively. Five μl of the final "nested" PCR reaction is removed for analysis of DNA products by agarose gel electrophoresis. If, as expected, the reaction contains only one prominent band of the predicted size for the desired product (~1.1 kb), and the yield is estimated to be sufficient to permit sequencing (i.e. -200 ng total), then 40 μl of the reaction is removed for purification of the DNA product using the MinElute PCR Purification Kit (Qiagen). The remaining ~5 μl of the reaction is stored at -20°C as a backup. Following elution of the purified DNA product, an appropoate volume (cooesponding to at least 40 ng of DNA) is transfeoed to two tubes, each containing a different sequencing primer (one for each strand of the DNA). The "+" strand sequencing primer (F-300) anneals approximately 300 bp upstream of the CA-SPl cleavage site. The "-" strand sequencing primer (R+275) anneals approximately 275 bp downstream of the CA-SPl cleavage site. The template/primer mixture is shipped for sequencing and analysis. The remainder of the purified DNA product is stored at -20°C as a backup. The resulting sequence analysis provides overlapping reads for each DNA strand to help resolve any ambiguities in any single sequencing reaction.
[0355] If ambiguities are found in both sequencing reactions, additional sequencing reactions are analyzed using alternate validated sequencing primers in case the problem lies in the heterogeneity of the region of gag to which the original sequencing primers anneal. These will include two "+" strand primers (F-375 and F-125) that anneal approximately 375 and 125 bp upstream and two "-" strand primers (R+100 and R+400) that anneal approximately 100 and 400 bp downstream of the CA-SPl cleavage site, respectively.
[0356] If the final "nested" PCR reaction contains significant background bands when analyzed on an agarose gel (i.e. greater than approximately 10% of the total yield), or if sequencing fails to yield clean sequence, then the desired DNA product is purified by running the entire PCR reaction (re- amplified from a backup sample if necessary) on an agarose gel and excising the desired band. The DNA is then purified from the agarose using the QIAEX II Gel Extraction Kit (Qiagen) and eluted product is prepared for sequencing as described above.
[0357] If the PCR reaction fails to yield sufficient product for sequencing, then additional RT-PCR or PCR reactions could be run, if necessary, using any of the backup samples outlined above and additional validated primer sets, including four forward primers that anneal approximately 550, 375, 300, and 125 bp upstream (F-550, F-375, F-300 and F-125) and three reverse primers that anneal approximately 400, 275, and 100 bp downstream (R+ 400, R+275 and R+100) of the CA-SPl cleavage site. For example, excellent results have been obtained using primer R+525 for reverse transcription and primers F-575 and R+450 for single-round PCR amplification. Primers F-550 and R+400 also work well for PCR amplification.
[0358] The resulting genotype is then matched with the genotype of virases identified elsewhere herein to determine whether the viras is inhibited or is not inhibited by DSB. Further confirmation of the genotyping results may be obtained by follow-up experiments such as by direct experiments on viral isolates.
Example 11
Genetic Change During Treatment With DSB
[0359] To determine the change in genotype of HIN-1 during a course of treatment with DSB, the genotype of the virus population in each patient prior to dosing and at the end of the study (day 28) is obtained to determine if any mutations have occurred during the course of treatment. If any mutations are identified in the end of study samples that were not present prior to dosing, then intermediate samples drawn on days 7 and 10 after dosing are also be genotyped to determine when the mutation occuoed.
[0360] Using this method, the mutations that may occur in the total viras population during the course of the study are determined. A mutation will be identified as a greater than 25% variation in the amino acid designation for a given codon. Once a mutation has been identified, a chromatogram of the raw data is reviewed to determine the identities of the amino acids at that position in the minor virus populations. If none of the resistance mutations listed above are identified using these criteria, then the chromatograms from each reaction are reviewed to determine if any minor species (less than 25% of the total population) are present at any of the relevant positions.
[0361] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, applications and publications cited herein are fully incorporated by reference in their entirety.

Claims

What is Claimed Is:
1. A method of treating HIN-1 infection in a patient, comprising administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps.
2. The method of claim 1, wherein said method does not significantly reduce the quantity of virions released from infected cells in said patient.
3. The method of claim 1, wherein said method has no significant effect on the amount of RΝA incorporation into virions released from cells of said patient.
4. The method of claim 1, wherein said compound inhibits the maturation of virions released from cells of said patient.
5. The method of claim 1, wherein a majority of virions released from cells of said patient exhibit an altered phenotype relative to wild type virions, wherein said phenotype is selected from the group consisting of:
(a) virions possessing spherical, electron-dense cores that are acentric with respect to the viral particle;
(b) virions possessing crescent-shaped electron-dense layers lying just inside the viral membrane;
(c) virions having reduced or no infectivity; and
(d) the combination of any one of (a) through (c)
. A method of treating HIN-1 -infection in a patient, comprising administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide having a sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ DD NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ D NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
7. The method of claim 6, wherein said polynucleotide has a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18; (f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
8. A method of treating HTV-1 -infection in a patient, comprising administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), and wherein said compound binds to a polypeptide having a sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTTLKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATTM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEvl (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATEVI; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ID NO: 26)
(g) SHKARTLAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATEVI (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATTM (SEQ ED NO: 119)
9. The method of claim 8, wherein said polypeptide has a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ DD NO: 21);
(b) KNWMTETLLVQNANPDCKTD KALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPAT (SEQ DD NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ DD NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ DD NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and 0) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
10. A method of treating HTV-1 -infection in a patient, composing administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), and wherein said compound binds to a polypeptide having a sequence selected from any one of SEQ ED NO: 121 to 443.
