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• W2090828745 abstract "Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-Å resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 Å with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C.(1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The Cα tracings of the two native structures are identical with a root-mean-square deviation of 0.5 Å, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (~2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 Å) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding. Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-Å resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 Å with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C.(1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The Cα tracings of the two native structures are identical with a root-mean-square deviation of 0.5 Å, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (~2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 Å) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding. INTRODUCTIONThe emergence of drug resistance remains a major bottleneck in the pursuit of a long lasting, antiviral treatment against AIDS(1Richman D.D. Nature. 1995; 374: 494Crossref PubMed Scopus (28) Google Scholar). When faced with selective pressure of an inhibitor, some 20 of the 99 amino acid residues of the HIV protease undergo mutations(2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar, 3Husson R.N. Shirasaka T. Butler K.M. Pizzo P.A. Mitsuya H. J. Pediatr. 1993; 123: 9-16Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 4Richman D.D. Broder S. Merigan T.C. Bolognesi D. Textbook of AIDS Medicine. Williams and Wilkins, Baltimore1994: 795-805Google Scholar). Sequence analyses of virus isolates from patients participating in clinical trials have revealed that various amino-acid substitutions in the HIV 1The abbreviations used are: HIVhuman immunodeficiency virus4Xthe quadruple variant of HIV-1 protease containing the mutations M46I, L63P, V82T, and I84Vr.m.s.root mean squareDTTdithiothreitolMES2-(N-morpholino)ethanesulfonic acid. protease, in combination with as many as 10 or more residues, are associated with the decrease in antiviral efficacy of the protease inhibitor MK639 upon its prolonged usage in the course of 1 year (2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar) (Fig. Z1).MK639 (previously L-735,524) is an inhibitor of peptide cleavages catalyzed by HIV-1 and HIV-2 proteases with Kivalues of 0.38 and 2.48 nM, respectively. It is effective against HIV replication in cell culture and is efficacious in reducing viral load in carriers of HIV-1(5Vacca J.P. Dorsey B.D. Schlief W.A. Levin R.B. McDaniel S.L. Darke P.L. Zugay J. Quintero J.C. Blahy O.M. Roth E. Sardana V.V. Schlabach A.J. Grahan P.I. Condra J.H. Gotlib L. Holloway M.K. Lin J. Chen I.-W. Vastag K. Ostovic D. Anderson P.S. Emini E.A. Huff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4096-4100Crossref PubMed Scopus (552) Google Scholar). Analyses of mutations in the HIV provirus have revealed (2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar) that as few as four amino acid substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) 2Amino acid residues of the protease are designated with the single-letter code, numerically for one subunit and numerically with a prime for the second subunit when a distinction between the two subunits is necessary. suffice to yield cross-resistance to a panel of protease inhibitors either in or being considered for clinical trials, including MK639. To aid in the design of inhibitors able to evade viral resistance, it is important to understand the structural basis of resistance. We have previously determined (6Chen Z. Li Y. Chen E. Hall D.L. Darke P.L. Culberson C. Shafer J. Kuo L.C. J. Biol. Chem. 1994; 269: 26344-26348Abstract Full Text PDF PubMed Google Scholar) the x-ray crystallographic structures of HIV-1 and HIV-2 proteases complexed to MK639 at 2.0- and 1.9-Å resolution, respectively. Although four active-site residues (V32I, I47V, L76M, V82I) are altered between the two enzymes, there are only subtle changes in the binding mode of the inhibitor, suggesting that the structural basis of resistance may not be discernible until the degree of resistance becomes greater.In this work, the native and bound structures of a mutant HIV-1 protease, with a 70-fold change in affinity (Ki = 26 nM) toward