JP2008504808A - Inhibition of HIV-1 replication by disruption of viral capsid spacer peptide 1 protein processing - Google Patents

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. An amino acid sequence comprising a mutation in the Gag p25 protein (with a mutation that results in reduced inhibition of p25 to p24 processing by dimethyl succinyl betulinic acid or dimethyl succinyl betulin), a polynucleotide encoding such a mutated sequence, Also included are antibodies that selectively bind to such mutated sequences. Methods of inhibition, inhibitory compounds, and methods of discovering inhibitory compounds that target proteolytic processing of 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 the protease enzyme. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the Gag proteolytic site can be used in screening assays to identify compounds that target proteolytic processing.

Description

The present invention includes methods of inhibiting HIV infection, inhibitors of HIV infection, and methods of discovery of inhibitors of HIV infection.

References to federal-funded research and development The U.S. Government has determined that other parties may agree to the lump sum license in this invention and reasonable conditions as provided by grant number 2R44AI051047-02 awarded by NIH / NIAID. May have the right in limited circumstances to require the patentee to license.

Background Human immunodeficiency virus (HIV) is a member of a lentivirus, a subfamily of retroviruses. The viral genome contains a number of regulatory elements that allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system; this destroys the body's immune system and makes the patient susceptible to opportunistic infections and neoplasms. Immune deficiency appears to be progressive and irreversible, with a high mortality rate reaching 100% over several years.

  HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system that express the cell surface antigen CD4 (also known as OKT4, T4, and leu3). Virus nutrition is due to the interaction between the viral envelope glycoprotein gpl20 and cell surface CD4 molecules (Dalgleish et al. Nature 312: 763-767 (1984)) (Non-Patent Document 1). These interactions not only mediate infection of sensitive cells with HIV, but are also responsible for virus-induced fusions of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytium, cell death, and progressive depletion of CD4 cells in HIV-infected patients. These events result in HIV-induced immunosuppression and subsequent sequelae, opportunistic infections, and neoplasms.

In addition to CD4 ⁺ T cells, the host range of HIV includes cells of the mononuclear phagocyte lineage (Dalgleish et al., Supra) (Non-Patent Document 1), which includes blood monocytes, tissue macrophages, skin Of Langerhans cells, and dendritic reticulum cells in lymph nodes. HIV is also neurotrophic and can infect monocytes and macrophages in the central nervous system, causing severe neurological damage. Macrophages and monocytes are the major reservoirs of HIV. These can interact and fuse with CD4 bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.

  Significant progress has been made in the development of drugs for the treatment of HIV-1. Treatments for HIV include, but are not limited to: AZT, 3TC, ddC, d4T, ddI, tenofovir, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir , Nelfinavir, lopinavir, amprenavir, atazanavir and phosamprenavir, or an antiretroviral agent or antibody that is combined with each other or biologically based (e.g., gp41-derived peptide, enfuvirtide ( enfuvirtide) (Fuzeon; Timeris-Roche) and T-1249 (Trimeris), or soluble CD4, antibodies to CD4, and CD4 or anti-CD4 conjugates, etc.) or further presented herein Any other antiretroviral agent or antibody, such as These drug combinations are particularly effective and can reduce the level of viral RNA to undetectable levels in plasma, indicating the development of viral resistance, resulting in improved patient health and longevity.

  Despite these advances, there are still problems with currently available drug regimens. Many of these drugs exhibit significant toxicity, exhibit other side effects (eg, fat redistribution), or require complex dosing schedules that reduce compliance, thereby limiting efficacy. Resistant strains of HIV often appear over time even for combination therapy. The high cost of these drugs is also a limitation on their widespread use, especially outside developed countries.

  There is still a widespread need for the development of additional drugs to circumvent these problems. Ideally, they target the different stages in the viral life cycle and have minimal toxicity but a lower manufacturing cost in addition to the equipment set required for combination therapy.

  HIV virion assembly occurs at the surface membrane of infected cells where viral Gag polyprotein accumulates, resulting in the assembly of immature virions that bud from the cell surface. In virions, Gag is cleaved by viral proteinase (PR) into matrix (MA) protein, capsid (CA) protein, nucleocapsid (NC) protein, and C-terminal p6 structural protein (Wiegers K. et al., J. Virol. 72: 2846-2854 (1998)) (non-patent document 2). Gag processing induces a process called “maturation”, the reorganization of internal virion structures. In mature HIV particles, MA aligns with the inner surface of the membrane, whereas CA forms a conical core that contains genomic RNA complexed with NC. Cleavage and maturation are not necessary for particle formation but are essential for infectivity (Kohl, N. et al., Proc. Natl. Acad. Sci. USA 85: 4686-4690 (1998)) (Non-Patent Document 3).

  CA and NC and NC and p6 are separated on the Gag polyprotein by short spacer peptides (p2) of 14 and 10 amino acids, respectively (spacer peptides 1 (SP1) and SP2 respectively) (Wiegers K. et al ., J. Virol. 72: 2846-2854 (1998), Pettit, SC et al., J. Virol. 68: 8017-8027 (1994), Liang et al. J. Virol. 76: 11729-11737 (2002 )) (Non-Patent Documents 2, 4, 5). These spacer peptides are released by PR-mediated cleavage at their N- and C-termini during particle maturation. Individual cleavage sites on HIV Gag and Gag-Pol polyproteins are processed at different sites, resulting in intermediates that appear transiently before the final product. Such intermediates may be important for virion morphogenesis or maturation but do not contribute to the structure of mature virions (Weigers et al. And Pettit, et al., Supra) (non-patent References 2, 4). The first Gag cleavage event occurs at the C-terminus of SP1, separating the N-terminal MA-CA-SP1 intermediate from the C-terminal NC-SP2-p6 intermediate. Subsequent cleavage separating CA-SP1 to MA and p6 from NC-SP2 occurs at a rate about 10 times slower. Cleavage of SP1 from the C-terminus of CA is a late event, occurring at a rate 400 times slower than cleavage at the SP1-NC site (Weigers et al. And Pettit, et al., Supra) (non- Patent Documents 2 and 4). The uncleaved CA-SP1 intermediate protein is alternatively named “p25”, whereas the cleaved CA protein is alternatively named “p24” and the cleaved SP1 peptide is alternatively Was named "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 show particles with a high electron density conical core. On the other hand, electron microscopic studies of viral particles lacking CA-SP1 cleavage show particles with a spherical high electron density ribonucleoprotein core and a semi-moon-like high electron density layer located just inside the viral membrane (Weigers et al (Supra) (Non-Patent Document 2). Mutations at or near the CA-SP1 cleavage site have been shown to disrupt Gag processing and normal maturation processes, thereby resulting in the production of non-infectious viral particles (Weigers et al. Supra) (non- Patent Document 2). Phenotypically, these particles are defective in Gag processing (which reveals itself in the presence of p25 (CA-SP1) in Western blot analysis), and the above abnormalities resulting from defective capsid condensation A good particle morphology.

Previously, betulinic acid and platanic acid were isolated from Syzigium claviflorum and determined to have anti-HIV activity. Betulinic acid and platanic acid show inhibitory activity against HIV-1 replication in H9 lymphocyte cells with EC ₅₀ values of 1.4 μM and 6.5 μM, respectively, and therapeutic index (TI) values of 9.3 and 14, respectively. It was. 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 TI value of 14 (Fujioka, T., et al., J. Nat. Prod. 57: 243-247 (1994)) (Non-Patent Document 6). Esterification of betulinic acid with certain substituted acyl groups (3 ′, 3′-dimethylglutaryl and 3 ′, 3′-dimethylsuccinyl groups) yields derivatives with enhanced activity (Kashiwada, Y , et al., J. Med. Chem. 39: 1016-1017 (1996)) (non-patent document 7). Acylated betulinic acid and dihydrobetulinic acid, which are potent anti-HIV agents, are also described in US Pat. No. 5,679,828. The anti-HIV assay shows extremely potent anti-HIV activity in H9 lymphocytes both acutely infected with 3-O- (3 ', 3'-dimethylsuccinyl) -betulinic acid (DSB) and dihydrobetulinic acid analogs. , Each with an EC ₅₀ value of less than 1.7 × 10 ⁻⁵ μM. These compounds showed significant TI values higher than 970,000 and higher than 400,000, respectively.

R=H（ベツリン酸） U.S. Pat. No. 5,468,888 describes a lupine.
Of 28 amide derivatives, which are described as having cytoprotective effects against HIV-infected cells.

R = H (Betulinic acid)

  Japanese Patent Application No. JP 01 143,832 (Patent Document 3) discloses betulin and its 3,28 diester useful in the field of anticancer agents.

ここでR₁はC₂-C₂₀置換型または非置換型のカルボキシアシルであり、R₂はC₂-C₂₀置換型または非置換型のカルボキシアシルであり;かつR₃は水素、ハロゲン、アミノ、置換されていてもよいモノアルキルアミノもしくはジアルキルアミノ、または--OR₄であり、ここでR₄は水素、C_1-4アルカノイル、ベンゾイル、またはC₂-C₂₀置換型もしくは非置換型のカルボキシアシルであり;ここで破線はC20とC29の間の任意の二重結合を表す。 US Pat. No. 6,172,110 discloses derivatives of betulinic acid and dihydrobetulin having the formula: embedded image or a pharmaceutically acceptable salt thereof.
Derivatives of betulin and dihydrobetulin

Wherein R ₁ is a C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl, R ₂ is a C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl; and R ₃ is hydrogen, halogen, Amino, optionally substituted monoalkylamino or dialkylamino, or --OR ₄ where R ₄ is hydrogen, C _1-4 alkanoyl, benzoyl, or C ₂ -C ₂₀ substituted or unsubstituted Where the dashed line represents any double bond between C20 and C29.

  US Patent Application No. 60 / 413,451 discloses 3,3-dimethylsuccinyl betulin, which is incorporated herein by reference. Zhu, YM. 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) (Non-Patent Documents 8-11) are dimethylsuccinyl. Disclosed are betulinic acid and dimethylsuccinyl oleanolic acid. Esterification of the 3 ′ carbon of betulin with succinic acid produced compounds capable of inhibiting HIV-1 activity (Pokrovskii, AG et al., Gos. Nauchnyi Tsentr Virusol. Biotekhnol. “Vector,” 9 : 485-491 (2001)) (Non-Patent Document 12).

  Published International Publication No. WO 02/26761 discloses the use of betulin and its analogs for treating fungal infections.

  There is a need for new HIV inhibition methods that are effective against drug resistant strains of viruses. The strategy of the present invention is to provide therapeutic methods and compounds that inhibit viruses in a manner different from the approved treatment.

  The compounds and methods of the present invention have a novel mechanism of action and are therefore active against HIV strains that are resistant to current reverse transcriptases and protease inhibitors. In this way, the present invention provides a completely new approach for treating HIV / AIDS.

U.S. Pat.No. 5,679,828 U.S. Pat.No. 5,468,888 Japanese Patent Application No. JP 01 143,832 U.S. Patent No. 6,172,110 U.S. Patent Application No. 60 / 413,451 International Publication No.02 / 26761 Dalgleish et al. Nature 312: 763-767 (1984) Wiegers K. et al., J. Virol. 72: 2846-2854 (1998) Kohl, N. et al., Proc. Natl. Acad. Sci. USA 85: 4686-4690 (1998) Pettit, S.C. et al., J. Virol. 68: 8017-8027 (1994) Liang et al. J. Virol. 76: 11729-11737 (2002) Fujioka, T., et al., J. Nat. Prod. 57: 243-247 (1994) Kashiwada, Y., et al., J. Med. Chem. 39: 1016-1017 (1996) 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) Kashiwada Y. et al., Chem. Pharm. Bull. 48: 1387-1390 (2000) Pokrovskii, A.G. et al., Gos. Nauchnyi Tsentr Virusol. Biotekhnol. “Vector,” 9: 485-491 (2001)

Brief Summary of the Invention In general, the present invention provides methods of inhibition, compounds of inhibition, and methods of identifying inhibitors that target proteolytic processing of HIV-1 Gag protein. In one embodiment, such a compound may directly or indirectly inhibit the interaction of a protease enzyme with HIV-1 Gag protein. In another embodiment, such inhibition of interaction occurs via binding of the compound to Gag. Inhibition of protease cleavage of the CA-SP1 protein of HIV-1 by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) is one example, but other proteolytic sites are It can be targeted by a similar approach using inhibitory compounds that interact with the substrate in a manner similar to interacting with Gag.

  Another aspect is directed to methods that inhibit the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but have no effect on other Gag processing steps.

  Further aspects are directed to methods for identifying compounds that inhibit the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but have no effect on other Gag processing steps.

  One aspect extends to a compound or pharmaceutical composition identified by the method for identifying a compound that inhibits HIV-1 replication as disclosed herein.

  Another aspect is directed to a polynucleotide comprising a sequence encoding an amino acid sequence comprising a mutation in the Gag p25 protein, wherein the mutation is p25 by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. Results in reduced inhibition of processing from to p24. This aspect of the invention is also directed to vectors, viruses, and host cells containing the polynucleotide, and methods of making the protein.

  A further aspect is directed to an amino acid sequence that includes a mutation in the Gag p25 protein, which reduces the inhibition of p25 to p24 processing by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. Bring.

  A still further aspect is directed to an antibody that selectively binds an amino acid sequence containing a mutation in the Gag p25 protein, which mutation is p25 to p24 by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. Resulting in reduced inhibition of processing. Also included in this aspect of the invention are methods of making the antibodies, hybridomas producing the antibodies, and methods of making the hybridomas.

  A still further aspect relates to a kit comprising a polynucleotide, polypeptide, or antibody disclosed herein.

  The invention further relates to a method of inhibiting HIV-1 infection in animal cells by contacting said cells with a compound that blocks the maturation of virus particles released from the treated infected cells. In one embodiment, the released virus particles exhibit a unique thin high electron density layer in the vicinity of the uncondensed core and virus membrane and have reduced infectivity. A method of contacting an animal cell with a compound that both inhibits processing of the viral Gag p25 protein and disrupts maturation of the viral particle is included. Also included are methods of treating HIV-infected cells, wherein HIV that infects the cells does not respond to other HIV treatments.

  The invention further includes a method for identifying compounds that inhibit the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but have no effect on other Gag processing steps. The method includes contacting HIV-1 infected cells with a test compound, and then analyzing the released viral particles to detect the presence of p25. Methods for detecting p25 include Western blotting of viral proteins and detection using antibodies to p25, gel electrophoresis, and imaging of metabolically labeled proteins. Methods for detecting p25 also include immunoassays using antibodies against epitope tags inserted into p25 or SP1 (p2) or SP1 sequences.

  The invention is further directed to a method for identifying a compound comprising contacting HIV-1 infected cells with a test compound, and then flaking and releasing viral particles released by the contacted cells. Analyzing by transmission electron microscopy and identifying whether virus particles with a unique thin high electron density layer near the uncondensed core and virus membrane are present.

  The present invention is also directed to compounds identified by the screening methods described above. In a further embodiment, the present invention provides by administering a compound that inhibits the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but does not significantly affect other Gag processing steps. Extends to methods of treating HIV-1 infection in patients. In related embodiments, such inhibition may be accompanied by a different observable phenotype. For example, inhibition may not need to significantly reduce the amount of virions released from treated infected cells; and / or the inhibition is significant relative to the amount of RNA incorporation into the released virions. It may have little or no effect; and / or the inhibition destroys the maturation of virions released from infected cells treated with the compound. In related embodiments, the virion structure can be affected, and the majority of virions released from the treated cells exhibit a spherical high electron density core that is non-centric with respect to the viral particle; and / or Has a semi-moon shaped electron density layer located just inside the viral membrane; and / or has reduced or no infectivity.

  In additional embodiments, the present invention results in inhibition of viral Gag p25 protein (CA-SP1) to p24 (CA) processing, but has no significant effect on other Gag processing processes. It extends to a method of treating HIV-1 infection in a patient by administering a compound that inhibits the interaction of HIV protease with CA-SP1. Such inhibition may be direct, or alternatively, indirect; and / or viral Gag protein such that the interaction of HIV protease with CA-SP1 is inhibited. The compound that binds may be included. The present invention also treats HIV in a patient with a compound that binds at or near the site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA), thereby treating the HIV protease CA- It extends to methods that inhibit the interaction with the SP1 cleavage site and result in inhibition of p25 to p24 processing.

。 In another embodiment, the present invention relates to a method of treating HIV-1 infection in a patient by administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), comprising: The compound has at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to or is identical to a sequence selected from the group consisting of Binds to a polypeptide having an amino acid sequence:

.

In another embodiment, the present invention extends to a method of treating HIV-1 infection in a patient by administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), comprising: The compound has or is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical or identical to a polynucleotide selected from the group consisting of: Binds to a polypeptide encoded by a polynucleotide sequence that is:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.

。 In another aspect, the invention extends to a method of inhibiting the processing of viral Gag p25 protein (CA-SP1). In related embodiments, such compounds are at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to a sequence selected from the group consisting of: Binds to a polypeptide having an amino acid sequence that has or is identical to:

.

In a related embodiment, the invention extends to a method of inhibiting viral Gag p25 protein (CA-SP1) processing by administration of a compound, wherein the compound is at least about a polynucleotide selected from the group consisting of: Binds to a polypeptide encoded by a polynucleotide sequence having, or identical to, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.

  The present invention may be useful in the treatment of HIV in patients who are not well treated by other HIV-1 therapies. Thus, the present invention also extends to methods of treating patients in need of treatment, where HIV-1 infected cells do not respond to other HIV treatments. In another embodiment, the methods of the invention are performed on a subject infected with HIV that is resistant to the drug used to treat the HIV infection. In one application, HIV is resistant to protease inhibitors, polymerase inhibitors, nucleoside analogs, vaccines, binding inhibitors, immunomodulators, or any other inhibitor. In another embodiment, the methods of the invention are performed on a subject infected with HV that is resistant to drugs used to treat HIV infection selected from the group consisting of: Zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, ampnavirta, amponavir Fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene, polymannoacetate, castanospermine, contracan, cream pharmatex ( creme pharmatex), CS-87, pen Cyclovir, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine, trisodium phosphonoformate, fusidic acid, HPA-23, efflornitine ( eflornithine), nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimethrexate, SK-818, suramin, UA001, and combinations thereof.

  The compounds of the present invention are also useful as part of a therapeutic combination. Accordingly, in one aspect, the invention extends to a method of treating HIV in a patient, wherein the patient is administered the compound in combination with at least one antiviral agent. Suitable antiviral agents include, but are not limited to: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, tenofovir , Adefovir, Atazanavir, Fosamprenavir, Hydroxyurea, AL-721, Ampurigen, Butylated hydroxytoluene, Polymannoacetate, Castanospermine, Contracan, Cream Pharmatex, CS-87, Penciclovir, Famciclovir, Acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine, trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxy , Pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, gp41-derived peptide, antibody against CD4, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof. In another embodiment, the patient is administered the compound in combination with an immunomodulator, anticancer agent, antibacterial agent, antifungal agent, or combinations thereof.

  The present invention is also directed to compounds. Such compounds are used in methods for treating patients infected with HIV; in methods for inhibiting the processing of viral Gag p25 protein (CA-SP1) to p24 (CA), or in human blood and human blood products. Is useful in methods for processing. Such compounds useful in the present invention include, but are not limited to, derivatives of dimethyl succinyl betulinic acid or derivatives of dimethyl succinyl betulin, or 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.

  The compounds of the present invention may be used alone or may be 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, Ampurigen, Butylated hydroxytoluene; Polymannoacetate, Castanospermine; Contracan; Cream Pharma Tex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine, trisodium phosphonoformate, fusidin , HPA-23, efflornitine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimethrexate, SK-818, suramin, UA001, enfuvirtide, gp41-derived peptide, CD4 Antibodies, soluble CD4, CD4-containing molecules, CD4-IgG2, and combinations thereof; antiviral agents, immunomodulators, anticancer agents, antibacterial agents, antifungal agents, or combinations thereof.

  In a further embodiment, the present invention is directed to a method of treating a human blood product comprising contacting the blood product with a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA). Is done. In one aspect, the compound has no significant effect on other Gag processing steps. In a related embodiment of this method, the inhibition does not significantly reduce the amount of virions released from the treated infected cells; and / or is significant relative to the amount of RNA incorporation into the released virions. Has little or no effect; and / or inhibits virion maturation released from infected cells treated with the compound; and / or affects the morphology of the virus. Such effects on viral morphology include, but are not limited to: virions with a spherical high electron density core that is non-centric with respect to the viral particle; and / or located just inside the viral membrane Virions with a semi-moon-like high electron density layer; and / or virions with reduced or no infectivity. In a related embodiment, this method results in inhibition of processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but has no significant effect on other Gag processing processes. Including administration. This may be direct or indirect inhibition of HIV protease with CA-SP1; and / or viral Gag protein so that the interaction of HIV protease with CA-SP1 is inhibited And / or the compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA), thereby allowing the CA- Inhibits interaction with the SP1 cleavage site and results in inhibition of p25 to p24 processing.

。 In a further embodiment, the invention comprises contacting the blood product with a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is selected from the group consisting of: Binds to a polypeptide having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to or the amino acid sequence of Extends to methods of processing human blood products:

.

In a related embodiment, the invention comprises contacting the blood product with a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound comprises the group consisting of Encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity or identity to the selected polynucleotide sequence Binds to the polypeptide to be:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.

  The present invention also embodies a method for identifying compounds that inhibit HIV-1 replication. Accordingly, the present invention also includes a method of identifying a compound that inhibits HIV-1 replication in an animal cell comprising the steps of: contacting a Gag protein comprising a CA-SP1 cleavage site with a test compound; Adding a labeled substance that selectively binds in the vicinity of the SP1 cleavage site; and measuring competition between binding of the test compound to the CA-SP1 cleavage site and binding of the labeled substance. In a further embodiment of this method, the compound inhibits the interaction of HIV-1 protease with the target site by binding to the target site.

  These methods also include a CA-SP1 cleavage site region included in the polypeptide fragment or recombinant peptide; and / or the labeled substance is a labeled antibody specific for CA-SP1, compared to a control And measuring the change in the amount of labeled antibody that binds to the protein in the presence of the test compound. Labels include, but are not limited to, enzymes, fluorescent materials, chemiluminescent materials, horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density materials, radioisotopes, or combinations thereof.

  A method for identifying a compound that inhibits HIV-1 replication in an animal cell is also in one embodiment, labeled 3-O- (3 ′, 3), bound to a protein in the presence of a test compound compared to a control. Including measuring the change in the amount of 3'-dimethylsuccinyl) betulinic acid, wherein the labeled substance is 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid.

  In an alternative embodiment, the invention includes a method for identifying a compound that inhibits HIV-1 replication in an animal cell, wherein the method comprises a polypeptide comprising a CA-SP1 cleavage site in the presence of a test compound. Contacting with a protease. Preferably, the protease is related to or is an HIV-1 protease. In one embodiment, the method comprises the steps of: contacting a polypeptide comprising a wild type CA-SP1 cleavage site with a protease in the presence of a test compound, and also providing a mutant CA-SP1 cleavage site. Contacting a polypeptide comprising a polypeptide or an alternative protease cleavage site with HIV-1 protease in the presence of a test compound, detecting cleavage, as well as the amount of cleavage of the mutant polypeptide or alternative protease cleavage Comparing the amount of cleavage of the native wild-type polypeptide against the amount of cleavage of the protein comprising the site. In a related aspect of this method, wild type CA-SP1 or mutant CA-SP1 or an alternative protease cleavage site is included in the polypeptide fragment or recombinant peptide. In a further related aspect, the polypeptide is labeled with a fluorescent moiety and a fluorescent quenching moiety, each binding to the opposite side of the CA-SP1 cleavage site, and the detecting step measures the signal from the fluorescent moiety. including. In another related embodiment, the polypeptide is labeled with two polypeptide moieties, each binding to the opposite side of the CA-SP1 cleavage site, and the detecting step is performed from one moiety in the presence of the test compound. Measuring the transfer of fluorescence energy to other parts. In a further embodiment, the effect of the test compound on polypeptide cleavage is detected by measuring the amount of labeled antibody bound to SP1 or p24 (CA). In a related aspect, a labeled antibody that binds CA or an antibody that binds SP1 is an enzyme, a fluorescent substance, a chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, a high electron density substance, a radioisotope And a molecule selected from the group consisting of combinations thereof.

The present invention is also directed to methods for identifying compounds that inhibit HIV-1 replication in animal cells. In one embodiment, the method comprises the following steps; cells significantly infected with wild-type virus isolate or significantly reduced against inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid Contacting a test compound with a cell infected with a virus isolate having a selected sensitivity; and a virus having a reduced sensitivity to 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid Selecting a test compound that is more active against the wild-type virus isolate as compared to the isolate. In another embodiment, the method comprises the steps of: contacting HIV-1 infected cells with a test compound; lysing infected cells or released virus particles to form a lysate; and lysis Analyzing the product to determine whether cleavage of the CA-SP1 protein has occurred. In this latter embodiment, the analyzing step may comprise measuring the presence or absence of p25; and / or performing a western blot of viral proteins and detecting p25 using antibodies to p25 And / or performing gel electrophoresis of viral proteins and imaging of metabolically labeled proteins; and / or performing immunoassays. Such immunoassays may be performed by any method known in the art, including but not limited to the following:
(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 present invention also includes modifications of the above assays as will be apparent to those skilled in the art.

  In a further embodiment, the method for identifying a compound according to the invention comprises the use of an epitope tag sequence inserted in SP1, and the selective detection of p25 is performed using an antibody against this epitope tag.

  The present invention is also directed to a method for identifying a compound that inhibits HIV-1 replication in an animal cell comprising the steps of: contacting the HIV-1 infected cell with a test compound, and thereafter And analyzing the virus particles using transmission electron microscopy. Such analysis includes, for example, exploring the presence of a spherical high electron density core that is non-centric with respect to the virus particles; and / or having a semi-moon shaped electron density layer located just inside the virus membrane .

  In an additional aspect, the invention includes a sequence encoding an amino acid sequence comprising a mutation in HIV Gag p25 protein (CA-SP1), wherein the mutation is 3-O- (3 ′, 3′-dimethylsuccinyl). It results in a decrease in the processing of p25 protein (CA-SP1) to p24 (CA) by betulinic acid (DSB). Inhibiting the processing of p25 reduces the inhibition of HIV-1 protease interaction with Gag; and / or decreases the binding of 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid to Gag; This may be due to decreased binding of DSB at or near the CA-SP1 cleavage site. Suitable polynucleotides also include: those encoding mutations in or near the CA-SP1 cleavage site or in the SP1 domain of CA-SP1; and / or the amino acid sequence G / SHKARV / ILAEAMSQV (SEQ ID NO: 1) encoding a mutation at or near it; and / or encoding the amino acid sequence GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and / or SEQ ID NO: 4, SEQ ID An isolated polynucleotide selected from the group consisting of NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and / or selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6 A polynucleotide having at least about 95% identity to the polynucleotide; and / or at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9 Polynuku with And / or a polynucleotide having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7; and / or a polynucleotide of SEQ ID NO: 10 A polynucleotide having at least about 80% identity to the nucleotide. In additional embodiments, the polynucleotide has greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% identity to the polynucleotide sequences listed above. Or are identical to these sequences.

  The invention also includes a vector comprising a polynucleotide as described above; a host cell comprising such a vector; and incubating a host cell comprising such a vector in a medium, and recovering the polypeptide from the medium Directed to a method of producing a polypeptide comprising the steps.

。 In one embodiment, the present invention is directed to antibodies. Such an antibody has at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to a sequence selected from the group consisting of: Or can bind to a polypeptide having an amino acid sequence identical to these sequences:

.

In further related embodiments, the invention provides at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, to a polynucleotide having a sequence selected from the group consisting of: Or an antibody that binds to a polypeptide encoded by a polynucleotide sequence that has 99% identity or is identical to this polynucleotide:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(f) about 1370-1413 nucleotides of SEQ ID NO: 18;
(g) about 1857 to 1899 nucleotides of SEQ ID NO: 19;
(h) about 1372-1419 nucleotides of SEQ ID NO: 18;
(i) about nucleotides 1858 to 1905 of SEQ ID NO: 19;
(j) nucleotides from about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.

  In one embodiment, the antibody binds to an amino acid of the CA-SP1 region of HIV-1, which amino acid comprises SHKARILAEAMSQV (SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26).

  In one embodiment, the present invention extends to an antibody that inhibits the binding of 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid to the CA-SP1 region of a Gag polypeptide.