11. The method of any one of claims 1-10, wherein said HIV- 1 infection in a patient comprises infection with an HTV-1 which does not respond to other HIN-1 therapies.
12. The method of any one of claims 1-10, wherein said patient with said HIN-1 infection does not otherwise respond to other HTV-1 therapies.
13. The method of any one of claims 1-10, further comprising administering to said patient an additional compound which is an immunomodulating agent, an anticancer agent, an antibacterial agent, an antifungal agent, an antiviral agent, or a combination thereof.
14. The method of any one of claims 1-10, wherein said patient is administered said compound in combination with at least one other anti-viral agent.
15. The method of claim 14, wherein said other anti- viral agent is selected from the group consisting of: zidovudine; lamivudine; didanosine; zalcitabine; stavudine; abacavir; nevirapine; delavirdine; emtricitabine; efavirenz; saquinavir; ritonavir; indinavir; nelfinavir; amprenavir; tenofovir; adefovir; atazanavir; fosamprenavir; hydroxyurea; AL-721; ampligen; butylated hydroxytoluene; polymannoacetate; castanospermine; contracan; creme pharmatex; CS-87; penciclovir; famciclovir; acyclovir; cytofovir; ganciclovir; dextran sulfate; D- penicillamine trisodium phosphonoformate; fusidic acid; HPA-23; eflornithine; nonoxynol; pentamidine isethionate; peptide T; phenytoin; isoniazid; ribavirin; rifabutin; ansamycin; trimetrexate; SK- 818; suramin; UA001; enfuvirtide; gp41 -derived peptides; antibodies to CD4; soluble CD4; CD4-containing molecules; CD4-IgG2; and combinations thereof.
16. A method of inhibiting processing of the viral Gag p25 protein (CA- SPl) to p24 (CA), comprising administering a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps.
17. A method of inhibiting processing of the viral Gag p25 protein (CA- SPl) to p24 (CA), comprising administering a compound which binds to a polypeptide encoded by a polynucleotide having a sequence at least about 70%> identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and 0) about nucleotides 1858-1920 of SEQ ED NO: 19.
18. The method of claim 17, wherein said polynucleotide has a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19; (c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
19. A method of inhibiting processing of the viral Gag p25 protein (CA- SPl) to p24 (CA), comprising administering a compound which binds to a polypeptide having a sequence at least about 70%> identical to a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATΓM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATTM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQNTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ DD NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26) (g) SHKARILAEAMSQVTN (SEQ ED NO: 116);
(h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSAT1M (SEQ ED NO: 118); and
(j) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
20. The method of claim 19, wherein said polypeptide has a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTELKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATTM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and 0) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
21. A method of inhibiting processing of the viral Gag p25 protein (CA- SPl) to p24 (CA), comprising administering a compound which binds to a polypeptide having a sequence selected from any one of SEQ ED NO: 121 to 443.
22. A method of treating human blood products comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), but does not significantly affect other Gag processing steps.
23. The method of claim 22 wherein said inhibition does not significantly reduce the quantity of virions released from infected cells treated with said compound.
24. The method of claim 22, wherein said inhibition has no significant effect on the amount of RNA incorporated into the virions released from infected cells treated with said compound.
25. The method of claim 22, wherein said compound inhibits the maturation of virions released from infected cells treated with said compound.
26. The method of claim 22, wherein a majority of virions released from treated infected cells exhibit an altered phenotype relative to wild type virions wherein said phenotype is selected from the group consisting of:
(a) virions possessing spherical, electron-dense cores that are acentric with respect to the viral particle;
(b) virions possessing crescent-shaped electron-dense layers lying just inside the viral membrane;
(c) virions having reduced or no infectivity; and (d) the combination of any one of (a) through (c)
27. A method of treating human blood products, comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide encoded by a polynucleotide having a sequence at least about 70%> identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19;
(c) about nucleotides 1344- 1435 of SEQ DD NO: 18;
(d) about nucleotides 1828-1920 of SEQ DD NO: 19;
(e) about nucleotides 1370-1413 of SEQ DD NO: 18;
(f) about nucleotides 1857-1899 of SEQ DD NO: 19
(g) about nucleotides 1372- 1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
28. The method of claim 27, wherein said polynucleotide has a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828- 1920 of SEQ ED NO: 19; (e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and G) about nucleotides 1858-1920 of SEQ ED NO: 19.
29. A method of treating human blood products, comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide having a sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ DD NO: 24);
(e) SHKARELAEAMSQV (SEQ DD NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKAΪULAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
30. The method of claim 29, wherein said polypeptide has a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATEVI (SEQ DD NO: 119)
31. A method of treating human blood products, comprising contacting said blood products with a compound that inhibits processing of the viral Gag p25 protein (CA-SPl) to p24 (CA), wherein said compound binds to a polypeptide having a sequence selected from any one of SEQ ED NO: 121 to 443.