MK639, have been determined. The observed structural features of the MK639-bound mutant and wild-type proteases are consistent with the extent of resistance raised against MK639.MATERIALS AND METHODSThe pET-3b-HIVPR plasmid containing the synthetic gene for HIV-1 protease in the high expression vector pET3b was provided by Dr. Jordan Tang, Oklahoma Medical Research Foundation(7Ido E. Han H. Kezdy F.J. Tang J. J. Biol. Chem. 1991; 266: 24359-24366Abstract Full Text PDF PubMed Google Scholar). A 354 base pair cassette was designed based on the wild type sequence of pET-3b-HIVPR with appropriate nucleotide changes at positions 171 (G → C), 221 (T → C), 277 (T → A), 278 (T → C), 283 (A → G), and 285 (C → T) within the 333-base pair gene to yield the quadruple (4X) mutant protease (M46I/L63P/V82T/I84V). The 4X cassette was subcloned into the unique restriction sites Bpu1102I and BstEII within pET-3b-HIVPR to produce pET-3b-HIV1PR-4X. Sequence-verified clones were transformed into competent Escherichia coli BL21(DE3)pLysS (Ido and Tang cured) host cells(8Chen E. Methods Enzymol. 1994; 241: 29-46Crossref PubMed Scopus (6) Google Scholar). Cells were cultured, induced, and lysed as described(7Ido E. Han H. Kezdy F.J. Tang J. J. Biol. Chem. 1991; 266: 24359-24366Abstract Full Text PDF PubMed Google Scholar). Enzyme purification, activity and inhibition assays were conducted as described previously (7Ido E. Han H. Kezdy F.J. Tang J. J. Biol. Chem. 1991; 266: 24359-24366Abstract Full Text PDF PubMed Google Scholar, 9Darke P.L. Jordan S.P. Hall D.L. Zugay J.A. Shafer J.A. Kuo L.C. Biochemistry. 1994; 33: 98-105Crossref PubMed Scopus (71) Google Scholar).Crystallization of the mutant protease was accomplished at 4°C with use of the vapor diffusion method. The enzyme was prepared at 15 mg/ml in a pH 5 solution containing 10 mM MES, 1 mM EDTA, 1 mM DTT. The reservoir solution contained 0.6 M NaCl, 1 mM DTT, 3 mM NaN3, and 0.1 M imidazole at pH 7. Hanging drops were prepared by mixing the protein and reservoir solutions in 1:1 (v/v) ratio. Tetragonal bipyrimidal crystals (0.9 mm × 0.4 mm × 0.4 mm) of the mutant protease were obtained in 5-10 days. The cell constants were a = b = 49.98 Å, c = 108.1 Å with 1 dimer/asymmetric unit. A 2.4-Å resolution diffraction data set was collected on a RAXIS II imaging plate using CuKα irradiation (Rigagu RU200 rotating anode) generated at 50 kV and 100 mA. The data set, showing clearly that the space group was P41 and not P4122, encompassed 29,001 measurements with 8,651 unique reflections (82.3% completeness) and an Rmerge ([(Σ(I - 〈I〉) ÷ΣI]) of 3.3%. The coordinates (10Spinelli S. Liu Q.Z. Alzari P.M. Hirel P.H. Poljak R.J. Biochimie. 1991; 73: 1391-1396Crossref PubMed Scopus (182) Google Scholar) of the wild-type HIV-1 protease were used as the molecular model. The initial R factor, defined as Σ(∣Fo - Fc∣) ÷Σ∣Fo∣, was 0.308. Using the X-PLOR program package(11Brunger A.T. Kuriyan K. Karplus M. Biochimie Sci. 1987; 235: 458-460Google Scholar), rigid body refinements of the two monomers alternatively of the protease reduced the R value to 0.239 using the data in the 8-Å to 3.5-Å resolution range. Another cycle of X-PLOR refinement further reduced the R value to 0.226 at 2.4-Å resolution. A 2Fo - Fc map was calculated, and a mutant protease model was built by employing the program CHAIN on a Silicon Graphics system. The model was further refined; the final model included 82 solvent molecules with an R factor of 0.171 for a total of 8,101 reflections. The RF was 0.312, and the r.m.s. deviations, from ideal values of bond and angles, were 0.016 Å and 1.9°, respectively.Crystals of the 4X protease complexed with MK639 were obtained at ambient temperature in hanging drops under vapor diffusion conditions against a solution of 0.1 M NaAc, pH 5.4, 0.5 M NaCl, 1 mM DTT, and 3 mM NaN3. The cell constants of the crystals were a = 60.06 Å, b = 86.90 Å, and c = 46.68 Å in the space group P21212. There was one protease dimer per asymmetric unit. The diffraction data, collected as described for the native enzyme and extending to 2.0-Å resolution, included 41,085 measurements with 13,590 independent reflections (86% completeness) and an Rmerge of 5.02%. The structure of the inhibitor-bound HIV-1 mutant protease was determined with the difference Fourier method employing the structure of the HIV-1 protease MK639 as the model. The final model included 109 solvent molecules and an R factor of 0.170 for reflections in the 6-Å to 2.0-Å resolution. The Rmerge was 0.255. The r.m.s. deviations of atoms of the structure from ideal values of bond length and angle were 0.015 Å and 1.8°, respectively. The inhibitor MK639 was found to bind the mutant HIV-1 protease in a single orientation.RESULTS AND DISCUSSIONFig. 