EP0980388A1 - Inhibiteurs de la protease du vih derivant de la vif - Google Patents

Inhibiteurs de la protease du vih derivant de la vif

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Publication number
EP0980388A1
EP0980388A1 EP98924811A EP98924811A EP0980388A1 EP 0980388 A1 EP0980388 A1 EP 0980388A1 EP 98924811 A EP98924811 A EP 98924811A EP 98924811 A EP98924811 A EP 98924811A EP 0980388 A1 EP0980388 A1 EP 0980388A1
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EP
European Patent Office
Prior art keywords
vif
seq
hiv
protease
peptides
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EP98924811A
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German (de)
English (en)
Inventor
David J. Volsky
Moshe Kotler
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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Publication of EP0980388A1 publication Critical patent/EP0980388A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to novel inhibitors of an HIV or other lentiviral or retroviral protease which are capable of reducing, eliminating or preventing an HIV or other lentiviral or retroviral infection.
  • the inhibitors are peptides and polypeptides having an amino acid sequence which corresponds to the amino acid sequence of a lentiviral Vif protein (Vif-derived protease inhibitor).
  • the invention is also directed to compositions containing the inhibitors of the invention and to methods for using such compositions in the prevention or treatment of HIV or other lentiviral and retroviral infections.
  • the invention is also directed to methods for identifying such inhibitors in cellular model systems, HIV- infected cells, in vitro high flux assay systems, and animal model systems.
  • HIV-1 Human immunodeficiency virus type 1
  • auxiliary proteins which are not found in oncogenic retroviruses, but which influence HIV-1 replication (Cullen, Annu. Rev. Microbiol. 45:219-250,
  • Vif virion infectivity factor
  • lentiviruses a 23 kDa polypeptide which is conserved in lentiviruses (Oberste et al., Virus Genes 6:95-102, 1992; Lee et al., Science 231 :1546-1549, 1986) and among HIV-1 infected persons (Kan et al., Science 231 :1553-1555, 1986; Sova et al., J. Virol. 69:2557-2564, 1995).
  • Antibodies to Vif are found in HIV-1 infected individuals, and increasing levels of
  • Vif can be a marker of the progression of the viral infection (Schwander et al., J. Med. Virol. 36:142-146, 1992).
  • Vif is required for productive HIV-1 infection of primary blood lymphocytes (PBL) and macrophages in vitro (Chowdhury et al., J. Virol. 70:5336-5345, 1996; Courcoul et al., J. Virol. 69:2068-2074, 1995; Gabuzda et al., J. Virol. 66:6489-6495. 1992; Gabuzda et al., J. Acq. Imm. Def. Synd. 7: 908-915, 1994; Michaels et al, AIDS Res. and Hum. Retrovir. 9:1025-1030, 1993; Simm et al., J. Virol. 69: 4582-4586, 1995) and for in vivo pathogenesis in the SCID-hu mouse model of HIV-1 infection (Aldrovandi et al, J. Virol.70: 1505-1511, 1996).
  • HIV-1 infected nonpermissive cells produce noninfectious virions (Borman et al., J. Virol. 69:2058-2067, 1995; Chowdhury et al., J. Virol. 70:5336-5345, 1996; Courcoul et al., J. Virol. 69:2068-2074, 1995; Fisher et al., Science 237: 888-892. 1987; Simm et al., J. Virol. 69: 4582-4586, 1995; Von Schwedler et al., J. Virol. 67:4945-4955. 1993).
  • Such a virus can enter cells, but is unable to efficiently synthesize viral DNA (Chowdhury et al., J. Virol. 70:5336-5345, 1996; Courcoul et al., J. Virol. 69:2068-2074, 1995; Von Schwedler et al., J. Virol. 67:4945-4955, 1993; Sova et al., J. Virol. 67:6322-6326, 1993), displaying a 2,500-fold decrease in the number of reverse transcripts compared to wild-type virus when assayed in primary macrophages (Chowdhury et al., J. Virol.
  • Virions produced in the absence of Vif have aberrations in the composition of core proteins (Borman et al., J. Virol. 69:2058-2067, 1995; Simm et al., J. Virol. 69:4582-4586, 1995) and in core morphology as determined by electron microscopy (Borman et al., J. Virol. 69:2058-2067, 1995; Hoglund et al., Virology
  • Vif HIV- 1 Virions assembled in the absence of Vif in primary T lymphocytes carry unprocessed Gag polyproteins, which appears to correlate with their highly attenuated infectivity (Simm et al., J. Virol. 69:4582-4586, 1995). These findings suggest that one mechanism of action of Vif is to ensure proper processing or assembly of HIV- 1 core components in the production of mature infectious virions. Particles produced in the absence of functional Vif ( ⁇ Vif HIV- 1) can initiate efficient infection only in certain T lymphoid cell lines which can complement Vif function, such as SupT 1, but cannot in peripheral blood lymphocytes, which do not complement Vif function (Gabuzda et al., J. Virol. 66:6489-6495, 1992; Simm et al., J. Virol. 69:4582-4586, 1995).
  • HIV-1 core components are synthesized as two precursor polyproteins, Pr55 Gag and Prl60 Gag"Po1 , which consist of both overlapping and distinct polypeptides (Hunter et al., Sem. in Virol. 5:71-83, 1994; Willis et al, AIDS
  • the Gag gene encodes the core proteins pl7 matrix (MA), p24 capsid (CA), nucleocapsid (NC), p6 Gag , and two spacer peptides, p2 and pi .
  • Prl60 Gag"Po1 contains the viral protease (PR), reverse transcriptase, and integrase enzymes derived from the Pol gene. Both Pr55 Gag and Prl60 Gag"Po1 are processed to their mature forms present in infectious virions exclusively by viral protease (Dougherty et al., Microbiol. Rev 57:781-822, 1993; Hunter, Seminar in Virol. 5:71-83, 1994; Willis et al., AIDS 5:639-654, 1991).
  • the HIV protease one of four enzymes encoded by the virus, is an aspartic protease having a molecular weight of about 11 kDa, and containing 99 amino acids (Katz et al., Annu. Rev. Biochem. 63:133-173, 1994).
  • the three-dimensional structure of the enzyme has been determined (Navia et al., Nature 337:615-620, 1989; Miller et al., Nature 337:576-579, 1990).
  • Enzymatic activity requires dimerization of protease monomers to provide a functional enzymatic active site (Dougherty et al., Microbiol. Rev 57:781-822, 1993).
  • the HIV-1 protease cleaves the Gag and Gag-Pol precursor polyproteins at at least ten distinct cleavage sites, each having a distinct amino acid sequence (Debouck et al., Drug Development Research 21 :1-17, 1990). Domains adjacent to the cleavage sites in both Gag and Pol regulate protease-mediated proteolysis in cis (Goodbar-Larsson et al., Virology 206:387-394. 1995; Pettit et al., J. Virol. 68: 8017-8027, 1994; Quillent et al., Virology. 219:29-36, 1996; Zybarth et al., J. Virol.
  • RT reverse transcriptase
  • HIV protease has been the target for several antiviral compounds that have shown therapeutic promise, such as saquinavir, indinavir and ritonavir (Richman et al., Science 272:1886-1887, 1996).
  • Protease inhibitors have been used in combination therapies with RT inhibitors.
  • Most protease inhibitors developed to date have been based on classical substrate or transition-state analogue approaches (Roberts et al., Science 248:358-361, 1990; McQuade et al., Science 247:454-456, 1990). and are peptidomimetic compounds.
  • protease inhibitors in clinical treatment has documented the emergence of drug-resistant HIV-1 mutants which are no longer susceptible to one or more structurally diverse inhibitors with common targets, such as indinavir
  • the present invention is directed to novel inhibitors of an HIV or other lentiviral or retroviral protease which are capable of inhibiting HIV or other lentiviral or retroviral replication. As such, the inhibitors are also capable of reducing, eliminating or preventing viral infection.
  • the inhibitors are peptides and polypeptides having an amino acid sequence which corresponds to the amino acid sequence of a lentiviral Vif protein (Vif-derived protease inhibitor).
  • the invention is also directed to compositions containing the inhibitors of the invention and to methods for using such compositions to prevent, treat, or inhibit HIV or other lentiviral and retroviral infection.
  • the invention is also directed to methods for identifying such inhibitors in cellular model systems, HIV-infected cells, in vitro high flux assay systems, and animal model systems.
  • Figure 1 shows the structure of constructs used to express autoproteolytic Gag-PR fusion proteins in bacteria.
  • Figure 2 shows the structure of constructs used to express Vif and control proteins as fusions with GST.
  • Panel A shows a schematic diagram of the Gag-PR cleavage products based on known PR cleavage sites (arrows) and their detection by the HIV-1 antisera.
  • Panel B shows a Western blot of cells expressing Gag-PR with GST-Vif (V) or GST-N'terminal half of Vif (NN), GST (G) or GST-C'terminal half of Vif (CN).
  • T cells+HIV extracts of CR10/ ⁇ 1 T (Casaerale et al., Virology
  • Figure 4 shows a Western blot analysis of cleavage products derived from the Gag-PR polyproteins co-expressed with Vif proteins in bacterial cells. Blots were stained with antisera to CA, MA, PR, and Vif. UN: uninduced bacterial assay; 1 hour, 2 hour: time post-induction by IPTG.
  • Figure 5 shows a Western blot of bacterial extracts prepared from cells expressing Gag-PR and CA-PR with or without mutations at the cleavage sites between p6 Po1 and PR.
  • the polyproteins were co-expressed with pGST or pGST- Vif.