32. The method of any one of claims 1 to 10, 16 to 31, wherein said compound inhibits the interaction of HTV protease with CA-SPl.
33. The method of claim 32, wherein said compound directly inhibits the interaction of HIV protease with CA-SPl.
34. The method of claim 32, wherein said compound indirectly inhibits the interaction of HIN protease with CA-SPl.
35. The method of any one of claims 1 to 10, 16 to 31, wherein said compound binds to the viral Gag protein such that interaction of HIN protease with CA-SPl is inhibited.
36. The method of any one of claims 1 to 10, 16 to 31, wherein said compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SPl) to p24 (CA).
37. The method of any one of claims 1 to 10, 16 to 31, wherein said compound is a derivative of dimethylsuccinyl betulinic acid or a derivative of dimethylsuccinyl betulin.
38. The method of claim 37, wherein said compound is selected from the group consisting of: 3-O-(3',3'-dimethylsuccinyl) betulinic acid; 3-O- (3 ',3 '-dimethylsuccinyl) betulin; 3-O-(3',3'-dimethylglutaryl) betulin; 3- O-(3 ',3 '-dimethylsuccinyl) dihydrobetulinic acid; 3-O-(3',3'- dimethylglutaryl) betulinic acid; (3',3'-dimethylglutaryl) dihydrobetulinic acid; 3-O-diglycolyl-betulinic acid; 3-O-diglycolyl- dihydrobetulinic acid; and combinations thereof.
39. The method of any one of claims 1 to 10, 16 to 31, wherein said compound is not a derivative of betulin or dihydrobetulin of Formulae I to IE: or a pharmaceutically acceptable salt thereof, wherein, R is a C2-C20 substituted or unsubstituted carboxyacyl, R' is hydrogen, C2- 0 substituted or unsubstituted alkyl, or aryl group; Rt is a C2-C20 substituted or unsubstituted carboxyacyl, R2 is hydrogen, C(C6H5)3, or a C2-C20 substituted or unsubstituted carboxyacyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or -OR4, where R4 is hydrogen, CM alkanoyl, benzoyl, or C2-C2o substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29.
40. A mutant lentivims comprising a mutation in the nucleic acid sequence encoding Gag which renders the replication of said mutant lentiviras less sensitive to 3-<9-(3',3'-dimethylsuccinyl) betulinic acid (DSB).
41. The mutant lentiviras of claim 40, wherein the Gag polypeptide of said virus does not bind to 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
42. The mutant lentivirus of claim 40, wherein said mutation further renders said mutant lentiviras less sensitive to inhibition of processing of p25 (CA-SPl) to p24 (CA) by DSB.
43. The mutant lentiviras of claim 40, comprising a mutation in the nucleotide sequence encoding the Gag p25 protein (CA-SPl).
44. The mutant lentivims of claim 43, which encodes a Gag polypeptide wherein one or more amino acids are deleted from, or substituted in the amino acid sequence in the CA-SPl region.
45. The mutant lentivirus of claim 40, wherein said mutation occurs in the nucleotide sequence encoding an amino acid sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) KNWMTETFLNQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDC TLKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATEM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ DD NO: 26) (g) SHKARILAEAMSQVTN (SEQ ED NO: 116);
(h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and
0) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
46. The mutant lentiviras of claim 40, wherein said mutation occurs in the nucleotide sequence encoding the amino acid sequence selected from group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARE AEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
7. The mutant lentivirus of claim 40, wherein said mutation occurs in the nucleotide sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
48. The mutant lentiviras of claim 40, wherein said mutation occurs in the nucleotide sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19; (i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
49. The mutant lentiviras of claim 40, wherein said mutation in CA-SPl occurs in the codon encoding the amino acid selected from the group consisting of:
(a) Amino acid 1 (Glycine) in GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(b) Amino acid 2 (Histidine) in SHKAREAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(c) Amino acid 6 (Valine or Isoleucine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(d) Amino acid 7 (Leucine) in SHKARILAEAMSQVTN (SEQ DD NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(e) Amino acid 8 (Alanine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(f) Amino acid 10 (Alanine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(g) Amino acid 11 (Methionine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(h) Amino acid 12 (Serine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117); (i) Amino acid 13 (Glutamine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(j) Amino acid 14 (Valine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(k) Amino acid 15 (Threonine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(1) Amino acid 16 (Asparagine) in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);and
(m) Amino acid 45 (Alanine) in HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNC GKEGHIA (SEQ ED NO: 120).