1 depicts in stereoview the Cα tracings of the wild-type HIV-1 protease and its quadruple (4X) mutant. The r.m.s. deviation for all 198 Cα atoms between the 4X and the wild-type protease is 0.5 Å. 3This value is well within the average r.m.s. found for the protease x-ray structure determined independently in different laboratories(12Wlodawer A. Erickson J.W. Annu. Rev. Biochem. 1993; 62: 543-585Crossref PubMed Scopus (801) Google Scholar). The largest deviations in the Cα position of these two molecules occur at the tip and in the hinge of the flap loops. 4The displacements in the position of Cα atoms in the flap loops of the 4X mutant from those of the wild-type protease are: Pro-39 (0.98 Å), Pro-39′ (0.93 Å), Gly-40 (0.54 Å), Gly-40′ (1.19 Å), Gly-49 (1.50 Å), and Gly-49′ (1.27 Å). In contrast, the deviations for the four substituted residues are less drastic: Met-46 (0.66 Å), Met-46′ (0.51 Å), Leu-63 (0.74 Å), Leu-63′ (0.47 Å), Val-82 (0.62 Å), Val-82′ (0.78 Å), Ile-84 (0.31Å), and Ile-84′ (0.21 Å). The substituted side chains, Ile-46, Pro-63, Thr-82, and Val-84, are highlighted for both subunits of the enzyme as dictated by the 2Fo - Fc electron density map. Residues 82 and 84 are located in the active site of the enzyme. Residue 46 is in the flexible flap loop of the enzyme, while residue 63 is near the hinge region of the flap; both side chains point toward the bulk solvent.Figure 1:Stereo tracings of the Cα backbones of the wild-type HIV-1 protease (green) and its quadruple mutant (pink) as determined by x-ray diffraction data at 2.4-Å resolution. The mutated side chains M46I, L63P, V82T, and I84V are shown in yellow for both subunits of the enzyme. Only residues 82 and 84 of both subunits are directly involved in inhibitor binding.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Unlike the wild-type HIV-1 proteases, the two subunits of the 4X mutant are not related by a 2-fold crystallographic axis in the asymmetric unit. Nonetheless, the r.m.s. deviation for the Cα atoms of the two subunits is relatively small (0.37 Å). The greatest difference between the subunits lies mainly in the flap loop and in the peptide chains containing residues 15-20 and 64-72. 5For example, the largest displacements in the Cα positions between the two subunits in these regions are: Pro-39 (0.99 Å) and Gly-50 (1.11 Å) in the flap loop, Gly-17 (0.68 Å) in the 15-20 peptide segment, and Gly-68 (0.89 Å) in the 64-72 peptide segment. By excluding residues 15-20, 34-40, 48-52 and 64-72, the r.m.s. deviation for the Cα atoms in the two subunits is further reduced to 0.25 Å. For the four substituted residues between the subunits, the displacements of the Cα atoms are 0.2 Å for Met-46, 0.31 Å for Leu-63, 0.2 Å for Val-82, and 0.18 Å for Ile-84.Fig. 2 illustrates the electron density of the quadruple mutant HIV-1 protease surrounding residues 46 and 63 as defined by x-ray diffraction. It reveals clearly that residues 46 and 63 of the mutant enzyme are isoleucine and proline as dictated by its DNA sequence. Similar unambiguous electron density definitions are found for V82T and I84V (electron density not shown).Figure 2:The 2Fo - Fc electron density map, contoured at 1 σ level, of the quadruple mutant HIV-1 protease surrounding (A) residue 46 and (B) residue 63. The refined model of the mutant enzyme is shown in green and that of the wild-type HIV-1 protease is in red. The maps show that residues 46 and 63 of the mutant enzyme are, respectively, Ile and Pro as predicted by DNA sequencing of the mutant plasmid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3 shows a view down the two-fold axis of the active site of HIV-1 protease complexed with MK639. The r.m.s. deviation for all Cα atoms between the 4X and the wild-type protease in the bound mode is now only 0.2 Å. For clarity, the flaps of the enzyme are only partially included in this figure. Residues Val-82 and Ile-84 are highlighted in thick pink lines. The Ile-84 and Ile-84′ are symmetry-related by a two-fold rotation but Val-82 and Val-82′ are only pseudo symmetrically related because their propyl side chains are oriented differently about the