An Orally Bioavailable HIV1 Protease Inhibitor Containing an Imidazole-Derived Peptide Bond Replacement: Crystallographic and Pharmacokinetic Analysis (original) (raw)
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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
Proceedings of the National Academy of Sciences, 1993
The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor [BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide] has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6%. The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees. The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites. The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A. The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases. The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures. The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30. The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.
European Journal of Biochemistry, 1994
We report comprehensive NMR studies in solution of the human-immunodeficiency-virus (H1V)-1 protease. Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323. A variety of triple-resonance experiments provided essentially complete 'H, 13C and I5N NMR signal assignments of the protease. These assignments, together with short-range NOE constraints, coupling constants and hydrogen-exchange data, yielded the secondary structure of the protease in solution. The results reported herein open the way to the determination of the high-resolution three-dimensional solution structures of proteasehnhibitor complexes, as well as to studies of protease dynamics and solvent interactions.
Biochemistry, 1993
A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the a-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptidebased irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs. Simian immunodeficiency virus (SIV) * is a retrovirus closely related to the type 2 human immunodeficiency virus (HIV-2) and more distantly related to human immunodeficiency virus type 1 (HIV-1). Infectious clones of a strain isolated from macaque monkeys (SIVmac239) also produce an AIDS-like diseaseinrhesusmonkeys (Kestler et al., 1990). Theseinfected monkeys provide an animal model for testing therapeutic agents targeting HIV-1 or HIV-2. The structure of SIVmaC protease (SIV PR) was determined to facilitate the incorporation of data from in vivo testing with efforts to improve the design of drugs targeting the HIV-2 and HIV-1 PRs. The residues in the binding pocket of SIV PR differ from those of HIV-1 PR in 3 of 13 positions identified as major peptide binding determinants in HIV-1 PR (Miller et al., 1989). Despite these differences, SIV PR is capable of authentically processing the HIV-1 p53gag polyprotein in vitro (Grant et al., 1991). The sequence of HIV-2 PR is identical to SIV PR at these 13 positions. It has been shown in uivo that thevirus can develop resistance to drugs targeting reverse transcriptase (Richman, 1993). It has been proposed that the virus could develop resistance to antiprotease drugs as well (Cameron et al., 1993). A comparison of the HIV-1, HIV-2, and SIV protease structures will identify regions that are structurally conserved and may
Two-step binding mechanism for HIV protease inhibitors
Biochemistry, 1992
Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-PheW [ CH(OH)CHzN]Pro-0-t-Bu (R diastereomer = 1R; S diastereomer = 1s) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu\k(CHzNH)Leu-Gln-Arg-NHz (2), (carbobenzyloxy)-P~~*[CH(OH)CHZN]P~O-O-~-BU (3), and pepstatin were determined by trapping free enzyme with 1R as 2,3, and pepstatin dissociated from the respective enzyme-inhibitor complex. Association rate constants of lR, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO~)-Gln-Arg-NH~ (4). The kinetic data for binding of 1s to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values. Human immunodeficiency virus (HIV) * protease is a 22-kDa dimer of two identical 11-kDa subunits (Meek et al., 1989). The protease maturation from the gag-pol polyproteingeneproduct is autocatalytic (Deboucket al., 1987). Since the protease is required for maturation of infectious HIV particles (Kohl et al., 1988; Peng et al., 1989; Gottlinger et al., 1989), it is a potential target for chemotherapeutic treatment of AIDS. Numerous peptide-based inhibitors and nonpeptide inhibitors have been synthesized [recently reviewed by Huff (1991) and Norbeck and Kempf (1991)l. Some of these compounds inhibit maturation of viral particles in vitro
Conformationally constrained HIV-1 protease inhibitors
Bioorganic & Medicinal Chemistry Letters, 1994
The synthesis and structure activity relationships of conformationally constrained analogs of the HIV-1 protease inhibitor L-685,434 are described. In addition, the X-ray crystal structure of a complex between L700,497 and the HIV-l protease is shown. The human immun~e~ciency virus type-l (HIV-l) protease plays a key role in the HIV viral life cycle by posttranslational processing of gag and gag-pal polypmteins into viral core components.1 Genetic inactivation of the protease resulted in the production of non-infectious virions in cell culture.2
Biochemistry, 1996
To gain greater understanding of the structural basis of human immunodeficiency virus (HIV) protease ligand specificity, we have crystallized and determined the structures of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant and simian immunodeficiency virus (SIV) protease in complex with SB203386, a tripeptide analogue inhibitor containing a C-terminal imidazole substituent as an amide bond isostere. SB203386 is a potent inhibitor of HIV-1 protease (K i ) 18 nM) but shows decreased inhibition of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant (K i ) 112 nM) and SIV protease (K i ) 960 nM). Although SB203386 binds in the active site cavity of the triple mutant in a similar fashion to its binding to the wild-type HIV-1 protease [Abdel-Meguid et al. (1994) Biochemistry 33, 11671], it binds to SIV protease in an unexpected mode showing two inhibitor molecules each binding to half of the active site. Comparison of these two structures and that of the wild-type HIV-1 protease bound to SB203386 reveals that HIV protease ligand specificity is imparted by residues outside of the catalytic pocket, which causes subtle changes in its shape. Furthermore, this work illustrates the importance of structural studies in order to understand the structure-activity relationship (SAR) between related enzymes.
Biochemistry, 1993
Simian immunodeficiency virus (SIV) proteins have considerable amino acid sequence homology to those from human immunodeficiency virus (HIV); thus monkeys are considered useful models for the preclinical evaluation of acquired immune deficiency syndrome (AIDS) therapeutics. We have crystallized apd determined the three-dimensional structure of SIV protease bound to the hydroxyethylene isostere inhibitor SKF107457. Crystals of the complex were grown from 25-32% saturated sodium chloride, by the hanging drop method of vapor diffusion. They belong to the orthorhombic space group 1222, with a = 46.3 A, b = 101.5 A, and c = 118.8 A. The structure has been determined at 2.5-A resolution by molecular replacement and refined to a crystallographic discrepancy factor, R ( =clpol -IFcll/clFol), of 0.189. The overall structure of the complex is very similar to previously reported structures of HIV-1 protease bound to inhibitors. The inhibitor is bound in a conformation that is almost identical to that found for the same inhibitor bound to HIV-1 protease, except for an overall translation of the inhibitor, varying along the backbone atoms from about 1 .O A at the termini to about 0.5 %L around the scissile bond surrogate.