50. The mutant lentivims of claim 40, wherein said mutation in the CA- SPl sequence results in the change in the encoded amino acid selected from the group consisting of:
(a) Glycine in GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(b) Histidine to Glutamine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO:l 17);
(c) Histidine to Tyrosine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(d) Valine or Isoleucine to Leucine at amino acid 6 in in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117); (e) Leucine to Methionine at amino acid 7 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(f) Alanine to Valine at amino acid 8 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(g) Alanine to Valine at amino acid 10 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(h) Methionine to Leucine at amino acid 11 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) Serine to Lysine at amino acid 12 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(j) Glutamine to Glutamic acid at amino acid 13 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(k) Valine to Alanine at amino acid 14 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(1) Threonine to Leucine at amino acid 15 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(m) Asparagine to Alanine at amino acid 16 in SHKARILAEAMSQVTN (SEQ ED NO: 116) or GHKARVLAEAMSQVTN (SEQ ED NO: 117); (n) Alanine to Threonine at amino acid 45 in HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNC GKEGHLA (SEQ ED NO: 525).
51. The mutant lentiviras of claim 40, wherein said mutation occurs in the polynucleotide encoding the amino acid sequence selected from any one of SEQ D NO: 121 to 443.
52. The mutant lentivirus of any one of claims 40 to 51 , which is a mutant HTV-1.
53. A recombinant non-HIN-1 retroviras the replication of which is inhibited by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB).
54. The recombinant non-HIN-1 retroviras of claim 53, wherein DSB inhibits the processing of the viral Gag p25 protein (CA-SPl) to p24 (CA) but does not significantly inhibit other Gag processing steps.
55. The recombinant non-HIN-1 retroviras of claim 53, wherein the Gag polypeptide of said virus binds to 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
56. The recombinant non-HIN-1 retroviras of claim 53 which contains a p25 (CA-SPl) protein comprising an amino acid sequence at least about 70%) identical to the group consisting of
(a) KΝWMTETFLNQΝAΝPDCKTTLKALGPAATLEEMMTA CQGNGGPSHKARILAEAMSQVTΝSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATTM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and 0) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
57. The recombinant non-HIN-1 retroviras of claim 56 which contains a p25 (CA-SPl) protein comprising an amino acid sequence selected from the group consisting of
(a) KΝWMTETFLNQΝAΝPDCKTILKALGPAATLEEMMTA CQGNGGPSHKARILAEAMSQVTΝSATEVI (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATEvI; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26) (g) SHKARILAEAMSQVTN (SEQ ED NO: 116);
(h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and
(j) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119)
58. The recombinant non-HIV-1 retroviras of claim 53 which comprises a polynucleotide encoding CA-SPl, which is at least about 70%> identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828- 1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
59. The recombinant non-HTV-1 retroviras of claim 53 which comprises a polynucleotide encoding CA-SPl, which has a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19; (c) about nucleotides 1344-1435 of SEQ E) NO: 18;
(d) about nucleotides 1828-1920 of SEQ E) NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ DD NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and 0) about nucleotides 1858-1920 of SEQ ED NO: 19.
60. The recombinant non-HTV-1 retroviras of claim 53 which contains a p25 (CA-SPl) protein comprising an amino acid sequence selected from any of SEQ ED NO: 121 to 443.
61. The recombinant non-HTV-1 retroviras of claim 53 having a nucleotide sequence at least about 70% identical to the sequence selected from the group consisting of:
(a) SEQ ED NO: 90;
(b) SEQ ED NO: 92;
(c) SEQ ED NO: 94;
(d) SEQ ED NO: 96; and
(e) SEQ ED NO: 98;
62. The recombinant non-HTV-1 retroviras of claim 53 having a nucleotide sequence selected from the group consisting of:
(a) SEQ D NO: 90; (b) SEQ ED NO: 92;
(c) SEQ ED NO: 94;
(d) SEQ ED NO: 96; and
(e) SEQ ED NO: 98.
63. The recombinant non-HTV-1 retrovirus of claim 53, which is derived from a virus selected from the group consisting of HTV-2, HTLV-I, HTLV-II, STV, avian leukosis viras (ALV), endogenous avian retroviras (EAV), mouse mammary tumor viras (MMTV), feline immunodeficiency virus (FIN), Bovine immunodeficiency viras (BIN), caprine arthritis encephalitis viras (CAEV), Visna-maedi viras, and feline leukemia viras (FeLV).
64. The recombinant non-HIV-1 retroviras of claim 63 which is derived from a vims selected from the group consisting of group consisting of:
(a) SIV;
(b) FTV;
(c) EATV;
(d) BIV;
(e) CAEV; and
(f) Visna-Maedi viras.
65. An animal model of lentiviras infection comprising a suitable non- human animal host infected with the recombinant non-HIN-1 retrovirus of claim 53.
6. The animal model of claim 65, wherein said non-human animal host is selected from the group consisting of :
(a) a cat;
(b) a monkey;
(c) an ape;
(d) a horse;
(e) a sheep;
(f) a goat;
(g) a cow; (h) a mouse; (i) a rat; and (j) a bird.
67. A method of making a recombinant non-HIN-1 lentiviras sensitive to inhibition by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), comprising replacing the polynucleotide sequence encoding CA-SPl from a non-HIN-1 lentiviras that is not sensitive to DSB with the sequence encoding CA-SPl of a DSB-sensitive HIN-1 viras.
68. The method of claim 67, wherein the polynucleotide sequence encoding CA-SPl in said recombinant non-HIN-1 lentiviras comprises a sequence at least about 70% identical with a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19; (c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ E) NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
69. The method of claim 68, wherein the polynucleotide sequence encoding CA-SPl in said recombinant non-HTV-1 lentiviras comprises a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ E) NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ D NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ JD NO: 19.