Cα-Cβ bond. The dashed lines mark the distances, within van der Waals radii, between atoms of the inhibitor and the CD1 atoms of Ile-84/Ile-84′, as well as the CG2 and CG1 atoms of Val-82 and Val-82′, respectively. Related by a pseudo two-fold rotation, the t-butyl and indanyl groups of MK639 are bound in the S2 and S2′ pockets of the enzyme. Correspondingly, the pyridyl methyl piperidine and benzyl rings of the inhibitor are situated in the S3/S1 and S1′ pockets. A bound water molecule is seen to be cushioned tetrahedrally between the tips of the flap loops (Ile-50 and Ile-50′) and the 2 amide oxygen atoms of the inhibitor. (A second bridging water molecule is found between the N2 amide nitrogen of the inhibitor and the carboxyl oxygen of Asp-29′.) Not seen in Fig. 3 are interactions of the hydroxyethylene group of the inhibitor hydrogen bonded to Asp-25 and Asp-25′ (beneath the inhibitor structure). Altogether, seven atoms of MK639 are within hydrogen bonding distances from atoms of the enzyme, either directly or indirectly. These interactions have been reported in detail (6Chen Z. Li Y. Chen E. Hall D.L. Darke P.L. Culberson C. Shafer J. Kuo L.C. J. Biol. Chem. 1994; 269: 26344-26348Abstract Full Text PDF PubMed Google Scholar) for the HIV-2 protease. For comparison, the conformation of MK639 (as seen in the active sites of HIV-1 and HIV-2 protease) are shown in Fig. 4A.Figure 3:Stereo diagrams of the active site of HIV-1 protease complexed with MK639 (previously L-725,524) as defined by x-ray diffraction data at 2.0-Å resolution. The inhibitor is depicted in green and the protease in thin purple wire. Residues of Ile-50, Ile-50′, Val-82, Val-82′, Ile-84, and Ile-84′ are highlighted in thick pink lines. The dash lines mark the distances within van der Waals contacts between atoms of the inhibitor and the CD1 atoms of Ile-84/Ile-84′, the CG1 atom of Val-82′, and the CG2 atom of Val-82. Stick colored in yellow represents the positions of the side chains of Thr-82, Thr-82′, Val-84, and Val-84′ of the mutant protease in the MK639 complex as defined by its electron density seen at 2.0-Å resolution and by super-posing the Cα backbone of the MK639-bound mutant enzyme onto that of the wild type HIV-1 protease. The wild-type CG1 atom of Val-82′ is now the mutant OG1 atom of Thr-82′ introducing an unfavorable interaction with the inhibitor. The mutant OG1 atom of Thr-82 points away from the plane of the stereoview and is shielded in this diagram; but its CG2 atom is still within van der Waals distance (3.58-3.72 Å) from the phenyl group of MK639 as opposed to a distance of 3.54-3.70 Å seen for the corresponding wild-type atom.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Comparison of the binding conformation of MK639 as seen in the active sites of (A) the HIV-1 (green) and HIV-2 (yellow) proteases (see (6Chen Z. Li Y. Chen E. Hall D.L. Darke P.L. Culberson C. Shafer J. Kuo L.C. J. Biol. Chem. 1994; 269: 26344-26348Abstract Full Text PDF PubMed Google Scholar) for details) and (B) the HIV-1 protease (green) and the 4X mutant (purple) as defined by x-ray diffraction data at 2-Å resolution. The inhibitors were superimposed with the matrices derived by the least square fitting of all Cα atoms of each pair of the proteases. For clarity, the proteins are omitted in the stereoviews.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Fig. 3 are also shown thick lines colored in yellow representing the positions of the side chains of Thr-82, Thr-82′, Val-84, and Val-84′ in the MK639-complexed 4X mutant protease, as dictated by its electron density and by super-positioning its Cα backbone onto that of the MK639-complexed wild-type HIV-1 protease. Both mutations in the active site introduce small alterations (~0.3 Å) in their side chain positions. Most importantly, the V82T substitution, while isosteric, introduces an unfavorable hydroxyl moiety (OG1) at what was previously (wild type) the CG1 position of Val-82′ in the S1 pocket, within van der Waals distance (3.39 Å) to the pyridyl methyl piperidine group of MK639. The corresponding substitution (OG1 of V82T) in the S1′ site points away from the inhibitor and thus does not directly impact on binding because its CG2 atom is still within van der Waals distance (3.58-3.72 Å) from the phenyl moiety. The I84V and I84V′ substitutions, on the other hand, symmetrically create in both the S1 and S1′ pockets a small void in bulk (~25 Å3) that can be expected to lead to a decrease in van der Waals interactions with the piperidine group and with the benzyl moiety of the inhibitor, respectively. 