  • the blot was reacted with monoclonal anti-CA.
  • the presumptive processed polypeptides are indicated at the right.
  • Figure 6 shows a Western blot of bacterial cells carrying a p6 Pol -PR encoding plasmid (Almog et al., JNirol. 70:7228-7232, 1996) co-expressed with plasmids encoding either GST-Tat (T), GST- ⁇ Vif ( ⁇ V), GST-CNif (CN), GST-
  • Figure 7 shows a kinetic analysis of autoproteolysis of a minimal substrate.
  • Panel A shows a Western blot as described in Figure 6, extracts prepared at 30 minute intervals.
  • Panel B shows a densitometric analysis of Western blot in Figure 7A comparing the fraction of signal in PR versus the total signal in PR with p ⁇ 1 " 1 - PR for each time point.
  • Panel C shows a schematic diagram of the Vif regions relative to inhibition of proteolysis: the black bar indicates the GST protein fused to the N'terminus of each Vif construct, the open bar indicates the region of Vif which is expressed relative to the intact 192 amino acid protein, the dashed line indicates regions from the N terminus or the internal section of Vif deleted from the corresponding expression vector.
  • Panel A shows an in vitro cleavage assay of a chromophoric synthetic peptide substrate (S) by purified HIV-1 protease (P) in the presence or absence of the protease inhibitor Ro 31-8959 (PI) (10 ⁇ M) as determined by a decrease in absorbance at 310 nm (A 3 , 0 ).
  • Panel B shows an in vitro assay assessing effect of Vif on substrate cleavage by protease using purified, full length Vif, GST- N'Vif, purified Tat or GST-CNif.
  • C and D show a dose response of GST- ⁇ Nif
  • Panel A shows PR immobilized on Protein A agarose beads bearing rabbit anti-PR.
  • GST-follistatin, GST-Vif, GST- ⁇ Nif, or GST-CNif were mixed with beads, analysed in Western blot by staining with anti-GST. Purified GSTNNif was loaded in one lane as a positive control.
  • Panel B shows a repeat of the same experiment testing binding of GST- ⁇ Nif.
  • Panel A shows a sequence of peptides derived from the ⁇ '- terminus of the HIV-1 Vif protein that were synthesized and tested in this study.
  • Panels B and C show peptides that were screened for PR inhibition at a concentration of ImM (panel B) or at increasing concentrations (panel C) in standard reaction mixtures of lO ⁇ l. Roche RO 31-8959 (O.lmM) and Pepstatin A (0.2mM) were used as controls for PR activity.
  • Panel D shows pepsin studies.
  • Figure 11 Panel A shows Vif-derived peptides immobilized on nitrocellulose filters and exposed to p6 Pol -PR, PR, or mutated PR . Binding of PR was detected by anti-PR and fluorography.
  • Panel B shows the same experiments under different conditions of binding. Spots 2 and 3 in the last row contain GST- Vif or RSV peptide, respectively, as indicated.
  • Panel A shows an analysis of binding of PR to Vif-derived peptides on wells by ELISA.
  • the upper panel shows results of binding to peptide coated wells by PR, the lower panel shows results of preincubation of PR with the indicated peptide prior to addition of the mixture to peptide coated wells.
  • Panel B shows binding of purified PR to Vif-derived peptides, as determined by ELISA. Binding of PR to the Vif-derived peptides attached to the wells, competitive binding experiments, or preincubation with 14 nM of RO 31-5989.
  • Panel C shows dose-dependent binding of PR to Vif-derived peptides. The assay was carried as described in panel B with increasing concentrations of PR.
  • Panel D shows competitive inhibition of PR binding by Vif-derived peptides.
  • PR 50 ng
  • PR was preincubated for 18 hr in 200 ⁇ l with increasing concentrations of cognate peptide before adding the mixture to the peptide-coated microwells (without addition of peptide, l ⁇ g/ml, 10 ⁇ g/ml, and 50 ⁇ g/ml of the cognate peptides).
  • Panel E shows the binding of PR to short peptides in an ELISA assay; closed bars indicate binding to peptide-coated wells by PR, open bars show the results of preincubation of PR with indicated peptide.
  • Figure 13 shows the inhibition of PR activity and virion maturation by Vif peptides in an eucaryotic expression system using cells infected with the vaccinia vector expressing HIV-1 Gag-Pol, vVK-1, and cultured in the presence of the indicated Vif-derived peptide.
  • Cells and extracellular virus-like particles were harvested and analysed by Western blot for Gag proteins using monoclonal anti-
  • Panels A and B show Gag expression in Hut-78 cells and their particles
  • Panels C and D show the same analysis using vVK-1 infected CEM cells
  • Panels E and F show the same analysis using vVK-1 infected PBL.
  • the lane labelled pT5 contains a bacterial lysate from cells expressing the HIV-1 construct covering matrix through PR.
  • the mobility of p55 Gag , p41 Gag , and p24 Gag are indicated at the right of each autoradiogram.
  • Figure 14 shows inhibition of HIV- 1 maturation by Vif-derived peptides.
  • Hut 78 Cells (Panel A) and extracellular virions (Panel B) were harvested and analyzed by Western blotting using monoclonal anti-CA antibody.
  • Figure 15 shows reduction of virus production followed by treatment with
  • Vif derived peptides in chronically infected Hut 78 cells Panel A
  • newly infected cells Panel B
  • Panel A shows reduction of HIV- 1 production in PBL after exposure to Vif-derived peptides.
  • Panel B shows dose-dependent effects of a Vif- derived peptide on HIV-1 production in PBL.
  • the present invention is directed to novel inhibitors of an HIV or other lentiviral or retroviral protease which are capable of inhibiting HIV or other lentiviral or retroviral replication.
  • the inhibitors are capable of reducing, eliminating or preventing viral infection.
  • the inhibitors are peptides and polypeptides having an amino acid sequence which corresponds to the amino acid sequence of a lentiviral Vif protein (Vif-derived protease inhibitor).
  • the invention is also directed to compositions containing the inhibitors of the invention and to methods for using such compositions in the prevention or treatment of HIV or other lentiviral and retroviral infections.
  • the invention is also directed to methods for identifying such inhibitors in cellular model systems, HIV-infected cells, in vitro high flux assay systems, and animal model systems.
  • the Vif-derived protease inhibitors are derived from the amino acid sequence of a lentiviral Vif protein., and are capable of inhibiting the function of a lentiviral or retroviral protease. Such peptides and polypeptides are therefore comprised of amino acid sequences which are the same as, substantially correspond to, or are analogous or homologous to the sequences from the lentiviral Vif protein.
  • the peptide inhibitors are preferably derived from the HIV-1 Vif protein which is a protein that modulates the activity of the HIV-1 protease.
  • Vif-derived protease inhibitors are defined functionally by their ability to inhibit the proteolytic activity of an HIV or lentiviral or retroviral protease, Le., reduce or eliminate the ability of the enzyme to cleave a native or synthetic substrate. Analogs, homologs, derivatives, truncated fragments and chimeras of the Vif- derived protease inhibitors which retain this functional property are within the scope of the invention.
  • peptide refers to an oligomer of at least two contiguous amino acids, linked together by a peptide bond., and not greater than fifty amino acids.
  • polypeptide refers to an oligomer of at least fifty amino acids.
  • substantially corresponds means an inhibitor amino acid sequence having approximately 70% identity in amino acid sequence to a Vif- derived peptide or polypeptide, whether colinear or including gaps in the parent sequence, and which retain the functional capability of the parent peptide.
  • analog is meant substitutions, rearrangements, deletions, truncations and additions in the amino acid sequence of a Vif-derived protease inhibitor, so long as the structural and functional properties of the inhibitors are retained.
  • Analogs also include inhibitors which contain additional amino acids added to either end of the peptides that do not affect biological activity, e ⁇ , the presence of inert sequences added to a functional inhibitor which are added to prevent degradation.
  • conservative amino acid substitutions can be introduced into an inhibitor provided that the functional activity of the inhibitor is retained.
  • the criticality of particular amino acid residues in an inhibitor may be tested by altering or replacing the residue of interest.
  • the requirement for a cysteine residue, which can be involved in the formation of intramolecular or intermolecular disulfide bonds can be tested by mutagenesis of the cysteine to another amino acid, for example, tyrosine, which cannot form such a bond.
  • Vif or Vif-like protein which serves as the parent amino acid sequence for the inhibitors of the invention can be derived from any lentivirus or other retrovirus, including, but not limited to, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus and all isolates thereof.
  • SIV simian immunodeficiency virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • sequence of an inhibitor of the invention can include all or part of the amino acid sequence of the HIV-1 Vif protein, which contains 192 amino acids.
  • a preferred length for an inhibitor of the invention ranges from 4 amino acids to 35 amino acids.
  • the inhibitors of the invention are preferably derived from the N-terminal half of the Vif protein (N'Vif, amino acids 1-96), which has been shown to inhibit HIV-1 protease-mediated proteolysis.
  • the peptides may also be derived from the C-terminal half of the Vif protein (C'Vif, amino acids 97-192), which has been shown to mediate the membrane association of the Vif protein (Goncalves et al., J. Virol. 69:7196-7204, 1995).
  • specific peptide inhibitors of the invention are the following peptides which have an amino acid sequence derived from the HIV-1 Vif protein (numbering and sequence derived from strain HIV-1/NlT-A (Sakai et al., J. Virol. 65:5765-5773, 1991) or closely related HIV-1 BH10 (Ratner et al., Nature 313:277-284, 1985; SEQ ID NOs: 15-20).