  The present invention also extends to mutant HIV viruses. In one such embodiment, the invention provides that the inhibition of processing of viral Gag p25 protein (CA-SP1) to p24 (CA) in the virus is 3-O- (3 ′, 3′-dimethylsuccinyl) An isolated mutant recombinant HIV-1 virus that is not significantly inhibited by betulinic acid. In a related embodiment, the virus is not inhibited by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. In another embodiment, 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid does not inhibit the interaction of the protease with the Gag polypeptide in the virus. In another embodiment, the virus does not bind to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. In further embodiments, the invention extends to a virus in which an amino acid in the CA-SP1 region is replaced with an alternative amino acid, or an amino acid is added to the CA-SP1 region, or an amino acid is deleted. In one embodiment, one or more amino acids are deleted from the amino acid sequence AEAMSQV (amino acids 8-14 of SEQ ID NO: 26) in the CA-SP1 region.

  Mutant viruses may be used in the methods of the invention described elsewhere herein. For example, such viruses are useful in methods of identifying compounds that inhibit the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), which is a variant of the mutant virus outlined above. Comparing the ability of the compound to inhibit HIV-1 replication as compared to replication. Such inhibition may be tested in cells, or in animals, or in vitro.

  The present invention also extends to non-HIV-1 retroviruses that are sensitive to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. In certain embodiments, the retrovirus encodes a CA-SP1 polypeptide having an amino acid sequence comprising the sequence AEAMSQV (amino acids 8-14 of SEQ ID NO: 26) at or near the CA-SP1 cleavage site. In another embodiment, the retrovirus encodes a CA-SP1 polypeptide having an amino acid sequence comprising the sequence VLAEAMSQV (amino acids 6-14 of SEQ ID NO: 26) at or near the CA-SP1 cleavage site. In another embodiment, the retrovirus encodes a CA-SP1 polypeptide having an amino acid sequence comprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near the CA-SP1 cleavage site; Retroviruses against the sequence encoded by the polynucleotide of SEQ ID NO: 26; SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98 And comprising an amino acid sequence having at least 60%, 70%, 80%, 90% identity, or identical to that sequence; in another embodiment, the retrovirus comprises SEQ ID NO: 91; SEQ ID NO: 91; Has at least 60%, 70%, 80%, 90% identity to the sequence of ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97; or SEQ ID NO: 99, or Contains an amino acid sequence that is identical to the sequence. In another embodiment, the retrovirus comprises at least 70% relative to the sequence of SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98; Includes nucleic acid sequences that have 80%, 90% identity, or are identical to the sequence.

  Retroviruses of this aspect of the invention include HIV-2, HTLV-I, HTLV-II, SIV, avian leukemia virus (ALV), endogenous avian retrovirus (EAV), mouse mammary carcinoma virus (MMTV), cat These include but are not limited to immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), goat arthritis encephalitis virus (CAEV), visna-maedi virus, or feline leukemia virus (FeLV).

  In a related embodiment, the present invention is directed to a method of making a recombinant non-HIV-1 lentivirus that is sensitive to DSB. This method comprises the steps of deleting nucleotides corresponding to nucleotides 1370-1413 from SEQ ID NO: 18 in HIV-1 from the genome of the lentivirus; and in the region of the non-HIV-1 lentivirus, SEQ ID NO: Inserting nucleotides 1370-1413 from: 18 or nucleotides 1857-1899 of SEQ ID NO: 19.

  An example of a chimeric lentivirus constructed, constructed, or that can be constructed by this method is shown in FIG.

  Such viruses may be used in the methods herein as indicated elsewhere herein. For example, such a recombinant non-HIV-1 lentivirus may be used in a method of identifying a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), the method , Comparing the ability of the compound to inhibit replication of wild-type non-HIV-1 lentivirus with its DSB-sensitive recombinant variant. Such inhibition may occur in cells; in animals; or in vitro.

The invention also extends to animal models of lentiviral infection, including suitable non-human animal hosts infected with lentivirus that are sensitive to 3-O- (3′-3′-dimethylsuccinyl) betulinic acid. In such embodiments, the lentivirus is SIV;
May include, but is not limited to, FIV; EIAV; BIV; CAEV; and Bisna-Maedivirus.

  The invention also extends to isolated polypeptides. In one embodiment, the present invention extends to a polypeptide comprising a mutation of the HIV CA-SP1 protein, which mutation inhibits the processing of p25 by 3-O- (3′-3′-dimethylsuccinyl) betulinic acid. Cause a decrease. In related embodiments, the polypeptide comprises a mutation located at or near the CA-SP1 cleavage site, or the SP1 domain encoded by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10. And / or encoded by a polynucleotide 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 GHKARVLVEAMSQV ( SEQ ID NO: 2) or a sequence selected from the group consisting of SHKARILAEVMSQV (SEQ ID NO: 3); and / or the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 10 Encoded by an isolated nucleotide that hybridizes under stringent conditions to a more selected polynucleotide; and / or is part of a chimeric or fusion protein.

  The present invention also provides mutations in the HIV CA-SP1 protein that result in reduced inhibition of p25 (CA-SP1) to p24 (CA) processing by 3-O- (3'-3'-dimethylsuccinyl) betulinic acid. It extends to antibodies that selectively bind to amino acid sequences comprising In one such embodiment, the antibody selectively binds to a mutation located in or near the CA-SP1 cleavage site or in the SP1 domain of CA-SP1; in another embodiment, the antibody is GHKARVLVEAMSQV ( Selectively binds to a mutation comprising a sequence selected from the group consisting of SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment, the antibody comprises SEQ ID NO: 2 and SEQ ID NO It selectively binds to an amino acid sequence selected from the group consisting of NO: 3.

  In another embodiment, the present invention provides an antibody that selectively binds SP1 but not CA-SP1; another antibody that selectively binds CA-SP1 but does not bind CA; selectively binds CA Extends to another antibody that does not bind CA-SP1; and additional antibodies that selectively bind at or near the CA-SP1 cleavage site.

  The present invention is also directed to compounds identified by any of the methods described herein. In one embodiment, the compound comprises 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 not a compound selected from the group consisting of these.

  The present invention also extends to pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises dimethyl succinyl betulinic acid or a derivative of dimethyl succinyl betulin; in another embodiment, the pharmaceutical composition is 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 compounds selected from the group consisting of combinations thereof. In another embodiment, the pharmaceutical composition comprises one or more compounds identified according to the methods of the invention, otherwise listed; or any pharmaceutically acceptable salt, ester or prodrug thereof, And a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises an antiviral agent that may include any one of the following: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir , Ritonavir, Indinavir, Nelfinavir, Tenofovir, Amprenavir, Adefovir, Atazanavir, Fosamprenavir, Hydroxyurea, AL-721, Ampurigen, Butylated hydroxytoluene; Polymannoacetate, Castanospermine; Contracan, Cream Pharma Tex, CS-87, penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate, D-penicillamine trisodium phosphonoformate, fusidic acid, HPA -23, efflornitine, nonoxynol, pentamidine isethionate, peptide T, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimethrexate, SK-818, suramin, UA001, combinations thereof, any other antiviral agent, An immunomodulator, anticancer agent, antifungal agent, antibacterial agent, or a combination thereof.

  The present invention also extends to a method of determining whether an individual is infected with HIV-1 that is susceptible to treatment with a compound that inhibits p25 processing. In one embodiment, the method comprises collecting blood from a patient, genotyping viral RNA, and whether the viral RNA contains a mutation in the sequence encoding the CA-SP1 cleavage site region. Including the step of determining.

The invention also extends to a method of treating a disease in a patient in need thereof comprising the following steps:
Identifying compounds that inhibit the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but have no significant effect on other Gag processing steps;
Obtaining regulatory approval for the sale and use of the compound;
Packaging the compound for sale and treatment of disease in a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method of inhibiting HIV-1 replication in animal cells. More particularly, the present invention relates to HIV-1 in mammalian cells by contacting infected cells with a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 protein (CA). A method of inhibiting replication. More particularly, such compounds inhibit the processing of viral Gag p25 protein (CA-SP1) to p24 protein (CA) without significantly affecting other Gag processing steps.

  “A compound that does not have a significant effect on other Gag processing steps” means that the compound in question preferentially inhibits the processing of p25 to p24, but is a further secondary to other Gag processing steps. This means that it is not always necessary to exclude the possibility of having a positive effect.

  “Significant” or “significantly”, unless otherwise defined herein, means a change that is observable or measurable compared to a process in the absence of the compound. However, all observable or measurable changes may not necessarily be significant.

  A number of viral phenotypes may be observed when performing the methods of the invention. One result of contacting infected cells with a compound of the invention may be the formation of non-infectious viral particles. Alternatively or additionally, contacting infected cells with compounds that inhibit p25 to p24 processing results in the formation of non-infectious viral particles, where relative to other Gag processing steps There is no significant effect. This may not significantly reduce the amount of virus released from the treated cells and / or has little or no effect on the amount of RNA incorporation into the released virion. Does not have.

  Accordingly, the present invention also provides for HIV infection in an animal cell comprising contacting the cell with a compound that inhibits p25 processing and also affects the other viral phenotypes described above. A range of methods to inhibit.

  Mutant viruses deficient in CA-SP1 cleavage have been shown to be non-infectious (Wiegers K. et al., J. Virol. 72: 2846-2854 (1998)). 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB) is one example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-1 replication. The activity of this compound is specific for the p25 to p24 processing step and not the other steps of Gag processing. In addition, DSB treatment results in abnormal HIV particle morphology, as described in FIG.

Identification of HIV-1 determinants associated with sensitivity to 1.3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid
(a) Generation and selection of HIV-1 virus resistant to DSB
The amino acid sequence in the region of the CA-SP1 cleavage site was modified to generate mutant HIV-1 that reduces the sensitivity of these strains to compounds that disrupt CA-SP1 processing. In order to identify the amino acid residues of wild-type Gag responsible for the antiviral activity of these compounds, the data for these mutant viruses were used. In one embodiment, compounds that disrupt CA-SP1 processing directly or indirectly inhibit the interaction of HIV-1 protease with regions of the Gag protein that contain these amino acid residues. In another embodiment, compounds that disrupt CA-SP1 processing bind to a region comprising these amino acid residues. As used herein, the terms “bind”, “bound” or “binding” include, for example, bonds that include ionic interactions, electrostatic hydrophobic interactions, hydrogen bonds, and the like. Refers to attachment; and also includes bonds that may be covalent, eg, by chemical coupling. The covalent bond can be, for example, an ester, ether, phosphoester, thioester, thioether, urethane, amide, amine, peptide, imide, hydrazone, hydrazine, carbon-sulfur bond, carbon-phosphorus bond, and the like. The term “coupled” is broader than and includes these terms such as “coupled”, “conjugated” and “attached”.

  In another embodiment, a compound that disrupts CA-SP1 processing binds to another region of Gag, thereby inhibiting HIV-1 protease interaction with the CA-SP1 cleavage site region. In another embodiment, viruses or recombinant proteins that contain mutations in the CA-SP1 cleavage site region can be used in screening assays to identify compounds that disrupt CA-SP1 processing.

  In one set of experiments, amino acid residues in HIV-1 Gag involved in disrupting the processing of CA-SP1 by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) are resistant to DSB. Was identified by screening the gag-pol gene of the virus isolate selected for. The amino acid sequences from these resistant viruses were compared to the gag-pol gene sequence from the DSB sensitive HIV-1 isolate. Two single amino acid changes, alanine (Ala) to valine (Val) substitution (SEQ ID NO: 4) and second in residue 364 in the Gag polyprotein (see FIG. 4) A change at residue 366 (SEQ ID NO: 6) in an isolate of was identified in a DSB-resistant virus. These residues are located immediately downstream of the CA-SP1 cleavage site (SP1 N-terminus). Alanine is highly conserved at these positions throughout all HIV-1 subtypes described in the Los Alamos National Laboratory database. The five amino acid residues upstream and downstream of the CA-SP1 cleavage site are also highly conserved among the various subtypes. However, isoleucine is replaced by valine at two residues upstream of the cleavage site in many clades (see Figure 4, SEQ ID NO: 1) ("HIV Sequence Compendium 2002," edited by Kuiken et al. Los Alamos National Laboratory, Los Alamous, NM).

In order to more broadly map the viral genetic determinants for DSB resistance, further experiments were performed to select in vitro for viruses that are drug resistant. Multiple parallel cultures of Jurkat T cells (5 × 10 ⁵ each) were transfected with the proviral DNA clone pNLA4-3 in the presence or absence of 10-50 ng / ml DSB. Cells were passaged every 2 days and fresh drug was added at each passage. Viral replication was monitored by measuring reverse transcriptase activity in the culture supernatant. Virus was isolated from the culture supernatant collected at selected time points and genomic DNA was amplified by RT-PCR using primers spanning 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.

  In one experiment, an A366V mutation was identified in the SP1 region of NL4-3 virus cultured in the presence of DSB (note: numbering is for Gag polyprotein). Upon further passage, double mutants were identified that included the G357S mutation in CA as well as the A366V mutation in SP1. The A366V mutation was previously identified in experiments selecting resistant mutants of RF isolates. Interestingly, the wild type RF sequence also contains a serine residue at position 357 in CA (FIG. 4). Since serine is present at this position in isolates that are sensitive to DSB (eg, RF), the CA G357S mutation alone is not sufficient to confer resistance to DSB. To determine the contribution of each of these mutations to drug resistance, the A366V mutation and the A366V / G357S double mutation were reengineered into the wild-type NL4-3 backbone by site-directed mutagenesis. The resulting construct was transfected into Jurkat T cells and characterized in a virus replication assay as described above for resistance selection. SDS-PAGE analysis of the transfected cell lysate and virus released into the medium revealed that the A366V mutant Gag was processed and inefficiently released from the cells (data not shown), thus It proved to be very poorly replicated even in the presence (FIG. 11). However, the A366V / G357S double mutant replicated efficiently in the absence or presence of DSB. These data indicate that the resistant mutant, A366V, requires serine at position 357 in the CA region of Gag to compensate for the deleterious effects on viral replication (FIG. 11).

  In further experiments, 10 different resistant isolates were generated. Sequencing of these isolates identified four additional mutations that were 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 sequence for HIV-1 clade A-O, reflecting the breadth of DSB activity against HIV-1 genetically diverse clades. The L363M substitution in CA is found in the consensus sequence for HIV-2, which may in part explain the specificity of the DSB for HIV-1.

  These results demonstrate the presence of specific genetic determinants for DSB activity in HIV-1 and that these determinants are clustered around the CA-SP1 cleavage site.

(a) HIV-1 NL4-3 deletion and SIV insertion studies used to identify genetic determinants of DSB-sensitive viruses. Results from in vitro resistance selection experiments show determinants of DSB HIV-1 inhibitory activity Was mapped to the region of Gag adjacent to the CA-SP1 cleavage site. In order to better define the viral genetic determinants for DSB, HIV-1 point deletion mutagenesis and SIV insertion studies were undertaken to identify specific amino acid residues associated with compound activity. This study was performed as follows. A single residue deletion starting at residue E365 and continuing through residue M377 was engineered into the SP1 domain of infectious HIV-1 molecular clone NL4-3 (FIG. 12). The effects of these point deletions on virion production, infectivity, Gag processing and susceptibility to DSB were determined. The results of these experiments were used to identify Gag residues in the region of the CA-SP1 cleavage site that is associated with DSB activity. Residues associated with activity were inserted into the CA-SP1 cleavage site of DSB resistant virus SIV (Mac239 isolate) to generate HIV-1, SIV chimeric virus (SHIV). Point substitutions of HIV-1 residues from the N-terminus of the CA protein were made in this chimeric virus until the minimal sequence necessary to rescue DSB activity was identified. This minimal sequence required to obtain DSB activity is considered the major viral genetic determinant of DSB activity. This may suggest a molecular determinant of DSB activity.

1.Method
(a) Construction of NL4-3 single point deletion mutants Single point deletion constructs were generated using a PCR-ligation PCR (PLP) strategy as previously described. With the exception of the first residue of SP1, HIV-1 NL4-3 plasmid DNA was used as a template to perform all PCR reactions to generate point deletions across the complete Gag SP1.

  ΔE365 was generated by using a deletion specific downstream primer (Primer 1) with a universal upstream primer (Primer 2) using Vent DNA polymerase (NEB) using NL4-3 as template (Table 1). 1). The fragment derived from this was named the first flanking PCR fragment. The second flanking fragment was amplified using a deletion specific upstream primer (Primer 3) and a 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), the PCR procedure corresponds to each specific point deletion The same was done by changing the deletion specific downstream and upstream primers (Table 1).

  Each of these parallel two adjacent PCR fragments was gel purified, phosphorylated using T4 polynucleotide kinase (NEB), and ligated by using T4 DNA ligase (NEB). After inactivation at 65 ° C. for 15 minutes, the ligation reaction was used for subsequent amplification using universal upstream primer (Primer 2) and downstream primer (Primer 4). This product was gel purified, digested with SpeI and ApaI, and then ligated into the SpeI and ApaI sites of the NL4-3 proviral DNA clone.

Standard PCR conditions were used for the above reaction. These included one cycle of denaturation at 95 ° C for 1 minute 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. PCR reactions were set up using the following components:
5μL 10 × NEB thermophilic buffer
2μL 10mM dNTP
1μL 100nM MgSO ₂
1μL 50pmol upstream primer
1μL 50pmol downstream primer
1μL 50ng / μL template DNA
0.5μL Vent DNA polymerase
38.5 μL ddH ₂ O.

A 10 μL aliquot was run on a 1.0% agarose gel to confirm that the correct size product was amplified. The PCR product was then gel isolated and purified using the Qiaex II gel extraction kit (Qiagen). Two adjacent PCR fragments that were gel purified 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 10 × T4 polynucleotide kinase buffer
2μL 10mM ATP
1 μL T4 polynucleotide kinase.

The reaction was incubated for 1 hour at 37 ° C. with 15 μL gel purified DNA of each of these two adjacent PCR fragments. After inactivation at 65 ° C. for 10 minutes, the adjacent phosphorylated PCR fragments were then ligated together by using T4 DNA ligase (NEB) under the following conditions:
3μL 10 × T4 DNA ligase buffer
13 μL of each two adjacent PCR fragments
1 μL T4 DNA ligase.

After overnight incubation at 16 ° C., the ligation reaction product was used in a second round of PCR reaction to amplify a full-length PCR fragment spanning these two adjacent PCR products. The second round PCR reaction was performed as described above, except that only the universal upstream primer (Primer 2) and the downstream primer (Primer 4) were used. Again, a 10 μL aliquot was run on an agarose gel to confirm that the correct purified product was amplified. The full length PCR fragment was then gel isolated and purified using the Qiaex II kit. The purified full length PCR fragment was cleaved with NL4-3 using SpeI and ApaI under the following conditions:
2 μL 10 × NE buffer 4 (NEB)
1μL ApaI (NEB)
1μL SpeI (NEB)
16 μL full length PCR product (1 μg) or NL4-3 (500 ng).

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 the QiaexII kit. Digested vector NL4-3 and full-length PCR fragment were ligated using T4 DNA ligase under the following procedure:
1μL 10 × T4 DNA ligase buffer
1 μL (25-50 ng) digested NL4-3 vector
7 μL digested (200 ng to 400 ng) digested PCR fragment (700 bp)
1 μL T4 DNA ligase.

  The ligation reaction was incubated overnight at 16 ° C. and the ligation product was transformed into Escherichila coli Max Efficiency Stbl2 (Invitrogen) by heat shock according to the instructions (Invitorgen). Proviral DNA clones were then screened by automated sequencing using the Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems) and individually using internal primers (primers 29 and 30). After confirmation of the mutation, proviral DNA clones were used for various future studies.

1. Construction of SIV chimera mutant
A panel of SIV chimeric constructs with various residues in the NL4-3 CA-SP1 border region was generated by utilizing PCR and cloning procedures as described above, using SIVmac239 molecular clones. These constructs and their amino acid sequences in the CA-SP1 border region are shown in FIG. SIVmac239 was used to generate SIV DD and DE constructs. The SIV DD construct was used to generate SIV DM. Different SIV chimera constructs were produced in PCR by changing the respective mutagenic upstream and downstream primers corresponding to each chimera (Table 1). Each of these parallel two adjacent PCR fragments was gel purified and used directly for subsequent amplification with universal upstream primer (Primer 31) and downstream primer (Primer 32) without phosphorylation. This product was gel purified, digested with BamHI and SbfI and then ligated into the BamHI and SbfI sites of the SIVmac239 proviral DNA clone. Proviral DNA clones were then individually screened by automated sequencing using the internal primer (primer 39) using the Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems). After confirmation of the mutation, proviral DNA clones were used for various future studies.

2. Cell culture and DNA transfection
HeLa cells were maintained in DMEM (Invitrogen) (10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) and passaged during confluence. Jurkat cells were cultured in RPMI 1640 (Invitrogen) (10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin) and passaged every 2 or 3 days.

  To characterize the effects of deletions or substitutions on virion production and Gag polyprotein processing, wild-type HIV-1 NL4-3 or SIVmac239 and the respective mutant proviral DNA were transformed into FuGENE 6 transfection reagent (Roche) HeLa cells were transfected by using Briefly, cells were seeded at a concentration of 0.5 x 105 per well in 6-well plates (Corning) the day before transfection to reach 60-80% confluence on the day of transfection. For each transfection, 3 μl of FuGENE6 was diluted in 100 μl of serum-free DMEM and 1 μg of DNA was added. After gentle mixing, the DNA-lipid complex mixture was gently added dropwise to 2 ml of complete DMEM medium containing cells. Twenty-four hours after transfection, the medium containing the DNA-FuGENE6 complex was removed and 2 ml of fresh DMEM was added to the transfected cells. Forty-eight hours after transfection, the media containing virus particles was collected and clarified by centrifugation at 2,000 rpm, 4 ° C. for 20 minutes in a Sorvall RT 6000B centrifuge. The supernatant containing virus particles was then concentrated through a 20% sucrose cushion in a microcentrifuge at 13,000 rpm, 4 ° C. for 120 minutes, and the pellet was dissolved in lysis buffer (150 mM Tris-HCl, 5% TritonX-100 1% deoxycholic acid, pH 8.0). The level of virus particle production for wild type NL4-3 and point deletion mutants was determined by p24 antigen capture ELISA (ZeptoMetrix, Buffalo, NY).

  To determine the effect of deletions or substitutions on Gag polyprotein processing (in the absence of DSB), SDS-PAGE and Western blot were performed. Briefly, viral proteins are separated on a 12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane (Invitrogen), followed by PBS buffer containing 0.5% Tween and 5% dry milk. Blocking was performed. The membrane was incubated with immunoglobulin (HIV-Ig) from an HIV-1-infected patient (NIH AIDS research and reference reagent program) and hybridized with goat anti-human horseradish peroxidase (Sigma). For membranes containing SIV protein, the membrane was incubated with a reference polyclonal immune serum (NIH AIDS Research and Reference Reagent Program) from SIV infected monkeys and hybridized with goat anti-monkey horseradish peroxidase (Sigma). Immune complexes were visualized using the ECL system (Amersham Pharmacis Biotech) according to the instructions supplied by the manufacturer.

  To address the effects of deletions or substitutions on the ability of DSB to inhibit CA-SP1 processing, HeLa cells were transformed into wild type HIV-1 NL4-3 or SIVmac239 and their respective variants by utilizing the procedure described above. Transfected with proviral DNA. DSB and DMSO controls at a concentration of 1 μg / ml were maintained throughout the entire culture and SDS-PAGE / Western blots to analyze viral proteins derived from these transfections were performed as described in the previous paragraph. did.

The 50% -tissue culture infectious dose per ml (TCID ₅₀ ) was used as a measure of the infectivity of each deletion mutant. Mutant virus derived from transfection in HeLa cells was used to infect U87 CD4.CXCR4 cells. Each virus stock was tested in triplicate at a 1:10 starting dilution followed by a 4-fold serial dilution. The day before infection, cells were plated at a density of 3 × 10 ³ cells / well. On the day of infection, the culture medium was removed from the cell plate and 90 μl of diluted virus was added. On day 1 after infection, on days 3 and 6, virus was removed from the plate and 200 μl of culture medium was added. Supernatants were collected for p24 ELISA analysis on days 6 and 8 after infection. Virus dilution causing 50% of the cultures to infect (TCID ₅₀ ) was determined according to the method of Reed and Muench (Aldovini A. and B. Walker 1990; Dulbecco R. 1988).

3.Result
Viruses containing sequential point deletions in the Gag SP1 domain (Figure 12) were characterized for particle production, infectivity, Gag processing and susceptibility to DSB. Results from these experiments were used to identify SP1 residues associated with DSB activity.

  As expected, the effect of point mutations on virus 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 FIG. Viruses with deletions at residues E366, A367 and M368 were largely affected, producing <25% of the number of particles normally observed in wild-type virus infection. In vitro infectivity assays were used to characterize the ability of deletion mutants to assist virus replication. These experiments resulted in viruses in which the deletion of a single residue at any of the five positions from E365 to Q369 was either non-infectious or was significantly impaired for replication (FIG. 13). In contrast, starting with residue V370 and extending away from the CA-SP1 cleavage site, none of the characterized point deletions resulted in a reduction in viral infectivity (FIG. 13). With the exception of viruses with deletions at positions I376 and M377, all mutant viruses showed a normal or near normal Gag processing phenotype (FIG. 13). The results from these three sets of experiments allowed design and experimental interpretation to identify genetic determinants of DSB activity.

  Sensitivity to DSB was determined in an experiment characterizing the effect of DSB on CA-SP1 cleavage at a later stage of Gag processing. Specifically, these assays measured the ability of DSB to disrupt CA-SP1 processing. For example, as seen in Example 8, DSB-induced defects in Gag processing correlated with the ability of compounds to inhibit viral replication. Results from these experiments show that deletion of a single residue at any of the six positions from E365 to V370 significantly reduces the effect of DSB on CA-SP1 processing (Figure 14). . In contrast, when starting at residue T372 and extending away from the CA-SP1 cleavage site, all of the characterized point deletions are completely susceptible to DSB-induced disruption of CA-SP1 processing. Yes (Figure 14).

  The SP1 residue associated with DSB activity consists of contiguous residues from E365 to V370.

  Residues A364 to V370 were inserted at similar positions in the Gag SP1 domain in the DSB resistant retrovirus SIV (Mac239 isolate). In addition, the N-terminus of the CA protein of this chimeric virus was modified by cumulative substitution of residues found in SIV with HIV-1 specific residues. This approach is summarized in FIG. Next, the effect of DSB on each Gag processing phenotype of the chimeric virus was determined. As seen in FIG. 15, the SIV.DM virus exhibits a Gag processing phenotype that indicates susceptibility to DSB. Thus, the minimal sequence of HIV-1 CA-SP1 specific residues that need to be inserted to rescue DSB activity in SHIV extends from V362 to V370.

(Table 1) PCR mutagenesis primers

  The resistance and mutagenesis data presented above suggest that the GHKARVL-AEAMSQV amino acid sequence in the region of the HIV-1 Gag CA-SP1 cleavage site serves as a genetic determinant of virus susceptibility to DSB.

Extending the determinants of Dsb susceptibility to other lentiviruses: Ca-Sp1 chimeras as animal efficacy models for the development of maturation inhibitors The development of anti-HIV therapies has been hampered by the lack of animal efficacy models. This lack of animal model is mainly due to the inability of most HIV-1 strains to replicate and causes disease in non-human primates. In some instances, this problem has been overcome through the use of chimeric viruses that incorporate regions of interest from the HIV-1 viral target into the SIV viral backbone that aids replication in non-human primates. The most notable example of this approach is HIV-1 / SIV (SHIV), which is originally exclusive to SIV, with the exception that the protein that makes the infectious virus is Env (gp120 / gp41) derived from HIV-1 Includes chimeric viruses. These SHIV envelope chimeras have been used extensively in HIV-1 vaccine development.

  HIV-1 maturation inhibitors disrupt Gag CA-SP1 processing, which results in the formation and release of non-infectious viral particles that exhibit abnormal core morphology. See, for example, Li et al. Proc Natl Acad Sci USA 100.13555-60 (2003). The betulinic acid derivative DSB is an example of this class of inhibitors. The critical viral genetic determinants associated with the activity of maturation inhibitors are mapped to amino acid residues adjacent to the HIV-1 CA-SP1 cleavage site. When this determinant is introduced at the CA-SP1 cleavage site of a DSB resistant non-HIV-1 virus, a maturation inhibitor sensitive chimera results. These CA-SP1 chimeric viruses serve as the basis for animal efficacy models for HIV-1 maturation inhibitors.

  The region of HIV-1 CA-SP1 that is required for maturation inhibitor sensitivity is introduced into the selected lentivirus. Amino acid residues from the HIV-1 CA-SP1 junction, a determinant of DSB sensitivity, were used to replace the corresponding CA-SP1 amino acid in the simian immunodeficiency virus (SIV) genome. Similarly, amino acid residues from the HIV-1 CA-SP1 binding site, which is a determinant of DSB, are feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), visna -Can be replaced in Maedi, and goat arthritic encephalitis virus (CAEV). Table 2 shows the Gag polypeptide sequences for HIV-1, SIV, FIV, EIAV and BIV in the region of the CA-SP1 cleavage site.