6The cavity generated by the I84V mutation is too small to be occupied by a solvent molecule. There is no electron density seen to indicate any partial occupancy. In an apparently unsuccessful attempt to fill this void, the CD1 atom of Ile-50′ from the tip of the flap domain relocates by 1.29 Å toward this pocket by rotating −35° about the Cα-Cβ bond and 170° about the Cβ-Cγ bond; however, a similar change is not seen for Ile-50. The structures of the bound MK639 in the active sites of the wild-type and 4X mutant proteases are shown in Fig. 4B, revealing that the binding mode of MK639 is essentially unchanged in the active site of the two enzymes. Together, these results suggest that resistance against inhibitor binding, in the case of MK639, is caused by subtle changes of the substituted side chains (the introduction of an isosteric but unfavorable hydrophilic group and the introduction of a smaller side chain to decrease van der Waals contacts) rather than repositioning the bound inhibitor (due to the introduction of a spatially hindering, larger side chain). The side-chain positions of Thr-82 and Val-84 are also not different in the open and closed forms of the mutant protease (data not shown), indicating that these residues are not perturbed, within a r.m.s. deviation of ~0.2 Å, upon binding of MK639. The observed changes in binding interactions of MK639 in the active sites of the 4X protease are consistent with an attenuated affinity of this inhibitor by a factor 70 (~2.5 kcal/mol), 7The energetic contribution of a methyl moiety to binding has been estimated to be ~0.7-0.8 kcal/mol(13Hansch C. Coats E. J. Pharmacol. Sci. 1970; 59: 731-743Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 14Andrews P.R. Craik D.J. Martin J.L. J. Med. Chem. 1984; 27: 1648-1657Crossref PubMed Scopus (455) Google Scholar). That of a hydroxyl group, approximated from the Gibbs energy of transfer from n-octanol to water, is ~1.6 kcal/mol(13Hansch C. Coats E. J. Pharmacol. Sci. 1970; 59: 731-743Abstract Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the combined contribution of the V82T (but not V82T′, see text), I84V, and I84V′ substitutions should account for a total loss of ~3 kcal/mol. This rough estimate is in reasonable agreement with the 70-fold drop in binding affinity (i.e. -RT•lnΔK = 2.55 kcal/mol). Also, repositioning of the Ile-50′ side chain may partially alleviate loss in hydrophobic interactions due to the I84V′ replacement. a value sufficient to render a potent protease inhibitor ineffective as an antiviral agent against the HIV(2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar).Whereas structural contributions of V82T and I84V toward resistance against MK639 are accountable with the diffraction data shown here, the role of the M46I and L63P substitutions is not obvious. These mutations away from the immediate vicinity of the active site induce significant changes in the Cα positions of residues in the flap domain of the enzyme (see text above related to Fig. 1 and Footnote 4), but the significance of these changes is unclear. However, preliminary kinetic results from our laboratory suggest that the combination of M46I and L63P mutations affords the protease greater catalytic efficiency than the native enzyme, and the V82T/I84V double mutations render the HIV-1 protease a very poor enzyme. 8H. B. Schock and L. C. Kuo, unpublished data. Thus, it may be that the M46I and L63P modifications, by introducing fine adjustments to the protease conformation, compensate dynamically for deleterious effects of the mutations (V82T and I84V) in the active site.Our observations are in contrast to those reported by Baldwin et al.(15Baldwin E.T. Bhat T.N. Liu B. Pattabiraman N. Erickson J.W. Struct. Biol. 1995; 2: 244-249Crossref PubMed Scopus (108) Google Scholar). These authors have observed rearrangements of the HIV-1 protease Cα backbone around residues 81-84 by up to 0.6 Å on binding of a symmetrical inhibitor, A-77003, when Val-82 is substituted by an alanine. These changes lead to a repacking of enzyme and inhibitor atoms in the S1 but not S1′ subsite in a manner that would diminish the potential loss of binding affinity. The remainder of the mutant protease complex is little altered from that of the wild-type enzyme (supporting our interpretation that the changes seen in the flap domain of the 4X mutant are due to the M46I and L63P substitutions). It is difficult to generalize at this time the effect of resistance mutations of the protease based on limited available data thus far. To further extend our understanding of the structural basis of drug resistance in the HIV-1 protease, we are pursuing determination of additional inhibitor-bound structures of the 4X HIV-1 protease INTRODUCTIONThe emergence of drug resistance remains a major bottleneck in the pursuit of a long lasting, antiviral treatment against AIDS(1Richman D.D. Nature. 