  • YVSGKARGWFYRHHY (30-44) (SEQ ID NO: 5) ISSEVHIPLGDARLV (51-65) (SEQ ID NO: 6)
  • GKARGWFY (33-40) (SEQ ID NO: 8)
  • GWFYRHHY (37-44) (SEQ ID NO: 9)
  • LGDARLVITTYWGLHT (59-74) (SEQ ID NO: 13)
  • LGQGVSIE (81-88) (SEQ ID NO: 17)
  • LGQGVSIKWRKKRYS (81-95) (SEQ ID NO: 25) MENRWQVMIVWQVDRM (1-16) (SEQ ID NO: 26)
  • RHHYESPHPRISSEVHIPLGDARLV (41-65) (SEQ ID NO: 43)
  • the following peptides represent consensus sequences which are derived from Vif sequences contained in the Los Alamos database (Myers et al., eds., Human Retroviruses and AIDS, Los Alamos, New Mexico, 1993):
  • Peptide inhibitors of the invention also include, inter alia, the following peptides containing consensus sequences which correspond to the major conserved domains of Vif (Myers et al., eds., Human Retroviruses and AIDS, Los Alamos,
  • Chimeric protease inhibitors which combine one or more of the preferred peptide inhibitors or segments thereof or fragments of polypeptide inhibitors are within the scope of the invention.
  • Protease inhibitors of the present invention also include cyclic or derivatized peptides, and further include peptides containing D-amino acids as well as L-amino acids. Inhibitors which retain the functional properties of the Vif- derived protease inhibitors and are derived by techniques of combinatorial chemistry known to those skilled in the art are within the scope of the invention.
  • the peptide and polypeptide inhibitors of the invention can be synthesized according to Merrifield solid-phase synthesis techniques (Kotler et al., Proc. Natl. Acad. Sci. 85:4185-4189, 1985; Barany et al., in Gross et al., eds., The Peptides,
  • amino acid sequences of the peptides and polypeptides can be confirmed and identified by amino acid composition analysis as well as manual and automated Edman degradation and determination of each amino acid, HPLC analysis, or mass spectrometry.
  • Polypeptide inhibitors derived from the Vif protein may also be produced by chemical or enzymatic digestion of the full-length protein using techniques that are known to those skilled in the art.
  • the HIV protease which is used in assays to detect inhibitors of the invention may be recovered by purification from viral particles, synthesized in full, or may be produced by recombinant DNA technology using techniques known to those skilled in the art.
  • the N-terminal amino acid of the peptides may contain a free amino group or be acetylated, and the C-terminal amino acid of the peptide may be amidated or comprise a free carboxy group.
  • Other modifications of the peptide termini known to those skilled in the art are within the scope of the invention.
  • the invention is further directed to providing assays to identify protease peptide inhibitors.
  • Inhibitors are characterized by an ability to block the activity of protease in an active infection in vivo, a cellular model system, or in vitro assays.
  • the effects of an inhibitor may also be characterized by its effects in altering, reducing or eliminating viral morphogenesis or replication, or virion infectivity.
  • the native substrates for the HIV-1 protease are the Pr55 Gag and Prl60 Gag"Po1 polyproteins which are processed to yield the core proteins pi 7 matrix (MA), p24 capsid (CA), nucleocapsid (NC), p6 Gag , and two spacer peptides, p2 and pi from the Gag gene.
  • Prl60 Gag"Po1 also comprises the viral protease (PR), reverse transcriptase, and integrase enzymes encoded by the Pol gene.
  • Substrates for use in identifying the peptide inhibitors of the invention include these native polyprotein substrates, Pr55 Gag and Prl60 Gag " Po1 , substrates derived from these native substrates as truncated fragments containing one or more protease cleavage sites; and synthetic peptides encompassing all or part of the Gag and/or all of part of the Gag-Pol scissile (cleavable) sites. Also included are other lentiviral and retroviral Gag and Gag-Pol proteins which serve as substrates for their proteases.
  • Preferred substrates include HIV-1 Gag-PR, which includes a Gag-Pol fragment that contains PR and contains at least six cleavage sites for the protease; CA-PR, which includes truncated Gag polyprotein beginning at capsid p24 (CA) and a Pol fragment that includes PR; and p6 Pol -PR, containing only a segment of HIV-1 Gag-PR, which includes a Gag-Pol fragment that contains PR and contains at least six cleavage sites for the protease; CA-PR, which includes truncated Gag polyprotein beginning at capsid p24 (CA) and a Pol fragment that includes PR; and p6 Pol -PR, containing only a segment of HIV-1 Gag-PR, which includes a Gag-Pol fragment that contains PR and contains at least six cleavage sites for the protease; CA-PR, which includes truncated Gag polyprotein beginning at capsid p24 (CA) and a Pol fragment that includes PR
  • This latter substrate contains only one major protease cleavage site and allows for a simple determination of protease activity by assaying for generation of the 11 kDa protease upon a processing event.
  • Shorter length substrates may be designed to contain a specific protease cleavage site, e.g.. the site between Gag proteins matrix pi 7 and capsid p24, and the cleavage site between Pol proteins reverse transcriptase and integrase.
  • Substrates can be further designed to contain a defined subset of cleavage sites by site-specific mutagenesis of selected sites contained in the protein fragment.
  • the substrate Gag-PR can be modified to alter the cleavage site between p6 Po1 and PR, to prevent the release of PR from the polyprotein.
  • Studies have shown that truncated polyproteins containing the p ⁇ '-PR fusion undergo efficient autoprocessing mediated by the PR. Thus, the release of PR from its precursor is not a prerequisite for PR activity (Kotler et al., J. Virol. 66:6781-6783, 1992; Almog et al, J. Virol. 70:7228-7232, 1996; Zybarth et al., J.
  • the molecular weights of the expected processed products can be predicted, as well as intermediate products generated by incomplete processing.
  • Detection of the protein products resulting from an assay can be performed by standard techniques for protein detection, including Western blotting using anti-HIV-1 serum or monoclonal antibodies against specific proteins, immunoprecipitation, Coumassie blue staining or radiolabeling of SDS-PAGE protein gels, ELISA assays, as well as other methods known to those skilled in the art.
  • the resolution of lower molecular weight species can be accomplished using polyacrylamide gel electrophoresis, including gradient gels. Quantitation of protein levels following electrophoretic resolution can be performed using densitometry.
  • In vivo assays for identifying a Vif-derived protease inhibitor can be conducted in art-recognized cellular model systems in order to simulate the environment of an HIV-1 infection. Such assays can be performed in, for example, bacteria, yeast, insect cells, or mammalian cell culture.
  • the activity of a Vif- derived protease inhibitor can be assayed by its effect on viral protease activity in the cell-based system. This can be accomplished by the introduction of the genes encoding protease and the test substrates on separate plasmids into a cellular model system. Where the substrate itself includes the protease and is subject to autoproteolysis, no exogenous protease need be provided.
  • the gene encoding protease also must be provided in the assay.
  • a nucleic acid encoding a candidate Vif-derived protease inhibitor can also be engineered into the PR-containing plasmid downstream from the PR gene, using an oligonucleotide encoding the inhibitor and appropriate regulatory elements that ensure transcription and translation along with the PR gene.
  • a Vif- derived protease inhibitor can be provided exogenously in the growth medium. Analysis of the processed protein products produced in the presence of a Vif- derived protease inhibitor reveals the extent of the inhibition of protease activity.
  • the cells used in the assay can be engineered to constitutively or inducibly express an autoproteolyzable substrate (i.e.. containing protease), by stable transfection of a DNA sequence encoding such a substrate.
  • an autoproteolyzable substrate i.e.. containing protease
  • Such cells can be transfected with a Vif-derived protease inhibitor which is expressed in the cell, or supplied with a Vif-derived protease inhibitor in the growth medium and the nature of the processed protein products can be determined.
  • This assay can be accomplished using one plasmid that is engineered to contain protease and a Vif- derived protease inhibitor. Where a substrate does include protease, such a plasmid need only provide the DNA sequence encoding a Vif-derived protease inhibitor.
  • the cellular model assays can further be performed using a three-plasmid system where protease, substrate and a Vif-derived protease inhibitor are encoded on separate plasmids, each preferably containing different selectable markers to allow for the selection of cells transformed with all plasmids.
  • a bacterial two-plasmid expression system is used in which one plasmid carries DNA encoding a Vif- derived protease inhibitor and another plasmid is engineered to carry an HIV-1 Gag-Pol gene fragment that encodes a protein having protease-dependent cleavage sites.
  • the Gag-Pol fragment provides for production of the protease and also provides a protein substrate susceptible to cleavage by the protease in an autoproteolytic manner.
  • protease is equimolar to substrate.
  • Both plasmids transform a suitable bacterial host such as an E. coli BL21 (D ⁇ 3), where expression from the plasmids is controlled by using an inducible promoter in the plasmids or an inducible RNA polymerase.
  • the bacterial host cell is engineered to contain an RNA polymerase, such as the T7 polymerase, that is under the control of an inducible promoter.
  • the coding sequences in each plasmid are each under the regulatory control of a promoter that is responsive to the inducible polymerase engineered into the host.
  • the coding sequences for a Vif-derived protease inhibitor and a Gag-Pol substrate are engineered into a separate pGEX vector and a separate pT5 vector, respectively, (Pharmacia, Piscataway, NJ).
  • An E. coli BL21 (DE3) host cell carrying the T7 polymerase gene under the control of the lacUV5 promoter is transformed by both plasmids.
  • a nucleic acid encoding the specific substrate Gag-PR is used, which spans the Gag gene and contains the Pol gene up to the protease coding region and has a molecular weight of 68 kDa.