(Table 2) Sequence comparison with SIV, FIV, EIAV, and BIV in the CA-SP1 cleavage site region of HIV-1

The HIV-1 CA-SP1 sequence used for replacement is as follows:

  The above method for generating SHIV CA-SP1 chimeric proviral DNA clones comprises selected residues or extended regions from the CA-SP1 region of HIV-1 that replace the corresponding wild type sequence. Used to generate EIAV and BIV proviruses (FIG. 16).

  The SHIV CA-SP1 chimeric-proviral DNA clone was generated by site-directed mutagenesis using standard molecular biology techniques. Briefly, unique restriction enzyme sites in the SIV Gag surrounding the CA-SP1 region were identified, namely BamHI (in the matrix) and Sbf-I (in the NC). Starting from the CA-SP1 region intended for mutagenesis, two overlapping primers, sequences with mutations, a forward primer and a reverse primer incorporating HIV CA-SP1 at their 5 ′ ends were synthesized. Using wild-type SIV proviral DNA as a template, set up two separate PCR reactions to amplify the SIV-Gag fragment in either direction from the site of mutagenesis (CA-SP1 region), i.e. Two amplified fragments overlapping in the CA-SP1 region with mutations, BamHI-CA-SP1 fragment and CA-SP1-SbfI fragment were produced. In the third PCR reaction, the BamHI-SP1 and CA-SP1-SbfI fragments are annealed at their common HIV-CA-SP1 sequence and amplified using the forward SIV BamHI primer and the reverse SIV Sbf-I primer. A full-length chimeric SHIV CA-SP1 PCR fragment was generated. This chimeric SHIV CA-SP1 gag fragment was cloned into the BamHI-Sbf-I window of the SIV proviral clone replacing the SIV-Gag wild type sequence, producing a SHIV CA-SP1 proviral cDNA clone.

  Similarly, unique restriction enzyme cloning sites surrounding the CA-SP1 region in the FIV (Genbank accession number NC — 001482), EIAV (GA # AF016316), and BIV (GA # M32690) genomes were identified (FIG. 16). Specific FIV / EIAV / BIV-HIV1CA-SP1 chimeric primers are synthesized with FIV, EIAV or BIV primers incorporating a specific cloning site. These primers, together with the corresponding proviral DNA (FIV, EIAV or BIAV) as a template, generate a chimeric HIV-1 CA-SP1 fragment in the PCR reaction. The chimeric HIV-1 CA-SP1 fragment is digested with appropriate restriction enzymes and the Fac provirus SacI-EcoRI window; or (ii) the EIAV provirus KasI-EcoRV window; or (iii) the corresponding wild type sequence. Cloning into the BsrGI-ApaI window of the replacement BIV provirus (FIG. 16). Chimeric FIV / EIAV / BIV-HIV-1CA-SP1 proviral DNA clones are sequenced to confirm the presence of the intended mutation. Based on the observed results indicating DSB-sensitive migration, additional constructs are generated utilizing the above strategy to optimize the results.

  In summary, chimeric viruses were generated in which the HIV-1 maturation inhibitor sensitive CA-SP1 determinant was replaced with a similar region of Gag in simian immunodeficiency virus (SIV). Transfer of this region of HIV-1 to the genome of SIV results in a maturation inhibitor sensitive phenotype. Infection of non-human primates with this HIV-1 / SIV chimeric virus should result in an animal efficacy model for therapeutic development of maturation inhibitors.

  A similar approach is used to prepare and characterize HIV-1 CA-SP1 chimeras with FIV, BIV and EIAV. These additional DSB sensitive chimeric viruses should enable the development of additional animal potency monoclonal antibodies for the study of HIV-1 maturation inhibitors.

Use of Mutant Viruses and Chimeric Viruses The mutant viruses and chimeric viruses of the present invention as described above are useful in a variety of cell-based and animal-based assays.

  By comparing the phenotype associated with a virus that is resistant to DSB to a virus that is sensitive to DSB, it is possible to identify compounds that act by a mechanism similar to that of DSB. Accordingly, the present invention includes a method for identifying compounds that inhibit p25 to p24 cleavage in wild-type HIV-1 but do not inhibit CA-SP1 processing in HIV-1 containing a deletion in the CA-SP1 region. . Compounds obtained by such methods are also included in the present invention.

  SIV and other lentiviral chimeras that do not readily infect humans have additional advantages. First, these viruses are less safety issues that are caused to laboratory workers. As a result, for example, cell-based assays to identify new compounds that inhibit CA-SP1 processing can be performed at a lower risk. This lower risk can make it possible to perform assays that could not be performed easily or safely with HIV, and can reduce the cost of such assays.

  Furthermore, such chimeric viruses are useful in animal models. For example, chimeric SIVs that are sensitive to DSB identify, for example, pharmaceutical compositions, routes of administration and dosage regimens for the treatment of disease to identify novel compounds that inhibit CA-SP1 processing As well as to study the effects of combination therapy such as DSB with protease inhibitors.

  Since SIV is generally limited to monkey infections, the generation of additional lentiviral chimeras can make animal studies performed, for example, in cheaper animals, easier handling, faster disease It is possible to have a progression of or otherwise be more appropriate for certain aspects of human disease.

  In addition, animal models are used to identify suitable pharmaceutical compositions for the treatment of animal diseases of interest in the treatment of companion animals and other high value animals, such as agricultural breeders and racehorses. Can be used.

  The chimeric virus may be induced by any retrovirus. For example, HIV-2, HTLV-I, HTLV-II, SIV, avian leukemia virus (ALV), endogenous avian retrovirus (EAV), mouse mammary adenocarcinoma virus (MMTV), feline immunodeficiency virus (FIV), bovine immunity Derived from deficiency virus (BIV), goat arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), visna-maedi virus, or feline leukemia virus (FeLV).

  Such chimeric viruses may be used in the methods of the invention described elsewhere herein. For example, such a recombinant non-HIV-1 lentivirus may be used in a method of identifying a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), the method , Comparing the ability of the compound to inhibit replication of wild-type non-HIV-1 lentivirus with its DSB-sensitive recombinant variant. Such inhibition can occur in cells, animals; or in vitro.

Construction and Use of Viruses or Polypeptides with Epitope Tags The present invention also extends to recombinant retroviruses having an epitope tag in CA-SP1 of Gag. The epitope tag may be inserted into the CA domain and / or the SP1 domain. Suitable tags are well known to those skilled in the art and include the hemagglutinin epitope HA (YPYDVPDYA) (SEQ ID NO: 81), bluetongue virus epitope VP7 (QYPALT) (SEQ ID NO: 82), α-tubulin epitope (EEF ), Flag (DYKDDDDK) (SEQ ID NO: 83), and VSV-G (YTDIEMNRLGK) (SEQ ID NO: 84). An example of an SP1-containing epitope tag is illustrated in FIG.

  Such epitope-tagged viruses and fragments thereof are useful in cell-based assays and in vivo, including animal models, in identifying new compounds that inhibit CA-SP1 processing in vitro. Additional uses for such epitope-tagged viruses and fragments thereof are described elsewhere herein.

Polynucleotides, polypeptides and antibodies of the invention The invention also provides at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% to an amino acid sequence selected from the group consisting of: Or a polypeptide that is 99% identical:

  In another aspect, the invention includes a polynucleotide that encodes a polypeptide as described above. Polynucleotides of the invention include degenerate variants, eg, those that encode the same amino acid due to the difference in the third base of the codon but nevertheless encoding “degenerate”.

  The term “about” as used herein refers to a value that is 10% more or less than the value mentioned, preferably 5% more or less.

  The polypeptides and polynucleotides of the present invention are useful in the methods of the present invention. In one aspect, they may be used in in vitro assays to identify compounds that bind to the CA-SP1 region of Gag. In another aspect, they may be used in the production of antibodies that are useful in other methods described elsewhere herein. In another aspect, the polynucleotide may be inserted into a vector and consequently into a host cell for production of the polypeptide. The above embodiments are exemplary and are not intended to be limiting.

  The invention includes a polynucleotide comprising a sequence that encodes an amino acid sequence comprising a mutation in HIV Gag p25 protein (CA-SP1), wherein the mutation is from DS25 to p24 (CA) by p25 (CA-SP1). Results in reduced inhibition of processing. The polynucleotide of the present invention comprises a mutation that is optionally located at or near the CA-SP1 cleavage site, or optionally located in the SP1 domain of CA-SP1. The mutation may be present in an amino acid sequence selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) and SHKARILAEVMSQV (SEQ ID NO: 3). The polynucleotides of the invention also span the sequences shown as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. The invention also includes a vector comprising the polynucleotide, a host cell comprising the vector, and a method of producing the polypeptide comprising the steps of incubating the cell in a medium and recovering the polypeptide from the medium. Including.

  The present invention further hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. Polynucleotides. The invention also includes a polynucleotide that hybridizes to SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10 or 12, which comprises 3-O- (3 ′, 3′-dimethylsuccinyl ) Resulting in a decrease in inhibition of p25 to p24 processing by betulinic acid, and also where the mutation is optionally located in or near CA-SP1 or in the SP1 domain of CA-SP1. The present invention is also directed to a vector comprising the polypeptide, a host cell comprising the vector, and a method of producing the polypeptide, the method comprising incubating the host cell in a medium and the medium from the medium. Recovering the polypeptide.

  “Nearby” or “adjacent”, as used herein in reference to a polypeptide, means including about 50, about 25, about 20, or about 15 residues from the point of reference. To do. For example, about 50, about 25, about 20 or about 15 residues on either side of the HIV-1 Gag CA-SP1 cleavage site; more preferably on either side of the HIV-1 Gag CA-SP1 cleavage site About 10 residues; and most preferably about 7 residues on either side of the HIV-1 Gag CA-SP1 cleavage site. In reference to a polynucleotide, the term “neighboring” or “adjacent” refers 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 a composition or substance exists in nature and is found in an “isolated” form, it has been changed or removed from its natural environment, or both. An “isolated” nucleic acid molecule of the present invention also contemplates a nucleic acid molecule, DNA, or RNA and has been removed from its natural 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.

  “Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single and double stranded DNA, DNA that is a mixture of single and double stranded regions, single and double stranded RNA, and single and double stranded. Included are hybrid molecules comprising RNA, DNA and RNA, which are a mixture of single stranded regions, which may be single stranded or more typically double stranded, or a mixture of single and double stranded regions. Furthermore, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs that contain one or more modified bases and DNAs or RNAs that have a backbone modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA, and thus a “polynucleotide” is a chemically, enzymatically or metabolically modified version of a polynucleotide, as typically found in nature, and Includes the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also includes relatively short polynucleotides, often referred 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, ie, peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than those encoded by the 20 genes. “Polypeptide” includes amino acid sequences modified either by natural processes (eg, post-translational processing) or by chemical modification techniques well known in the art. Such modifications are well documented in basic textbooks, more detailed academic papers, and a vast research literature. Modifications can occur anywhere in the polypeptide, including the backbone, amino acid side chains, and the amino terminus or carboxy terminus. 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 can contain many types of modifications. Polypeptides can be branched as a result of ubiquitin binding and can be cyclic with or without branching. Cyclic polypeptides, branched polypeptides, and branched cyclic polypeptides can result from post-translation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond , Crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, Methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition (eg arginylation) to proteins, and ubiquitin binding.

  “Variant”, as the term is used herein, refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively. A typical variant of a polynucleotide differs from another reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the 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 variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. In general, the differences are limited such that the sequences of the reference polypeptide and variant as a whole are closely similar and are identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more, any combination of substitutions, additions, deletions. The substituted or inserted amino acid residue may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring (eg, allelic variants) or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or direct synthesis.

  Accordingly, a variant (or fragment, derivative or analog thereof) of a polypeptide encoded by any one of the polynucleotides described herein is (i) at least one or more amino acid residues. Substituted with a conservative or non-conservative amino acid residue (one conservative amino acid residue, or at least one but not more than 10 conservative amino acid residues), and such substituted amino acids The residue may be encoded or not encoded by the genetic code, (ii) one or more amino acid residues contain a substituent, (iii) the mature polypeptide is another compound ( For example, fused with a compound (e.g., polyethylene glycol) to increase the half-life of the polypeptide, or (iv) additional amino acids are fused to the mature polypeptide. Such as IgG: Fc fusion region peptides or leader or secretory sequences or sequences utilized for the purification of mature polypeptide or proprotein sequences. Such variants are deemed to be within the scope of those skilled in the art from the teachings herein. Polynucleotides encoding these variants are also included in the present invention. “Variant” as used herein is equivalent to the term “variant”.

  Substitution of a charged amino acid with another charged amino acid and substitution with a neutral or negatively charged amino acid is included. In addition, one or more of the amino acid residues (e.g., arginine and lysine residues) of the polypeptides of the present invention remove unwanted processing by proteases such as furin or kexin. Can be deleted or substituted with another residue. The prevention of agglomeration is highly desirable. Protein aggregation not only results in a loss of activity, but is also a problem in 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 10: 307-377 (1993)). Thus, the polypeptides of the present invention may include one or more amino acids, substitutions, deletions or additions, either from natural mutations or artificial manipulation.

  As indicated, the changes are probably minor properties such as conservative amino acid substitutions that do not significantly affect protein folding or activity (see Table 3).

Table 3 Conservative amino acid substitutions

  However, in certain embodiments, it may be desirable to use non-conservative substitutions of amino acids. For example, non-conservative substitutions of amino acids are used to make DSB sensitive viruses resistant to DSB.

  A polynucleotide encompassed by the present invention encodes an amino acid whose reference polynucleotide comprises a mutation in the CA-SP1 protein, wherein the mutation is p25 to p24 due to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. At least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% Or it may have 100% identity. Polynucleotides also encompassed by the present invention contain mutations that are “silent”, where different codons encode the same amino acid (fluctuations).

  “Identity” is a measure of the identity of a nucleotide or amino acid sequence. The term “identity” is used herein interchangeably with the term “homology”. In general, the sequences are aligned to give the best alignment match. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. Although there are a number of ways to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to those skilled in the art. Commonly used methods for determining identity or similarity between two sequences include Baxevanis and Oullette, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, 2nd edition, Wiley-Interscience, This includes, but is not limited to, the methods disclosed in New York, (2001). The method for determining identity and similarity is codified in a computer program. Preferred computer program methods for determining identity and similarity between two sequences include the GCS program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387, (1984)), BLASTP. , BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403, (1990)).

  A polynucleotide having a nucleotide sequence with at least, for example, 95% “identity” relative to a reference nucleotide sequence, wherein the polynucleotide sequence may comprise up to 5 point mutations for each 100 nucleotides of the reference nucleotide sequence, see Polynucleotide nucleotides, except that up to 5% of the nucleotides of the sequence can be deleted or replaced with another nucleotide, or multiple nucleotides up to 5% of the total nucleotides of the reference sequence can be inserted into the reference sequence It is intended that the sequence is identical to the reference sequence. These variations of the reference sequence can occur at the 5 ′ or 3 ′ end position of the reference nucleotide sequence, or individually between nucleotides in the reference sequence, or one or more consecutive in the reference sequence It can occur between these terminal positions, dispersed in the group.

  Similarly, a polypeptide having an amino acid sequence that is at least, for example, 95% “identical” to a reference amino acid sequence, except that the polypeptide sequence may contain up to 5 changes for each 100 amino acids of the reference amino acid. Is intended that the amino acid sequence of the polypeptide is identical to the reference sequence. To obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be deleted or replaced with another amino acid, or of the reference sequence Multiple nucleotides up to 5% of all amino acid residues can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino-terminal or carboxy-terminal position of the reference amino acid sequence, or individually between residues in the reference sequence, or one or more consecutive groups in the reference sequence Can occur between these terminal positions. The reference (query) sequence can be the entire nucleotide sequence of any one of the sequences of the invention or any polynucleotide fragment (eg, a polynucleotide encoding an amino acid sequence of the invention and / or a C-terminal deletion).

  Any particular nucleic acid molecule is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97, for example, relative to the nucleotide sequence of the invention. Whether or not they have the same, or identical, is determined by the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and can be routinely determined using known computer programs. 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. Using BESTFIT or other sequence alignment programs, if a particular sequence is 95% identical to a reference sequence, e.g., according to a sequence of the invention, the parameters are calculated over the entire length of the reference nucleotide sequence. And a homology gap of up to 5% of the total number of nucleotides in the reference sequence is allowed.

  In a particular embodiment, the identity (also referred to as global sequence alignment) between the sequences of the invention and the subject sequence is determined by the algorithm of Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1999)). Determined using the FASTDB computer program based. The preferred parameters used in FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0 Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 500, or the length of the target nucleotide sequence, whichever is shorter. In accordance with this embodiment, if the subject sequence is shorter than the reference sequence due to a 5 ′ or 3 ′ deletion rather than an internal deletion, the FASTDB program will calculate 5% of the subject sequence in calculating percent identity. Manual corrections are made to the results to account for the fact that they do not account for 'cuts and 3' cuts. For subject sequences cleaved at the 5 'end or 3' end relative to the query sequence, the percent identity is 5 'of the subject sequence that is not matched / aligned as the percent of total bases in the query sequence. And is corrected by calculating the number of bases in the query sequence that is 3 '. The determination of whether nucleotides are matched / aligned is determined by the results of FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. The corrected score is what is used for the purposes of this aspect. Only bases outside the 5 'and 3' bases of the subject sequence as shown by the FASTDB sequence that are not matched / aligned with the query sequence are for the purpose of manually adjusting the percent identity score. Calculated. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. A deletion is present at the 5 'end of the subject sequence and the FASTDB alignment does not show a match / alignment of the first 10 bases at the 5' end. Ten unpaired bases represent 10% of the sequence (number of unmatched bases at the 5 'and 3' ends / total number of bases in the query sequence), thus 10% is the same calculated by the FASTDB program Subtracted from the percent sex score. If the remaining 90 bases are a perfect match, the final percent identity is 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. This time the deletion is an internal deletion, so that there are no bases 5 'or 3' of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Only the 5 ′ and 3 ′ bases of the subject sequence that are not matched / aligned with the query sequence are manually corrected. No other manual correction is made for the purposes of this aspect.

  This application has at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity To nucleic acid molecules that are identical to the nucleic acid sequences disclosed herein or fragments thereof, whether or not they encode the polypeptides having the disclosed functional activity Oriented. This is how a person skilled in the art has previously used nucleic acid molecules (e.g., hybridization probes or polymerases) even if the particular nucleic acid molecule does not encode a polypeptide having the disclosed functional activity. This is because we know the chain reaction (PCR) primer. Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having the disclosed functional activity include, inter alia, (1) isolation of a variant thereof in a cDNA library; (2) intracellular expression of the disclosed sequence. In situ hybridization (eg, “FISH”) to determine localization or presence, and (3) Northern blot analysis to detect mRNA expression in specific tissues.

  As used herein, the term “PCR” refers to the polymerase chain reaction that is the subject of US Pat. Nos. 4,683,195 and 4,683,202 to Mullis et al., As well as improvements currently known in the art. In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art can be utilized. Such techniques are explained fully in the literature. See, for example, Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  The term “stringent conditions” as used herein is a combination of salt, temperature, organic solvent, and other parameters that are typically controlled in a hybridization reaction. And well known in the art (Sambrook, et al., Supra). The present invention provides the coding sequence of any polynucleotide or polynucleotide fragment as described herein, eg, as described herein, under stringent conditions, in a nucleic acid molecule of the invention as described above. And / or an isolated nucleic acid molecule comprising a polynucleotide that hybridizes to a sequence that is complementary to a non-coding sequence (ie, a transcribed and untranslated sequence). Depending on `` stringent hybridization conditions '', 50% formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / An overnight incubation at 42 ° C. in a solution containing, or consisting of ml denatured salmon sperm DNA, followed by a wash at about 65 ° C. in 0.1 × SSC is contemplated. Polypeptides encoded by these polynucleotides are also included in the present invention.

  The invention also includes a virus comprising the polynucleotide of the invention, wherein the virus comprises a retrovirus comprising the polynucleotide, wherein the retrovirus comprises HIV-1, HIV-2, HTLV-I, HTLV -A member of the group consisting of II, SIV, avian leukemia virus (ALV), endogenous avian retrovirus (EAV), mouse mammary adenocarcinoma virus (MMTV), feline immunodeficiency virus (FIV), or feline leukemia virus (FeLV) possible.

  The present invention further includes a polypeptide comprising a mutation in the CA-SP1 protein, wherein the mutation reduces the inhibition of p25 to p24 processing by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. And the mutation is optionally located at or near the CA-SP1 cleavage site, or SEQ ID NO: 5 or SEQ ID NO: 7 encoding the CA-SP1 protein (parent polynucleotide sequence). ) Located in the SP1 domain. The polypeptide can be encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9, or GHKARVLVEAMSQV (SEQ ID NO : 2) and a sequence selected from the group consisting of SHKARILAEVMSQV (SEQ ID NO: 3). The polypeptide of the present invention is further encoded by a polynucleotide that hybridizes to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. obtain. The present invention also provides a polypeptide encoded by an isolated polynucleotide that hybridizes to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10 or 12. Which includes a mutation that results in reduced inhibition of p25 to p24 processing by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid, and the mutation is optionally CA- Located at or near SP1 or in the SP1 domain of CA-SP1. The polypeptides of the present invention further include polypeptides that are part of a chimeric or fusion protein. The chimeric protein may be derived from species including but not limited to: primates (including apes and humans); rodents (including rats and mice); cats; cows; goats and sheep Ovine; dog; or pig. The fusion protein may comprise a synthetic peptide sequence, a bifunctional antibody, a protein from the above species or a peptide linked to a linker peptide. The polypeptides of the present invention may comprise a detectable label; a metal compound; a cofactor; a chromatographic separation tag (eg, but not limited to histidine, protein A, etc.), or a linker; a blood stabilizing component (eg, transferrin) But is not limited to these); and can be further linked to therapeutic agents and the like.

The present invention also includes a mutation in the CA-SP1 protein that results in a decrease in the processing of p25 (CA-SP1) to p24 (CA) by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. Including antibodies that selectively bind amino acids, wherein the mutation is optionally located in or near CA-SP1 or in the SP1 domain of CA-SP1. The invention also includes an antibody that selectively binds a polypeptide having a mutation comprising a sequence that is one of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3). The antibody can selectively bind a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. . The antibody also hybridizes under highly stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. The polypeptide encoded by the polynucleotide to be selectively bound. The present invention also includes antibodies that selectively bind SP1, which makes it possible to distinguish SP1 from CA-SP1 (p25). The present invention also includes antibodies that selectively bind CA (p24), which makes it possible to distinguish CA from CA-SP1. The present invention also includes antibodies that selectively bind CA-SP1, which makes it possible to distinguish CA from CA-SP1. The invention further includes antibodies that selectively bind to or near the CA-SP1 cleavage site. The antibodies of the invention can be polyclonal antibodies, monoclonal antibodies, or the antibodies can be chimeric or bifunctional, or part of a fusion protein. The invention further includes a portion of an antibody of the invention, including a single chain, light chain, heavy chain, CDR, F (ab ′) ₂ , Fab, Fab ′, Fv, sFv, or dsFv, or Any combination of these is included.

  As used herein, an antibody “selectively binds” if it binds to the target peptide and does not significantly bind to unrelated proteins. The term “selectively binds” also includes determining whether an antibody selectively binds to a target variant sequence as compared to a native target sequence. An antibody that “selectively binds” to a target sequence, as used herein, is equivalent to an antibody that is “specific for” the target peptide. An antibody is still selective for a peptide so long as such protein has homology to the peptide target fragment or domain of the antibody, even if it binds to other proteins that are not substantially homologous to the target peptide. Is considered to be coupled. In this case, it is understood that antibodies that bind to the peptide are still selective despite some degree of cross-reactivity. In another embodiment, determining whether an antibody selectively binds to a mutant target sequence comprises (a) determining the binding affinity of the antibody for the mutant target sequence and the natural target sequence, and (b) Comparing the binding affinity so determined, the presence of an affinity of the variant target sequence higher than the natural affinity indicates that the antibody selectively binds to the variant target sequence.

  The present invention further extends to antibodies immobilized on an insoluble carrier comprising any of the antibodies disclosed herein. Antibodies immobilized on an insoluble carrier include multi-well plates, culture plates, culture tubes, test tubes, beads, spheres, filters, electrophoretic materials, electron microscope slides, membranes, or affinity chromatography media.

  The present invention also includes a labeled antibody, which includes a detectable signal. The labeled antibody of the present invention is labeled with a detectable molecule, which is an enzyme, a fluorescent material, a chemiluminescent material, horseradish peroxidase, alkaline phosphatase, biotin, avidin, a high electron density material, and a radioisotope, or Any combination of these is included.

  The present invention further provides a method for producing a hybridoma comprising the steps of inducing a mammalian B cell that produces a monoclonal antibody that selectively binds an amino acid sequence containing a mutation in the CA-SP1 protein, and a mammalian myeloma cell. The mutations include p25 to p24 processing by hybridomas producing 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid and any of the monoclonal antibodies disclosed herein. Results in a decrease in inhibition. The present invention further includes the steps of growing a hybridoma producing the monoclonal antibody disclosed herein in an appropriate medium and isolating the antibody from the medium, as is well known in the art. Including. The present invention also includes any animal known in the art useful for the production of polyclonal antibodies, including but not limited to mice, rats, hamsters, rabbits, goats, sheep, deer, guinea pigs, or primates. ), Either single or multiple injections of any of the polypeptides of the invention, and the production of polyclonal antibodies comprising recovering the antibodies in the serum produced therefrom. The present invention includes high binding activity or high affinity antibodies produced therefrom. The invention also includes B cells produced from the listed species that are further used in cell fusion procedures for the production of monoclonal antibody producing hybridomas as disclosed herein.

  The invention further includes a kit comprising an antibody or portion thereof as disclosed herein, a container containing the antibody, and instructions for use, the polypeptides herein and instructions for use. As well as kits containing the polynucleotides of the invention and instructions for use, and any combination thereof. These kits include, but are not limited to, nucleic acid detection kits that may or may not use PCR kits and immunoassay kits. Such kits are useful for clinical diagnosis and provide standardized reagents as required in current clinical practice. These kits can either provide information about the presence or absence of the mutation prior to treatment, or monitor patient progression during treatment. The kits of the invention can also be used to provide standardized reagents for use in experimental studies.

Compounds of the invention
In one aspect, the invention is also directed to compounds, methods of using the compounds, methods for identifying compounds, and the like.

  The terms “one”, “one” or “one” (“a” “an” or “one”) as used in the present invention refer to either the singular or plural. Also good. For example, “a compound” includes one or more compounds.

  Compounds useful in the methods of the present invention are presented in US Pat. Nos. 5,679,828 and 6,172,110, respectively, and US Patent Applications Nos. 60 / 443,180 and 10 / 670,797, which are hereby incorporated by reference. Betulinic acid or derivatives of betulin. Further useful compounds include oleanolic acid derivatives disclosed by Zhu et al. (Bioorg. Chem. Lett. 11: 3115-3118 (2001)); Kashiwada et al. (J. Nat. Prod. 61: 1090- 1095 (1998)) and the oleanolic acid and promolic acid derivatives disclosed by Kashiwada et al. (J. Nat. Prod. 63: 1619-1622 (2000)). Acyl ursolic acid derivatives; and 3-alkylamido-3-deoxy-betulinic acid derivatives disclosed by Kashiwada et al. (Chem. Pharm. Bull. 48: 1387-1390 (2000)) (all references are Incorporated herein by reference).

式中、
RはC₂-C₂₀置換型または非置換型のカルボキシアシルであり、
R'は水素またはC₂-C₁₀置換型もしくは非置換型のアルキル基もしくはアリール基である。好ましい化合物は、Rが以下の表4の置換基の1つであり、R'が水素である化合物である。 In certain embodiments, compounds useful in the present invention include dihydrobetulinic acid derivatives having the following general formula I and dihydrobetulinic acid derivatives of formula II, or pharmaceutically acceptable salts thereof: Not limited to:

Where
R is C ₂ -C ₂₀ substituted or unsubstituted carboxy acyl,
R ′ is hydrogen or a C ₂ -C ₁₀ substituted or unsubstituted alkyl or aryl group. Preferred compounds are those in which R is one of the substituents in Table 4 below and R ′ is hydrogen.