0. The method of claim 67, wherein the CA-SPl protein of said recombinant DSB-sensitive non-HIN-1 lentivims comprises an amino acid sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) KΝWMTETFLVQΝAΝPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTΝSATEV1 (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and 0) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
71. The method of claim 67, wherein the CA-SPl protein of said DSB- sensitive recombinant non-HTV-1 lentiviras comprises an amino acid sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 21); (b) KNWMTETLLNQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ DD NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKAREAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
72. The method of claim 67, wherein the CA-SPl protein of said recombinant DSB-sensitive non-HIV-1 lentiviras has an amino acid sequence selected from any of SEQ ED NO: 121 to 443.
73. The method of claim 67, comprising:
(a) deleting from the genome of said lentiviras the nucleotides which cooesponds to nucleotides 1370-1413 from SEQ ED NO: 18; and
(b) inserting nucleotides 1370-1413 from SEQ ID NO: 18 or nucleotides 1857-1899 of SEQ DD NO: 19 into said region of said non-HTV-1 lentiviras.
74. A recombinant non-HIN-1 lentiviras produced by the method of any one of claims 67 to 73.
75. An antibody that selectively binds at or near the CA-SPl cleavage site of Gag.
76. The antibody of claim 75, which binds an amino acid sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) KΝWMTETFLNQΝAΝPDCKTILKALGPAATLEEMMTA CQGNGGPSHKARILAEAMSQNTΝSATEVI (SEQ DD NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ DD NO: 26)
(g) SHKARILAEAMSQVTN (SEQ DD NO: 116); (h) GHKARVLAEAMSQVTN (SEQ DD NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119).
77. The antibody of claim 75, which binds to an amino acid sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTTLKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARTLAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 119).
78. The antibody of claim 75, which binds to an amino acid sequence selected from any of SEQ ED NO: 121 to 443.
79. The antibody of claim 75, which selectively binds a polypeptide containing a mutation in HTV CA-SPl polypeptide which results in a decrease in the inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-O-(3'3'-dimethylsuccinyl) betulinic acid.
80. The antibody of claim 79, wherein said mutation is located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl.
81. The antibody of claim 79, wherein said polypeptide is selected from the group consisting of:
(a) GHKARVLVEAMSQV (SEQ ED NO: 2);
(b) SHKARILAEVMSQV (SEQ ED NO: 3);
(c) SQKARILAEAMSQVTN;
(d) GQKARVLAEAMSQVTN;
(e) SYKARILAEAMSQVTN;
(f) GYKARVLAEAMSQVTN;
(g) SHKARLLAEAMSQVTN; (h) GHKARLLAEAMSQVTN; (i) SHKARTMAEAMSQVTN; (j) GHKARVMAEAMSQVTN; (k) SHKARILAEVMSQVTN; (1) GHKARVLAEVMSQVTN; (m) SHKARILVEAMSQVTN; (n) GHKARVLVEAMSQVTN; (o) SHKARTLAEALSQVTN; (p) GHKARVLAEALSQVTN; (q) SHKARTLAEAMLQVTN; (r) GHKARVLAEAMLQVTN; (s) SHKARILAEAMSEVTN;
(t) GHKARVLAEAMSEVTN;
(u) SHKARE AEAMSQATN;
(v) GHKARVLAEAMSQATN;
(w) SHKARD AEAMSQVLN;
(x) GHKARVLAEAMSQVLN;
(y) SHKARILAEAMSQVTA;
(z) GHKARNLAEAMSQVTA; and
(aa) HKARILAEAMSQVTΝPATIMIQKGΝFRΝQRKTVKCFΝC GKEGHIT
82. The antibody of claim 79, which selectively binds an amino acid sequence selected from the group consisting of SEQ ED NO: 2 and SEQ ED NO: 3.
83. The antibody of claim 75, which is selected from the group consisting of:
(a) an antibody that selectively binds SPl but not CA-SPl ;
(b) an antibody that selectively binds CA-SPl but not CA; and
(c) . an antibody that selectively binds C A but not C A-SP 1.
84. The antibody of claim 75 that inhibits the binding of 3-O-(3',3'- dimethylsuccinyl) betulinic acid to Gag.
85. An isolated CA-SPl polypeptide which is at least about 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATEVI (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARTLAEAMSQVTNS ATM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ DD NO: 119)
86. The isolated CA-SPl polypeptide of claim 85, which is identical to a sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116);
(h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKAREAEAMSQVTNS ATEVI (SEQ ED NO: 118); and
(j) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
An isolated CA-SPl polypeptide, encoded by a polynucleotide sequence at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729- 1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18;
(h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and (j) about nucleotides 1858-1920 of SEQ ED NO: 19.