  • a frameshift mutation is introduced at the Gag-Pol junction to permit translation in a single reading frame.
  • This substrate has five or six protease cleavage sites.
  • the mature proteins can be predicted as well as all combinatorial possibilities from intermediates in the processing events (see Fig. 3).
  • the single polyprotein includes MA, CA, p2, NC, p6 Po1 and PR. IPTG is used to induce the lacUV5-dependent expression of the T7 polymerase.
  • the polymerase in turn, generates expression of the genes under the control of the T7 promoter.
  • the results of any cleavage of the protein substrate is observed by recovering the proteins in a cell lysate and analyzing the proteolytic processing events by, for example, a Western blot using anti-HIV antibodies or other standard techniques of protein identification known to those skilled in the art.
  • plasmids which could be used in such assays, include, but are not limited to, PQE (Qiagen, Chatsworth, CA), pET (Navagene, Madison, WI) as well as any plasmid which supports expression of the heterologous DNA sequences.
  • the plasmids used in the bacterial expression assays also contain regulatory sequences permitting expression in eucaryotic cells, so that inhibitors identified in a procaryotic assay system can be tested in a eucaryotic assay system.
  • Eucaryotic cellular model systems which can be used to assay the inhibitors of the invention simulate the natural environment of the viral infection to which the inhibitors are directed.
  • An assay system which employs a vector-delivered full or partial HIV genome into a eucaryotic cell can be used to simulate the production of viral proteins and virion production.
  • the vaccinia virus vector vVK- 1 containing the
  • Gag-Pol DNA fragment is used to infect suitable host cells.
  • vVK-1 infection of many human cell types, including primary T lymphocytes results in high levels of Gag-Pol production within 24h of infection and also permits the native PR- mediated processing of Gag-Pol and the assembly of virion-like particles which can be isolated from the cell supernatants (Karacostas et al., Proc. Natl. Acad. Sci.
  • Infection of the cells can be conducted in medium containing an inhibitor of the invention, and the effects on proteolytic processing and virion production can be determined. Protein analysis can be performed with standard techniques known to those skilled in the art. Virion-like particle production can assayed by recovering viral-like particles from cell supernatants, and determining their structural or compositional properties.
  • Cells which can be used in these assays include macrophages, peripheral blood lymphocytes (PBL), including T cells, or established cell lines such as the known T-cell leukemia cell lines Hut-78, CEM, H4/CD4, and H9.
  • eucaryotic cellular model systems include any plasmid or viral vector-based DNA delivery system which can be used to transfer the DNA for any combination of protease, substrate, inhibitor and/or viral genome that is necessary for an assay of virion production and/or infectivity.
  • An example of such a delivery system which can be used in the invention is the pIND/pVgRXR inducible plasmid eucaryotic expression system (Invitrogen, Carlsbad, CA).
  • Vif-derived protease inhibitors can also be performed in HIV-1 infected cells.
  • Vif-derived protease inhibitors can be identified by their ability to inhibit or reduce HIV infection in eucaryotic cells, with or without detectable alteration in virion or intracellular virus protein structure or composition. Where the inhibitor is incubated with HIV infected cells, the production of infectious virus progeny is determined relative to control experiments without inhibitor.
  • HIV strains which can be used in the assays of the invention include HIV/ADA, HIV-1 IIIB , CR10/N1T-A, NL4-3, NDK, and others.
  • the cells in such assays are infected with HIV or other lentiviral or other retroviral virions or transfected with proviral DNA comprising a desired virus genome.
  • the Vif-derived protease inhibitor is added to the culture medium, or, alternatively, the DNA therefor is contained in an expression plasmid which is transfected into the virally-infected cell.
  • the level and type of infectious progeny are assayed at suitable times post-infection.
  • Evidence of microscopically observed viral spread, cytopathic effect, and increased amounts of the p24 capsid protein can provide an assessment as to whether infectious progeny are being generated.
  • the assessment of progeny virus infectivity may be determined further by recovery of infected cells and co-cultivation with suitable cells (e.g.. PBL or macrophages) or by the recovery of supernatant from the infected cells and cell-free infection of suitable cells.
  • suitable cells are defined as cells which require Vif for infection (e.g., PBL and macrophages but not Sup Tl).
  • Progeny virus with aberrant morphology may be observed, e ⁇ ., by electron microscopy, in the presence of a Vif-derived protease inhibitor.
  • Analysis of the protein composition of progeny virus produced in the presence of Vif-derived protease inhibitors shows alteration in the normal composition of viral proteins, e.g.. altering the wild-type protein profile.
  • the mature proteins found in normal virus particles may be absent or reduced (e.g... capsid p24, matrix pi 7).
  • increased levels of higher molecular weight species e.g.. Gag-specific p55, p41 and p38
  • Gag-specific p55, p41 and p38 may be observed, which indicate incomplete polyprotein processing, and therefore the presence of a functional Vif-derived protease inhibitor.
  • Quantitative assessment of an HIV-1 infection conducted in the presence of a Vif-derived protease inhibitor can also be determined using molecular markers, for example, by assaying viral p24 production by ELISA assay, reverse transcriptase activity, or viral DNA synthesis by quantitative PCR using standard techniques known to those skilled in the art.
  • Primary cells or cell lines which can be used for Vif-derived protease inhibitor studies in the context of an HIV-1 infection are preferably those that are susceptible to such viral infection.
  • Such cells include, for example, PBL, macrophages, and the H4/CD4 and H9 cell lines.
  • H4/CD4 cells a human glioma cell line engineered to express the cell surface protein CD4, is used for assays of HIV- 1 infection in the presence of a Vif-derived protease inhibitor in eucaryotic cells.
  • H4/CD4 cells are highly susceptible to infection with HIV-1, both by standard infection methods and by transfection with pro viral DNA. Efficient transfectability of these cells permits the evaluation of candidate Vif-derived protease inhibitors which have shown protease-inhibiting activity in bacteria, eucaryotic or in vitro assays.
  • Vif-derived protease inhibitors of the invention can also be identified and characterized using in vitro assays.
  • Cell-free assays using protease and native or synthetic substrates can be used to identify Vif-derived protease inhibitors which exert their effect by altering, reducing, or inhibiting the effect of Vif on protease activity.
  • the inhibitor is added to the combination of protease and substrate, and the effect of the inhibitor on protease-mediated cleavage of one or more substrates is determined.
  • Substrates for such assays include any protein or peptide which contains one or more of the HIV-1 protease cleavage sites, including those described infra. Because the molecular weights of the processed products can be predicted, deviation from the expected proteolytic products indicates that a Vif-derived protease inhibitor is capable of altering, reducing or inhibiting protease activity.
  • proteolytic products any techniques for protein identification can be used in these assays to detect the proteolytic products, including Western blots using antiserum to the mature proteins, immunoprecipitation and Coumassie blue-stained SDS-PAGE analysis.
  • In vitro assays can also be performed in high flux evaluation in order to screen large numbers of candidate inhibitors.
  • Specific substrates can be designed for use in a high flux assay system, where cleavage is monitored by a signal system amenable to ready detection.
  • a protease substrate can include a chromophoric tag which is detectable by spectrophometry, and which changes its absorption properties upon substrate hydrolysis, e,, hydrolysis produces a detectable chromophore.
  • a fluorogenic substrate can be designed for detection by fluorescent emission by linking a fluorophore to a residue in the substrate and linking a chromophore to a nearby residue on the opposite side of a cleavage site. Upon cleavage of the substrate, the residues are separated and the chromophore no longer quenches the fluorescent emission, and such emission can be detected in a fluorometer.
  • the use of other signal systems which facilitate high flux identification of candidate inhibitors is within the scope of the invention.
  • Minimal protease-susceptible substrates can be used for the in vitro or high flux assays.
  • substrates include, for example, a chromophoric peptide encoding the cleavage site between reverse transcriptase and integrase: Acetyl- Arg-Lys-Ile-Leu'Phe(NO2)-Leu-Asp-GLy-NH2, where the peptide is cleaved between the Leu and Phe residues, and such cleavage can be detected by a decrease in spectrophotometric absorbance at 310 nm.
  • Another substrate for an in vitro assay includes a chromogenic peptide encoding the cleavage site at the junction of p6 Po1 and protease in the Pol gene: Ser-Phe-Asn-Phe'Pro-Gln-Ile-Thr, where nitrophenylalanine is substituted for the phenylalanine flanking the cleavage site. Cleavage by the protease is also detected by a decrease in spectrophotometric absorbance at 310 nm.
  • Another substrate includes a peptide that contains the cleavage site between the pi 7 matrix (MA) and p24 capsid (CA) proteins, and comprises the sequence Arg-Gln-Ser-Gln-Asn-Tyr'Pro-Ile-Val-Lys-Arg, in which a fluorophore is linked to the glutamine residue and an acceptor chromophore is linked to the lysine residue. Fluorescence is quenched by the proximity of the chromophore; upon cleavage, the residues separate and the dequenched emission can be detected in a fluorometer. In vitro assays also include binding studies which determine the physical interaction between protease and a Vif-derived protease inhibitor.
  • Such assays are performed by incubating protease and an inhibitor and determining complex formation by, for example, Western blot of immunoprecipitated proteins.
  • an inhibitor may be applied to nitrocellulose filters or microtiter well which are then incubated with protease, and binding can be detected in an ELISA format, e.g.. incubation with anti-protease antiserum, followed by a secondary antiserum which contains a signal that is readily assayed, e.g.. by cleavage of a chomophoric substrate.