式中、
R₁はC₂-C₂₀置換型または非置換型のカルボキシアシル、またはそのエステルであり;
R₂は水素、C(C₆H₅)₃、またはC₂-C₂₀置換型もしくは非置換型のカルボキシアシルであり;かつ
R₃は水素、ハロゲン、アミノ、置換されていてもよいモノアルキルアミノもしくはジアルキルアミノ、または-OR₄であり、ここでR₄は水素、C₁-₄アルカノイル、ベンゾイル、またはC₂-C₂₀置換型もしくは非置換型のカルボキシアシルであり;
ここで破線はC20とC29の間の任意の二重結合を表す。 In other embodiments, useful compounds include betulin and dihydrobetulin derivatives of the following formula III, or pharmaceutically acceptable salts thereof:

Where
R ₁ is a C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl, or ester thereof;
R ₂ is hydrogen, C (C ₆ H ₅ ) ₃ , or C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl; and
R ₃ is hydrogen, halogen, amino, optionally substituted monoalkyl amino or dialkylamino, or a -OR _4, wherein R ₄ is hydrogen, C ₁ - ₄ alkanoyl, benzoyl or C ₂ -C _20, Substituted or unsubstituted carboxyacyl;
Here, the broken line represents an arbitrary double bond between C20 and C29.

Preferred compounds useful in the present invention are those in which R ₁ 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 ₁ , R ₂

  More preferred compounds are 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid, 3-O- (3 ′, 3′-dimethylsuccinyl) dihydrobetulinic acid, 3-O- (3 ′, 3 '-Dimethylsuccinyl) betulin, and 3-O- (3', 3'-dimethylsuccinyl or glutaryl) dihydrobetulin.

  A particularly preferred compound is 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid.

R₁₁=-OR₁₄または-NHR₁₅であり;
R₁₂=COOR₁₇、COO^-A⁺、またはCH₂OR₁₇であり
R₁₃=-H、ハロゲン、アミノ、置換されてもよいモノ-もしくはジ-アルキルアミノ、または-OR₁₆であり;
R₁₄=-H、C₂-C₂₀置換型または非置換型のカルボキシアシルであり;
R₁₅=-H、C₂-C₂₀置換型または非置換型のカルボキシアシルであり;
R₁₆=-H、C₄-C₇アルカノイル、ベンジロイル、またはC₂-C₂₀置換型もしくは非置換型のカルボキシアシルであり;
R₁₇=-H、C(C₆H₅)₃、またはC₂-C₂₀置換型もしくは非置換型のカルボキシアシルであり;
ここで破線はC₂₀とC₂₉の間の任意の二重結合を表し、および
A=Na+、K⁺または他のカチオンであり、

ここで、R₂₁=

である。

In certain embodiments, compounds useful in the present invention are illustrated by formulas IV, V, VI and VII below.

R ₁₁ = -OR ₁₄ or -NHR ₁₅ ;
R ₁₂ = COOR ₁₇ , 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 ₂₀ substituted or unsubstituted carboxyacyl;
R ₁₅ = -H, C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl;
R ₁₆ = -H, C ₄ -C ₇ alkanoyl, benzyloyl, or C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl;
R ₁₇ = -H, C (C ₆ H ₅ ) ₃ , or C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl;
Where the dashed line represents any double bond between C ₂₀ and C ₂₉ , and
A = Na +, K ⁺ or other cation,

Where R ₂₁ =

It is.

The R ₃₈ moiety other than hydrogen is bonded to the R ₃₃ oxygen by a covalent bond to the carbonyl carbon.

Preferred compounds are those in which R ₃₈ is not hydrogen.

In a further embodiment, any of R ₃₈ , R ₄₀ and / or R ₄₁ is methyl.

  In certain embodiments, compounds useful in the methods of the invention also include the methods described in US Provisional Application No. 60 / 559,358, which is hereby incorporated by reference in its entirety. In one aspect, these compounds are described by reference to compounds VIII to XI below.

またはその薬学的に許容される塩またはエステルを有し、
ここでAは下記式の縮合環であり:

ここでAの式においてxおよびyと名付けられた環炭素は、式VIIIにおけるxおよびyと名付けられた環炭素と同じであり;
R₅₁はカルボキシアルカノイルであり、ここで、アルカノイル鎖は、1つまたは複数のヒドロキシもしくはハロによって任意に置換されることができ、または窒素、硫黄、もしくは酸素原子、またはそれらの組み合わせが挿入され得;
R₅₂、R₅₃およびR₅₄は、独立して、水素、メチル、ハロゲン、またはヒドロキシル、カルボニルもしくは-COOR₆₆であり、ここでR₆₆はアルキルまたはカルボキシルアルキルであり、ここで、アルキル鎖は、1つもしくは複数のヒドロキシルもしくはハロによって任意に置換されることができ、または窒素、硫黄、もしくは酸素原子、またはそれらの組み合わせが挿入され得;
R₅₅はカルボキシアルコキシカルボニル、アルコキシカルボニル、アルカノイルオキシメチル、カルボキシアルカノイルオキシメチル、アルコキシメチルまたはカルボキシルアルコキシメチルであり、それらのいずれかは、1つもしくは複数のヒドロキシもしくはハロであり、またはR₅₅はカルボキシルもしくはヒドロキシメチルであり;
R₅₆は水素、メチル、ヒドロキシまたはハロゲンであり;
R₅₇およびR₅₈は、独立して、水素またはC_1-6アルキルであり;
R₅₉はCH₂またはCH₃であり;
R₆₀は水素、ヒドロキシまたはメチルであり;
R₆₁はメチル、メトキシカルボニル、カルボキシアルコキシカルボニル、アルカノイルオキシメチル、アルコキシメチルまたはカルボキシアルコキシメチルであり、これらのいずれかが1つまたは複数のヒドロキシまたはハロによって任意に置換され;
R₆₂は水素またはメチルであり;
R₆₃は水素またはメチルであり;
R₆₄は水素またはヒドロキシであり;
R₆₅は、C12およびC13が単結合を形成する場合には水素であり、またはR₆₅は、C12およびC13が二重結合を形成する場合には存在せず;および
ここで、直線の破線は、C12とC13、またはC20とC29の間の任意の二重結合を表し;
Aが

であるならば、R₅₁は、二重結合がC12とC13の間に存在するときに、グルタリルまたはスクシニルではあり得ず;
Aが(ii)でありかつR₆₁がメチルであるならば、R₅₁はスクシニルではあり得ず;
Aが(iii)でありかつR₅₂、R₅₃およびR₆₃が各々水素であるならば、R₅₁はスクシニルではあり得ないという条件を伴い;ならびに
R₅₂およびR₅₃が両方ともメチルであり、C12とC13の間に二重結合が存在するならば、A(i)は

ではあり得ないという条件を伴う。 In certain embodiments, compounds useful in the present invention have the general formula VIII:

Or having a pharmaceutically acceptable salt or ester thereof,
Where A is a fused ring of the formula:

Where the ring carbons named x and y in the formula of A are the same as the ring carbons named x and y in formula VIII;
R ₅₁ is carboxyalkanoyl, wherein the alkanoyl chain can be optionally substituted with one or more hydroxy or halo, or a nitrogen, sulfur, or oxygen atom, or combinations thereof can be inserted. ;
R ₅₂ , R ₅₃ and R ₅₄ are independently hydrogen, methyl, halogen, or hydroxyl, carbonyl or —COOR ₆₆ , wherein R ₆₆ is alkyl or carboxyalkyl, wherein the alkyl chain is May be optionally substituted by one or more hydroxyl or halo, or nitrogen, sulfur, or oxygen atoms, or combinations thereof may be inserted;
R ₅₅ is carboxy alkoxycarbonyl, alkoxycarbonyl, alkanoyloxy methyl, carboxymethyl alkanoyloxymethyl, alkoxymethyl or carboxyl alkoxymethyl, they either are one or more hydroxy or halo, or R ₅₅ is carboxyl Or is hydroxymethyl;
R ₅₆ is hydrogen, methyl, hydroxy or halogen;
R ₅₇ and R ₅₈ are independently hydrogen or C _1-6 alkyl;
R ₅₉ is CH ₂ or CH ₃ ;
R ₆₀ is hydrogen, hydroxy or methyl;
R ₆₁ is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted with one or more hydroxy or halo;
R ₆₂ is hydrogen or methyl;
R ₆₃ is hydrogen or methyl;
R ₆₄ is hydrogen or hydroxy;
R ₆₅ is hydrogen when C 12 and C 13 form a single bond, or R ₆₅ is absent when C 12 and C 13 form a double bond; and where the straight dashed line is Represents any double bond between C12 and C13, or C20 and C29;
A is

R ₅₁ cannot be glutaryl or succinyl when a double bond is present between C12 and C13;
If A is (ii) and R ₆₁ is methyl, then R ₅₁ cannot be succinyl;
If A is (iii) and R ₅₂ , R ₅₃ and R ₆₃ are each hydrogen, with the condition that R ₅₁ cannot be succinyl; and
If R ₅₂ and R ₅₃ are both methyl and there is a double bond between C12 and C13, then A (i) is

With the condition that it is impossible.

In some embodiments, R ₅₁ is a carboxy (C _2-6 ) alkylcarbonyl group or a carboxy (C _2-6 ) alkoxy (C _1-6 ) alkylcarbonyl group. Suitable groups are selected from the group consisting of:

ここで、R₅₁、R₅₄、R₅₅、R₅₆、R₅₇、R₅₈およびR₆₄は、式VIIIについて上記に定義したのと同様である。1つの態様において、R₅₆はβ-メチルであり、R₅₈は水素であり、R₅₅はヒドロキシメチルであり、およびR₅₁は3',3'-ジメチルグルタリル、3',3'-ジメチルスクシニル、グルタリル、またはスクシニルである。別の態様では、R₅₆は水素であり、R₅₇およびR₅₈は共にメチルであり、R₅₅はカルボキシルであり、およびR₅₁は3'3-ジメチルグルタリル、3'3-ジメチルスクシニル、グルタリル、またはスクシニルである。 According to the present invention, in certain embodiments, the compound has the following formula IX:

Here, R ₅₁ , R ₅₄ , R ₅₅ , R ₅₆ , R ₅₇ , R ₅₈ and R ₆₄ are as defined above for formula VIII. In one embodiment, R ₅₆ is β-methyl, R ₅₈ is hydrogen, R ₅₅ is hydroxymethyl, and R ₅₁ is 3 ′, 3′-dimethylglutaryl, 3 ′, 3′-dimethyl. Succinyl, glutaryl, or succinyl. In another embodiment, R ₅₆ is hydrogen, R ₅₇ and R ₅₈ are both methyl, R ₅₅ is carboxyl, and R ₅₁ is 3′3-dimethylglutaryl, 3′3-dimethylsuccinyl, glutaryl. Or succinyl.

In certain embodiments, R ₅₅ is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which may be substituted with one or more hydroxy, Or R ₅₅ is carboxyl or hydroxymethyl. In certain embodiments, R ₅₅ is carboxyl, hydroxymethyl, -C0 ₂ (CH ₂ ) _n COOH, -C0 ₂ (CH ₂ ) _n CH ₃ , -CH ₂ OC (O) (CH ₂ ) _n CH ₃ ,- It is selected from the group consisting of CH ₂ OC (O) (CH ₂ ) _n COOH, —CO (CH ₂ ) _n CH ₃ and —CO (CH ₂ ) _n COOH. In certain embodiments, R ₅₅ is selected from the group consisting of:

In certain embodiments, R ₅₅ is hydroxymethyl. In certain embodiments, R ₅₅ is carboxyl. In some embodiments, n is 0-20, and preferably 0-6. In some embodiments, n is 1-10. In some embodiments, n is 2-8. In some embodiments, n is 1-6. In some embodiments, n is 2-6.

ここで、R₅₁、R₅₉、R₆₀、およびR₆₁は、式VIIIについて上記に定義したのと同様である。1つの態様において、R₅₁は3',3'-ジメチルグルタリル、3',3'-ジメチルスクシニル、グルタリル、またはスクシニルである。 In certain embodiments, the compounds useful in the present invention have the following formula X:

Here, R ₅₁ , R ₅₉ , R ₆₀ , and R ₆₁ are the same as defined above for formula VIII. In one embodiment, R ₅₁ is 3 ′, 3′-dimethylglutaryl, 3 ′, 3′-dimethylsuccinyl, glutaryl, or succinyl.

In certain embodiments, R ₆₁ is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which may be substituted with one or more hydroxy or halo. In certain embodiments, R ₆₁ is methyl, -C0 ₂ (CH ₂ ) _n COOH, -COC (O) (CH ₂ ) _n CH ₃ , -CO (CH ₂ ) _n CH ₃ and -CO (CH ₂ ) _n Selected from the group consisting of COOH.

In some embodiments, n is 0-20, and preferably 0-6. In some embodiments, n is 1-10. In some embodiments, n is 2-8. In some embodiments, n is 1-6. In some embodiments, n is 2-6. In certain embodiments, R ₆₁ is methyl. In certain embodiments, R ₆₁ is methoxycarbonyl. In certain embodiments, R ₆₁ is selected from the group consisting of methoxymethyl and ethoxymethyl. In certain embodiments, the methyl group found in R ₆₁ can be substituted with halogen or hydroxy.

ここで、R₅₁、R₅₂、R₅₃、R₅₄、およびR₆₃は、式VIIIについて上記に定義したのと同様である。1つの態様において、R₅₁は3',3'-ジメチルグルタリル、3',3'-ジメチルスクシニル、グルタリル、またはスクシニルである。1つの態様において、R₅₂とR₅₃の両方がメチルである。 In certain embodiments, the compounds useful in the present invention have the following formula XI:

Here, R ₅₁ , R ₅₂ , R ₅₃ , R ₅₄ , and R ₆₃ are as defined above for Formula VIII. In one embodiment, R ₅₁ is 3 ′, 3′-dimethylglutaryl, 3 ′, 3′-dimethylsuccinyl, glutaryl, or succinyl. In one embodiment, both R ₅₂ and R ₅₃ are methyl.

  Any triterpene that falls within the scope of Formula VIII can be used. In accordance with the present invention, in certain embodiments, the compound of Formula VIII comprises: ubaol, ursolic acid, erythrodiol, oleanolic acid, sumaresinolic acid, lupeol, dihydrolupeol, methyl betulinate, dihydrobetulin It is selected from the group consisting of acid methyl esters, 17-α-methylandrostanediol, androstanediol, and derivatives of 4,4-dimethylandrostanediol.

である。 In certain embodiments, the compounds of the invention are defined in formula VIII, wherein R ₅₂ and R ₅₃ are both methyl. In certain embodiments, the compounds of the present invention are defined as in Formula VIII, wherein R ₅₁ is 3 ′, 3′-dimethylsuccinyl. In certain embodiments, the compounds of the invention are defined in Formula VIII, wherein R ₅₁ is succinyl, ie,

It is.

According to the present invention, in some embodiments, the stereochemistry of the side chain substituents is important. In certain embodiments, the compounds of the present invention are defined in formula VIII, wherein A is (i) and R ₅₅ is in the β-position. In certain embodiments, the compounds of the present invention are defined in Formula VIII, A is (i), and R ₅₆ is in the β position. In certain embodiments, the compounds of the present invention are defined in Formula VIII, A is (i), and R ₆₄ is in the α position. In certain embodiments, the compounds of the invention are defined in Formula VIII, A is (i), R ₅₇ is α-methyl, and R ₅₈ is hydrogen. In certain embodiments, the compounds of the invention are defined in Formula VIII, A is (i), R ₅₈ is α-methyl, and R ₅₇ is hydrogen. In certain embodiments, the compounds of the present invention are defined in Formula VIII, A is (i), and R ₅₇ and R ₅₈ are both methyl. In certain embodiments, the compounds of the present invention are defined in Formula VIII, A is (ii), and R ₆₁ is in the β position.

  In some embodiments, 3 ′, 3′-dimethylsuccinyl is in the C3 position. In some embodiments, the compound of Formula IX is 3-O- (3 ′, 3′-dimethylsuccinyl) ubaol; 3-O- (3 ′, 3′-dimethylsuccinyl) erythrodiol; 3-O- (3 ′ 3,3'-dimethylsuccinyl) echinocystic acid or 3-O- (3 ', 3'-dimethylsuccinyl) sumarecinoleic acid. In some embodiments, the compound of formula X is 3-O- (3 ′, 3′-dimethylsuccinyl) lupeol; 3-O- (3 ′, 3′-dimethylsuccinyl) dihydrolpeol; 3-O- (3 ', 3'-dimethylsuccinyl) 17β-methyl ester betulinic acid; or 3-O- (3', 3'-dimethylsuccinyl) 17β-methyl ester dihydrobetulinic acid. In some embodiments, the compound of formula XI is 3-O- (3 ′, 3′-dimethylsuccinyl) 4,4-dimethylandrostanediol; 3-O- (3 ′, 3′-dimethylsuccinyl) 17α-methyl. Androstanediol; 3-O- (3 ′, 3′-dimethylsuccinyl) androstanediol.

ここでR₇₂は以下のうちの1つであり:

ここで、
Zはヒドロキシまたはハロゲンであり;および
R₇₃は、メチル、エチルまたはプロピルなどの低級アルキルである。 In further embodiments, the present invention includes compounds and methods using compounds of formula XII:

Where R ₇₂ is one of the following:

here,
Z is hydroxy or halogen; and
R ₇₃ is lower alkyl such as methyl, ethyl or propyl.

ここで、
R₅₁は、C₃-C₂₀アルカノイル、カルボキシアルカノイル、カルボキシアルケノイル、アルコキシカルボニルアルカノイル、アルケニルオキシカルボニルアルカノイル、シアノアルカノイル、ヒドロキシアルカノイル、アミノカルボニルアルカノイル、ヒドロキシアミノカルボニルアルカノイル、モノアルキルアミノカルボニルアルカノイル、ジアルキルアミノカルボニルアルカノイル、ヘテロアリールアルカノイル、ヘテロシクリルアルカノイル、ヘテロシクリルカルボニルアルカノイル、ヘテロアリールアミノカルボニルアルカノイル、シアノアミノカルボニルアルカノイル、アルキルスルホニルアミノカルボニルアルカノイル、アリールスルホニルアミノカルボニルアルカノイル、スルホアミノカルボニルアルカノイル、ホスホノアミノカルボニルアルカノイル、テトラゾリルアルカノイル、ホスホノ、スルホ、ホスホノアルカノイル、スルホアルカノイル、アルキルスルホニルアルカノイル、もしくはアルキルホスホノアルカノイルであり;
R₈₂は、ホルミル、カルボキシアルケニル、ヘテロシクリル、ヘテロアリール、-CH₂SR₉₄、

であり; In a further embodiment, the compound useful in the present invention is a betulin derivative compound of formula XIII, or a pharmaceutically acceptable salt thereof, or a prodrug thereof:

here,
R ₅₁ is C ₃ -C ₂₀ alkanoyl, carboxyalkanoyl, carboxyalkenoyl, alkoxycarbonylalkanoyl, alkenyloxycarbonylalkanoyl, cyanoalkanoyl, hydroxyalkanoyl, aminocarbonylalkanoyl, hydroxyaminocarbonylalkanoyl, monoalkylaminocarbonylalkanoyl, dialkylamino Carbonylalkanoyl, heteroarylalkanoyl, heterocyclylalkanoyl, heterocyclylcarbonylalkanoyl, heteroarylaminocarbonylalkanoyl, cyanoaminocarbonylalkanoyl, alkylsulfonylaminocarbonylalkanoyl, arylsulfonylaminocarbonylalkanoyl, sulfoaminocarbonylalkanoyl, phosphonoaminocarbonyl Rubonylalkanoyl, tetrazolylalkanoyl, phosphono, sulfo, phosphonoalkanoyl, sulfoalkanoyl, alkylsulfonylalkanoyl, or alkylphosphonoalkanoyl;
R ₈₂ is formyl, carboxyalkenyl, heterocyclyl, heteroaryl, —CH ₂ SR ₉₄ ,

Is;

であり、
ここで、Yは-SR₁₁₁もしくは-NR₁₁₃R₁₁₄であり;
R₁₁₁はメチルであり;
R₁₁₂は水素もしくはヒドロキシルであり;
R₁₁₃およびR₁₁₄は、独立して、水素、アルキル、アルカノイル、アリールアルキル、ヘテロアリールアルキル、アリールスルホニルもしくはアリールカルボニルであり;または
R₁₁₃およびR₁₁₄は、それらが結合してヘテロ環を形成する窒素と一緒になることができ、ここで、ヘテロ環は、1つもしくは複数のさらなる窒素、硫黄もしくは酸素原子を任意に含むことができ;
mは0〜3であり;
R₈₄は水素であり;または
R₈₃およびR₈₄は、一緒になって、オキソ、アルキルイミノ、アルコキシイミノもしくはベンジルオキシイミノを形成することができ;
R₈₅は、C₂-C₂₀アルキル、アルケニル、C₂-C₂₀カルボキシアルキル、アミノ、アミノアルキル、モノアルキルアミノアルキル。ジアルキルアミノアルキル、アルコキシアルキル、シアノ、シアノアルキル、アルキルチオアルキル、シクロアルキル、シクロアルキルアルキル、アリール、アリールアルキル、ヘテロシクリル、ヘテロアリール、ヘテロシクリルアルキル、ヘテロアリルアルキル、スルホ、ホスホノ、スルホアルキル、ホスホノアルキル、アルキルスルホニル、アルキルホスホノ、アルカノイルアミノアルキル、アミノカルボニルアルキル、アルキルアミノカルボニルアルキル、ジアルキルアミノカルボニルアルキル、ヘテロシクリルカルボニルアルキル、シクロアルキルカルボニルアルキル、ヘテロアリールアルキルアミノカルボニルアルキル、アリールアルキルアミノカルボニルアルキル、ヘテロシクリルアルキルアミノカルボニルアルキル、カルボニルアルキルアミノカルボニルアルキル、アリールスルホニルアミノカルボニルアルキル、アルキルスルホニルアミノカルボニルアルキル、アリールホスホノアミノカルボニルアルキル、アルキルホスホノアミノカルボニルアルキル、もしくはヒドロキシイミノ(アミノ)アルキルであり;
R₈₆は、水素、ホスホノ、スルホ、シアノ、アルキル、スルホアルキル、ホスホノアルキル、アルキルスルホニル、アルキルホスホノ、シクロアルキル、ヘテロシクリル、アリール、ヘテロアリール、カルボキシアルキル、アルコキシカルボニルアルキル、もしくはシアノアルキルであり;
R₈₇もしくはR₈₈は、独立して、水素、アルキル、アミノアルキル、モノアルキルアミノアルキル、ジアルキルアミノアルキル、カルボキシアルキル、アルコキシアルキル、アルコキシアルコキシアルキル、アルコキシカルボニルアミノアルコキシアルキル、アルコキシカルボニルアミノアルキル、アミノアルコキシアルキル、アルキルカルボニルアミノアルキル、ヘテロシクリル、ヘテロシクリルアルキル、アリール、アリールアルキル、アリールカルボニルアミノアルキル、もしくはシクロアルキルであり、またはR₈₇およびR₈₈は、それらが結合する窒素原子と一緒になって、ヘテロシクリルもしくはヘテロアリール基を形成することができ、ここで、ヘテロシクリルまたあはヘテロアリールは、1つもしくは複数のさらなる窒素、硫黄もしくは酸素原子を任意に含むことができ;
R₈₉は、水素、ホスホノ、スルホ、シアノ、アルキル、アルキルシリル、シアノアルキル、カルボキシアルキル、アルコキシカルボニルオキシアルキル、アミノアルキル、モノアルキルアミノアルキル、ジアルキルアミノアルキル、シアノアルキル、ホスホノアルキル、スルホアルキル、アルキルスルホニル、アルキルホスホノ、アリール、ヘテロアリール、ヘテロシクリル、アリールアルキル、ヘテロアリールアルキル、ヘテロシクリルアルキル、もしくはジアルコキシアルキルであり;
R₉₀およびR₉₁は、独立して、水素、ヒドロキシル、シアノ、アルキル、アミノ、アミノアルキル、モノアルキルアミノアルキル、ジアルキルアミノアルキル、カルボキシル、カルボキシアルキル、アルカノイルオキシアルキル、アルコキシアルキル、アルコキシアルコキシアルキル、アルコキシカルボニルアミノアルコキシアルキル、アルコキシカルボニルアミノアルキル、アミノアルコキシアルキル、アルキルカルボニルアミノアルキル、ヘテロシクリル、ヘテロシクリルアルキル、アリール、アリールアルキル、アリールカルボニルアミノアルキル、アリールスルホニル、もしくはシクロアルキル、または1つもしくは複数の酸素原子が挿入されたアルキルであり、あるいはR₉₀およびR₉₁は、それらが結合する窒素原子と一緒になってヘテロシクリル基を形成することができ、ここで、このヘテロシクリルは、1つもしくは複数のさらなる窒素、硫黄、もしくは酸素原子を任意に含むことができ;
R₉₂およびR₉₃は、独立して、水素、アルキル、アルコキシカルボニル、アルコキシアミノアルキル、シクロアリールオキシ、ヘテロシクリルアミノアルキル、シクロアルキル、シアノアルキル、シアノ、スルホ、ホスホノ、スルホアルキル、ホスホノアルキル、アルキルスルホニル、アルキルホスホノ、アルコキシアルキル、ヘテロシクリルアルキルであり、またはR₉₂およびR₉₃は、それらが結合する窒素原子と一緒になってヘテロシクリル基を形成することができ、ここで、このヘテロシクリルは、1つもしくは複数のさらなる窒素、硫黄もしくは酸素原子を任意に含むことができ、またはR₉₂およびR₉₃は、それらが結合する窒素原子と一緒になってアルキルアゾ基を形成することができ、ならびにbは1〜6であり;
R₉₄は、水素、アルキル、アルケニル、アリールアルキル、カルボキシアルキル、カルボキシアルケニル、アルコキシカルボニルアルキル、アルケニルオキシカルボニルアルキル、シアノアルキル、ヒドロキシアルキル、カルボキシベンジル、アミノカルボニルアルキルであり;
R₉₅およびR₉₆は、独立して、水素、アルキル、アルコキシカルボニル、アルコキシアミノアルキル、シクロアルキルオキシ、ヘテロシクリルアミノアルキル、シクロアルキル、シアノアルキル、シアノ、スルホ、ホスホノ、スルホアルキル、ホスホノアルキル、アルキルスルホニル、アルキルホスホノ、アルコキシアルキル、ヘテロシクリルアルキルであり、またはR₉₅およびR₉₆は、それらが結合する窒素原子と一緒になってヘテロシクリル基を形成することができ、ここで、このヘテロシクリルは、1つもしくは複数のさらなる窒素、硫黄もしくは酸素原子を任意に含むことができ、またはR₉₅およびR₉₆は、それらが結合する窒素原子と一緒になってアルキルアゾ基を形成することができ;
R₉₇は、水素、アルキル、アルケニル、カルボキシアルキル、アミノ、アミノアルキル、モノアルキルアミノアルキル、ジアルキルアミノアルキル、アルコキシアルキル、アルコキシカルボニル、シアノアルキル、アルキルチオアルキル、シクロアルキル、シクロアルキルアルキル、アリール、アリールアルキル、ヘテロシクリル、ヘテロアリール、ヘテロシクリルアルキル、ヘテロシクリルアルキル、ヘテロアリールアルキル、アルカノイルアミノアルキル、アミノカルボニルアルキル、アルキルアミノカルボニルアルキル、ジアルキルアミノカルボニルアルキル、ヘテロシクリルカルボニルアルキル、シクロアルキルカルボニルアルキル、ヘテロアリールアルキルアミノカルボニルアルキル、アリールアルキルアミノカルボニルアルキル、ヘテロシクリルアルキルアミノカルボニルアルキル、カルボキシアルキルアミノカルボニルアルキル、アリールスルホニルアミノカルボニルアルキル、アルキルスルホニルアミノカルボニルアルキル、もしくはヒドロキシイミノ(アミノ)アルキルであり;
R₉₈およびR₉₉は、独立して、水素、メチルもしくはエチルであり、好ましくは水素もしくはメチルであり;ならびにdは1〜6である。 R ₈₃ is hydroxyl, isopropenyl, isopropyl, 1′-hydroxyisopropyl, 1′-haloisopropyl, 1′-thioisopropyl, 1′-trifluoromethylisopropyl, 2′-hydroxyisopropyl, 2′-haloisopropyl, 2 '-Thioisopropyl, 2'-trifluoromethylisopropyl, 1'-hydroxyethyl, 1'-(alkoxy) ethyl, 1 '-(alkoxyalkoxy) ethyl, 1'-(arylalkoxy) ethyl; 1 '-(aryl Carbonyloxy) ethyl, 1 '-(oxo) ethyl, 1'-(hydroxyl) -1 '-(hydroxyalkyl) ethyl, 1' (oxo) oxazolidinyl, 1 ', 2'-epoxyisopropyl, 2'-haloiso Propenyl, 2'-hydroxyisopropenyl, 2'-aminoisopropenyl, or