88. The isolated CA-SPl polypeptide of claim 87, encoded by a polynucleotide sequence selected from the group consisting of:
(a) about nucleotides 1243-1435 of SEQ ED NO: 18;
(b) about nucleotides 1729-1920 of SEQ ED NO: 19;
(c) about nucleotides 1344-1435 of SEQ ED NO: 18;
(d) about nucleotides 1828-1920 of SEQ ED NO: 19;
(e) about nucleotides 1370-1413 of SEQ ED NO: 18;
(f) about nucleotides 1857-1899 of SEQ ED NO: 19
(g) about nucleotides 1372-1419 of SEQ ED NO: 18; (h) about nucleotides 1858-1905 of SEQ ED NO: 19;
(i) about nucleotides 1372-1434 of SEQ ED NO: 18; and 0") about nucleotides 1858-1920 of SEQ ED NO: 19.
89. An isolated polypeptide from the CA-SPl region of HIV-l, consisting of an amino acid sequence selected from any one of SEQ ED NO: 121 to 443.
90. An isolated polypeptide from the HIV CA-SPl polypeptide which contains a mutation that results in a decrease in inhibition of processing of p25 by 3 -O-(3 ',3 '-dimethylsuccinyl) betulinic acid.
91. The isolated polypeptide of claim 90, wherein said mutation is located in the CA-SPl cleavage site or in the SPl domain.
92. The isolated polypeptide of claim 90, which is encoded by a polynucleotide selected from the group consisting of SEQ DD NO: 4, SEQ DD NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
93. The isolated polypeptide of claim 90, containing a mutation in that results in a decrease in inhibition of processing of p25 by 3-O-(3',3'- dimethylsuccinyl) betulinic acid; wherein said mutation occurs in the nucleic acid sequence encoding a polypeptide selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTEJ ALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQNTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119) The isolated polypeptide of claim 90 selected from the group consisting of:
(a) GHKARVLVEAMSQV (SEQ ED NO: 2);
(b) SHKARILAEVMSQV (SEQ ED NO: 3);
(c) SQKARELAEAMSQVTN;
(d) GQKARVLAEAMSQVTN;
(e) SYKARE EAMSQVTN;
(f) GYKARVLAEAMSQVTN;
(g) SHKARLLAEAMSQVTN;
(h) GHKARLLAEAMSQVTN;
(i) SHKARIMAEAMSQVTN;
(j) GHKARVMAEAMSQVTN;
(k) SHKARELAEVMSQVTN;
(1) GHKARVLAEVMSQVTN;
(m) SHKARELVEAMSQVTN;
(n) GHKARVLVEAMSQVTN;
(o) SHKARILAEALSQVTN;
(p) GHKARVLAEALSQVTN;
(q) SHKARE EAMLQVTN;
(r) GHKARVLAEAMLQVTN;
(s) SHKARILAEAMSEVTN;
(t) GHKARVLAEAMSEVTN; (u) SHKARILAEAMSQATN;
(v) GHKARVLAEAMSQATN;
(w) SHKARILAEAMSQVLN;
(x) GHKARVLAEAMSQVLN;
(y) SHKARILAEAMSQVTA;
(z) GHKARVLAEAMSQVTA; and
(aa) HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNC GKEGHIT.
95. The isolated polypeptide of claim 90, encoded by an isolated polynucleotide which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ED NO: 5, SEQ ED NO: 7, and 10.
96. The isolated polypeptide of claim 90, which is part of a chimeric or fusion protein.
97. An isolated polynucleotide which encodes an amino acid sequence containing a mutation in an HTV Gag p25 protein (CA SPl), said mutation resulting in a decrease in inhibition of processing of p25 (CA-SPl) to p24 (CA) by 3-O-(3',3'-dimethylsuccinyl) betulinic acid.
98. The isolated polynucleotide of claim 97, wherein said decrease in inhibition of processing of p25 is due to a decrease in inhibition of the interaction of HTV-1 protease with Gag.
99. The isolated polynucleotide of claim 97, wherein said decrease in inhibition of processing of p25 is due to a decrease in the binding of 3- O-(3',3'-dimethylsuccinyl) betulinic acid to Gag.
100. The isolated polynucleotide of claim 97, wherein said decrease in inhibition of processing of p25 is due to a decrease in the binding of DSB at or near the CA-SPl cleavage site of Gag.
101. The isolated polynucleotide of claim 97, wherein said mutation is located at or near the CA-SPl cleavage site or in the SPl domain of CA-SPl.
102. The isolated polynucleotide of claim 97, wherein said mutation is located at or near the polynucleotide encoding the amino acid sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26)
(g) SHKARILAEAMSQVTN (SEQ ED NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and 0') GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
103. The isolated polynucleotide of claim 97 selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, SEQ ED NO: 8 and SEQ ED NO: 9.
104. The isolated polynucleotide of claim 97, having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ED NO: 4, and SEQ ED NO: 6.
105. The isolated polynucleotide of claim 97, having at least about 80%> identity to a polynucleotide selected from the group consisting of SEQ ED NO: 8 and SEQ ED NO: 9.
106. The isolated polynucleotide of claim 97, having at least about 95%> identity to a polynucleotide selected from the group consisting of SEQ NO: 5 and SEQ ED NO: 7.