  • the in vitro assays are performed by incubating protease, Vif-derived protease inhibitor and substrate, using a range of molar ratios between inhibitor and protease in order to assess the degree of inhibition by inhibitor on protease activity. Determination of the kinetic parameters of protease-catalyzed proteolysis such as Km and Kcat of cleavage sites is carried out by using increasing concentrations of a specific substrate, e__g_, a chromophoric substrate. The inhibition constant (Ki) can be determined under identical conditions using a fixed concentration of substrate (Tomaszak et al., Biochem. Biophys. Res. Comm. 168:274, 1980).
  • the reagents for the in vitro assays can be synthesized using peptide synthetic techniques or recombinant DNA technology, described infra. Where recombinant DNA techniques are used to produce a peptide or protein reagent for these assays, they may be produced as fusion products which permit ready recovery and purification.
  • the gene for an HIV protease can be engineered into an expression plasmid and transformed into bacterial cells such that it is expressed as a fusion protein containing a molecular tag such as glutathione- S-transferase (GST) or 6 histidine residues.
  • GST glutathione- S-transferase
  • the tag allows purification of the expressed protein using beads or columns coated with glutathione to purify GST-tagged proteins or coated with nickel to purify histidine- tagged proteins.
  • Purified protease obtained by standard biochemical purification techniques from viral particles can also be used.
  • Candidate inhibitors of the invention can also be obtained using peptide libraries which contain large numbers of peptides for screening.
  • An oligonucleotide endoding a Vif-derived protease inhibitor can be used to engineer cell lines which constitutively express the inhibitor in order to test the effect of an inhibitor on different isolates of HIV- 1 or other HIV strains.
  • isolates include lymphotropic and macrophage-tropic strains, primary strains derived from blood cells or tissues, and North American, European, African and Asian isolates.
  • the peptide inhibitors of the invention can be tested in animal models of HIV infection, including the SCID-Hu mouse model of HIV- 1 infection
  • the inhibitors of the invention can be assayed to determine the concentration required to achieve an antiviral effect against a target virus.
  • a convenient variable for measurement is the concentration of an inhibitor required to inhibit 50% of viral replication (IC 50 ), whether assayed in cell culture or with the use of a molecular marker such as the measurement of viral p24 production by ELISA assay, presence of viral RNA, reverse transcriptase activity, or viral DNA synthesis by quantitative PCR using standard techniques known to those skilled in the art.
  • Peptides of the invention can be evaluated for cytotoxic effects using standard assays that measure cell viability. Such assays include 14 C protein hydrolysate, 3 H thymidine uptake, MTT reduction, and cell growth.
  • TD 50 toxic dose to 50% of the tested culture
  • IC 50 inhibitor concentration required to inhibit 50% of the viral marker being tested or viral replication
  • TI therapeutic index for a particular compound
  • a prospective inhibitor of the invention has an IC 50 that is at least ten times higher than the TD 50 , and the IC 50 is effective at a minimum concentration of 10 "6 M in culture.
  • an inhibitor of the invention exhibits an IC 50 of 10 "7 M or 10 "8 M.
  • the inhibitors of the invention are useful in the isolation of HIV or other lentiviral and retroviral protease mutants which can be used in subsequent screens to identify other antiviral agents to which they are susceptible.
  • the inhibitors are also useful in the three-dimensional analysis of the Vif-protease interaction.
  • the inhibitors of the invention can also be used to inhibit any viral protein in addition to or instead of a viral protease, including the Vif protein, in order to achieve an antiviral effect which is useful in the prevention or treatment of HIV and other lentiviral and retroviral infections.
  • inhibitors of the invention may be used in compositions for the prevention or treatment of an HIV or other lentiviral and retroviral infections, and the treatment of consequent pathologic conditions such as AIDS.
  • Another aspect of the invention is directed to methods for preventing and treating an HIV or other lentiviral or retroviral infection by administering a composition containing one or more of the inhibitors of the invention to a patient for a time and under conditions to accomplish such result.
  • the inhibitors, compositions and methods of the invention can be used in the treatment of HIV-positive individuals, including those exhibiting the conditions of AIDS -related complex (ARC) and AIDS, as well as those who are asymtomatic.
  • ARC AIDS -related complex
  • AIDS AIDS -related complex
  • inhibitors, compositions and methods can also be used in the prophylaxis of HIV or other lentiviral and retroviral infections, and can also be used the treatment or prophylaxis of veterinary infections caused by lentiviruses and other retroviruses.
  • the peptides of the invention may be used in combination with other inhibitors of HIV replication, including, but not limited to, other antiviral compounds, immunomodulators, antibiotics, vaccines, chemokines and other therapeutic agents.
  • agents which can be used in combination with the inhibitors of the invention include agents such as azidothymidine (AZT), dideoxyinosine (DDI), dideoxycytosine, saquinavir, indinavir, ritonavir, (DDC), and other antiviral compounds.
  • the inhibitors of the invention may also be used in combination with agents which are used to treat secondary complications of HIV infection, e-g., gancyclovir used in the treatment of cytomegalovirus retinitis. Combination therapy may retard the development of drug-resistant mutants by requiring multiple mutation events for the emergence of a fully drug-resistant isolate.
  • the inhibitors of the present invention may be administered to a host as a composition in an amount effective to inhibit viral replication and/or infection, together with a physiologically acceptable carrier.
  • the inhibitors of the invention may be administered systemically for preventing or treating an HIV or other lentiviral or retroviral infection.
  • the inhibitor compositions may be formulated as liquids, pills, tablets, lozenges or the like, for enteral administration, or in liquid form for parenteral injection.
  • the peptides and/or polypeptides (or inhibitor-protein conjugates) may be combined with other ingredients such as carriers and/or adjuvants.
  • An inhibitor can also be covalently attached to a protein carrier, such as albumin, so as to minimize diffusion of the inhibitor.
  • physiologically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The use of such media and agents are well-known in the art.
  • the forms of the compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the ultimate solution form must be sterile and fluid.
  • Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e..
  • biocompatible buffers examples include ethanol, polyol such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization can be accomplished by an art-recognized technique, including but not limited to, filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • sterile injectable solutions containing the subject inhibitors is accomplished by incorporating these compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization. To obtain a sterile powder, the above solutions are vacuum-dried or freeze-dried as necessary.
  • the peptide compositions also may be impregnated into transdermal patches, plasters and bandages, preferably in a liquid or semi-liquid form.
  • compositions thereof containing an effective dosage of the peptide may also contain an inert diluent, an assimilable edible carrier and the like, be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.
  • the subject inhibitors are thus compounded for convenient and effective administration in an amount effective to inhibit viral replication and/or infection, together with a physiologically acceptable carrier.
  • the precise therapeutically effective amount of inhibitor to be used in the methods of this invention to prevent or treat an HIV infection cannot be stated because of the nature of the infectious process. It must be noted that the amount of inhibitor to be administered will vary with the degree of infection in an individual, as determined by such parameters as viral load and CD4 cell counts. Individual-specific variables such as age, weight, general health, gender, diet, and intake of other pharmaceuticals factor into the dosage range. The design of an optimal protocol for an infected individual may further consider the identity of the viral isolate(s) isolated from an infected individual with an infection for optimal result.
  • protocol design would be the presence of a viral strain which is resistant to existing protease inhibitors or RT inhibitors.
  • an inhibitor of the invention per unit volume of combined medication for administration is also very difficult to specify, because it depends upon the amount of active ingredients that are afforded directly to the site of infection.
  • a peptide or polypeptide inhibitor of the invention should preferably be present in an amount of at least about 1.0 nanogram per milliliter of combined composition, more preferably in an amount up to about 1.0 milligram per milliliter.
  • Systemic dosages depend on the age, weight and condition of the individual and on the administration route. For example, a suitable dosage for the administration to adult humans ranges from about 0.01 to about 100 mg per kilogram body weight. The preferred dosage ranges from about 0.5 to about 5.0 mg per kilogram body weight.
  • peptide and polypeptide compositions of this invention are effective in reducing or eliminating the ability of HIV or other lentiviruses and other retroviruses to replicate and/or generate infectious progeny, a continual application or periodic reapplication of the compositions is indicated and preferred.
  • the peptide and polypeptide inhibitors of the invention can also be delivered to an individual by gene transfer techniques.
  • DNA coding for a Vif- derived protease inhibitor of the invention can be delivered to the cells of an individual in need of such an inhibitor by any method of gene transfer known to those skilled in the art, including, but not limited to, viral vectors, lipid-mediated delivery, transfection, electroporation, as well as other methods.
  • Viral vectors which can be used to deliver such inhibitors include those derived from DNA and RNA viruses, including, but not limited to, adenovirus, herpesvirus, poxvirus, retrovirus, and adeno-associated virus. (See PCT publication No. WO 95/05851, published March 2, 1995, which is incorporated herein by reference).
  • Parameters which are used to monitor the effect of an inhibitor of the invention administered to an individual with an established HIV infection or administered to an individual for prophylaxis include the use of CD4 counts, plasma viral RNA concentration, viral phenotype, p24 antigen concentration, viral phenotype, level of anti-HIV antibodies as well as other markers of the clinical progression of an HIV infection known to those skilled in the art.
  • inhibitors and methods of the invention can be used in the treatment or prevention of any other lentiviral or retroviral infection, including, but not limited to, those resulting from HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus and all strains and isolates thereof.
  • SIV simian immunodeficiency virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • Example 1 Bacterial Assays of Vif Function Using A Two Plasmid System
  • primer 15 (5 ,789 GGCCATGGGTGCGAGAGCGTCAGTATTAAGC-3 T ) and primer 15 (5' 2558 - GCTAATGGCAAAATTTAAAGTGCACCC-3') were used to amplify the region encompassing MA-PR to create substrate Gag-PR.