And
Where Y is -SR ₁₁₁ or -NR ₁₁₃ R ₁₁₄ ;
R ₁₁₁ is methyl;
R ₁₁₂ is hydrogen or hydroxyl;
R ₁₁₃ and R ₁₁₄ are independently hydrogen, alkyl, alkanoyl, arylalkyl, heteroarylalkyl, arylsulfonyl or arylcarbonyl; or
R ₁₁₃ and R ₁₁₄ can be taken together with the nitrogen they combine to form a heterocycle, where the heterocycle optionally contains one or more additional nitrogen, sulfur or oxygen atoms. Can;
m is 0-3;
R ₈₄ is hydrogen; or
R ₈₃ and R ₈₄ can be taken together to form oxo, alkylimino, alkoxyimino or benzyloxyimino;
R ₈₅ is C ₂ -C ₂₀ alkyl, alkenyl, C ₂ -C ₂₀ carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl. Dialkylaminoalkyl, 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, heterocyclylalkylamino Carbonyl alkyl, carboni Alkylaminocarbonylalkyl, arylsulfonylaminocarbonyl alkyl, alkylsulfonyl aminocarbonyl alkyl, aryl phosphono aminocarbonylalkyl, alkylphosphono aminocarbonyl alkyl, or be a hydroxyimino (amino) alkyl;
R ₈₆ is hydrogen, phosphono, sulfo, cyano, alkyl, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyalkyl, alkoxycarbonylalkyl, or cyanoalkyl. ;
R ₈₇ or R ₈₈ is independently hydrogen, alkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl, alkoxycarbonylaminoalkoxyalkyl, alkoxycarbonylaminoalkyl, aminoalkoxy Alkyl, alkylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, or cycloalkyl, or R ₈₇ and R ₈₈ together with the nitrogen atom to which they are attached, heterocyclyl or A heteroaryl group can be formed, wherein heterocyclyl or heteroaryl is one or more additional nitrogen, sulfur, or It can contain an oxygen atom optionally;
R ₈₉ is hydrogen, phosphono, sulfo, cyano, alkyl, alkylsilyl, cyanoalkyl, carboxyalkyl, alkoxycarbonyloxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, cyanoalkyl, phosphonoalkyl, sulfoalkyl, Alkylsulfonyl, alkylphosphono, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, or dialkoxyalkyl;
R ₉₀ and R ₉₁ are independently hydrogen, hydroxyl, cyano, alkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyl, carboxyalkyl, alkanoyloxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl, alkoxy Carbonylaminoalkoxyalkyl, alkoxycarbonylaminoalkyl, aminoalkoxyalkyl, alkylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, arylsulfonyl, or cycloalkyl, or one or more oxygen atoms the insertion alkyl, or R ₉₀ and R _91, together with the nitrogen atom to which they are attached het It can form a cyclyl group in which the heterocyclyl, one or more further nitrogen, can contain sulfur, or oxygen atom optionally;
R ₉₂ and R ₉₃ are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloaryloxy, heterocyclylaminoalkyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkyl Sulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R ₉₂ and R ₉₃ together with the nitrogen atom to which they are attached can form a heterocyclyl group, wherein the heterocyclyl is 1 Can optionally contain one or more additional nitrogen, sulfur or oxygen atoms, or R ₉₂ and R ₉₃ together with the nitrogen atom to which they are attached can form an alkylazo group, and b is 1-6;
R ₉₄ is hydrogen, alkyl, alkenyl, arylalkyl, carboxyalkyl, carboxyalkenyl, alkoxycarbonylalkyl, alkenyloxycarbonylalkyl, cyanoalkyl, hydroxyalkyl, carboxybenzyl, aminocarbonylalkyl;
R ₉₅ and R ₉₆ are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloalkyloxy, heterocyclylaminoalkyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkyl Sulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R ₉₅ and R ₉₆ together with the nitrogen atom to which they are attached can form a heterocyclyl group, wherein the heterocyclyl is 1 One or more additional nitrogen, sulfur or oxygen atoms can optionally be included, or R ₉₅ and R ₉₆ can be taken together with the nitrogen atom to which they are attached to form an alkylazo group;
R ₉₇ is hydrogen, alkyl, alkenyl, carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, cyanoalkyl, alkylthioalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl , Heterocyclyl, heteroaryl, heterocyclylalkyl, heterocyclylalkyl, heteroarylalkyl, alkanoylaminoalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, heterocyclylcarbonylalkyl, cycloalkylcarbonylalkyl, heteroarylalkylaminocarbonylalkyl, Arylalkylaminocarbonylalkyl, Heterocyclylalkylaminocarbonylalkyl, carboxyalkylaminocarbonylalkyl, arylsulfonylaminocarbonylalkyl, alkylsulfonylaminocarbonylalkyl, or hydroxyimino (amino) alkyl;
R ₉₈ and R ₉₉ are independently hydrogen, methyl or ethyl, preferably hydrogen or methyl; and d is 1-6.

The alkyl groups and alkyl-containing groups of the compounds of the present invention can be linear or branched alkyl groups and preferably have 1 to 10 carbon atoms. In certain embodiments, the alkyl and alkyl-containing groups of the compounds of the invention can be substituted with a C _3-7 cycloalkyl group. In certain embodiments, the cycloalkyl group may include, but is not limited to, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group.

  Non-toxic pharmaceutically acceptable salts of the compounds of the present invention are also included within the scope of the present invention. These salts are formed in situ during final isolation and purification of the compound, and thus by reacting the free acid form of the purified compound separately with a suitable organic or inorganic base, and thus Can be prepared by isolating the salt. These include cations based on alkali metals and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, N- Non-toxic ammonium, quaternary ammonium and amine cations can be included, including but not limited to methylglucamine and the like.

Non-toxic pharmaceutically acceptable esters of the compounds of the present invention are also included within the scope of the present invention. The ester group is preferably of the type that is relatively easily hydrolyzed under physiological conditions. Examples of pharmaceutically acceptable esters of the compounds of the present invention include C _1-6 alkyl esters wherein the alkyl group is straight or branched. 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 preferred. In certain embodiments, the ester is selected from the group consisting of alkyl carboxylic acid esters, such as acetate esters, and mono- or dialkyl phosphate esters, such as methyl phosphate esters or dimethyl phosphate esters. Esters of the compounds of the present invention can be prepared according to conventional methods.

  Certain compounds are the derivatives listed above called “prodrugs”. This includes, for example, compounds that fall within the scope of formulas VIII to XI. The expression “prodrug” refers to compounds that are rapidly transformed in vivo by enzymatic or chemical processes, for example by hydrolysis in blood, to give the parent compound of the above formula. A thorough discussion is given by Higuchi, T. and V. Stella in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association, Given by Pergamon Press, 1987. Useful prodrugs can be, for example, esters of compounds of formula VIII, IX, X, and XI. In certain prodrug embodiments, the lower alkyl group is substituted with one or more hydroxyl or halo groups with a suitable acid. Suitable acids include, for example, carboxylic acids, sulfonic acids, phosphoric acids or lower alkyl esters thereof; and phosphonic acids or lower alkyl esters thereof. For example, suitable carboxylic acids include alkyl carboxylic acids such as acetic acid, aryl carboxylic acids and arylalkyl carboxylic acids. Suitable sulfonic acids include alkyl sulfonic acids, aryl sulfonic acids and arylalkyl sulfonic acids. Suitable phosphoric acids and phosphate esters are methyl or ethyl esters.

  In certain embodiments, a C3 acyl group having a dimethyl group or oxygen at the C3 ′ position may be the most active compound. This observation suggests that these types of acyl groups may be important for enhanced anti-HIV activity.

  In one embodiment, the present invention inhibits the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), a compound of formulas I to XIII, but has a significant effect on other Gag processing steps Extends to a method of treating HIV-1 infection in a patient by administering a compound that does not give.

  In one embodiment, the present invention inhibits the processing of viral Gag p25 protein (CA-SP1) to p24 (CA), but other Gag processing, which are compounds of formulas I to XIII with the exception of DSB. It extends to a method of treating HIV-1 infection in a patient by administering a compound that does not significantly affect the stage.

  In one embodiment, the present invention inhibits viral Gag p25 protein (CA-SP1) to p24 (CA) processing that is not a compound of formula I to VII or a compound of formula I to XIII Extends to methods of treating HIV-1 infection in patients by administering compounds that do not significantly affect other Gag processing steps. In one embodiment, the invention relates to a viral Gag p25 protein (CA-SP1) to p24 (CA) that is not a compound of formula I to XI, or in another embodiment is not a compound of formula I to XIII. It extends to methods of treating HIV-1 infection in patients by administering compounds that inhibit processing but do not significantly affect other Gag processing steps.

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 () in a cell without significantly affecting other Gag processing steps, wherein the compound is a compound of formula I to XIII. Extends to methods of inhibiting processing to CA).

  In another embodiment, the invention provides that the compound is not a compound of formula I to VII, or in other embodiments is not a compound of formula I to XIII, without significantly affecting other Gag processing steps, It extends to methods of inhibiting the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) in cells.

  In another embodiment, the invention provides that the compound is not a compound of formula I to XI, or in other embodiments is not a compound of formula I to XIII, without significantly affecting other Gag processing steps, It extends to methods of inhibiting the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) in cells.

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 () in a cell without significantly affecting other Gag processing steps, wherein the compound is a compound of formula I to XIII. It extends to methods of processing human blood or blood products by inhibiting processing to CA).

  In another embodiment, the invention provides that the compound is not a compound of formula I to VII, or in other embodiments is not a compound of formula I to XIII, without significantly affecting other Gag processing steps, It extends to methods of treating human blood or blood products by inhibiting the processing of viral Gag p25 protein (CA-SP1) to p24 (CA) in cells.

Synthesis of Dsb and related compounds
The reaction of betulinic acid and dihydrobetulinic acid with dimethyl succinic anhydride was performed using 3-O- (2 ', 2'-dimethylsuccinyl) and 3-O- (2', 2'-dimethylsuccinyl) -betulin respectively. A mixture of acid and dihydrobetulinic acid was produced. The mixture was successfully separated by preparative scale HPLC producing a pure sample. The structures of these isomers were given by the long range ¹ H- ¹³ C COZY test.

The derivatives of betulinic acid and dihydrobetulinic acid according to the present invention can be prepared by mixing betulinic acid or dihydrobetulinic acid, dimethylaminopyridine (1 equimolar), and a suitable anhydride (2.5-10 All equimolar solutions were synthesized by refluxing. The reaction mixture was then diluted with ice water and extracted with CHCl ₃ . The organic layer was washed with water, dried over MgSO ₄ and concentrated under reduced pressure. The residue was subjected to chromatography using a silica gel column or semi-preparative scale HPLC to collect the product.

Preparation of 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid:
Yield 70% (starting with 542 mg betulinic acid); crystallization from MeOH gave colorless needles.

3-O- (3 ', 3'-dimethylsuccinyl) dihydrobetulinic acid:
24.5 percent yield (starting at dihydro betulin acid 155.9mg); crystallized from MeOH-H ₂ O gave colorless needles.

  The synthesis of 3-O- (3 ′, 3′-dimethylglutaryl) betulinic acid was disclosed in US Pat. No. 5,679,828 as COMPOUND No. 4.

3-O- (3 ', 3'-dimethylglutaryl) dihydrobetulinic acid:
93.3% yield (starting at dihydro betulin acid 100.5 mg); crystallized from MeOH-H ₂ O gave colorless needles.

  The synthesis of 3-O-diglycolyl-betulinic acid was disclosed as COMPOUND No. 5 in US Pat. No. 5,679,828.

3-O-Diglycolyl-dihydrobetulinic acid:
Yield 79.2% (starting with 103.5 mg of dihydrobetulinic acid); off-white amorphous powder.

  The synthesis of 3-O- (3 ′, 3′-dimethylsuccinyl) betulin and 3-O- (3 ′, 3′-dimethylglutaryl) betulin was disclosed in US patent application Ser. No. 10 / 670,797.

Methods of Inhibiting HIV with Compounds The methods of “inhibiting HIV” or “inhibiting HIV,” as used herein, use the methods of the present invention to provide any interference, inhibition, and inhibition of HIV, and Means prevention. As such, methods of inhibition are useful in inhibiting HIV infectivity, inhibiting p25 processing, inhibiting viral maturation, forming virions that exhibit altered phenotypes, and the like. Preferably, the method of the invention acts on p25 processing in animal cells, but is not limited by the method of action.

  The method of inhibiting HIV using a compound may be related to the method of HIV infection in the patient. Therefore, methods of inhibiting HIV using compounds can be used to treat patients as well.

  A method of inhibiting HIV-1 replication in an animal cell comprises contacting the infected cell with a compound of formula I to XIII above. A related aspect is a method of treating an HIV-1 infection in a patient comprising administration of a compound of formulas I through XIII; either in a cell, in vivo, and / or in vitro by administration of a compound that inhibits p25 processing. And a method of treating human blood or blood products by administering a compound of formulas I to XIII. Also included are methods of identifying compounds that inhibit any one of p25 processing, HIV maturation, HIV infectivity, HIV virion phenotype, and the like.

  In one embodiment, the compound of the invention is betulinic acid, betulin, or dihydrobetulinic acid or dihydrobetulin, and these include the preferred substituents of Table 4. Preferred compounds include, but are not limited to: 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, and 3-O-diglycolyl-dihydrobetulinic acid.

  In one embodiment, the present invention inhibits the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), a compound of formulas I to XIII, but has a significant effect on other Gag processing steps The invention extends to methods of inhibiting HIV-1 replication in animal cells by contacting the infected cells with a compound that does not confer.

  In one embodiment, the present invention inhibits the processing of viral Gag p25 protein (CA-SP1) to p24 (CA), which is a compound of formula I to XIII, with the exception of DSB, but other Gag It extends to methods of inhibiting HIV-1 replication in animal cells by contacting infected cells with compounds that do not significantly affect the processing stage.

  In one embodiment, the present invention inhibits viral Gag p25 protein (CA-SP1) to p24 (CA) processing but excludes other Gag processing steps, excluding compounds of formulas I to VI. The invention extends to methods of inhibiting HIV-1 replication in animal cells by contacting infected cells with compounds that do not significantly affect. In one embodiment, the present invention inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA) other than compounds of formulas I to XIII, but is significant for other Gag processing steps. It extends to methods of inhibiting HIV-1 replication in animal cells by contacting infected cells with non-influenced compounds.

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 () in a cell without significantly affecting other Gag processing steps, wherein the compound is a compound of formula I to XIII. Extends to methods of inhibiting processing to CA).

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 (CA) in cells without significantly affecting other Gag processing steps, wherein the compound is not a compound of formula I to VI. Extends to methods of inhibiting processing to CA).

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 (CA-SP1) in a cell without significantly affecting other Gag processing steps, wherein the compound is not a compound of formula I to XIII. Extends to methods of inhibiting processing to CA).

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 (CA) in cells without significantly affecting other Gag processing steps, wherein the compound is not a compound of formula I to XI. Extends to methods of inhibiting processing to CA). In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) to p24 () in a cell without significantly affecting other Gag processing steps, wherein the compound is a compound of formula I to XIII. It extends to methods of processing human blood or blood products by inhibiting processing to CA).

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) in a cell without significantly affecting other Gag processing steps, wherein the compound is a compound other than compounds of Formulas I through VI. It extends to methods of treating human blood or blood products by inhibiting the processing of p24 (CA).

  In another embodiment, the present invention relates to viral Gag p25 protein (CA-SP1) in cells without significantly affecting other Gag processing steps, wherein the compound is a compound other than compounds of Formulas I through VIII. It extends to methods of treating human blood or blood products by inhibiting the processing of p24 (CA).

  The methods disclosed herein comprise one or more drugs selected from the group consisting of antiviral agents, antifungal agents, antibacterial agents, anticancer agents, immunostimulants, and combinations thereof, and the cells. The method further includes contacting. The method can include processing of a human blood product.

  The present invention can also be used in combination with methods of treating cancer, including administering one or more anti-neoplastic agents to an animal, exposing the animal to a cancer cell killing dose of radiation, or a combination of both.

Methods for Identifying Compounds The present invention further includes methods for identifying compounds that inhibit HIV-1 replication in the cells of an animal disclosed herein. In one embodiment, the method includes the following steps:
(a) contacting a Gag polypeptide comprising a CA-SP1 cleavage site with a test compound;
(b) adding a labeled substance that selectively binds at or near the CA-SP1 cleavage site; and
(c) measuring the binding of the test compound at or near the CA-SP1 cleavage site.

  Labeled materials include labeled antibodies or labeled DSBs, which include enzymes, fluorescent materials, chemiluminescent materials, horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density materials (e.g. , Gold, osmium tetroxide, lead, or uranyl acetate), and radioisotopes, antibodies labeled with such substance molecules, or combinations thereof. Assay methods may include, but are not limited to, ELISA, single and double sandwich techniques, immunodiffusion techniques, or immunoprecipitation techniques, as known in the art (`` Immunoassay Handbook, 2nd Edition '' D. Wild, Nature Publishing Group, (2001)). The method of identification may also include, but is not limited to, Western blot assays, colorimetric assays, light and electron microscopy techniques, confocal microscopy, or other techniques known in the art.

The method of identifying a compound that inhibits HIV replication in an animal cell further comprises the following steps:
(a) contacting a Gag protein comprising a wild-type CA-SP1 cleavage site with HIV-1 protease in the presence of a test compound;
(b) separately contacting a Gag protein comprising a mutant CA-SP1 cleavage site or a protein comprising an alternative protease cleavage site with HIV-1 protease in the presence of a test compound;
(c) A step of comparing the cleavage amount of the natural wild-type Gag protein against the cleavage amount of the mutant Gag protein or the cleavage amount of the protein containing an alternative protease cleavage site.

  Step (b) above is performed as a control to remove compounds that can bind directly and thus inhibit the protease enzyme. The above methods also include methods in which wild type CA-SP1, mutant CA-SP1, or an alternative protease cleavage site is included in a polypeptide fragment or recombinant peptide.

  The methods for identifying compounds that inhibit HIV-1 disclosed herein also include labeling the peptide or protein with a fluorescent moiety and a fluorescent quenching moiety, each on the opposite side of the CA-SP1 cleavage site. The step of binding and detecting here measures the signal from the fluorescent moiety, or the peptide or protein is labeled with two fluorescent moieties, each bound to the opposite side of the CA-SP1 cleavage site and detected here Measuring the transfer of fluorescence energy from one moiety to another in the presence of the test compound and HIV-1 protease, and including or otherwise comprising a sequence containing a mutation in the CA-SP1 cleavage site Comparing the transfer of fluorescence energy observed when the same procedure is applied to a peptide comprising a sequence comprising a cleavage site of Examples of fluorescence-based assays for protease activity are well known in the art. In one such example, the protease substrate is labeled with a green fluorescent dye molecule that fluoresces when the substrate is cleaved by the protease enzyme (Molecular Probes, Protease Assay Kit).

  The method of comparing cleavage above also includes using a labeled antibody that selectively binds to CA or SP1 or CA-SP1 to determine the extent to which the test compound inhibits CA-SP1. The antibody can be labeled with a molecule selected from enzymes, fluorescent materials, chemiluminescent materials, horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density materials, and radioisotopes, or combinations thereof. This method also includes the use of antibodies directed against a specific epitope tag sequence to selectively detect CA-SP1 (p25) or SP1 in which the amino acid sequence for that epitope tag is engineered according to standard methods in the art. Including use. Suitable tags are well known to those skilled in the art and include the hemagglutinin epitope HA (YPYDVPDYA) (SEQ ID NO: 81), bluetongue virus epitope VP7 (QYPALT) (SEQ ID NO: 82), α-tubulin epitope (EEF ), Flag (DYKDDDDK) (SEQ ID NO: 83), and VSV-G (YTDOEMNRLGK) (SEQ ID NO: 84). An example of an SP1-containing epitope tag is illustrated in FIG.

  As an example, a FLAG epitope tag (Sigma-Aldrich) is inserted into the p2 (SP1) region of Gag by oligonucleotide-specific mutagenesis of a Gag expression plasmid. The presence of the SP1 domain in the cell expressed protein is then detected using a commercially available anti-FLAG monoclonal antibody (Sigma-Aldrich) (Hopp, T.P. Biotechnology 6: 1204-1210 (1988)).

  Methods to identify compounds that disrupt CA-SP1 cleavage also add the compound to cells infected with HIV-1 and detect CA-SP1 cleavage products by lysing and analyzing the cells or released virions including. The methods included in the present invention can be performed using Western blot analysis of viral proteins and detection of p25 using an antibody against p25, or wherein the mixture is of gel electrophoresis of viral proteins Performed and analyzed by imaging of metabolically labeled proteins or the mixture uses an immunoassay that selectively binds p25 or uses an antibody that selectively binds to distinguish p25 from p24 And analyzed. The present invention involves the use of antibodies to specific epitope tags inserted into the C-terminal domain of SP1 to selectively detect p25 or SP1. For example, a sandwich ELISA can be performed in which antibodies that selectively bind to the CA region of Gag where p25 and p24 in a virus solubilized by a detergent are bound to a multi-well plate. Captured using. After the washing step, bound p25 is detected using an antibody against the epitope tag inserted in SP1, conjugated to an appropriate detection reagent (e.g., alkaline phosphatase for enzyme-linked immunosorbent assay). . Viruses released by cells treated with compounds that act through this mechanism generally have increased levels of p25 compared to untreated virions.

  The disclosed method extends to an antibody that selectively binds p25, or an antibody that selectively binds SP1, or an antibody against an epitope tag sequence inserted into SP1, which comprises an enzyme, a fluorescent substance, a chemiluminescent substance , Labeled with a molecule selected from the group consisting of horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density material, and radioisotopes, or combinations thereof.

  “Infected cell” as used herein refers to the transfection of a cell naturally infected by membrane fusion and subsequent insertion of the viral genome into the cell, or using viral genetic material through artificial means. Including. These methods include, but are not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, lipid mediated transfection, electroporation, or infection.

  The present invention can be practiced by infecting target cells in vitro using appropriate infectious strains of HIV and in the presence of a test compound for various periods of time. Infected cells or supernatant fluid can be processed by methods well known in the art and loaded onto polyacrylamide gels for detection of virus levels. Uninfected and untreated cells can be used as negative and positive infection controls, respectively. Alternatively, the present invention can be practiced by culturing target cells in the presence of a test compound prior to infection of the cells with an HIV strain.

The invention also includes a method for identifying a compound that inhibits HIV-1 replication in an animal cell, the method comprising the following steps:
(a) contacting the test compound with the wild-type virus isolate and separately with a virus isolate having reduced sensitivity to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid. ; And
(b) A test that is more active against wild-type virus isolates compared to virus isolates with reduced sensitivity to 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid Selecting a compound.

  The present invention further includes a method for identifying a compound that acts by any of the above-described mechanisms, which can be performed by standard methods well known in the art by HIV-1 infected cells or HIV-1 Treating the transfected cells with the compound, and then analyzing the released viral particles by slicing and transmission electron microscopy. If particles are detected that show non-centric spherical aggregated cores with respect to viral particles and / or a semi-moon-like high electron density layer just inside the viral membrane, the compound acts by the mechanism described above.

  For electron microscopy studies, infected cells or centrifuged virus pellets obtained from the supernatant liquid are used in fixatives such as glutaraldehyde, freshly prepared paraformaldehyde, and / or osmium tetroxide, or in the art. It can be contacted with other known electron microscope compatible fixatives. Virus from the supernatant liquid or cells is dehydrated, embedded in a low electron density polymer such as an epoxy resin or methacrylate, sliced using an ultramicrotome, and high in the uranyl acetate and / or lead citrate It was stained using electron density staining and observed with a transmission electron microscope. Uninfected and untreated cells can be used as negative and positive infection controls, respectively. Alternatively, the present invention can be practiced by culturing target cells in the presence of a test compound prior to infection of the cells with an HIV strain. Maturation defects caused by the compounds of the present invention are determined by the presence of morphologically abnormal viral particles compared to controls, as described herein.

  For cell culture studies, virus-infected cells can be observed for syncytium formation or the supernatant can be tested for the presence of HIV particles. Virus present in the supernatant can be collected to infect other untreated cultures to determine infectivity.

  Also included in the present invention is a method for determining whether an individual is infected with HIV-1 and is sensitive to treatment with a compound that inhibits p25 processing, which comprises drawing blood from a patient. Performing genotyping of the viral RNA and determining whether the viral RNA contains a mutation in the CA-SP1 cleavage site.

  The invention also includes a method for identifying compounds that act by the mechanisms described above, the method comprising testing by a combination of the methods disclosed herein.

  HIV Gag proteins and fragments thereof for use in the above-described assays can be expressed or synthesized using various methods well known to those skilled in the art. Gag can be produced in an in vitro transcription and translation system using 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, SC et al. J. Virol. 76: 10226-10233 ( 2002)). Furthermore, Gag expressed by this method can be assembled into immature virions when fused to a heterologous D-type retroviral cytoplasmic self-assembly domain (Sakalian, M. et al., J. Virol 76: 10811-10820 (2002)). Plasmid pDAB72, available from the NIH AIDS Reagent Program, can be used for this purpose (Erickson-Viitanen, S. et al., AIDS Res. Hum. Retroviruses. 5: 577-91 (1989); Sidhu MK 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. HIV Gag can also be expressed in transfected cells using a variety of commercially available expression vectors. The plasmid p55-GAG / GFP, available from the NIH AIDS Reagent Program, can be used to express Gag-green fluorescent protein fusion proteins in mammalian cells in drug interaction studies (Sandefur, S. et al. , J. Virol. 72: 2723-2732 (1998)). This construct allows capture and purification of Gag fusion proteins using GFP specific monoclonal antibodies. In addition, Gag can be expressed in cells using recombinant viral vectors, such as those used in vaccinia virus systems, adenovirus systems, or baculovirus systems. Gag can also be expressed by infecting cells with HIV or by transfecting cells with proviral DNA. Finally, Gag can be expressed in yeast cells or bacterial cells transformed with an appropriate expression vector.

  In addition to Gag proteins expressed in cells or in vitro using cell lysates, peptides corresponding to various regions of Gag are synthesized commercially using standard peptide synthesis techniques. obtain.

  The present invention further includes compounds identified by the methods of the invention, and / or compounds that inhibit HIV-1 replication according to the methods of the invention, and one or more compounds as disclosed herein. A pharmaceutical composition comprising, or a pharmaceutically acceptable salt, ester, or prodrug thereof, and a pharmaceutically acceptable carrier.

Pharmaceutical compositions It has been found that the compounds according to the invention have antiretroviral activity, in particular anti-HIV-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 is easier to formulate the salts of the invention into pharmaceutical preparations. Furthermore, the compounds according to the invention are expected to have improved biodistribution properties.

  In one embodiment, the compounds of the invention are compounds of formulas I to XIII, and in another embodiment they are compounds other than the compounds of formulas I to XIII.

  The present invention also provides compounds that inhibit the processing of viral Gag p25 protein (CA-SP1) to p24 (CA), but have no significant effect on other Gag processing steps, or treated infected cells A pharmaceutical composition comprising a compound that inhibits the maturation of virus particles released from, for example, a compound of formula I to XIII. The present invention provides a pharmaceutical composition comprising one or more compounds disclosed herein, or a pharmaceutically acceptable salt, ester, or prodrug thereof, and a pharmaceutically acceptable carrier. Wherein the compound is a compound of formula I to XIII above, or preferably the compound is 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, and 3-O-diglycolyl-dihydrobetulin Selected from the group consisting of acids. The pharmaceutical composition according to the present invention further comprises one or more drugs selected from antiviral agents, antifungal agents, anticancer agents, or immunostimulants.

  The pharmaceutical composition of the present invention may comprise at least one of the compounds of formulas I to XIII disclosed herein. The pharmaceutical compositions according to the present specification also include, for example, but not limited to, other antiviral agents: AZT (zidovudine, RETROVIR®, GlaxoSmithKline), 3TC (lamivudine, EPIVIR®), GlaxoSmithKline), AZT + 3TC (COMBIVIR (R), GlaxoSmithKline), AZT + 3TC + Abacavir (TRIZIVIR (R), GlaxoSmithKline), ddI (Didanocin, VIDEX (R), Bristol-Myers Squibb), ddC (Zarcibin) HIVID (registered trademark), Hoffmann-La Roche), D4T (stabudine, ZERIT (registered trademark), Bristol-Myers Squibb), Abacavir (ZIAGEN (registered trademark), GlaxoSmithKline), Tenofovir (VIREAD (registered trademark), Gilead Sciences) Nevirapine (VIRAMUNE®, Boehringer Ingelheim), delavirdine (Pfizer), emtricitabine (EMTRIVA®, Gilead Sciences), efavirenz (SUSTIVA®, DuPont Pharmaceuticals), saquinavir (INVIRASE®), FORTOVASE ), Hoffmann-La Roche), Ritonavir (NORVIR®, Abbott Laboratories), Indinavir (CRIXIVAN®, Merck and Company), Nelfinavir (VIRACEPT®, Pfizer), Amprenavir (AGENERASE) (Registered trademark), GlaxoSmithKline), Adefovir (PREVEON®, HEPSERA®, Gilead Sciences), Atazanavir (Bristol-Myers Squibb), Fosamprenavir (LEXIVA®, GlaxoSmithKline) and Hydroxyurea (HYDREA®, Bristol-Meyers Squibb), or any other antiretroviral drug or antibody combined with each other or combined with a biologically-based therapeutic agent, such as gp41-derived peptide enfuvirtide ( FUZEON®, Roche and Trimeris) and T-1249, or soluble CD4, an antibody to CD4, and a CD4 or anti-CD4 conjugate, or as described herein Further presented in.