107. The isolated polynucleotide of claim 97, having at least about 80%> identity to a polynucleotide of SEQ ED NO: 10.
108. A vector comprising the isolated polynucleotide of claim 97.
109. A host cell comprising the vector of claim 108.
110. A method of producing a polypeptide composing incubating the host cell of claim 109 in a medium and recovering the polypeptide from said medium.
111. A method of identifying a compound that inhibits HIV-l replication in cells of an animal, comprising:
(a) contacting a polypeptide comprising a CA-SPl cleavage site with a test compound; (b) adding a labeled substance that selectively binds at or near the CA-SPl cleavage site; and
(c) measuring the binding of said labeled substance to said polypeptide.
112. The method of claim 111, wherein said compound binds at or near the CA-SPl cleavage site.
113. The method of claim 111, wherein said compound inhibits the interaction of HTV-1 protease with the CA-SPl cleavage site.
114. The method of claim 111, wherein said polypeptide comprising a CA- SPl cleavage site is a polypeptide fragment or recombinant peptide.
115. The method of claim 111, wherein said labeled substance is a labeled antibody specific for CA-SPl; and wherein said measuring comprises measuring the change in the amount of labeled antibody bound to the protein in the presence said test compound compared with a control.
116. The method of claim 111, wherein the labeled substance is 3-O-(3',3'- dimethylsuccinyl) betulinic acid, and wherein the method comprises measuring the change in the amount of labeled 3-O-(3',3'- dimethylsuccinyl) betulinic acid bound to the protein in the presence of test compound, compared with a control.
117. The method according to claim 111, wherein the label on said labeled substance is an enzyme, a fluorescent substance, a chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, an electron dense substance, radioisotope, or a combination thereof.
118. A method for identifying a test compound that inhibits HTV-1 replication in the cells of an animal comprising: (a) contacting a polypeptide composing a CA-SPl cleavage site, with a protease in the presence of a test compound; and
(b) detecting the interaction of protease with said polypeptide.
119. The method of claim 118, wherein said protease consists essentially of HIN-1 protease.
120. The method of claim 118, wherein said polypeptide is labeled with two fluorescent moieties, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the transfer of fluorescent energy from one moiety to the other in the presence of the test compound.
121. The method of claim 118, wherein the effect of the test compound on cleavage of said polypeptide is detected by measuring the binding of an antibody to at least one of SPl, p24 (CA), or CA-SPl (p25).
122. The method of claim 121, wherein said labeled antibody is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electron dense substance, radioisotope, and combinations thereof.
123. The method of claim 118 comprising:
(a) contacting a first polypeptide comprising an HIN-1 wild-type CA-SPl cleavage site, with HTV-1 protease in the presence of said test compound;
(b) contacting a second polypeptide, comprising a mutant CA-SPl cleavage site or an alternative protease cleavage site with HIN- 1 protease in the presence of said test compound; wherein the cleavage of said mutant cleavage site or alternative protease cleavage site is not substantially inhibited by 3-O-(3',3'- dimethylsuccinyl) betulinic acid;
(c) detecting the interaction of HIN-1 protease with said first and second peptides; and
(d) comparing the cleavage of said first polypeptide to the cleavage of said second polypeptide.
124. The method of claim 123, wherein the wild-type CA-SPl, mutant CA- SPl, or alternative protease cleavage site region is contained within a polypeptide fragment or recombinant peptide.
125. The method of claim 123, wherein said polypeptide is labeled with a fluorescent moiety and a fluorescence quenching moiety, each bound to opposite sides of the CA-SPl cleavage site, and wherein said detecting comprises measuring the signal from the fluorescent moiety.
126. The method of claim 123, wherein at least one of said first or said second polypeptide further comprises an epitope tag.
127. The method of claim 123, wherein (a) and (b) occur in a cell.
128. The method of claim 123, wherein said detecting the interaction of said compound with said first and second polypeptides comprises gel electrophoresis.
129. A method for identifying compounds that inhibit HIN-1 replication in the cells of an animal comprising:
(a) contacting a test compound with cells infected with a first viras, the replication of which is sensitive to inhibition by 3-O- (3',3'-dimethylsuccinyl) betulinic acid (DSB); (b) contacting a test compound with cells infected with a second viras having significantly reduced sensitivity to DSB;
(c) determining the phenotype of treated cells infected with the first viras and the second viras; and
(d) selecting a test compound that is more active against the first virus compared with second viras.
130. The method of claim 129, wherein said phenotype is selected from the group consisting of:
(a) viral replication;
(b) cleavage of C A-SP 1 ; and
(c) production of an altered virion phenotype.
131. A method for identifying a compound that inhibits HIV-l replication in the cells of an animal, comprising:
(a) applying a test compound to cells infected with a lentiviras, the replication of which is sensitive to inhibition by 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB); and
(b) analyzing a lysate derived from said infected cells to determine whether cleavage of the CA-SPl protein has occuoed.
132. The method of claim 131, wherein said lysate is obtained by lysing at least one of said
(a) infected cells; or
(b) the released viral particles.
133. The method of claim 131, wherein said analyzing to determine whether cleavage of the CA-SPl protein has occuoed comprises measuring the presence or absence of p25.
134. The method of claim 131, wherein said analyzing to determine whether cleavage of the CA-SPl protein has occuoed comprises performing a western blot of viral proteins and detecting p25 using an antibody to p25.