  • a frameshift mutation was at position 2088 by inserting a single base, A, thus placing Gag and Pol in the same reading frame using primer 2
  • a construct of substrate CA-PR was created by amplification using primer 1 (5" 185 -CCTATAGTGCAGAACATCCAGGGGCAAATGG-3 * ) and primer 15.
  • primer 1 5" 185 -CCTATAGTGCAGAACATCCAGGGGCAAATGG-3 *
  • the amplified Gag-PR or MA-PR DNA was cloned through pUC12N (Norrander et al., J. Biotechnol. 2:157-175, 1985; Vieria et al., Gene 19:259-268,
  • p6 Po '-PR was amplified using primer 4 (5' 2080 AGGGAAGATCTGGCCTTCCTACAAGGG-3') and primer
  • a cleavage site mutation between p6 Po1 and PR was generated by replacing the Phe codon, normally found at the carboxyl terminus of p6 Po1 (position 2249) with an lie codon, abrogating cleavage using primers 4 and 5
  • the pGEX-2T plasmid (Pharmacia, Piscataway, NJ), which carries the GST gene under the control of the P t ⁇ c promoter, was modified by cloning the 1 kbp Pstl fragment from pKan r into Pstl site in the ampicillin resistance gene, generating plasmid PGEX-T Kan (Fig. 2).
  • the pGEX-2T plasmid contains the kanamycin resistance gene (Kan r ) to permit co- selection of two different expression plasmids in the same bacterial cell.
  • Vif gene or Vif gene segments were amplified from pKS242 plasmid (Sakai et al., J. Virol. 65:5765-5773, 1991) using Pfu polymerase (Stratagene, La Jolla, CA) and primers containing BamHI and EcoRI restriction endonuclease sites at 5' and 3' ends, respectively.
  • vfgex5 (5'-)
  • vfgex3 5'- ACACAATGAATGGACACTAGAATTCATTAGAGG-3'
  • N-terminal Vif was amplified using vfgex5 and vfgexl (5'-CACATAAGAATTCCCTGAACTAGCA-3') and C'terminal Vif was amplified using vfgex2 (5'-GGAAAAAGGGATCCAGGATGGAAG-3') and vfgex3.
  • the amplified DNA was cloned into the corresponding sites in pGEX- 2T ⁇ an for translation in frame with the GST gene.
  • pGEX-2TTat (Rhim et al., AIDS 7: 1116-1121, 1994) was obtained from A. Andrew Rice through AIDS Research and Reference Reagent Program, Bethesda, MD. The sequences and residue coordinates for N'Vif, C'Vif and ⁇ Vif are shown in Table 1.
  • E. coli BL21 (DE3) PlysS which carries an integrated T7 polymerase gene under the control of the lacUV5 promoter (Novagen, Madison, WI) were co-transformed with pGag-PR, pGag x PR, PCA-PR, PC A X PR, p6 Pol -PR, p6 Pol x PR and either pGEX-2T Vif, pGEX-2TNNif, pGEX- 2TCNif, pGEX-2T ⁇ Vif, pGEX-2TTat, or pGEX-2T (Fig. 2), and co-transformants were selected in media containing chloramphenicol, ampicillin, and kanamycin.
  • HIV-1 protein analysis Bacteria were propagated overnight in ampicillin and kanamycin, induced in the presence of 0.1 mM IPTG for 1 h, washed, and cell pellets were lysed directly in Laemmli sample buffer containing 4% SDS. Samples were subjected to electrophoresis in a 12% SDS-polyacrylamide gel, proteins were blotted unto a ⁇ ytran membrane (BioRad, Hercules, Calif), and blots were exposed to an AIDS patient serum or specific antibodies to visualize HIV-1 specific proteins; bound antibody was detected by luminescence ( ⁇ E ⁇ DuPont, Boston, Mass).
  • anti-CA mouse monoclonal AG3.0 antibody which detects both mature HIV-1 CA protein and its CA precursor proteins (Simm et al., J. Virol. 69: 4582-4586, 1995) provided by J.S. Allan
  • anti-MA rabbit polygonal antibody prepared against recombinant MA produced in bacteria, provided by D. Trono
  • anti-Vif rabbit polygonal antibody prepared against recombinant Vif produced in bacteria, donated by D. Gabuzda.
  • the anti-Vif antibody detects GST-Vif and GST-CNif
  • Lysis resistant CR10 cells were infected with HIV-1/ ⁇ 1T and chronic infection was established as previously described (Casareale et al., Virology 155:40-49, 1987). These cells were used as control to demonstrate the pattern of Gag and Gag-Pol proteins in infected eucaryotic cells
  • the HIV-1 protease inhibitor, Ro 31-8959 was obtained from Roche Products, Ltd (London, UK) and was titered for inhibition of proteolysis by Western blotting of supernatants of infected cells. Results
  • Fig. 3A Based on the known protease cleavage sites (Debouck et al., Drug Development Research 21 :1-17, 1990), the predicted proteolytic products of Gag- PR and their detection by specific antibodies are shown schematically in Fig. 3A.
  • bacteria were induced to co-express Gag-PR and either GST-Vif (V), GST-N-terminal or C-terminal halves of Vif protein (N * V and CN, respectively), or GST alone (G), and the Gag-PR autoprocessing products were analyzed by Western blotting using an AIDS patient serum (Fig. 3B).
  • p24/26 and p41 are major products of synthesis and autoprotolysis of Gag-PR in systems containing no inhibitory proteins (G,T,CN, and ⁇ V lanes) indicating that, as previously reported (Tritch et al., J. Virol. 65:922-930, 1991), cleavage between MA and CA is the first to occur. Vif-related changes in the levels of intermediate Gag cleavage products detectable with anti-CA (anti-CA panel) or with anti-MA antibody (not shown) were not pronounced (Fig. 4) and are more easily detected during expression of different PR containing polypeptides, as shown below. Anti-
  • Polyproteins containing PR and representing cleavage products mainly in the C terminal portion of the precursor were visualized by anti-PR antibody staining (Fig. 4, anti-PR panel). The proteolytic patterns in the presence and absence of Vif polypeptides were similar, suggesting that all cleavages on the precursor polyprotein are not affected equally by Vif.
  • Anti-PR staining is less suitable than is anti-CA staining to demonstrate the cleavages flanking CA, since the N'terminal fragments would not be detected, predicted changes in the levels of PR-containing p24/26 were difficult to resolve, and a major background band obscured PR-containing p5O (Fig. 4, anti-PR panel).
  • the intensity and mobility of pi 7, a polyprotein composed of NC-p6 Pol -PR are decreased in the presence of inhibitory Vif proteins, possibly reflecting secondary cleavage sites in the region following the frameshift.
  • HIV-1 Tat (as GST fusion), or GST protein alone did not affect autoprotolysis of Gag-PR (CN, ⁇ V, T, and G lanes, respectively).
  • Vif modifies PR-mediated cleavage of HIV- 1 Gag-PR primarily at the junction p2/p7 ⁇ C, resulting in selective inhibition of release of mature CA proteins.
  • the analyses shown in Fig. 4 do not exclude that other cleavage sites can also affected by Vif, but suggest that there is a ranking of Vif activity at specific sites. PR and Vif specificity of proteolysis of Gag-Pol in bacteria.
  • proteolysis was more efficient in cells expressing either cleavage site mutant than in cells expressing wildtype Gag-PR or CA-PR, confirming that these cleavages were carried out by HIV-1 PR; the p24/26 bands were more pronounced in lane 2 (CA X PR) than in lane 4 (CA-PR).
  • Expression of any of the four truncated Gag-PR vectors in the presence of GST-Vif resulted in an inhibition of cleavage, indicating that Vif acts specifically on PR-mediated proteolysis.
  • the reduction in p24/26 in the presence of Vif indicates that the cleavages flanking p2 are inhibited by Vif.
  • Vif sensitive site is apparent in the increased intensity in the p3 band consisting of CA-p2-NC in cells expressing wildtype CA- PR in the absence of GST-Vif (lane 4) compared to the presence of GST-Vif (lane 3). That Vif affected the processing of p33 from the p50 precursor indicates that Vif can retard cleavage at the site between NC and p6 Po1 . The effects of Vif were most pronounced when co-expressed with vectors carrying the less active, wildtype PR than with those carrying cleavage site mutations.
  • the final product p24 was most easily detected in cells carrying CA X PR or Gag x PR in the absence of Vif (lanes 2 and 6) and was least abundant in cells expressing wildtype Gag constructs in the presence of GST-Vif (lanes 3 and 7).
  • the presence of Vif tended to favor accumulation of incompletely processed proteins, while processing went to completion in the absence of Vif.
  • Mature pi 1 PR was undetectable in the presence of either full length GST-Vif and N'Vif indicating that they totally inhibited proteolysis at the p6 Pol -PR junction.
  • GST-Tat, GST- ⁇ Vif, GST-C'-Vif and GST alone yielded similar ratios of precursor p6 Pol -PR to PR, indicating that none of these polypeptides affect proteolysis more than the control, GST.
  • PR were standardized prior to electrophoresis. This facilitated the comparison of precursor to product ratios among systems.
  • proteolysis of p ⁇ - PR was evident within i hour of induction in the presence of GST or C Vif and the level of product PR increased with time after induction (Fig. 7 A).