  Additional suitable antiviral agents for optimal use with one of the compounds of Formulas I through XIII of the present invention may include, but are not limited to: amphotericin B (FUNGIZONE®); Hemispherx Ampligen (mismatch RNA) developed by Biopharma; BETASERON® (β-interferon, Chiron); Butylated hydroxytoluene; Carrosyn (polymannoacetate); Castanospermine (Castanospermine); Contracan (stearic acid derivative); Creme Pharmatex (containing benzalkonium); 5-unsubstituted derivative of zidovudine; penciclovir (DENAVIR®, Novartis); famciclovir (famciclovir) (FAMVIR®, Novartis); acyclovir (ZOVIRAX®, GlaxoSmithKline); cytofovir (VISTIDE®, Gilead); Lovir (CYTOVENE®, Hoffman LaRoche); dextran sulfate; D-penicillamine (3-mercapto-D-valine); FOSCARNET® (trisodium phosphonoformate; Astra Zeneca); fusidin Acid; glycyrrhizin (component of licorice root); HPA-23 (ammonium-21-tungst-9-antimonate); ORNIDYL® (eflornithine; Aventis); nonoxynol; pentamidine isethionate (PENTAM-300) ); Peptide T (octapeptide sequence; Peninsula Laboratories); phenytoin (Pfizer); INH or isoniazid; ribavirin (VIRAZOLE®, Valeant Pharmaceuticals); rifabutin, ansamycin (MYCOBUTIN®) CD4-IgG2 (Progenics Pharmaceuticals) or other CD4-containing or CD4-based molecules; Trimetrexate (Medimmune); Suramin and its analogs (Bayer); and WELLF ERON® (α-interferon, GlaxoSmithKline).

  The pharmaceutical composition of the present invention may further comprise an immunomodulator. In accordance with the present invention, suitable immunomodulators for optimal use with the betulinic acid or betulin derivative of the present invention may include, but are not limited to: ABPP (Bropririmine); Ampligen (mismatch RNA) Hemispherx Biopharma Anti-human interferon-α-antibody; ascorbic acid and its derivatives; interferon-β; Ciamexon; cyclosporine; cimetidine; CL-246,738; colony stimulating factors including GM-CSF; dinitrochlorobenzene; HE2000 (Hollis-Eden Pharmaceuticals); interferon -γ; glucan; hyperimmune γ-globulin (Bayer); imthiol (sodium diethylthiocarbamate); interleukin-1 (Hoffmann-La Roche; Amgen), interleukin-2 (IL-2) (Chiron) Isoprinosine (Inosine Planovex); Krestin; LC-9018 (Yakult); Lentinan (LF); LF-1695; Methionine-enkephalin; Mino Argen C; Muramyl tripeptide, MTP-PE; Naltrexone (Barr Laboratories); RNA immunomodulator (Nippon Shingaku); REMUNE (registered trademark) (Immune Response Corporation); RETICULOSE (registered trademark) (Advanced Viral Research Corporation); Ginseng; Ginseng; Thymic Factor; Thymopentin; Thymosin Factor 5; Thymosin 1 (ZADAXIN®, SciClone), Thythymtimulin, TNF (Tumor Necrosis Factor, Genentech), and vitamin preparations.

  The pharmaceutical composition of the present invention may further comprise an anticancer therapeutic agent. Anti-cancer therapeutics for optimal use include anti-cancer compositions that are effective for inhibiting neoplasms, which include compounds that can be used for combination therapy, or pharmaceutically acceptable. Or alkylating agents (e.g., busulfan), cisplatin, mitomycin C, and carboplatin, mitotic inhibitors (e.g., colchicine), vinblastine, taxol (e.g., paclitaxel (TAXOL (TAXOL)) Registered trademark), Bristol-Meyers Squibb), docetaxel (TAXOTERE®, Aventis)), topoI inhibitors (e.g., camptothecin, irinotecan, and topotecan (HYCAMTIN®, GlaxoSmithKline), topoII inhibitors (e.g., doxorubicin, Daunorubicin, and etoposide (e.g., VP16)); RNA / DNA antimetabolites (e.g., 5-azacytidine, 5 -Fluorouracil, and methotrexate), DNA antimetabolites (e.g., 5-fluoro-2'-deoxy-uridine), ara-C, hydroxyurea, thioguanine, and antibodies (e.g., trastuzumab (HERCEPTIN®, Genentech) And rituximab (RITUXAN®, Genentech and Biogen-Idec)), melphalan, chlorambucil, cyclophosphamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone, ellipticine, fludarabine, octreotide, retinoin Acids, tamoxifen, alanosine, and combinations thereof include, but are not limited to.

  The present invention further provides methods for providing antibacterial, antiparasitic, and antifungal therapeutics for use in combination with the compounds of the present invention and pharmaceutically acceptable salts thereof. Examples of antibacterial therapeutic agents include penicillin, ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin, cephalazine, cefaximol, cefacoxicefetine, , Ceftriaxone, moxalactam, imipenem, clavulanate, timentin, sulbactam, erythromycin, neomycin, gentamicin, streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin, quinolone, rifampin, sulfoneid, bacitracin, polymyxin B , Vancomycin, doxycycline, metacycline Compounds such as phosphorus, minocycline, tetracycline, amphotericin B, cycloserine, ciprofloxacin, norfloxacin, isoniazid, ethambutol, and nalidixic acid, and derivatives and variants of each of these compounds are included.

  Examples of antiparasitic therapeutic agents include bithionol, diethylcarbamazine citrate, mebendazole, metriphonate, niclosamine, niridazole, oxamniquin, and other quinine derivatives, pierazine citrate, praziquantel, pyrantelpamoate, and thiabendazole, and these Derivatives and variations of each of these compounds are included.

  Examples of antifungal therapeutic agents include amphotericin B, clotriazole, econazole nitrate, flucytosine, griseofulvin, ketoconazole, and miconazole, and derivatives and variants of each of these compounds. Antifungal compounds also include acureacin A and papulocandin B.

  A preferred animal subject of the present invention is a mammal. By the term “mammal” is meant an individual belonging to the class Mammalia. The present invention is particularly useful in the treatment of human patients.

  The term “treat, treat, treat” refers to a compound of formulas I through XIII, or a compound identified by one or more assays in the present invention, for the prevention, amelioration, or By administering to a subject for purposes that may include treatment. The compound for treating a subject identified by one or more assays in the present invention is identified as a compound that has the ability to disrupt Gag processing as described herein.

  The term “inhibit interaction” as used herein prevents direct or indirect binding of one or more molecules, peptides, proteins, enzymes, or receptors, or It means reducing its rate, or impeding or reducing the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors.

  Medications are delivered “in combination” with each other when they are delivered to the patient at the same time, or when the time between administration of each medication is such that it allows for overlapping biological activities.

  In one preferred embodiment, at least one compound of formulas I through XIII above comprises a single pharmaceutical composition.

  A pharmaceutical composition for administration according to the invention may comprise at least one compound of formulas I to XIII above, or a compound identified by one or more assays in the present invention. The compound for treating a subject identified by one or more assays in the present invention is identified as a compound having the ability to disrupt Gag processing as described herein. The compounds according to the invention are further comprised in a pharmaceutically acceptable form, optionally in combination with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purpose. The amount and dosing schedule for administration of the compounds of Formulas I through XIII according to the present invention can be readily determined by those skilled in the clinical arts of treating retroviral pathology.

  For example, administration can be parenteral, eg, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, transmucosal, ocular, rectal, intravaginal, or buccal routes. Alternatively or simultaneously, administration can be by the oral route. Administration can be as an oral or nasal spray or topically, such as a powder, ointment, drop, or patch. The dosage to be administered depends on the age, health status, and weight of the recipient, the type of treatment to be performed previously or simultaneously, the frequency of treatment, if any, and the nature of the effect desired.

  The compounds and methods of the invention are useful in a further manner. For example, such compounds may be used prophylactically to minimize the risk of infection. In another embodiment, the compound may be used to minimize the transmission of disease from an infected person.

  The present invention is also directed to a novel method of treating HIV in infected individuals. In one embodiment, the present invention is particularly useful in stimulating an immune response in a person infected with HIV. For example, by allowing non-infectious virions to be released from infected cells, such infected cells will continue to expose the antigen and by the immune system or other therapy directed against such antigens. It may be effectively targeted. In another example, by continuing to allow the release of non-infectious viruses, infected individuals continue to generate an immune response against the virus without suffering from the deleterious effects of such viruses.

  The present invention is also useful in expanding the scope of treatment and provides a novel means of treating disease in patients in need thereof. In another aspect, the invention may be practiced in patients who do not respond to other treatments for reasons other than viral resistance. For example, as is known in the art, conventional methods of treating HIV are associated with deleterious side effects. In one embodiment, the methods and compositions of the invention are useful in treating patients who do not have a reduction in one or more adverse side effects. In one embodiment, the present invention includes a method of treating a patient with a compound that has no or fewer specific side effects.

  Drug bioavailability is also relevant in therapy. In one embodiment, the present invention may be practiced such that the compound is efficiently absorbed by infected cells. In one embodiment, the present invention includes an improved method of delivering a drug to cells infected with HIV.

  Compositions within the scope of the present invention include all compositions comprising at least one compound of formula I to 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, the dosage may comprise from about 0.0001 mg to about 10 g / kg body weight. Exemplary dosages include about 0.1 to about 100 mg / kg body weight. Preferred dosages comprise from about 1 to about 100 mg / kg body weight of active ingredient. More preferred dosages include about 5 to about 50 mg / kg body weight.

  Administration of the compounds of the present invention may also optionally include prior, simultaneous, subsequent or additional treatments using immune system boosters or immunomodulators. In addition to pharmacologically active compounds, the pharmaceutical compositions of the present invention may also be suitable pharmacological agents that include excipients and auxiliaries that facilitate the processing of the active compounds into pharmaceutically usable preparations. May include an acceptable carrier. Preferably, preparations, in particular preparations that can be administered orally and can be used for preferred forms of administration (e.g. tablets, dragees and capsules) and also preparations which can be administered rectally ( Solutions such as suppositories and the like, as well as by injection or oral administration, contain from about 0.01 to 99%, preferably from about 20 to 75%, of the active compound, along with excipients.

  The pharmaceutical preparations according to the invention are produced in a manner known per se, for example by conventional mixing, granulating, dragee-mixing, dissolution or lyophilization processes. Therefore, pharmaceutical preparations for oral use are desirable or necessary to mix the solid excipient with the active compound, optionally mill the resulting mixture, and obtain a tablet or dragee core. If so, it can be obtained by processing the mixture of granules after adding the appropriate auxiliaries.

  Suitable excipients include, for example, bulking agents such as saccharides (e.g., lactose, mannitol, or sorbitol); cellulose preparations and / or calcium phosphates (e.g., tricalcium phosphate or calcium hydrogen phosphate); and binding Agents such as starch paste (eg, using corn starch, wheat starch, rice starch, potato starch), gelatin, tragacanth, cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents can be added, such as the starches mentioned above, and also carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Auxiliaries are all of the above, flow regulators and lubricants, such as silica, talc, stearic acid and its salts (eg 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 include gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. . To make a coating that is resistant to gastric juice, a solution of a suitable cellulose preparation (eg, acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate) is used. Dyestuffs and pigments can be added to the tablets or dragee coatings, for example, for identification or to characterize combinations of active compound doses.

  Other pharmaceutical preparations 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. Push-in capsules can contain the active compound in granular form that can be mixed with a bulking agent such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and, optionally, a stabilizer. In soft capsules, the active compounds are dissolved or suspended in suitable liquids such as fatty oils or liquid paraffin. In addition, 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. It is also possible to use gelatin capsules consisting of a combination of base and active compound. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

  Suitable formulations for parenteral administration include aqueous solutions of water-soluble active compounds (eg, water-soluble salts). In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils (eg, sesame oil), or synthetic fatty acid esters (eg, ethyl oleate), triglycerides, or glycol-400. Aqueous injection suspensions that may contain substances increasing the viscosity of the suspension include, for example, carboxymethyl cellulose, sorbitol, and / or dextran. Optionally, this suspension may contain a stabilizer.

  Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms are inert diluents commonly used in the art (e.g., water or other solvents), solubilizers and emulsifiers (e.g., water or other solvents), stable Agents and emulsifiers (e.g. ethyl alcohol, isopropyl alcohol, ethyl carbonate, 1,3-butylene glycol, dimethylformamide, oils (e.g. cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), Glycerol, tetrahydrofurfuryl alcohol, polypropylene glycol, and sorbitan fatty acid esters, and mixtures thereof).

  Suspensions include, in addition to the active compound, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, cellulose, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, and combinations thereof Can be included.

  Pharmaceutical compositions for topical administration include formulations suitable for administration to the skin, mucosa, lung surface, or eye. The composition can be prepared as a dry powder, liquid, or suspension, pressurized or non-pressurized. The active ingredient in a non-pressurized powder formulation can be mixed in finely divided form in a pharmaceutically acceptable inert carrier. Such carriers include, but are not limited to, mannitol, fructose, dextrose, sucrose, lactose, saccharin, or other sugars or sweeteners.

  The pressurized composition can include a compressed gas (eg, nitrogen or a liquefied gas propellant). The propellant may also include a surface active component that is a liquid or solid nonionic or anionic agent. The anionic agent can be in the form of a sodium salt.

  Formulations for use in the eye include pharmaceutically acceptable ophthalmic carriers such as ointments, oils (such as vegetable oils), or encapsulated materials. The area of the eye to be treated includes the corneal area or the internal area (e.g., 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 can be in the form of suppositories, creams, foams, or indwelling intravaginal inserts.

  The compositions of the invention can be administered in the form of liposomes. Liposomes can be made from natural or artificial phospholipids, phosphatidylcholines (lecithins) or other lipidic compounds, as is known in the art. Any non-toxic pharmacologically acceptable lipid capable of forming liposomes can be used. Liposomes can be multilamellar or monolayer membranes.

  Pharmaceutical formulations for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. In fact, all three types of formulations 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 inhalants, and their controlled release Contains type.

  A compound described above, or a compound identified by one or more assays in the present invention and having the ability to disrupt Gag processing, can also be used in combination with a biodegradable delayed release carrier. It can be administered in the form. Alternatively, the compounds of the present invention can be formulated as transdermal patches for continuous release of the active ingredient.

  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 method of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of the present invention provided they come within the scope of the appended claims and their equivalents.

Example Example 1
Antiviral activity against primary HIV-1 isolates A robust virus inhibition assay was used to assess the antiviral activity of DSB against primary HIV-1 isolates grown in PMBC. Briefly, serial dilutions of DSB were made in 96 well tissue culture plates. 25-250 TCID ₅₀ of virus and 5 × 10 ⁵ PHA-stimulated PBMC were added to each well. At 1, 3 and 5 days after infection, the medium was removed from each well and replaced with fresh medium containing DSB at the appropriate concentration. Seven days after infection, culture supernatants for p24 detection of viral replication were removed and 50% inhibitory concentration (IC50) was calculated by standard methods.

  Table 5 shows the potent antiviral activity of DSB against a panel of primary HIV-1 isolates. DSBs show similar levels of activity as approved drugs tested in parallel. Importantly, DSB activity was not limited by co-receptor use.

*で示された臨床的HIV-1単離物はPanacosで単離された。すべての他のウイルス単離物はNIH AIDS Reference Repositoryから得られた。
注記:R5およびX4はそれぞれケモカインレセプターCCR5およびCXCR4を示す。 Table 5: Inhibitory activity (IC ₅₀ ) of DSB and two approved drugs against a panel of primary Clade B HIV-1 isolates

The clinical HIV-1 isolate indicated with * was isolated in Panacos. All other virus isolates were obtained from the NIH AIDS Reference Repository.
Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4, respectively.

  DSB toxicity was analyzed by incubating with PHA-stimulated PBMC for 7 days at a range of concentrations, and then determining cell viability using the XTT method. The 50% cytotoxic concentration was> 30 μM, corresponding to an in vitro therapeutic index of about 5000.

Example 2
Antiviral activity of DSB against drug-resistant HIV-1 isolate
The activity of DSB was tested against a panel of HIV-1 isolates that are resistant to approved drugs. These viruses were obtained from the NIH AIDS Research and Reference Reagent Program. The assay was performed using virus grown in PBMC with the p24 endpoint (above) or using cell line targets (MT-2 cells) and cell kill endpoint. The MT-2 assay format was as follows. Serial dilutions of DSB or each approved drug were prepared in 95 well plates. To each sample well was added medium containing 3 × 10 ⁵ cells / mL MT-2 cells and the virus inoculum at the concentration required to produce 80% killing of the cell target 5 days after infection (PI) . On day 5 post infection, virus-induced cell killing was determined by the XTT method and the inhibitory activity of the compounds was determined.

  Table 6 shows the potent antiviral activity of DSB against a panel of drug resistant HIV-1 isolates. These results are not significantly different from those obtained using a panel of wild type isolates (Table 5), and DSB is resistant to all major classes of approved drugs It was demonstrated that it retains its activity against virus strains.

アッセイ法は、NNRTI-耐性単離物がMT-2細胞中で細胞生存度(XTT)終点を用いて実行された以外は、p24終点を用いて新しいPBMC中で行われた。
*耐性の倍数
注記:R5およびX4はそれぞれケモカインレセプターCCR5およびCXCR4を示す。 Table 6: DSB inhibitory activity against a panel of drug resistant HIV-1 (nM IC ₅₀ )

The assay was performed in fresh PBMC using the p24 endpoint except that the NNRTI-resistant isolate was performed in MT-2 cells using the cell viability (XTT) endpoint.
* Multiplicity of resistance Note: R5 and X4 indicate chemokine receptors CCR5 and CXCR4, respectively.

Example 3
Inhibition of HIV-1 replication in the late stages of the viral life cycle by DSB Multinuclear activation of the galactosidase indicator (MAGI) assay was used to distinguish the inhibitory activity of DSB on early and late replication targets. In this assay, the target is HeLa cells, a reporter consisting of a galactosidase gene (modified to localize in the nucleus) driven by CD4, CXCR4, CCR5, and a truncated HIV-1 LTR. Expresses the construct stably. Infection of these cells results in the 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, entry inhibitors T-20, NRTI AZT, and NNRTI nevirapine are associated with HIV-1 infection due to their ability to disrupt the early stages of viral expression affecting Tat protein expression. Caused significant decrease in β-galactosidase gene expression in MAGI cells. In contrast, the protease inhibitor indinavir targets the late stages of viral replication (after Tat expression) and does not interfere with β-galactosidase gene expression in this system. Similar results were obtained with DSB as with indinavir, indicating that DSB blocks viral replication at a time after completion of proviral DNA integration and synthesis of viral transactivating proteins. (Table 7).

DMSO対照は薬物を含まなかった。 Table 7. Effects of DSB and entry (gp41 peptide T-20), RT (AZT and nevirapine), and protease (indinavir) inhibitors on the expression of β-galactosidase in HIV-1 infected MAGI cells

The DMSO control contained no drug.

  Kanamoto et al. (Antimicrob. Agents Chemother. April; 45 (4): 1255-30, (2002)) also reported that DSB acts at a late stage of HIV replication. However, they reported that this compound inhibits virus release from chronically infected cells. In contrast, our data show that DSB has no significant effect on virus release using various experimental systems (eg, Example 6).

Example 4
DSB does not inhibit HIV-1 protease activity Previously, DSB was effective against HIV-1 protease function using a cell-free fluorometric assay that characterized enzyme activity by following the cleavage of synthetic peptide substrates. It was decided not to have. The results of these experiments indicated that DSB had no effect on protease function at concentrations up to 50 μg / mL. As a result of the observation that DSB blocks viral replication at a late stage, studies using recombinant Gag protein, a more relevant system than the synthetic peptide substrate used in the initial assay, were also Executed. The use of recombinant Gag protein as a substrate yielded similar experimental results. 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 (FIG. 1).

Example 5
A deficiency in the final stage of Gag processing (CA-SP1 cleavage) associated with defective viral maturation caused by DSB
In order to better define the mechanism of action of DSB, a detailed study of viruses produced from HIV-1 infected cells treated with DSB was performed. Briefly, H9 cells chronically infected with HIV-1 _IIIB isolate were treated with 1 μg / mL DSB for 48 hours. Indinavir was used as a control. At 48 hours, spent medium was removed and fresh medium containing the compound was added. Both cells and supernatants were collected for analysis at 24, 48, and 72 hours after addition of new compound. The level of virus in the culture supernatant was determined and Western blot was used to characterize viral protein production in both cell-bound and cell-free viruses. As observed in previous experiments, DSB did not cause a significant reduction in the amount of virus produced by chronically infected H9 cells, but Gag processing in both cell-bound and cell-free viruses. There was a deficiency. This defect took the form of an additional band corresponding to p25 in the Western blot (Figure 2). This p25 band results from incomplete processing of the capsid CA-SP1 precursor. DSB treatment of cell lines chronically infected with HIV-2 and SIV showed normal Gag processing consistent with the observation of a lack of antiviral activity against these viruses. The Gag processing deficiency seen in the presence of DSB is completely distinguished from that observed with the protease inhibitor indinavir (FIG. 2). As discussed above, mutations in p25 to the p24 cleavage site that interfere with processing are associated with viral maturation and infectious defects (Wiegers K. et al., J. Virol. 72: 2846-54 ( 1998)).

  As previously discussed (CT Wild et al., XIV Int. AIDS Conf. Barcelona, Spain, Abstract MoPeA3030, (July 2002)), abnormal p25 to p24 processing is also found in other mature budding defects. It can be seen. These include Gag late domain (PTAP) mutations or defects in TSG-101 mediated viral assembly that disrupt budding (Garrus, JE et al., Cell 107: 55-65, (2001); Demirov, DG et al., J. Virology 76: 105-117, (2002)). However, these mutations cause inhibition of virus release, whereas DSB treatment has no significant effect on virus release. The morphology of these mature / budding variants is also completely distinguished from that after DSB treatment (see Example 6).

  Furthermore, mutations that interfere with viral RNA dimerization and result in the production of immature viruses with defective core structures give a similar Gag processing phenotype (Liang, C. et al., J. Virology, 73: 6147-6151, (1999)). However, in these cases, RNA uptake is inhibited and the morphology of the released particles is distinct from that after DSB treatment (see Example 6).

Example 6
HIV-1 maturation as determined by electron microscopy (EM) provided by DSB treatment
Mutations in HIV-1 Gag that disrupt p25 to p24 processing have been demonstrated to yield non-infectious viral particles characterized by an internal morphology that is distinct from normal viruses (Wiegers K. et al., J. Virol. 72: 2846-54 (1998)). In order to determine whether the virus produced in the presence of DSB exhibits this unique morphology, the following experiment was performed.

  HeLa cells were infected with the HIV-1 infectious molecular clone pNL4-3 and treated with DSB as previously described. After treatment, DSB-treated infected cells were fixed in glutaraldehyde and analyzed by EM. The results of this analysis are shown in FIG.

  These results are consistent with compounds that disrupt p25 to p24 processing, which produces non-infectious morphologically abnormal virus particles.

  3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB) is an example of a compound that disrupts p25 to p24 processing and potently inhibits HIV-1 replication. However, this compound does not inhibit PR activity and its action is specific to the p25 to p24 processing step and not to other steps of Gag processing. In addition, DSB treatment results in the abnormal HIV particle morphology described above.

Example 7
In vitro selection of HIV-1 isolates resistant to compounds that disrupt the processing of viral Gag capsid (CA) protein from the CA spacer peptide 1 protein precursor
A series of experiments was performed to select viruses that were resistant to inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB), an inhibitor of HIV-1 maturation. For each experiment, two cell cultures were infected using either NL4-3 or RF isolates. After infection, one culture was maintained in growth medium containing DSB, while the other culture was maintained in parallel in growth medium lacking DSB.

  In one experiment, H9 cells infected with RF virus were maintained in the presence or absence of increasing concentrations of DSB (0.05 to 1.6 μg / mL). Cells were passaged every 2-3 days with the addition of fresh drug. Viral replication was monitored by p24 ELISA every 7 days. At this point, DSB-treated cultures with high levels of p24 were co-cultured at a 1: 1 cell ratio with new uninfected H9 cells in the presence of 1x or 2x of the original DSB concentration. Passaged through. After 8 weeks of co-culture, cell-free virus was collected from cultures containing DSB at a concentration of 1.6 μg / mL and used to infect new H9 cells. Every 7 days, virus from medium containing high levels of p24 was passaged by cell-free infection in the presence of 1x or 2x of the original DSB concentration. Five weeks after cell-free passage, virus from cultures containing 3.2 μg / mL DSB was collected and used to infect MT-2 cells. Viral replication in MT-2 cells was monitored by observation with a syncytium forming microscope. Every 1-3 days, they were washed to remove the input virus and new drug was added to the culture under selection. Every 3-4 days after the appearance of extensive syncytium in the culture under selection, the supernatant from each culture was collected and cell debris removed through a 0.45 μm filter. This filtered viral supernatant was then used to infect new MT-2 cells in the presence or absence of a new drug. After 4 cell-free infections (about 2 weeks of culture), the drug concentration was 3.2 μg / mL and the virus stock was collected and frozen for further analysis.

In a second experiment, a stock of virus derived from the molecular clone pNL4-3 (5.7 × 10 ⁴ TCID ₅₀ ) was used to infect MT-2 cells (6 × 10 ⁶ cells) and the culture was 1.6 μg Maintained in the presence or absence of DSB at a concentration of / ml. Every 1-3 days, they were washed to remove the input virus and new drug was added to the culture under selection. Viral replication was monitored by observation with a syncytium forming microscope. Every 3-7 days, after the appearance of extensive syncytium in the culture under selection, the supernatant from each culture was collected and cell debris was removed through a 0.45 μm filter. This filtered viral supernatant was then used to infect new MT-2 cells in the presence or absence of a new drug. The drug concentration was doubled after 5 cell-free infections and every other subsequent time. After 10 cell-free infections (about 7 weeks of culture), the drug concentration reached 12.8 μg / mL and virus stock was collected and frozen for further analysis.

Example 8
Characterization of HIV-1 isolates selected as resistant to compounds that disrupt the processing of the viral Gag capsid (CA) protein from the CA-spacer peptide 1 protein precursor. Further analysis was performed both phenotypically and genotypically to characterize the nature of drug resistance. Viral resistance to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB) was determined in a viral replication assay. Briefly, virus stocks were titrated (by ELISA) by first quantifying the level of p24 in cultures at 8 days post infection using a 4-fold serial dilution of the virus in H9 cells. The virus input was then normalized for a second assay in which each virus was cultured for 8 days in the presence of a 4-fold serial dilution of the drug. The IC50 for each virus was determined as the drug dilution that reduced the p24 endpoint by 50% when compared to the no drug control. In these assays, two independently derived virus stocks had an IC ₅₀ higher than 2 μM for DSB compared to an IC ₅₀ of 0.02 μM for virus cultured in parallel in the absence of drug. Yielded value. In a series of subsequent experiments, the A364V mutation was engineered into HIV-1 NL4-3 proviral DNA, which was subsequently transfected into HeLa cells. The resulting virus was collected and used to test the activity of DSB in a virus replication assay as described above. In these assays, DSB resistant virus yielded an IC ₅₀ value of 0.1 μM, whereas wild type NL4-3 gave an IC ₅₀ value of 0.01 μM.

  Each virus grown either in the presence or absence of a drug to determine whether the resistant virus is able to circumvent the CA-SP1 cleavage defect caused by DSB in the wild-type virus Stocks were analyzed by Western blot. Virus was pelleted through a 20% sucrose cushion from filtered culture supernatant collected 60 hours after infection and 18 hours after washing the cells and adding new drug. Viruses were lysed and the amount of each virus was normalized by quantifying the p24 level in each sample. Western blot analysis of viral proteins in each sample demonstrated that drug-resistant viruses do not contain the CA-SP1 product in the presence of DSB, and these viruses were responsible for the drug's effect on this cleavage event. I confirmed that.

  Finally, to identify genetic determinants of DSB resistance, the entire Gag and PR coding region of the viral genome was amplified by high fidelity RT-PCR for sequencing. Viral RNA was purified from each virus lysate prepared as described above and digested with DNase to remove any contaminating DNA. The RT-PCR product was then gel purified to remove any non-specific PCR product. Finally, both strands of the resulting DNA fragment were sequenced using overlapping sets of primers. Two amino acid mutations that can confer resistance to DSB independently have been identified. These are alanine to valine substitutions in the Gag polyprotein at residue 364 in the NL4-3 isolate and residue 366 in the RF isolate. These are the first and third residues, respectively, downstream of the CA-SP1 cleavage site (SP1 N-terminus). Alanine is highly conserved at each of these positions throughout all HIV-1 clades in the database.

