Inhibition of HIV1 replication by a peptide dimerization inhibitor of HIV1 protease (original) (raw)
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Research in Virology, 1992
The processing of the human immunodeficiency virus (HIV) gag and gag-pol precursor proteins by the virus-encoded protease is an essential step in maturation of Infectious virus particles. Like most retroviral proteeses, the HIV protease belongs to the aspertyl-protease family and can be inhibited by specific inhibitors. Twenty-four synthetic peptides known to be inhibitors of human renin were tested for inhibition of HIV replication in tissue cultures. One of them, a synthetic peptide analogue, SR41476, which has been s~town to be a specific inhibitor of purified recombinant HIV 1 protease in vitro, totally blocked infection with different isolates including the HIV1 LAV prototype, the highly cytopathic Zairian isolate HIV1 NDK, and HIV2 ROD, both in primary blood lymphocytes (PBL) and in the lymphoid cell lines MT4 and CEM, for at least 3 weeks. It also significantly reduced virus replication in chronically infected CEM cells, without any effect on cell proliferation. Radioimmunoprecipitation assay revealed that the inhibitor blocked processing of polyprotein precursors p55 gag and p40 gag into a mature form of gag proteins, p25 and p18. Synthetic peptide analogue SR 41476, when added before infection, efficiently inhibited formation of HIV DNA provirus and successfully suppressed synthesis of HIV-specific proteins. These results imply that the HIV protease inhibitor not only inhibited virus maturation in the late phase of the HIV replication cycle, but also interfered in the eady phase, before the provirus was formed. This mechanism of antiviral activity provides new possibilities and strategies for AIDS chemotherapy.
Journal of the American Chemical Society, 1997
Agents have been designed and synthesized which target the dimerization interface of HIV-1 protease. These agents, which contain cross-linked peptides from the N-and C-termini of the protease, both inhibit HIV-1 protease activity and decrease the amount of protease dimer in solution as measured by size exclusion chromatography, protein crosslinking, and protease fluorescence studies. Additionally we have shown that active site-targeted agents inhibit HIV-1 protease activity but have little effect on protease dimerization. These data support the claim that inhibition with the crosslinked agents is based on a decrease in the amount of protease homodimer in solution which in turn is responsible for a decrease in the activity of the protease.
Proceedings of the National Academy of Sciences, 1996
Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit selfassociation of the defective monomers. Expression of the designed PR monomers inhibits activity ofwild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.
Synthetic “interface” peptides alter dimeric assembly of the HIV 1 and 2 proteases
Protein Science, 1992
Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N-and C-terminal residues of both monomers, which form a four-stranded 6-sheet. Peptides mimicking one 6-strand (residues 95-99), or two &strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIVl) interface were shown to inhibit the HIVl and 2 proteases (PRs) with ICso's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIVl P R dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIVl PR. Both one-and two-strand peptides alter the monomer/dimer equilibrium of both HIVl and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIVl and H1V2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one-and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.
Structural Basis of HIV-1 and HIV-2 Protease Inhibition by a Monoclonal Antibody
Structure, 2001
virus (HIV) are obtained by posttranslational cleavage of the Gag, Gag/Pol, and Env viral polyprotein precur-Institute of Molecular Genetics Academy of Sciences of the Czech Republic sors. The Gag and Gag/Pol polyproteins, in particular, are cleaved by a viral protease, which is itself contained Flemingovo nam. 2 166 37 Prague 6 within the Gag/Pol polyprotein chain. Since the demonstration that the human immunodeficiency virus prote-Czech Republic 2 Centre de Recherches sur les Macromolé cules ase (HIV Pr) is essential in the viral life cycle [1-3], this enzyme has become one of the primary targets for anti-Vé gé tales (affiliated with Université Joseph Fourier)-CNRS BP53 viral drug design. This has led to the development of many active-site inhibitors, some of which are currently F38041 Grenoble Cedex France in use as therapeutic agents for AIDS treatment [4]. In order to be proteolytically active, two protease monomers 3 European Synchrotron Radiation Facility BP220 must assemble into a homodimer, with each subunit contributing residues to the substrate binding pocket, F38043 Grenoble Cedex France including residue Asp-25, which is directly involved in the catalysis [5]. The requirement for protease dimeriza-4 Unité d'Immunologie Structurale (URA 1961 CNRS) tion to achieve proteolytic activity has led several authors to propose alternative noncompetitive inhibitors Dé partement d'Immunologie Institut Pasteur that would provoke the dissociation of the active homodimeric assembly [6-8]. 75724 Paris Cedex 15 France With the objective of probing the structural stability of HIV Pr and the eventual design of potential inhibitors of the enzyme that are directed to regions other than the active site, we have examined the effects of anti-Summary HIV-1 Pr monoclonal antibodies (mAbs) on the catalytic activity of the protease [9, 10]. We have recently reported Background: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is a preliminary study of mAb1696, which, although raised against the HIV-1 Pr, inhibits the catalytic activity of both essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The the HIV-1 and HIV-2 enzymes with inhibition constants of 0.6 nM and 1.5 nM, respectively, at pH 7.4 [11]. This murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the study also showed that mAb1696 cross-reacts with peptides containing the N terminus of the HIV protease. The catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nano-N-terminal region accounts for a large percentage of the interface between the two HIV Pr monomers because it molar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that interdigitates with the C-terminal segment from the other monomer, thus forming a 4-stranded intermolecu-is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimer-lar -pleated sheet in the active homodimer. We proposed that mAb1696 inhibits HIV Pr by perturbing the ization to the active enzymatic form. native structure of the enzyme at the dimer interface [11]. In addition, a clustering of negatively charged resi-Results: We report here the crystal structure at 2.7 Å resolution of a recombinant single-chain Fv fragment of dues at the antigen binding site was observed in the unliganded Fab crystal structure, which suggested that mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions electrostatic forces play an important role in the interaction between mAb1696 and HIV Pr. observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-To investigate further the mechanism of HIV Pr inhibition by mAb1696, we have now expressed a single-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. chain Fv fragment (scFv) in E. coli, which contains both 1696 variable domains joined by a flexible linking peptide. Such scFv constructs, which retain the full antigen Conclusion: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the binding capacities, are the object of very active research. Firstly, they are of interest for structural studies design of inhibitors aimed at binding inactive monomeric species. because they usually yield crystals diffracting to higher resolution than the corresponding Fab fragments on
Biochemical Society Transactions, 2007
Mutations that occur in response to the HIV-1 protease inhibitors are responsible for the development of multidrug cross-resistance to these antiproteases in AIDS treatment. One alternative to inhibiting the active site of HIV-1 protease is to target the dimer interface of the homodimeric enzyme at the antiparallel β-sheet formed by the interdigitation of the C- and N-ends of each monomer. This region is highly conserved and is responsible for approx. 75% of the dimer-stabilization energy. The strategies that have been used to design small molecules to target the interface antiparallel β-sheet have produced lipopeptides, guanidinium derivatives and peptides (or peptidomimetics) cross-linked with spacers. The mechanism of inhibition was determined using a combination of kinetic and biophysical methods. These dimerization inhibitors proved equally active in vitro against both wild-type and mutated proteases. They are therefore promising alternatives to active-site-directed inhibitors ...