  • the levels of PR-containing proteins were expressed as a ratio of 11 kDa PR to total stained protein present in each lane to display the proteolysis of precursor to final product over time (Fig. 7B).
  • the fraction of PR increased with time in the presence of GST or GST-CNif to approximately 40% hydrolysis, but was insignificant in the presence of GST-
  • HIV-1 PR was purified as described previously (Kotler et al., J. Virol. 66:6781-6783, 1992). Bacterial cells (E. coli BL-21 strain) expressing PR were collected by centrifugation and the pellets were suspended in
  • the dissolved proteins were fractionated by reverse-phase high-pressure liquid chromatography on a 19X150 mm ⁇ -Bondapack C lg column (Water Associates, Inc.) using a linear gradient (0- 60%) of acetonitrile in the presence of 0.05% TFA.
  • Fractions collected from the HPLC column were lyophilized and dissolved in 2M guanidine-hydrochloride, 100 mM Tris-HCl (pH 8.0), to a concentration of 200-300 ⁇ g/ml and refolded as follows: 1 vol of protein solution was diluted with 2 vol of refolding buffer (20 mM Pipes, 100 mM NaCl, ImM EDTA, 10% glycerol), and dialyzed against refolding buffer for 4 hr at 4°C. Dialyzed fractions having the highest specific activity were collected and the protein concentration was determined, using a commercial Bio-Rad Kit.
  • This minimal PR enzymatic assay system consisted of recombinant PR and the chromophoric octapeptide substrate Ac-Arg-Lys-lle-Leu 1 - Phe(NO 2 )-Leu-Asp-Gly-NH 2 , which mimics the cleavage site between reverse transcriptase and integrase in the Gag-Pol polyprotein (Tomaszak et al., Biochem. Biophys. Res. Comm. 168:274-280, 1990) and is absent from the truncated Gag- PR substrates employed above (Fig. 1).
  • HIV-1 PR cleaves the peptide between the Leu and Phe(NO 2 ) residues and the reaction can be measured spectro- photometrically by following the decrease in absorbance at 310 nm (Tomaszek et al., Biochem. Biophys. Res. Comm. 168:274-280, 1990).
  • E. coli were transformed separately with each listed plasmid: pGEX-2T Vif, pGEX-2TNNif, pGEX-2TCNif, pGEX- 2T ⁇ Vif and pGEX-2TTat.
  • pGEX-2T Vif pGEX-2TNNif
  • pGEX-2TCNif pGEX- 2T ⁇ Vif
  • pGEX-2TTat One hour after induction, bacteria were harvested, resuspended in a buffer containing 10 mM TrisHCI, pH 8.0, 150 mM ⁇ aCl, 1 mM EDTA, 1% Triton X-100, 5 mM DTT, lOO ⁇ g/ml lysozyme, 1 mM PMSF on ice for 15 min, frozen and thawed, and lysed by sonication.
  • Spectrophotometric assay of synthetic peptide cleavage The conditions of the assay were essentially those described (Tomaszek et al, Biochem. Biophys.
  • the chromogenic synthetic peptide substrate Ac-Arg-Lys-lle-Leu l -Phe( ⁇ O 2 )-Leu-Asp-Gly- ⁇ H 2 was suspended at 250 ⁇ M in a buffer containing 80 mM sodium acetate, ImM EDTA, ImM dithiothreitol, and 0.8 M NaCl (pH 5.0) in a total reaction volume of 200 ⁇ l. After equilibration of substrate at 37 °C for 10 min, 10 ng protease alone or mixed with proteins at the indicated doses or Ro 31-8959 (Roberts et al., Science 248:358-361, 1990) were added.
  • a 310 was read in Beckman DU-62 spectrophotometer, the zero time point was taken at the addition of protease.
  • the substrate, the HXB-2 protease, and purified full-length Vif and Tat proteins were all provided by the AIDS Research and Reference Reagent Program.
  • the GST-NNif and GST-CNif proteins were purified from bacteria transformed with either pGEX-2T ⁇ Nif or pGEX-2TCNif (Fig. 2).
  • Fig. 8 A The kinetics of substrate cleavage by PR in the presence and absence of Vif are depicted in Fig. 8.
  • the A 310 of substrate alone (250 ⁇ M) remained unchanged during a 35 min incubation at 37 °C.
  • the molar ratio of protease inhibitor to PR in this system was about 2000: 1 ; no inhibition was seen at a ratio of 400:1.
  • Vif (either as a purified 23 kDa protein or a purified 35 kDa GST- ⁇ Nif protein) is a specific inhibitor of HIV- 1 PR in cell free proteolytic assay using a selected synthetic peptide substrate. Since Vif efficiently blocked PR activity at approximately 1 :1 molar ratio to the enzyme (Fig. 8B).
  • the H4/CD4 cell line is a human glioma cell line which has been engineered to express CD4, making it susceptible to infection with HIV-1.
  • An experiment to determine whether H4/CD4 can support a productive HIV-1 infection in the absence of the Vif protein was conducted.
  • 5 x 10 4 H4/CD4 cells were transfected with 5 ⁇ g of either full-length wild-type HIV-1 proviral DNA or a mutant Vif (vif) proviral DNA ( ⁇ Vif-NL4-3) by CaPO 4 precipitation.
  • ⁇ Vif-NL4- 3 contains a 35 bp deletion in Vif (Simm et al., J. Virol.69:4582-4586, 1992).
  • This virus was made by exchanging the Vif open reading frame in NL4-3 with the corresponding open reading frame from N1T-E (Sakai et al., J. Virol.65:5765- 5773, 1991), which is the natural clone carrying this deletion in Vif.
  • Cells were cultured in DMEM medium with 5% FCS and antibiotics, and tested on the designated days post-infection for the expression of HIV- 1 antigens by indirect immunofluorescence staining using an AIDS patient serum (Table 2).
  • transfected cells and culture supernatants were evaluated from their infectivity by cocultivation and cell-free virus infection. 14 days after transfection, samples of transfected cells and supernatants were collected for the evaluation of virus progeny infectivity. Virus in culture supernatants used for infection was standardized by p24 content; infectious dose was 1 ⁇ g p24/10 6 cells. Cocultures were set up at a 1 :10 ratio of infected to uninfected cells for vif transfectants and at a 1 :50 ratio for wild-type transfectants, to account for the difference in the % HIV- 1 antigen positive cells (Table 3).
  • Infected/cocultivated cells were evaluated for HIV-1 replication by testing the proportion of HIV- 1 antigen expressing cells at the designated times post- infection by an indirect immunofluorescence assay using an AIDS patient serum. It was clear that the replication of Vif-mutant HIV in H4/CD4 cells results in the production of progeny virus that is noninfectious in H4/CD4 cells by both cocultivation and cell-free virus transmission assays (Table 3).
  • HIV-1 vz/mutant cell-free virus ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 ⁇ 0.1 DNA-transfected infection H4/CD4
  • Mutated DNAs constructed for the experiment included a NL4-3 provirus in which both cysteines in Vif were replaced with leucines (Cysvif) resulting in the synthesis of full-length nonfunctional Vif that does not support production of infectious virions, and a provirus which encodes a truncated Vif protein, ⁇ Vif, described in Example 1, infra. (Simm et al., J. Virol. 69:4582-4586, 1995). Transfections were performed using 1 ⁇ g/DNA per 10 6 cells.
  • Vif-derived protease inhibitor peptides were manually assembled on a Boc-Val-PAM resin according to the HBTU activation in situ neutralization protocol for BOC solid phase peptide synthesis (Barany and Merrifield in The Peptides: Analysis. Synthesis. Biology. Gross et al., eds., Vol. 2, pp. 1-225,
  • the purified material was deemed to be >98% homogenous as indicated by both analytical HPLC and electrospray mass spectrometry.
  • Peptides were designed from the amino acid sequence of HIV-1/NlT-A (Sakai et al., J. Virol. 65:5765- 5773, 1991). The sequence and location of the peptides is shown in Table 5.
  • peptides were synthesized according to the SPPS method, using an Applied Biosystems Peptide Synthesizer, model 433A on Rink amide resin (loading 0.5 mmol/g) by standard Fmoc chemistry. They were cleaved from the resin with trifluoroacetic acid (TFA) containing 5% anisole as a scavenger, precipitated from cold ether, dissolved in water and lyophilized. Crude peptides were analyzed by reverse-phase HPLC [C3 column 5-60% acetonitrile : water gradient containing 0.1% TFA, 45 min] and characterized by TOF-MS and amino acid analysis. TABLE 5
  • TFA trifluoroacetic acid
  • Protein A agarose beads were saturated with rabbit anti-PR antiserum, washed extensively with 0.1 M phosphate buffer, pH 8.5 (PB) and 250 ⁇ l of 50% suspension beads in PB were mixed with PR and washed with PBS. The suspended beads were divided into 4 equal parts and into each the following proteins were added: GST Vif, GST N'Vif, GST CNif and GST folistatin at 60 ng per ml, pH 8.5, and immune complexes allowed to form for 1.5 h at 4° C.
  • the beads were washed three times in PBS, pelleted, mixed with 50 ⁇ l Laemmli buffer, boiled, and proteins were analysed by electrophoresis and Western blot, followed by staining with rabbit anti-GST antiserum with detection as described above.
  • Fig. 9B is a repeat of the same experiment showing that ⁇ Nif bound to the anti-PR immunoadsorbent in the presence, but not the absence of PR. The results lead to a conclusion that ⁇ Nif directly binds PR, an activity which may account for its ability to block PR-mediated proteolysis.