Example 9
Determinants of the activity of the HIV-1 maturation inhibitor DSB map to the Gag protein CA-SP1 domain
To further define the molecular determinants of DSB activity, residues close to the CA-SP1 cleavage site from a DSB-sensitive virus are inserted into a similar region of the Gag protein of the DSB resistant retrovirus, SIV A series of chimeric viruses was prepared. Characterization of these SIV / HIV chimeras (SHIV) for DSB activity allowed for further identification of the minimal HIV-1 Gag sequence that is both necessary and sufficient for DSB activity.

Materials and methods
Construction of SHIV DNA clone
Three panels of SHIV were generated in combination with residues from the Gag CA-SP1 region of DSB sensitive HIV-1 pNL4-3 using SIVmac239 as the backbone. The CA-SP1 sequence for these SHIV constructs is shown in FIG. All DNA mutagenesis can be performed using PCR overlapping PCR strategies (Ho, SN, HD Hunt, RM Horton, JK Pullen, and LR 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., EF Fritsch, and T. Maniatis (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) And executed.

Cell culture and DNA transfection
HeLa cells were maintained in DMEM (Invitrogen) (supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) and passaged during confluence. All plasmid DNAs were prepared using a miniprep kit (Qiagen). HeLa cells were transfected with wild type SIVmac239, HIV-1 pNL4-3 or SHIV proviral DNA by utilizing FuGENE 6 transfection reagent (Roche). Briefly, cells were seeded at a concentration of 1.5 × 10 ⁵ per well in 6-well plates (Corning) the day before use to reach 60-80% confluence on the day of transfection. For each transfection, 3 μl FuGENE6 was diluted in 100 μl serum-free DMEM, followed by addition of 1 μg DNA. After gentle mixing, the DNA-lipid complex mixture was added gently dropwise to cells in 1.5 ml complete DMEM medium. Twenty-four hours after transfection, the medium containing the DNA-FuGENE6 complex was removed and 1.5 ml of fresh DMEM was added to the transfected cells. 48 hours after transfection, both cells and culture supernatant were collected for further analysis.

SDS-PAGE and Western Blot To characterize the effect of incorporation of residues from the CA-SP1 domain of HIV-1 into SIV on virus particle production and Gag polyprotein processing, Western blots were performed. Briefly, media containing virus particles were collected and clarified by centrifugation at 2,000 rpm, 4 ° C. for 20 minutes in a Sorvall RT 6000B centrifuge 48 hours after transfection. The supernatant containing virus particles was then concentrated through a 20% sucrose cushion in a microcentrifuge at 13,000 rpm, 4 ° C. for 120 minutes, and the pellet was dissolved in lysis buffer (150 mM Tris-HCl, 5% Triton X- 100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], pH 8.0). For cell lysates, 48 hours after transfection, cells were washed once with PBS, lysed (150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholic acid, pH 8.0) followed by nuclei. In order to remove the fraction, centrifugation was performed at 13,000 rpm, 4 ° C. for 5 minutes. Virus pellets and cell lysates are separated on a 12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane (Invitrogen), followed by PBS buffer containing 0.5% Tween and 5% dry milk powder Blocking was performed. This membrane was incubated with anti-SIVmac251 p27 McAb (NIH AIDS Research and Reference Reagent Program) and hybridized with goat anti-mouse horseradish peroxidase (Sigma). For membranes containing HIV-1 protein, the membrane is incubated with immunoglobulin from HIV-1-infected patients (HIV-Ig) (NIH AIDS Research and Reference Reagent Program) and goat anti-human horseradish peroxidase (Sigma) And hybridized. Immune complexes were visualized using the ECL system (Amersham Pharmacia Biotech) according to the instructions supplied by the manufacturer.

Effect of DSB on SHIV Gag processing
To address the effect of Gag substitution on the ability of DSB to inhibit CA-SP1 processing, HeLa cells were transduced with wild-type SIVmac239, HIV-1 pNL4-3 or SHIV proviral DNA by utilizing the procedure described above. I did it. SDS-PAGE / Western blots for analysis of viral proteins from these cultures were maintained as described above, with DSB and DMSO (drug-free controls) at a concentration of 1 μg / ml maintained throughout the entire culture. Carried out.

  Only viruses that were completely 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 the previous scoring in that the virus that was not completely resistant was scored susceptible. For this reason, SHIV.DM was scored as DSB resistant in Experiment 9, but was previously scored as DSB sensitive.

result
Characterization of Gag SHIV
Three panels of HIV-1 / SIV Gag chimeras were prepared (Figure 20). Panel 1 consisted of a virus containing a SIV backbone with residues from the HIV-1 SP1 domain inserted. The HIV-1 insert in these chimeras ranged in size from a single point mutation (SHIV DA) to a complete replacement of the SIV SP1 domain with the HIV-1 derived SP1 sequence (SHIV DN) . Panel 2 consisted of a virus containing the same SP1 substitution plus two C-terminal CA residues (LM to VL) from HIV-1. Panel 3SHIV is identical to that in panel 2 except for mutations in the two C-terminal CA residues (LM to VL) from HIV-1 in addition to the SP1 domain, and each of these chimeras is also CA -A change from Q (SIV) to H (HIV-1) was incorporated into the sixth position (P6) upstream from the SP1 cleavage site.

  The level of released virus particles from the transfected cells and the Gag processing profile for each of the SHIVs were determined (Figure 21). All panel 1 SHIVs showed similar behavior with respect to particle production and Gag processing; these chimeric viruses showed a near-wild-type level of particle production and a normal Gag processing profile compared to the parental SIV. It was. Most of the chimeras in panel 2 were characterized by a normal Gag processing profile, whereas SHIV FC and 11 cell expression was somewhat reduced (FIG. 21A). For all Panel 2 SHIVs, the amount of virus production compared to cell-related expression was comparable to the parental SIV. In contrast to SHIV in panels 1 and 2, the majority of the chimeras in panel 3 showed a lack of Gag processing. Of these SHIVs, only GH, GI and 23 showed normal Gag processing profiles. It is clear that the change from Q to H 6 residues upstream from the CA-SP1 cleavage site affects the ability of SIV PR to process the chimeric Gag protein.

  The results from SHIV panel 2 consisting of viruses containing HIV-1 residues in both SP1 and CA domains are shown in FIG. As previously described, this panel of SHIV shows that, in addition to substitutions in the SP1 domain, each of these chimeras also incorporates two HIV-1 CA C-terminal residues (VL from HIV-1). Is the same as Panel 1 except that it replaces the LM from SIV). Similar to the virus in panel 1, the chimera in panel 2 was characterized by a normal Gag processing profile (Figure 21), whereas the cellular expression of SHIV FC and 11 was somewhat reduced (Figure 21A), and these The percentage of virus found in the supernatant of cells transfected with DNA encoding SHIV was proportional to the level of virus release observed for all panel 2 viruses.

  Results from SHIV panel 3 with viruses containing HIV-1 residues in both the GagSP1 and CA domains are shown in FIG. This panel of SHIV shows that in addition to substitutions in the SP1 domain, each of these chimeras also has two C-terminal residues from HIV-1 (VL from HIV-1 replaces LM from SIV) It is the same as panel 1 except that a change from Q (SIV) to H (HIV-1) is incorporated at the sixth position upstream from the plus CA-SP1 cleavage site. Unlike the first two panels, the majority of the chimeras in panel 3 showed a lack of Gag processing (Figure 21). Of these SHIVs, only GH, GI, and 23 showed normal Gag processing profiles. It is clear that the change from Q to H 6 residues upstream from the CA-SP1 cleavage site affects the ability of the SIV protease to process the resulting chimeric Gag protein.

Sensitivity of Gag SHIV to DSB Each of the SHIVs in panels 1, 2 and 3 was characterized for their sensitivity to DSB. As mentioned above, DSB disrupts HIV-1 CA-SP1 cleavage and results in the release of non-infectious virions exhibiting abnormal core morphology (Li, F., R. Goila-Gaur, K. Salzwedel, NR Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, DE Martin, JM Orenstein, GP Allaway, EO Freed, and CT Wild. (2003) PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc. Natl. Acad. Sci. USA 100: 13555-13560). In the current study, SHIV expressing cells were cultured in the presence of DSB at a concentration of 1 μg / ml for a 48 hour period. At the end of that time, the virus was collected from the culture supernatant and the Gag processing profile for each chimeric virus was analyzed and compared to Gag protein processing in the absence of compound (Figure 22).

As can be seen in FIG. 22, none of SP1 SHIV (panel 1) showed sensitivity to DSB. Even SHIV DN containing the complete HIV-1 SP1 domain showed a normal Gag processing profile in the presence of compounds at concentrations above the IC ₅₀ determined in tissue culture (Figure 22) (Li et al.) . These results from the panel 1 chimera demonstrate that the determinants of DSB activity include a region of HIV-1 Gag outside the SP1 domain. The results from panel 2 SHIV are identical to the results observed for panel 1. Specifically, all of these CA-SP1 showed DSB sensitivity (FIG. 22). Since the virus in panel 2 contained a C-terminal VL amino acid residue in addition to the SP1 sequence from HIV-1, these results indicate that HIV-1 Gag residues other than those immediately adjacent to the CA-SP1 cleavage site Demonstrate that the group plays a role in DSB sensitivity.

  As shown in FIG. 22A, DSB disrupts CA-SP1 processing for a subset of panel 3 SHIV. Of these chimeric viruses, SHIV 23 and GI showed a level of DSB sensitivity comparable to the prototype HIV-1 isolate NL4-3 (FIG. 22A). In replicate experiments, the magnitude of the compound's effect on each of these viruses was nearly identical, as determined by the relative ratio of CA-SP1 to CA. Also, in panel 3, SHIV GH showed some level of DSB sensitivity, but at the qualitative level, the activity observed against this virus was observed with SHIV23 and GI. Compared to decrease. For SHIV GH, the effect of DSB on Gag processing was reduced to the point where the very faint CA-SP1 band observed in the immunoblot was not seen in FIG. 22A. A 5-fold increase in the amount of viral protein loaded on the gel enhanced the sensitivity of the Western blot assay to a level that allowed DSB-mediated processing defects for SHIV GH to be observed (FIG. 22B). For the rest of the virus in panel 3, the effect of the compound could not be determined by the lack of sequence-related Gag processing (FIG. 21B). Panel 3 Results from SHIV show that the His residue at position 6 upstream from the CA-SP1 cleavage site plays an important role in DSB sensitivity and that both CA and SP1 just across the cleavage site It shows that a significant part is required for DSB activity.

Consideration
Three panels of SIV / HIV-1 chimeras were prepared (Figure 20) and characterized. All SHIV in panels 1 and 2 behaved similarly with respect to the effect of SP1 or CA / SP1 substitution on Gag protein processing, viral particle release and susceptibility to DSB (FIGS. 21 and 22). Some of the viruses in panel 2 (i.e., FD and 11) were characterized by a decrease in the level of virus particle production, but this effect is an overall indication of the amount of virus produced by the transfected cells. Most likely due to a significant decrease (Figure 21A). The fact that none of these panel 1 and 2 SHIVs were sensitive to DSB, as determined by the effect of the compound on CA-SP1 processing, was found immediately following the HIV-1 CA-SP1 cleavage site. We show that the outer outer Gag sequence plays a decisive role in DSB activity.

  In contrast to the viruses in panels 1 and 2, the three members of panel 3 SHIV showed some DSB sensitivity. Among these viruses, SHIV 23 and GI showed NL4-3 like sensitivity to DSB, whereas SHIV GH showed a somewhat reduced level of sensitivity to this compound. In an additional panel 3 virus, the effect of CA-SP1 substitution on Gag processing made it impossible to determine the effect of the compound on the remaining chimeras.

  The effect of DSB on these three panels of the CA-SP1 SHIV Gag processing profile suggests that the determinants of compound activity include a relatively large region of Gag adjacent to the CA-SP1 cleavage site. Panel 2 Comparison of activity of DSB against SHIV 11 (insensitive) with panel 3 SHIV 23 (sensitive), the His residue located at the 6th CA position upstream from the cleavage site is critical for DSB activity This is shown (FIG. 22B). Consistent with this observation, in vitro resistance selection studies have identified mutations at this position that confer some degree of DSB insensitivity.

  The results in this example provide for the presence of both high and low affinity binding sites in the CA-SP1 region. Low affinity interactions result in partial DSB activity (ie SIVm3), whereas high affinity binding confers full compound sensitivity (ie SHIV23).

Example 10
Genotyping of virus isolates As indicated above, sequence polymorphisms in HIV have been demonstrated to correspond to the ability of the virus to replicate in the presence of DSB. Many sequence polymorphisms are clustered in gag, particularly in the region encoding CA-SP1. Therefore, genotyping of virus isolates can be used to easily determine whether such viruses can be inhibited by DSB or any other compound that interferes with p25 processing. Also good.

  The results of such genotyping are, for example, in determining whether such viruses can be treated with DSB or any other compound that interferes with p25 processing in a similar manner, or with DSB It is useful in determining the appearance of resistant variants during the course of treatment with.

  Genotyping may be performed by a number of methods. In certain embodiments, genotyping is performed by sequencing.

Methods A single frozen aliquot of plasma (approximately 1.2 ml volume) is obtained from each patient. Plasma samples are stored at -70 ° C. until ready for processing. Each sample is identified using a 3-digit patient ID number.

  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 remaining plasma sample was transferred to another tube and briefly centrifuged at low speed (3 minutes, 8,000 rpm) to clarify the plasma. Then 1 ml of the clarified supernatant is transferred to a fresh tube and centrifuged for 2 hours at high speed (13,000 rpm) to pellet the virus. Carefully remove the supernatant using a pipette and transfer to a separate tube for storage at -70 ° C as a precaution against possible disruption of the virus pellet. The virus pellet was resuspended in 140 μl PBS and stored at −70 ° C. as a backup sample in case sufficient RT-PCR product was not obtained from an initial aliquot of unpelleted plasma.

(Table 8) HIV-1 Gag primer

  After purification, the viral RNA is eluted in a final volume of approximately 60 μl. Only 7 μl of this stock is initially used as a template for reverse transcription using the StrataScript First Strand Synthesis System (Stratagene). Store the remaining RNA stock at -70 ° C as a backup. The primer for reverse transcription (R + 625) anneals approximately 625 bp downstream of the CA-SP1 cleavage site. All primers used for RT-PCR and sequencing in this project were designed to anneal to a region of gag that is highly conserved among clade B HIV isolates, 42 different patients Was confirmed using plasma samples from

  Reverse transcription reactions are performed in a total volume of 50 μl. Only 5 μl of this reaction is initially used as a template for PCR amplification of the CA-SP1 region using the PicoMaxx High Fidelity PCR Master Mix (Stratagene). Store the remainder of the reaction at -20 ° C as a backup. A two-step “nested” PCR strategy is used that has been found to provide a high yield of very clear DNA product. The forward and reverse primers (F-625 and R + 525) for the first round of PCR amplification anneal about 625 bp upstream and 525 bp downstream of the CA-SP1 cleavage site, respectively. After this first PCR reaction, no product is typically seen by agarose gel analysis.

  The initial PCR reaction is performed in a total volume of 50 μl. After cycling, remove 5 μl of this reaction and anneal to the region of gag that is inside the region where the first primer pair anneals, a second round of “nested” PCR using primers F-575 and R-450 Used as a template in the reaction. 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-SP1 cleavage site, respectively. 5 μl of the final “nested” PCR reaction is removed for analysis of the DNA product by agarose gel electrophoresis. As expected, the reaction contains only one prominent band of the expected size (~ 1.1 kb) for the desired product, and the yield is estimated to be sufficient to allow sequencing If so (ie, ~ 200 ng total), 40 μl of the reaction is removed for purification of the DNA product using the MinElute PCR Purification Kit (Qiagen). The remaining ˜5 μl reaction is stored at −20 ° C. as a backup. After elution of the purified DNA product, the appropriate amount (corresponding to at least 40 ng of DNA) is transferred to two tubes, each containing a different sequencing primer (one for each strand of DNA). The “+” strand sequencing primer (F-300) anneals approximately 300 bp upstream of the CA-SP1 cleavage site. The “-” strand sequencing primer (R + 275) anneals approximately 275 bp downstream of the CA-SP1 cleavage site. The template / primer mixture is shipped for sequencing and analysis. Store the remainder of the purified DNA product at -20 ° C as a backup. The resulting sequence analysis provides duplicate readings for each DNA strand to help resolve ambiguities in any single sequencing reaction.

  If ambiguity is found in both sequencing reactions, and there is a problem with the heterogeneity of the gag region where the original sequencing primer anneals, use alternating sequencing primers to further sequence Analyze the decision reaction. These are two “+” strand primers (F-375 and F-125) that anneal about 375 bp and 125 bp upstream of the CA-SP1 cleavage site, respectively, and about 100 bp and 400 bp downstream of the CA-SP1 cleavage site. Two “−” strand primers (R + 100 and R + 400).

  If the final "nested" PCR reaction contains a significant background band when analyzed on an agarose gel (i.e., greater than about 10% of the overall yield) or the sequencing yields a clear result If unsuccessful, the desired DNA product is purified by running the entire PCR reaction (re-amplified from the backup sample, if necessary) on an agarose gel and excising the desired band. The DNA is then purified from agarose using the QIAEX II Gel Extraction Kit (Qiagen) and the eluate is prepared for sequencing as described above.

  If the PCR reaction fails to produce sufficient product for sequencing, perform additional RT-PCR or PCR reactions, if necessary, using the backup samples outlined above and additional confirmed primer sets be able to. These primer sets include four forward primers (F-550, F-375, F-300 and F-125) that anneal approximately 550, 375, 300, and 125 bp upstream of the CA-SP1 cleavage site and CA- Three reverse primers (R + 400, R + 275 and R + 100) are included that anneal approximately 400, 275, and 100 bp downstream of the SP1 cleavage site. For example, excellent results were obtained using primer R + 525 for reverse transcription and using 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 to the viral genotype identified elsewhere in this specification to determine whether the virus is or is not inhibited by DSB. Further confirmation of genotyping results may be obtained by follow-up experiments, for example by direct experiments on virus isolates.

Example 11
Genetic changes during treatment with DSB
To determine HIV-1 genotype changes during the course of treatment with DSB, obtain the genotype of the viral population in each patient prior to dosing and at the end of the study (day 28), and optionally Determine whether any changes occurred during the course of treatment. If a mutation was identified in the last sample of the study and it was not present before dosing, an intermediate sample taken on days 7 and 10 after dosing was also used to determine when the mutation occurred Genotyped.

  This method is used to determine mutations that may occur in the entire virus population during the course of the study. Mutations are identified as higher than a 25% variation in amino acid assignments for a given codon. Once the mutation is identified, the chromatogram of the raw data is reviewed to determine the identity of the amino acid at that position in the secondary virus population. If none of the resistance mutations listed above are identified using these criteria, the chromatograms from each reaction will be related to any subspecies (less than 25% of the total population). Is reviewed to determine if it exists in any of the

  Although the present invention has been fully described so far, the invention has been implemented within a broad and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any aspect thereof. It will be appreciated by those skilled in the art. All patents, patent applications and publications cited herein are fully incorporated herein by reference in their entirety.

DSB does not destroy the activity of HIV-1 at a concentration of 50 μg / mL. In DSB-containing samples, recombinant Gag is processed accurately. In contrast, indinavir blocks protease activity at a concentration of 5 μg / mL as evidenced by the absence of bands corresponding to p24 and MA-CA precursors. Virions from chronically infected H9 / HIV-1 _IIIB , H9 / HIV-2 _ROD , and H9 / SIVmac251 in the presence of DSB (1 μg / mL), indinavir (1 μg / mL) or control (DMSO) Western blot of bound Gag. Gag protein was visualized using HIV-Ig (HIV-1) or monkey anti-SIVmac251 serum (HIV-2 and SIV; NIH AIDS Research and Reference Reagent Program). EM analysis of DSB-treated HIV-1-infected cells. EM data shows two main differences between DSB treated and untreated samples. Virions produced in the presence of DSB are characterized by the absence of a conical mature core. In these samples, the core is uniformly spherical and often non-centric. Second, many virions exhibit a high electron density layer that is a lipid bilayer but outside the core (indicated by the arrows in the panel of DSB treated samples). No mature virus particles were observed in the DSB treated samples. DSB sensitive HIV-1 isolates NL4-3 and RF (# 1; SEQ ID NO: 1) and DSB resistant HIV-1 isolates (# 2; SEQ ID NO: 2 (NL4-3) and # 3; The amino acid sequence in the region of the CA-SP1 cleavage site from SEQ ID NO: 3 (RF)) is shown. The difference between the native and DSB resistant sequences is the change from alanine to valine in the first downstream residue (# 2), and the third downstream residue from the CA-SP1 cleavage site (-|-). Includes a change from alanine to valine in group (# 3). These residues are underlined and bold for ease of identification. A + sense consensus sequence for the A364V DSB resistant NL4-3 variant (SEQ ID NO: 4) is shown, starting at the first part of gag, followed by pol, and containing the entire protease coding sequence. Missense mutations not found in the wild type NL4-3 GENBANK M19921 sequence are bold and shaded in gray. The coding sequence for the consensus CA-SP1 cleavage site region is underlined. The shaded area including the cleavage site represents the SP1 sequence. The first mutation is the A364V mutation. A second amino acid change (in the protease) was also found in the parental clone and confirmed to correspond to the sequencing error in the original GENBANK entry. Therefore, the mutation was not actually present in the protease. In parallel with the A364V mutant isolate, the + sense consensus sequence is shown for the DSB-sensitive NL4-3 parental isolate (SEQ ID NO: 5) passaged in the absence of drug. The + sense consensus sequence for the A366V DSB resistant HIV-1 RF variant (SEQ ID NO: 6) starting at the start of gag and continuing to pol and containing the entire protease coding sequence is shown. Missense mutations not found in the wild-type HIV-1 RF GENBANK M17451 sequence are bold and shaded in gray. The CA-SP1 cleavage site is underlined. The only missense mutation that is also not found in DSB sensitive isolates passaged to be identical is the A366V mutation at the CA-SP1 cleavage site. In parallel with the A366V mutant isolate, the + sense consensus sequence for the parental isolate of DSB sensitive HIV-1 RF (SEQ ID NO: 7) passaged in the absence of drug is shown. The polynucleotides SEQ ID NO: 8 and SEQ ID NO: 9 are shown encoding the polypeptides named herein SEQ ID NO: 2 and SEQ ID NO: 3, respectively. SEQ ID NOs: 10 and 12 show the nucleotide sequence encoding the parent polypeptide sequence named SEQ ID NO: 1. SEQ ID NO: 1 is a consensus sequence based on the sequence of regions from NL4-3 and RF. 10A. Amino acid sequences in the CA-SP1 region of a lentivirus (SEQ ID NO: 13; SEQ ID NO: 11; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 20; SEQ ID NO, respectively) SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 30). 10B: Amino acid sequence of CA-SP1 region in HIV-1 strains RF (SEQ ID NO: 11) and NL4-3 (SEQ ID NO: 13). 10C-10D: nucleotide sequence of gag gene chimeric SIV. 42 nucleotide sequence encoding 7 amino acids upstream and 7 amino acids downstream of the CA-SP1 cleavage site. 10E-H: Chimeric FIV, EIAV and BIV gag gene nucleotide sequences made according to the present invention. The 42 nucleotide sequence encoding 7 amino acids upstream and 7 amino acids downstream of the CA-SP1 cleavage site is underlined and shown in bold (nucleotide sequence: SEQ ID NO: 16; amino acid sequence SEQ ID NO : 17). 10F. Nucleotide sequence of the chimeric feline immunodeficiency virus (FIV) GAG gene containing the HIV CA-SP1 region: nucleotides 1-1353 of the chimeric FIV-GAG gene correspond to nucleotides 628-1980 of the chimeric FIV genome. The nucleotide sequence SEQ ID NO: 94 encodes the amino acid SEQ ID NO: 95. 10G.HIG1 is the nucleotide sequence of the chimeric equine infectious anemia virus (EIAV) GAG gene containing the CA-SP1 region: nucleotides 1-1587 of the chimeric EIAV-GAG gene correspond to nucleotides 450-1910 of the chimeric EIAV genome. The nucleotide sequence SEQ ID NO: 96 encodes the amino acid SEQ ID NO: 97. 10H. The nucleotide sequence of the GAG gene of the chimeric bovine immunodeficiency virus (BIV) containing the HIV CA-SP1 region: nucleotides 1-1471 of the chimeric BIV-GAG gene correspond to nucleotides 316-1746 of the chimeric BIV genome. The nucleotide sequence SEQ ID NO: 98 encodes the amino acid SEQ ID NO: 99. It is the replication kinetics of PA-457 (DSB) resistant mutant. Sequential SP1 point deletion in the context of NL4-3, used to identify residues that are required for DSB activity. The amino acid sequence of SP1 domain in NL4-3 is shown. `` Δ '' indicates deletion and ``-'' means residues that are identical between the point deletion mutant and NL4-3 (SEQ ID NO: 13; SEQ ID NO: 33; SEQ ID, respectively) NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 100; SEQ ID NO: 101; SEQ ID NO: 102; SEQ ID NO: 103). 2 is a summary of particle production and infectivity of point deletion mutants. Western blot for viruses containing a point deletion in SP1 in the presence (+) and absence (−) of DSB. Substitution of HIV-1 CA-SP1 residue VL-AEAMSQV (SEQ ID NO: 32) to SIVmac239 sensitizes SIVmac239 to DSB (SEQ ID NO: 14; SEQ ID NO: 15; SEQ, respectively) ID NO: 20; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 13). (Upper panel) The amino acid sequences in the vicinity of the CA-SP1 cleavage site (including the entire SP1 region) are shown for SIVmac239, HIV-1 NL4-3 and a series of SIV variants, which contain various NL4-3 residues. The base (underlined) was inserted. A dash (“-”) indicates the same as in SIVmac239. (Lower panel) Western blot showing CA and CA-SP1 proteins for this series of viruses in the presence (+) and absence (-) of DSB. Sequence conservation in the CA-SP1 region of lentivirus: replace HIV-1 specific CA-SP1 residues in the corresponding Gag region of FIV, EIAV or BIV. HIV-1 NL4-3 SP1 tagged with an epitope. The sequence of the SP1 peptide with the inserted peptide tag is shown. `` Δ '' indicates a deletion and ``-'' means that the residue is identical to that in NL4-3 SP1 (Figure 17 (1); SEQ ID NO: 15; SEQ ID NO: 104; respectively) (SEQ ID NO: 105; SEQ ID NO: 106; SEQ ID NO: 107); (Figure 17 (2); SEQ ID NO: 15; SEQ ID NO: 108; SEQ ID NO: 109); (3); respectively, SEQ ID NO: 15; SEQ ID NO: 110; SEQ ID NO: 111); (Fig. 17 (4); respectively, SEQ ID NO: 15; SEQ ID NO: 112; SEQ ID NO: 113; SEQ ID NO: 114); (Figure 17 (5); SEQ ID NO: 15; SEQ ID NO: 115, respectively). 18A-C: HIV-1 strain RF polynucleotide sequence. The nucleotide sequence of Gag polyprotein is underlined and shown in bold. The 42 nucleotide sequence encoding 7 amino acids upstream and 7 amino acids downstream of the CA-SP1 cleavage site is highlighted in green. The additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (7 amino acid residues) in SP1 are highlighted. 19A-E: HIV-1 strain NL4-3 polynucleotide sequence. The nucleotide sequence of Gag polyprotein is underlined and shown in bold. The 42 nucleotide sequence encoding 7 amino acids upstream and 7 amino acids downstream of the CA-SP1 cleavage site is highlighted in green. The additional 129 nucleotides (43 amino acid residues) upstream of the cleavage site in CA and the remaining 21 nucleotides (7 amino acid residues) in SP1 are highlighted. It is a schematic diagram of GIV protein and CA-SP1 sequence of SHIV used in this study. The sequences adjacent to the CA-SP1 cleavage site for SIV Mac239 and HIV-1 NL4-3 are shown at the top and bottom of the sequence table. Dashed line (-) represents residues identical to parental SIV MAC239, and delta (Δ) represents SIV residues missing in SHIV. Western blot analysis of Gag processing profiles for SHIV in panels 1-3. FIG. 21A shows Gag processing from cell-bound virus, while FIG. 21B shows Gag processing profile for cell-free virions. Normal Gag processing is indicated by a plus sign (+), whereas a defective processing profile is indicated by a minus sign (-). Western blot analysis of the effect of 1 μg / ml DSB on the conversion of the capsid precursor CA-SP1 to the mature capsid protein. In panel A, virus for Western blotting was obtained using a constant volume of cell culture supernatant. This resulted in variability in the intensity of the viral protein band due to differences between SHIVs at the level of virus production. Panel B shows the Gag processing profile obtained when increasing amounts of viral protein are used for Western blot analysis. Only SHIV showing normal Gag processing was included here. The asterisk ( ^* ) in panel A failed to see the faint CA-SP1 band for SHIV GI observed in the autoradiograph, but the DSB sensitivity for this virus used increasing amounts of protein. Based on the results observed when the analysis was performed, it is scored +/- (panel B). Figure 23A-H. "HIV Sequence Compendium 2002" CA in HIV-1 clinical isolates obtained from Kuiken et al. Eds. Los Alamos National Laboratory, Los Alamos, NM. (Www.hiv.lanl.gov) -SP1 area alignment.