135. The method of claim 131, wherein said analyzing to determine whether cleavage of the CA-SPl protein has occuoed comprises performing a gel electrophoresis of viral proteins and imaging of metabolically labeled proteins.
136. The method of claim 131, wherein said analyzing to determine whether cleavage of the CA-SPl protein has occuoed comprises performing an immunoassay.
137. The method of claim 133, wherein said immunoassay comprises:
(a) capturing p25 and p24 on a substrate using an antibody that selectively binds 24;
(b) detecting the presence or absence of p25 on the substrate with an antibody that selectively binds p25.
138. The method of claim 131, wherein an epitope tag sequence is inserted into SPl, and wherein said analyzing comprises selective detection of p25 using an antibody to the epitope tag.
139. A method of identifying a compound that inhibits HTV-1 replication in the cells of an animal comprising: applying a test compound to cells infected with a lentivirus, the replication of which is sensitive to inhibition by 3 -O-(3 ',3 '-dimethylsuccinyl) betulinic acid (DSB), and thereafter analyzing viras particles released by the cells for an altered virion phenotype.
140. The method of claim 139, wherein said analyzing involves transmission electron microscopy.
141. The method of claim 139, wherein said altered phenotype is selected from the group consisting of:
(a) virions possessing spherical, electron-dense cores that are acentric with respect to the viral particle;
(b) virions possessing crescent-shaped electron-dense layers lying just inside the viral membrane;
(c) virions having reduced or no infectivity; and
(d) the combination of any one of (a) through (c).
142. The method of any one of claims 129, 131 or 139, wherein said lentivirus, the replication of which is sensitive to inhibition by 3-O- (3*,3'-dimethylsuccinyl) betulinic acid (DSB), is HTV-1.
143. The method of any one of claims 129, 131 or 139, wherein said lentiviras, the replication of which is sensitive to inhibition by 3-O- (3',3'-dimethylsuccinyl) betulinic acid (DSB), is a recombinant non- HIN-1 lentiviras comprising the nucleotide encoding the CA-SPl from HTV-1, and wherein said lentivims is derived from a lentivirus selected from the group consisting of.
(a) SIN;
(b) FTV;
(c) EAIV; (d) BIV;
(e) CAEV;
(f) Visna-Maedi virus; and
(g) HTV-2.
144. The method of claim 131, wherein said viras having significantly reduced sensitivity to DSB is selected from the group consisting of:
(a) STV;
(b) FTV;
(c) EAIV;
(d) BTV;
(e) CAEV;
(f) Visna-Maedi viras;
(g) HIN-2; and (h) mutant HIN-1.
145. A method of identifying a compound, comprising:
(a) measuring disease progression in treated animals infected with a lentiviras, wherein said lentiviras is sensitive to 3-0-(3',3'- dimethylsuccinyl) betulinic acid (DSB);
(b) measuring disease progression in untreated animals infected with said lentiviras,
(c) comparing the response of said treated and said untreated animals.
146. A method of identifying a compound, comprising
(a) administering said compound to animals infected with a first lentivims, wherein the replication of said first lentiviras is inhibited by 3-0-(3',3'-dimethylsuccinyl) betulinic acid (DSB);
(b) administering said compound to animals infected with a second lentiviras, wherein the replication of said second lentivirus is not substantially inhibited by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB);
(c) measuring and comparing disease progression in animals infected with said first lentiviras, compared with animals infected with said second lentivims.
147. A method of identifying a compound, comprising measuring the interaction of said compound with an amino acid sequence selected from the group consisting of:
(a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATM (SEQ ED NO: 21);
(b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATEVI (SEQ ED NO: 22);
(c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ED NO: 23);
(d) TACQGVGGPGHKARVLAEAMSQVTNPATEvϊ (SEQ ED NO: 24);
(e) SHKARILAEAMSQV (SEQ ED NO: 25);
(f) GHKARVLAEAMSQV (SEQ ED NO: 26) (g) SHKARILAEAMSQVTN (SEQ ED NO: 116);
(h) GHKARVLAEAMSQVTN (SEQ ED NO: 117);
(i) SHKARILAEAMSQVTNSATIM (SEQ ED NO: 118); and
(j) GHKARVLAEAMSQVTNPATIM (SEQ ED NO: 119)
148. A method of determining if a patient is infected with HIV-l that is susceptible to treatment by a compound that inhibits p25 processing, comprising:
(a) obtaining a specimen from said patient;
(b) obtaining viral nucleic acid encoding CA-SPl from the specimen;
(c) genotyping said viral nucleic acid;
(d) determining if the nucleic acid encodes a CA-SPl variant, the processing of which is sensitive to inhibition by to 3-O-(3',3'- dimethylsuccinyl) betulinic acid (DSB).
149. A method of treating a disease in a patient in need thereof comprising:
(a) identifying a compound which inhibits the processing of viral Gag p25 protein (CA-SPl) to p24 (CA), but has no significant effect on other Gag processing steps;
(b) obtaining regulatory approval for the sale and use of said compound; packaging the compound for sale and treatment of a disease in a patient in need thereof.
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