  • a proteolysis reaction was set up consisting of 0.5 ⁇ pepsin in 50 mM ⁇ a acetate pH 3.5, 0.1 M ⁇ aCl, 1 mM decapeptide protease substrate corresponding to the HIV-1 MA/CA cleavage site in the presence or absence of 1 mM Vif derived peptides.
  • the mixture was incubated at 37 °C for 30 minutes and was stopped by addition of guanidium chloride to a final concentration of 6 M.
  • the reaction products were analysed by reverse phase HPLC and the extent of cleavage in the presence of Vif peptides is expressed relative to cleavage in the absence of Vif peptides.
  • Pepstatin A was used as a control inhibitor of pepsin.
  • Vif-derived peptides inhibit HIV-1 PR activity.
  • a set of partially overlapping peptides were synthesized, based on the sequence of HIV-1 BH10 Vif (Simon et al., JNirol.
  • the IC 50 of Peptide 4 was in the range of 230-250 ⁇ M, whereas the IC 50 value of Peptides 6 and 7 was in the order of 110 and 25 ⁇ M, respectively.
  • Peptides 41 and 43 each composed of 15 amino acid residues, were less active than Peptide 4 (Fig. 10C). Overlapping Peptides 6 and 7, containing the Arg-Lys-Lys motive, proved to be efficient inhibitors. However, none of these peptides was as active as N'-Vif (Kotier et al.,
  • Vif-derived peptides inhibit pepsin activity.
  • the inhibition of pepsin activity is shown in Figure 10D.
  • Peptides 4, 5, 412 and 413 exhibited significant inhibition of pepsin-mediated proteolysis.
  • Vif-derived peptides/ PR binding assay Each well of a 96 microwell ELISA plate (M 129B, Dynatech) was coated with 200 ⁇ l of a solution containing 20 ⁇ g/ml peptides in 100 mM Tris-HCI (pH 8.8) for 18 hr at 4°C. The wells were aspirated, incubated with low fat milk for 1 hr and washed with PBS containing 0.05% Tween 20. A volume of 200 ⁇ l containing 100 ng PR in 0.1 M NaCl and 50 mM sodium phosphate buffer (pH 7.4) was then added to each well and the microplates were incubated for 2 hr at room temperature.
  • the specific PR inhibitor Ro 31-5989 which blocks the active site of PR, was used as a control.
  • Preincubation of PR with the inhibitor reduced the binding of the enzyme to Vif-derived peptides (Fig. 12B), showing the specificity of the reaction.
  • Peptide 6 blocked the binding of PR to the cognate peptides more efficiently than did Ro 31-5989, whereas the PR inhibitor successfully competed with Peptides 41, 42 and 43.
  • Peptide 4 and its derivatives 41, 42 and 43 compete with Ro 31-5989 for the same site on the PR molecule.
  • the data suggest that Peptide 6 binds to a different site on PR, or that it binds to the same site with a higher affinity than does the commercial inhibitor.
  • Example 8 The results presented in Example 8 indicate that at least some of the interaction of Vif with PR is preserved in small peptides fragments of the N- terminal domain. It was reasoned that peptides of this size may be taken up by eucaryotic cells in culture, offering the means to test their activity of PR function in cells susceptible to HIV-1 infection. To maximize the resolution of PR activity, the vaccinia vector of HIV- 1 Gag-Pol, vVK-1, designed by Dr. B. Moss and colleagues (Karacostas et al., Proc. Natl. Acad. Sci. USA 86:8964-8967, 1989), was used.
  • vVK-1 infection of many human cell types results in high levels of Gag-Pol production within 24 h of infection and also permits the native PR-mediated processing of Gag-Pol and the assembly of virion-like particles which can be isolated from the cell supernatants.
  • the effects of Vif derived peptides on Gag-Pol processing were tested in two human cell lines, Hut-78 and CEM, which are T leukemia cell lines, and phytohemagglutinin stimulated peripheral blood lymphocytes (PBL). PBL have been shown to be the cell type most dependent upon Vif for productive HIV-1 infection. Cell lines were obtained from the ATCC.
  • CV-1 monkey kidney cells were cultured in DMEM supplemented with 5% fetal calf serum (FCS) as monolayers.
  • Hut-78 and CEM human T leukemia cells were cultured in suspension in RPMI 1640 supplemented with 10% FCS.
  • Peripheral blood was obtained from healthy donors by venipuncture and lymphocytes were isolated by Ficoll-Paque density sedimentation.
  • Peripheral blood lymphocytes (PBL) were cultured in RPMI supplemented with 10% FCS and 5 ⁇ g per ml phytohemagglutin.
  • Vaccinia virus expressing HIV-1 gag-pol, vVK-1 was kindly supplied by Dr. B. Moss (NIH).
  • NHI Newcastle disease virus
  • subconfluent CV-1 cultures were infected with vVK-1 at an MOI (multiplicity of infection) of 1.
  • MOI multipleplicity of infection
  • cells were cultured for 24-48 hours prior to harvest of virus.
  • CV-1 cells and culture supernatants were collected and sonicated to prepare virus stocks.
  • T cell lines or PBL cultures were infected with vVK-1 at an MOI of 5 in the presence of the designated peptide at 100 ⁇ g/ml.
  • One hour after exposure to virus cells were washed and then were incubated in complete medium containing the same concentration of peptide for 24 h prior to harvesting the cells and medium for HIV-1 protein expression.
  • Extracts prepared from vVK-1 -infected Hut 78 and CEM cells incubated for 24 hr with Peptides 42 and 43 contained the same HIV-1 polyproteins present in the control vVK-1 infected cells. However, lysates of cells incubated with Peptides 41, 6 and 7 contained the unprocessed MA-CA and p55 Gag polyproteins and only small amounts of mature CA protein (panels A and C).
  • Example 10 Vif-Derived Peptides Reduce The Production of Infectious Viruses
  • Peptides 41, 6 and 7 may have caused undetectable inhibition of Gag and Gag-Pol autoprocessing in the cells, which was sufficient to interfere with assembly and/or release of particles, as shown previously for other PR inhibitors (Kaplan et al., JNirol. 67:4050-4055, 1993; Kaplan et al., JNirol. 67:6782-6786, 1994).
  • a quantity of 7.5x10 4 chronically infected Hut 78 cells/well (5 days postinfection) were cultivated for 9 days in a 96-well-microwell-plate in 200 ⁇ l of RPMI 1640 containing 100 ⁇ g/ml of Vif-derived peptides.
  • Half of the culture medium was replaced daily with fresh medium containing 50 ⁇ g/ml of the tested peptide.
  • Aliquots removed at the indicated days were quantified for p24 CA antigen, using a Vironostika HIV-1 Antigen Microelisa System (Organon Teknika, USA).
  • Figure 15A shows that prolonged treatment of HIV-l ⁇ B chronically infected Hut 78 cells with Peptides 41 and 6, but not with Peptides 42 and 43, caused a reduction in the total amount of particulate and soluble p24 antigen present in the culture media. Similarly, treatment of newly infected Hut 78 cultures (at an HIV-l ⁇ B chronically infected Hut 78 cells with Peptides 41 and 6, but not with Peptides 42 and 43, caused a reduction in the total amount of particulate and soluble p24 antigen present in the culture media. Similarly, treatment of newly infected Hut 78 cultures (at an HIV-
  • the data indicates that Peptides 41 and 6, and to a lesser extent Peptides 43 and 7, interfere with the autoprocessing of viral precursors in human cells infected with vVK-1 or HIV-1 and cause a significant reduction in the number of infectious virions released from the HIV-1 infected cells.
  • PBL were stimulated by cultured with phytohemagglutin-interleukin 2 and were infected with 0.1 pg p24 per cell HIV-/ ADA. After infection, cells were cultured with 100 ⁇ g Vif peptide A (residues 21-65 HIV-1/NlT-A Vif), Vif peptide B (residues 41-65 N1T-A Vif), control peptide, or 1 ⁇ M Ro 31-8959
  • HIV- 1 infection was monitored by measurement of extracellular core antigen p24by ELISA on days 3, 7 and 10 after infection.
  • PBL were also cultured, infected, and exposed to peptides as described above, except that graded doses of peptides were used.
  • the Vif peptide employed was N1T-A Vif residues 41-65 (Peptide B). HIV-1 infection was monitored by measurement of extracellular p24 on days 7, 9 and 12 after infection.
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 212-705-5000 (B) TELEFAX: 212-705-5020

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Abstract

L'invention porte sur de nouveaux inhibiteurs d'une protéase du VIH ou d'autres protéases lentivirales ou rétrovirales capables d'inhiber la réplication du VIH et des lentivirus et rétrovirus, ce qui, à ce titre, les rend capables de réduire, supprimer ou prévenir les infections virales. Lesdits inhibiteurs sont des peptides et des polypeptides présentant une séquence d'acide aminé correspondant à celle de la protéine lentivirale Vif (inhibitrice de la protéase dérivant de la Vif). L'invention porte également sur des compositions contenant les inhibiteurs de l'invention et sur leurs procédés d'utilisation pour la prévention ou le traitement des infections par le VIH ou les lentivirus ou les rétrovirus; elle porte en outre sur des procédés d'identification desdits inhibiteurs dans des systèmes de modèles de cellules, dans des cellules infectées par le VIH, dans des systèmes d'essais in vitro à flux intense, et dans des systèmes de modèles animaux.
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WO2014066382A2 (fr) * 2012-10-22 2014-05-01 University Of Rochester Dosage pour le criblage de composés antiviraux qui inhibent les interfaces d'interaction spécifique entre la culline 5 et un complexe d'élongine b/élongine c/cbf-bêta/vif du vih-1

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