Claims (149)

  HIV-1 infection in patients, including administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but does not significantly affect other Gag processing steps How to treat.   2. The method of claim 1, wherein the method does not significantly reduce the amount of virions released from infected cells in the patient.   2. The method of claim 1, wherein the method has no significant effect on the amount of RNA incorporation into virions released from the patient's cells.   2. The method of claim 1, wherein the compound inhibits the maturation of virions released from the patient's cells. The method of claim 1, wherein a majority of the virions released from the patient's cells exhibit an altered phenotype compared to the wild-type virion, wherein the phenotype is selected from the group consisting of:
(a) a virion with a spherical high electron density core that is non-centric with respect to the viral particle;
(b) a virion having a semi-moon-like high electron density layer located just inside the viral membrane;
(c) virions with or without reduced infectivity; and
(d) Any one combination of (a) to (c). Administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is at least about 70% identical to a sequence selected from the group consisting of: A method of treating HIV-1 infection in a patient that binds to a polypeptide encoded by a polynucleotide having a sequence:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The method of claim 6, wherein the polynucleotide has a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. Administering a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is at least about 70% identical to a sequence selected from the group consisting of: A method of treating HIV-1 infection in a patient that binds to a polypeptide having a sequence:

. The method of claim 8, wherein the polypeptide has a sequence selected from the group consisting of:

.   Administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound comprises a sequence selected from any one of SEQ ID NOs: 121-443. A method of treating HIV-1 infection in a patient that binds to a polypeptide comprising.   11. The method of any one of claims 1-10, wherein the HIV-1 infection in the patient comprises an HIV-1 infection that does not respond to other HIV-1 treatments.   11. The method of any one of claims 1-10, wherein the patient with HIV-1 is otherwise unresponsive to other HIV-1 treatments.   11. The method of any one of claims 1-10, further comprising administering to the patient an additional compound that is an immunomodulator, anticancer agent, antibacterial agent, antifungal agent, antiviral agent, or a combination thereof. the method of.   1 1. The method of any one of claims 1-10, wherein the patient is administered the compound in combination with at least one other antiviral agent.   Other antiviral agents are zidovudine; lamivudine; didanosine; sarcitabine; stavudine; abacavir; nevirapine; delavirdine; emtricitabine; efavirenz; saquinavir; ritonavir; indinavir; virnavir; Adefovir; atazanavir; fosamprenavir; hydroxyurea; AL-721; ampligen; butylated hydroxytoluene; polymannoacetate; castanospermine; contracan ( contracan); cream pharmatex; CS-87; penciclovir; famciclovir; acyclovir; cytofovir; ganciclovir; dextran sulfate; D-penicillamine; trisodium phosphonoform Mate (trisodium phosphon oformate); fusidic acid; HPA-23; eflornithine; nonoxynol; pentamidine isethionate; peptide T; phenytoin; isoniazid; ribavirin; rifabutin; ansamycin; trimethrexate; SK-818; 15. The method of claim 14, wherein the method is selected from the group consisting of suramin; UA001; enfuvirtide; gp41-derived peptide; antibody against CD4; soluble CD4; CD4-containing molecule; CD4-IgG2;   Viral Gag p25 protein (CA) comprising administering a compound that inhibits processing of viral Gag p25 protein (CA-SP1) to p24 (CA) but does not significantly affect other Gag processing steps -A method of inhibiting the processing from SP1) to p24 (CA). Viral Gag p25 protein (CA-SP1) comprising administering a compound that binds to a polypeptide encoded by a polynucleotide having a sequence that is at least about 70% identical to a sequence selected from the group consisting of To inhibit the processing from p24 (CA) to:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The method of claim 17, wherein the polynucleotide is selected from a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. From a viral Gag p25 protein (CA-SP1) to p24 (CA) comprising administering a compound that binds to a polypeptide having a sequence that is at least about 70% identical to a sequence selected from the group consisting of: Methods to inhibit the processing of:

. The method of claim 19, wherein the polypeptide has a sequence selected from the group consisting of:

.   Processing of viral Gag p25 protein (CA-SP1) to p24 (CA) comprising administering a compound that binds to a polypeptide having a sequence selected from any one of SEQ ID NOs: 121-443 How to inhibit.   Human blood comprising contacting a blood product with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but does not significantly affect other Gag processing steps A method of processing a formulation.   23. The method of claim 22, wherein the inhibition does not significantly reduce the amount of virions released from infected cells treated with the compound.   23. The method of claim 22, wherein the inhibition has no significant effect on the amount of RNA incorporation into virions released from infected cells treated with the compound.   23. The method of claim 22, wherein the compound inhibits virion maturation released from infected cells treated with the compound. The method of claim 22, wherein a majority of the virions released from the treated infected cells exhibit an altered phenotype compared to wild-type virions, wherein the phenotype is selected from the group consisting of:
(a) a virion with a spherical high electron density core that is non-centric with respect to the viral particle;
(b) a virion having a semi-moon-like high electron density layer located just inside the viral membrane;
(c) virions with or without reduced infectivity; and
(d) Any one combination of (a) to (c). Contacting the blood product with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is at least about 70 to a sequence selected from the group consisting of: A method of processing a human blood product that binds to a polypeptide encoded by a polynucleotide having a sequence that is% identical:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The method of claim 27, wherein the polynucleotide has a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. Contacting the blood product with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is at least about 70 to a sequence selected from the group consisting of: A method of processing a human blood product that binds to a polypeptide having a sequence that is% identical:

. The method of claim 29, wherein the polypeptide has a sequence selected from the group consisting of:

.   Contacting the blood product with a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), wherein the compound is selected from any one of SEQ ID NOs: 121-443 A method of treating a human blood product that binds to a polypeptide having a sequence of:   32. The method of any one of claims 1-10, 16-31, wherein the compound inhibits the interaction of HIV protease with CA-SP1.   35. The method of claim 32, wherein the compound directly inhibits the interaction of HIV protease with CA-SP1.   35. The method of claim 32, wherein the compound indirectly inhibits the interaction of HIV protease with CA-SP1.   32. The method of any one of claims 1-10, 16-31, wherein the compound binds to a viral Gag protein such that the interaction of HIV protease with CA-SP1 is inhibited.   32. The method of any one of claims 1-10, 16-31, wherein the compound binds at or near the site of cleavage of the viral Gag p25 protein (CA-SP1) to p24 (CA).   32. The method according to any one of claims 1 to 10, and 16 to 31, wherein the compound is a derivative of dimethyl succinyl betulinic acid or a derivative of dimethyl succinyl betulin.   The compound is 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) 38. The method of claim 37, wherein the method is selected from the group consisting of:) dihydrobetulinic acid; 3-O-diglycolyl-betulinic acid; 3-O-diglycolyl-dihydrobetulinic acid; The method of any one of claims 1-10, 16-31, wherein the compound is not a betulin or dihydrobetulin derivative of formulas I-III below, or a pharmaceutically acceptable salt thereof:

Where
R is C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl,
R ′ is hydrogen, a C ₂ -C ₁₀ substituted or unsubstituted alkyl, or aryl group;
R ₁ is C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl,
R ₂ is hydrogen, C (C ₆ H ₅ ) ₃ , or C ₂ -C ₂₀ substituted or unsubstituted carboxyacyl; and
R ₃ is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or —OR ₄ , where R ₄ is hydrogen, C _1-4 alkanoyl, benzoyl, or C ₂ -C ₂₀ Substituted or unsubstituted carboxyacyl,
The dotted line represents any double bond between C20 and C29.   A mutant lenti, comprising a mutation in a nucleic acid sequence encoding Gag, that makes the replication of the mutant lentivirus less sensitive to 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) Virus.   41. The mutant lentivirus of claim 40, wherein the viral Gag polypeptide does not bind to 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid.   41. The mutant lentivirus of claim 40, wherein the mutation makes the mutant lentivirus replication even less sensitive to inhibition of processing of p25 (CA-SP1) to p24 (CA) by DSB.   41. The mutant lentivirus according to claim 40, comprising a mutation in the nucleotide sequence encoding Gag p25 protein (CA-SP1).   44. The mutant lentivirus of claim 43, encoding a Gag polypeptide, wherein one or more amino acids are deleted from or substituted in the amino acid sequence in the CA-SP1 region. The mutant lentivirus of claim 40, wherein the mutation occurs in a nucleotide sequence encoding an amino acid sequence that is at least about 70% identical to a sequence selected from the group consisting of:

. The mutant lentivirus of claim 40, wherein the mutation occurs in a nucleotide sequence encoding an amino acid sequence selected from the group consisting of:

. The mutant lentivirus of claim 40, wherein the mutation occurs in a nucleotide sequence that is at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The mutant lentivirus of claim 40, wherein the mutation occurs in a nucleotide sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The mutant lentivirus of claim 40, wherein the mutation in CA-SP1 occurs in a codon encoding an amino acid selected from the group consisting of:
(a) amino acid 1 (glycine) in GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(b) amino acid 2 (histidine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(c) amino acid 6 (valine or isoleucine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(d) amino acid 7 (leucine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(e) amino acid 8 (alanine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(f) amino acid 10 (alanine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(g) amino acid 11 (methionine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(h) amino acid 12 (serine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(i) amino acid 13 (glutamine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(j) amino acid 14 (valine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(k) amino acid 15 (threonine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(l) amino acid 16 (asparagine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); and
(m)

Inside amino acid 45 (alanine). The mutant lentivirus of claim 40, wherein the mutation in the CA-SP1 sequence results in a change in the encoded amino acid sequence selected from the group consisting of:
(a) Glycine in GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(b) histidine to glutamine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(c) histidine to tyrosine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(d) valine or isoleucine to leucine at amino acid 6 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(e) leucine to methionine at amino acid 7 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(f) Alanine to valine at amino acid 8 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(g) alanine to valine at amino acid 10 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(h) methionine to leucine at amino acid 11 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(i) serine to leucine at amino acid 12 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(j) glutamine to glutamic acid at amino acid 13 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(k) valine to alanine at amino acid 14 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(l) Threonine to leucine at amino acid 15 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(m) Asparagine to alanine at amino acid 16 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117);
(n)

Threonine from alanine at amino acid 45 in.   41. The mutant lentivirus of claim 40, wherein the mutation occurs in a polynucleotide encoding an amino acid sequence selected from any one of SEQ ID NOs: 121-443.   52. The mutant lentivirus according to any one of claims 40 to 51, which is mutant HIV-1.   A recombinant non-HIV-1 retrovirus whose replication is inhibited by 3-O- (3′-3′-dimethylsuccinyl) betulinic acid (DSB).   54. The recombinant non-HIV-1 retrovirus of claim 53, wherein the DSB inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but does not significantly inhibit other Gag processing steps.   54. The recombinant non-HIV-1 retrovirus of claim 53, wherein the viral Gag polypeptide binds to 3-O- (3 ',-3'-dimethylsuccinyl) betulinic acid. The recombinant non-HIV-1 retrovirus of claim 53, comprising a p25 (CA-SP1) protein comprising an amino acid sequence that is at least about 70% identical to the group consisting of:

. The recombinant non-HIV-1 retrovirus of claim 56, comprising a p25 (CA-SP1) protein comprising an amino acid sequence selected from the group consisting of:

. The recombinant non-HIV-1 retrovirus of claim 53, comprising a polynucleotide encoding CA-SP1 that is at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The recombinant non-HIV-1 retrovirus of claim 53, comprising a polynucleotide encoding CA-SP1, having a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.   54. The recombinant non-HIV-1 retrovirus of claim 53, comprising p25 (CA-SP1) comprising an amino acid sequence selected from any of SEQ ID NOs: 121-443. The recombinant non-HIV-1 retrovirus of claim 53, having a nucleotide sequence that is at least about 70% identical to a sequence selected from the group consisting of:
(a) SEQ ID NO: 90;
(b) SEQ ID NO: 92;
(c) SEQ ID NO: 94;
(d) SEQ ID NO: 96; and
(e) SEQ ID NO: 98. The recombinant non-HIV-1 retrovirus of claim 53, having a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO: 90;
(b) SEQ ID NO: 92;
(c) SEQ ID NO: 94;
(d) SEQ ID NO: 96; and
(e) SEQ ID NO: 98.   HIV-2, HTLV-I, HTLV-II, SIV, avian leukemia virus (ALV), endogenous avian retrovirus (EAV), mouse mammary carcinoma virus (MMTV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus The recombinant non-HIV-I retro of claim 53, derived from a virus selected from the group consisting of (BIV), goat arthritis encephalitis virus (CAEV), visna-maedi virus, and feline leukemia virus (FeLV) Virus. The recombinant non-HIV-I retrovirus of claim 63, derived from a virus selected from the group consisting of:
(a) SIV;
(b) FIV;
(c) EAIV;
(d) BIV;
(e) CAEV; and
(f) Bisna-Maedivirus.   54. An animal model of lentiviral infection comprising a suitable non-human animal host infected with the recombinant non-HIV-1 retrovirus of claim 53. The animal model of claim 65, wherein the non-human animal host is selected from the group consisting of:
(a) cat;
(b) monkeys;
(c) an ape;
(d) Horse
(e) sheep;
(f) goat;
(g) cattle;
(h) mouse;
(i) a rat; and
(j) Birds.   A polynucleotide sequence encoding CA-SP1 from a non-HIV-lentivirus that is not sensitive to 3-O- (3 ',-3'-dimethylsuccinyl) betulinic acid (DSB) is expressed as a DSB sensitive HIV-1 virus. A method of producing a recombinant non-HIV-1 lentivirus that is sensitive to inhibition by DSB, comprising the step of replacing with a CA-SP1 encoding sequence. 68. The method of claim 67, wherein the polynucleotide sequence encoding CA-SP1 in the recombinant non-HIV-1 lentivirus comprises a sequence that is at least about 70% identical to a sequence selected from the group consisting of: :
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The method of claim 68, wherein the polynucleotide sequence encoding CA-SP1 in the recombinant non-HIV-1 lentivirus comprises a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The method of claim 67, wherein the CA-SP1 protein of the recombinant DSB sensitive non-HIV-1 lentivirus comprises an amino acid sequence that is at least about 70% identical to a sequence selected from the group consisting of:

. The method of claim 67, wherein the CAB-SP1 protein of the DSB sensitive recombinant non-HIV-1 lentivirus comprises an amino acid sequence selected from the group consisting of:

.   68. The method of claim 67, wherein the recombinant DSB sensitive non-HIV-1 lentiviral CA-SP1 protein has an amino acid sequence selected from any of SEQ ID NOs: 121-443. The method of claim 67, comprising the following steps:
(a) removing nucleotides corresponding to nucleotides 1370-1413 of SEQ ID NO: 18 from the lentiviral genome; and
(b) Inserting nucleotides 1370-1413 of SEQ ID NO: 18 or nucleotides 1857-1899 of SEQ ID NO: 19 into a region of non-HIV-1 lentivirus.   74. A recombinant non-HIV-1 lentivirus produced by the method of any one of claims 67-73.   An antibody that selectively binds at or near the CA-SP1 cleavage site of Gag. The antibody of claim 75, wherein the antibody binds an amino acid sequence that is at least about 70% identical to a sequence selected from the group consisting of:

. The antibody of claim 75, wherein the antibody binds to an amino acid selected from the group consisting of:

.   76. The antibody of claim 75, wherein the antibody binds to an amino acid sequence selected from any of SEQ ID NOs: 121-443.   Select polypeptides containing mutations in the HIV CA-SP1 polypeptide that result in reduced inhibition of p25 (CA-SP1) to p24 (CA) processing by 3-O- (3'3'-dimethylsuccinyl) betulinic acid 76. The antibody of claim 75, wherein said antibody binds.   80. The antibody of claim 79, wherein the mutation is located at or near the CA-SP1 cleavage site or the SP1 domain of CA-SP1. The antibody of claim 79, wherein the polypeptide is selected from the group consisting of:

.   80. The antibody of claim 79, which selectively binds an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. The antibody of claim 75, selected from the group consisting of:
(a) an antibody that selectively binds SP1 but not CA-SP1;
(b) an antibody that selectively binds CA-SP1 but does not bind CA; and
(c) An antibody that selectively binds CA but does not bind CA-SP1.   76. The antibody of claim 75, which inhibits binding of 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid to Gag. An isolated CA-SP1 polypeptide that is at least about 70% identical to a sequence selected from the group consisting of:

. The isolated CA-SP1 polypeptide of claim 85, which is identical to a sequence selected from the group consisting of:

. An isolated CA-SP1 polypeptide encoded by a polynucleotide sequence that is at least about 70% identical to a sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19. The isolated CA-SP1 polypeptide of claim 87, encoded by a polynucleotide sequence selected from the group consisting of:
(a) about nucleotides 1243 to 1435 of SEQ ID NO: 18;
(b) about 1729 to 1920 nucleotides of SEQ ID NO: 19;
(c) nucleotides about 1344 to 1435 of SEQ ID NO: 18;
(d) nucleotides about 1828-1920 of SEQ ID NO: 19;
(e) about 1370-1413 nucleotides of SEQ ID NO: 18;
(f) SEQ ID NO: 19 nucleotides from about 1857 to 1899;
(g) nucleotides about 1372-1419 of SEQ ID NO: 18;
(h) about 1858-1905 nucleotides of SEQ ID NO: 19;
(i) nucleotides about 1372 to 1434 of SEQ ID NO: 18; and
(j) nucleotides about 1858-1920 of SEQ ID NO: 19.   A polypeptide isolated from the CA-SP1 region of HIV-1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 121 to 443.   A polypeptide isolated from an HIV CA-SP1 polypeptide comprising a mutation that results in decreased inhibition of p25 processing by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid.   92. The isolated polypeptide of claim 90, wherein the mutation is located at a CA-SP1 cleavage site or SP1 domain.   93. The isolated polypeptide of claim 90, encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. In a nucleic acid sequence encoding a polypeptide selected from the group consisting of a mutation that results in reduced inhibition of p25 processing by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid The isolated polypeptide of claim 90, which occurs:

. The isolated polypeptide of claim 90, selected from the group consisting of:

.   93. Encoded by an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and 10. Isolated polypeptide.   92. The isolated polypeptide of claim 90, which is part of a chimeric or fusion protein.   It encodes an amino acid sequence containing a mutation in the HIV Gag p25 protein (CA SP1), which mutation is from p25 (CA-SP1) to p24 (CA) by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid. An isolated polynucleotide that results in a decrease in the inhibition of processing.   98. The isolated polynucleotide of claim 97, wherein the reduced inhibition of p25 processing is due to a reduced inhibition of HIV-1 protease interaction with Gag.   98. The isolated polynucleotide of claim 97, wherein the decreased inhibition of p25 processing is due to a decreased binding of 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid to Gag.   98. The isolated polynucleotide of claim 97, wherein the decreased inhibition of p25 processing is due to a decreased binding of DSB at or near the CA-SP1 cleavage site of Gag.   98. The isolated polynucleotide of claim 97, wherein the mutation is located at or near the CA-SP1 cleavage site, or the SP1 domain of CA-SP1. The isolated polynucleotide of claim 97, wherein the mutation is located at or near a polynucleotide encoding an amino acid sequence selected from the group consisting of:

.   98. The isolated polynucleotide of claim 97, selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9.   98. The isolated polynucleotide of claim 97, having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6.   98. The isolated polynucleotide of claim 97 having at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.   98. The isolated polynucleotide of claim 97, having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7.   99. The isolated polynucleotide of claim 97, having at least about 80% identity to the polynucleotide of SEQ ID NO: 10.   98. 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 comprising the steps of incubating the host cell of claim 109 in a medium and recovering the polypeptide from the medium. A method of identifying a compound that inhibits HIV-1 replication in an animal cell comprising the following steps:
(a) contacting a polypeptide comprising a CA-SP1 cleavage site with a test compound;
(b) adding a labeled substance that selectively binds at or near the CA-SP1 cleavage site; and
(c) measuring the binding of the labeled substance to the polypeptide.   112. The method of claim 111, wherein the compound binds at or near the CA-SP1 cleavage site.   112. The method of claim 111, wherein the compound inhibits HIV-1 interaction with the CA-SP1 cleavage site.   112. The method of claim 111, wherein the polypeptide comprising a CA-SP1 cleavage site is a polypeptide fragment or a recombinant peptide.   Claims wherein the labeled substance is a labeled antibody specific for CA-SP1, and measuring the change in the amount of labeled antibody bound to the protein in the presence of the test compound compared to a control. Item 111. The method according to Item 111.   The labeled substance is 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid, and the method is labeled 3-O- (conjugated to the protein in the presence of the test compound compared to the control. 112. The method of claim 111, comprising measuring the change in the amount of (3 ', 3'-dimethylsuccinyl) betulinic acid.   111. The label on the labeled material is an enzyme, fluorescent material, chemiluminescent material, horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density material, radioisotope, or a combination thereof. Method. A method for identifying a compound that inhibits HIV-1 replication in an animal cell comprising the following steps:
(a) contacting a polypeptide comprising a CA-SP1 cleavage site with a protease in the presence of a test compound; and
(b) detecting the interaction of the protease with the polypeptide.   119. The method of claim 118, wherein the protease consists essentially of HIV-1 protease.   The polypeptide is labeled with two fluorescent moieties, each binding to the opposite side of the CA-SP1 cleavage site, where the detecting step is the fluorescence energy from one moiety to the other in the presence of the test compound. 119. The method of claim 118, comprising measuring movement of   119. The method of claim 118, wherein the effect of the test compound on polypeptide cleavage is detected by measuring antibody binding to at least one of SP1, p24 (CA) or CA-SP1 (p25).   The labeled antibody is labeled with a molecule selected from the group consisting of enzymes, fluorescent materials, chemiluminescent materials, horseradish peroxidase, alkaline phosphatase, biotin, avidin, high electron density materials, radioisotopes, and combinations thereof. 122. The method of claim 121. The method of claim 118, comprising the following steps:
(a) contacting a first polypeptide comprising an HIV-1 wild type CA-SP1 cleavage site with HIV-1 protease in the presence of a test compound;
(b) contacting a second polypeptide comprising a mutant CA-SP1 cleavage site or alternative protease cleavage site with HIV-1 protease in the presence of the test compound, wherein the mutant cleavage site or alternative Cleavage of the protease cleavage site of the step is not substantially inhibited by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid;
(c) detecting HIV-1 interaction with the first and second peptides; and
(d) comparing the cleavage of the first polypeptide with the cleavage of the second polypeptide.   124. The method of claim 123, wherein wild type CA-SP1, mutant CA-SP1, or an alternative protease cleavage site region is included in a polypeptide fragment or recombinant peptide.   124. The method of claim 123, wherein the polypeptide is labeled with a fluorescent moiety and a fluorescent quenching moiety, each binding to the opposite side of the CA-SP1 cleavage site, and detecting comprises measuring a signal from the fluorescent moiety. .   124. The method of claim 123, wherein at least one of the first polypeptide or the second polypeptide further comprises an epitope tag.   124. The method of claim 123, wherein (a) and (b) are performed in a cell.   124. The method of claim 123, wherein detecting the interaction of the compound with the first polypeptide and the second polypeptide comprises gel electrophoresis. A method for identifying a compound that inhibits HIV-1 replication in an animal cell comprising the following steps:
(a) contacting the test compound with a cell infected with a first virus whose replication is sensitive to inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB);
(b) contacting a test compound with a cell infected with a second virus having a significantly reduced sensitivity to DSB;
(c) determining the phenotype of the treated cells infected with the first virus and the second virus; and
(d) selecting a test compound that is more active against the first virus compared to the second virus. The method of claim 129, wherein the phenotype is selected from the group consisting of:
(a) virus replication;
(b) cutting of CA-SP1; and
(c) Production of altered virion phenotype. A method for identifying a compound that inhibits HIV-1 replication in an animal cell comprising the following steps:
(a) applying a test compound to cells infected with lentivirus, wherein replication is sensitive to inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB); and
(b) analyzing the lysate from the infected cells to determine whether cleavage of the CA-SP1 protein has occurred; The method of claim 131, wherein the lysate is obtained by dissolving at least one of the following:
(a) infected cells; or
(b) Released virus particles.   132. The method of claim 131, wherein the step of analyzing to determine whether cleavage of the CA-SP1 protein has occurred comprises measuring the presence or absence of p25.   132. The step of analyzing to determine whether cleavage of the CA-SP1 protein has occurred comprises performing a Western blot of the viral protein and detecting p25 using an antibody against p25. the method of.   132. The step of analyzing to determine if cleavage of the CA-SP1 protein has occurred comprises performing gel electrophoresis of viral proteins and imaging of metabolically labeled proteins. Method.   132. The method of claim 131, wherein the step of analyzing to determine whether cleavage of the CA-SP1 protein has occurred comprises performing an immunoassay. The method of claim 133, wherein the immunoassay comprises the following steps:
(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 using an antibody that selectively binds p25.   132. The method of claim 131, wherein an epitope tag sequence is inserted into SP1 and the analyzing comprises selective detection of p25 using an antibody to the epitope tag.   A method for identifying a compound that inhibits HIV-1 replication in animal cells comprising the following steps: against replication inhibition by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) Applying a test compound to lentivirus-infected cells that are susceptible; and then analyzing the virus particles released by the cells for an altered virion phenotype.   140. The method of claim 139, wherein analyzing comprises transmission electron microscopy. The method of claim 139, wherein the altered phenotype is selected from the group consisting of:
(a) a virion with a spherical high electron density core that is non-centric with respect to the viral particle;
(b) a virion having a semi-moon-like high electron density layer located just inside the viral membrane;
(c) a virion with reduced or no infectivity; and
(d) A combination of any one of (a) to (c).   139. Any one of claims 129, 131 or 139, wherein the lentivirus whose replication is sensitive to inhibition by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) is HIV-1. The method described. A lentivirus whose replication is sensitive to inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB) contains a non-recombinant non-recombinant containing a nucleotide encoding CA-1 The method of any one of claims 129, 131, or 139, wherein the lentivirus is derived from a lentivirus selected from the group consisting of:
(a) SIV;
(b) FIV;
(c) EAIV;
(d) BIV;
(e) CAEV;
(f) visna-maedivirus; and
(g) HIV-2. The method of claim 131, wherein the virus having a significantly reduced sensitivity to DSB is selected from the group consisting of:
(a) SIV;
(b) FIV;
(c) EAIV;
(d) BIV;
(e) CAEV;
(f) visna-maedivirus;
(g) HIV-2; and
(h) Mutant HIV-1. A method of identifying a compound comprising the following steps:
(a) measuring disease progression in a treated animal infected with a lentivirus that is sensitive to 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB);
(b) measuring disease progression in an untreated animal infected with the lentivirus;
(c) comparing the response of the treated and untreated animals. A method of identifying a compound comprising the following steps:
(a) a compound is administered to an animal infected with a first lentivirus, and replication of the first lentivirus is inhibited by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB) Stage;
(b) administering a compound to an animal infected with a second lentivirus, wherein replication of the second lentivirus is substantially caused by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB). Uninhibited stage;
(c) measuring and comparing the progression of disease in the animal infected with the first lentivirus compared to the animal infected with the second lentivirus. A method of identifying a compound comprising measuring the interaction of a compound having an amino acid sequence selected from the group consisting of:

. A method of determining whether a patient is infected with HIV-1 that is susceptible to treatment with a compound that inhibits p25 processing, comprising the following steps:
(a) obtaining a sample from a patient;
(b) obtaining a viral nucleic acid encoding CA-SP1 from the sample;
(c) genotyping the viral nucleic acid;
(d) whether the nucleic acid encodes a CA-SP1 variant whose processing is sensitive to inhibition by 3-O- (3 ′, 3′-dimethylsuccinyl) betulinic acid (DSB). The stage to decide. A method of treating a disease in a patient in need thereof, including the following steps:
(a) identifying a compound that inhibits processing of the viral Gag p25 protein (CA-SP1) to p24 (CA) but has no significant effect on other Gag processing steps;
(b) obtaining regulatory approval for the sale and use of the compound;
Packaging the compound for sale and treatment of disease in a patient in need thereof.

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