1995; 374: 494Crossref PubMed Scopus (28) Google Scholar). When faced with selective pressure of an inhibitor, some 20 of the 99 amino acid residues of the HIV protease undergo mutations(2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar, 3Husson R.N. Shirasaka T. Butler K.M. Pizzo P.A. Mitsuya H. J. Pediatr. 1993; 123: 9-16Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 4Richman D.D. Broder S. Merigan T.C. Bolognesi D. Textbook of AIDS Medicine. Williams and Wilkins, Baltimore1994: 795-805Google Scholar). Sequence analyses of virus isolates from patients participating in clinical trials have revealed that various amino-acid substitutions in the HIV 1The abbreviations used are: HIVhuman immunodeficiency virus4Xthe quadruple variant of HIV-1 protease containing the mutations M46I, L63P, V82T, and I84Vr.m.s.root mean squareDTTdithiothreitolMES2-(N-morpholino)ethanesulfonic acid. protease, in combination with as many as 10 or more residues, are associated with the decrease in antiviral efficacy of the protease inhibitor MK639 upon its prolonged usage in the course of 1 year (2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar) (Fig. Z1).MK639 (previously L-735,524) is an inhibitor of peptide cleavages catalyzed by HIV-1 and HIV-2 proteases with Kivalues of 0.38 and 2.48 nM, respectively. It is effective against HIV replication in cell culture and is efficacious in reducing viral load in carriers of HIV-1(5Vacca J.P. Dorsey B.D. Schlief W.A. Levin R.B. McDaniel S.L. Darke P.L. Zugay J. Quintero J.C. Blahy O.M. Roth E. Sardana V.V. Schlabach A.J. Grahan P.I. Condra J.H. Gotlib L. Holloway M.K. Lin J. Chen I.-W. Vastag K. Ostovic D. Anderson P.S. Emini E.A. Huff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4096-4100Crossref PubMed Scopus (552) Google Scholar). Analyses of mutations in the HIV provirus have revealed (2Condra J.H. Schleif W.A. Blahy O.M. Gadryelski L.J. Graham D.J. Quintero J.C. Rhodes A. Robbins H.L. Roth E. Shivaprakash M. Titus D. Yang T. Teppler H. Squires K.E. Deutsch P.J. Emini E.A. Nature. 1995; 374: 569-571Crossref PubMed Scopus (934) Google Scholar) that as few as four amino acid substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) 2Amino acid residues of the protease are designated with the single-letter code, numerically for one subunit and numerically with a prime for the second subunit when a distinction between the two subunits is necessary. suffice to yield cross-resistance to a panel of protease inhibitors either in or being considered for clinical trials, including MK639. To aid in the design of inhibitors able to evade viral resistance, it is important to understand the structural basis of resistance. We have previously determined (6Chen Z. Li Y. Chen E. Hall D.L. Darke P.L. Culberson C. Shafer J. Kuo L.C. J. Biol. Chem. 1994; 269: 26344-26348Abstract Full Text PDF PubMed Google Scholar) the x-ray crystallographic structures of HIV-1 and HIV-2 proteases complexed to MK639 at 2.0- and 1.9-Å resolution, respectively. Although four active-site residues (V32I, I47V, L76M, V82I) are altered between the two enzymes, there are only subtle changes in the binding mode of the inhibitor, suggesting that the structural basis of resistance may not be discernible until the degree of resistance becomes greater.In this work, the native and bound structures of a mutant HIV-1 protease, with a 70-fold change in affinity (Ki = 26 nM) toward MK639, have been determined. The observed structural features of the MK639-bound mutant and wild-type proteases are consistent with the extent of resistance raised against MK639." @default.
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• W2090828745 title "Three-dimensional Structure of a Mutant HIV-1 Protease Displaying Cross-resistance to All Protease Inhibitors in Clinical Trials" @default.
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