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W2108093903 abstract "The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1–P4 residues. Both inhibitors bind in an extended backbone conformation, forming an anti-parallel β-sheet with one enzyme β-strand. The P1 residue contributes most to the binding energy, whereas P2–P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals. The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1–P4 residues. Both inhibitors bind in an extended backbone conformation, forming an anti-parallel β-sheet with one enzyme β-strand. The P1 residue contributes most to the binding energy, whereas P2–P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals. hepatitis C virus root mean square deviation high pressure liquid chromatography Hepatitis C virus (HCV)1infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals estimated at 100–200 million worldwide. Immunotherapy or other effective treatments for HCV infection are not yet available, and administration of interferon in combination with ribavirin has several limitations because of toxicity (1.Weiland O. FEMS Microbiol. Rev. 1994; 14: 279-288Crossref PubMed Scopus (23) Google Scholar). One of the best characterized targets for HCV therapy is the serine protease of NS3 protein. The NS3 protease domain constitutes the N terminus of the NS3 protein, which, when associated to the NS4A polypeptide, gets activated and therefore is responsible for maturation of the viral polyprotein (2.Bartenschlager R. Intervirology. 1997; 40: 378-393Crossref PubMed Scopus (53) Google Scholar). The structure determination of the HCV NS3 protease complexed with a truncated NS4A cofactor (residues 21–34) revealed a shallow, nonpolar P1 specificity pocket. Because of the unusual substrate specificity of this enzyme it has been inferred that the design of highly selective inhibitors that could bind to the NS3 protease would be unlikely (3.Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar,4.Yan Y. Li Y. Munshi S. Sardana V. Cole J.L. Sardana M. Steinkuehler C. Tomei L. De Francesco R. Kuo L.C. Chen Z. Protein Sci. 1998; 7: 837-847Crossref PubMed Scopus (263) Google Scholar). We have found that capped tri-peptide α-ketoacids, incorporating difluoro aminobutyric acid in the P1 position, are potent, slow binding inhibitors of this enzyme (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar). Their mechanism of inhibition is biphasic. The first kinetic phase involves the rapid formation of a noncovalent collision complex with association rate constants >0.2 s−1, and the second kinetic phase consists of a slow isomerization with rate constants between 5 × 10−3and 7.5 × 10−3 s−1. This results in the formation of a very tight complex with dissociation rate constants between 1.2 × 10−5 and 1.8 × 10−5s−1 and with half-lives of 11–16 h. The overallK i values are between 27 and 67 nm(5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar). The inhibitors described here span the P1–P4 residues and contain an activated carbonyl in an α-ketoacid moiety as the active-site serine trap. To investigate the binding mode of these compounds, an hexagonal crystal form of the NS3 protease domain (J strain) complexed with the truncated NS4A cofactor, amenable to soaking experiments, was obtained. The crystal structures of the noninhibited NS3/4A complex (2.4 Å) and with two inhibitors (Fig. 1 A), inhibitor I, α-ketoacid t, BOC-L, Glu-L, Leu-L(difluoro)aminobutyric acid (2.1 Å), and inhibitor II, α-ketoacid Z-L, Ile-L, Leu-L(difluoro)aminobutyric acid (2.4 Å), were solved. A DNA fragment encoding the serine protease domain of NS3J (amino acids 1–187) was obtained by polymerase chain reaction amplification of full-length cDNA and cloned in the pT7–7 vector. The NS3 protein was expressed inEscherichia coli BL21(DE3) as described previously (6.De Francesco R. Urbani A. Nardi M.C. Tomei L. Steinkühler C. Tramontano A. Biochemistry. 1996; 35: 13282-13287Crossref PubMed Scopus (85) Google Scholar). The purified protein was characterized by N-terminal sequencing, electrospray ionization mass spectrometry, dynamic light scattering, and HPLC activity assay. Results obtained from the N-terminal sequencing were confirmed by the molecular mass determined by electrospray ionization mass spectrometry (Met-NS3J (1–187)). Molecular size estimation, determined by light scattering, showed NS3J and NS3J/4A complex to have hydrodynamic radii (R H) of 2.6 and 2.2 nm, respectively, which correspond to estimated molecular masses of 29 kDa for NS3J and 20 kDa for the NS3J/4A complex, respectively. These results indicate a more compact protein state for the complex compared with NS3 alone and correlate with the activation of the protease because of complex formation with the cofactor, as determined by HPLC activity assay following the protocol previously described (7.Steinkühler C. Biasol G. Brunetti M. Urbani A. Koch U. Cortese R. Pessi A. De Francesco R. Biochemistry. 1998; 37: 8899-8905Crossref PubMed Scopus (218) Google Scholar). The kinetics of inhibition of the NS3 protease by the α-ketoacids were determined by stopped flow experiments, as described by Narjes et al. (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar). The NS3 protein (1 mg·ml−1) was incubated (4 °C) with the NS4A cofactor peptide, containing a solubilizing lysine tag at its N and C termini (KGSVVIVGRIILSGRK) at a molar ratio of 1:2 and concentrated by ultrafiltration to 290 μm. NS3J/4A crystals, with a maximum size of 0.6 × 0.3 × 0.2 mm3, were obtained by both “hanging” and “sitting drop” vapor diffusion methods, after 2 weeks at room temperature, with 3.4 m NaCl, 4.8 mmcyclohexyl-pentyl-β-d-maltoside, 5 mmdithiothreitol, and 0.02% NaN3 in 0.1 mcitrate buffer, pH 5.1. The ternary complexes with inhibitors were prepared by adding to the stabilized NS3J/4A crystals (in 4.5m NaCl, 10 mm dithiothreitol, 0.1 mcitrate buffer, pH 5.1), 5 mm inhibitor I, or 2.5 mm inhibitor II and equilibrated for 2–3 weeks before mounting. Inhibitor binding inside the crystal was confirmed by mass spectrometry. The x-ray diffraction data were collected at 100 K at beam lines X11 and X31 (EMBL, Deutsches Elektronen Synchrotron, Hamburg, Germany) and ID14/EH3 (European Synchrotron Radiation Facility, Grenoble, France), using 30% glycerol as cryoprotectant. Data were integrated and scaled with the HKL suite (8.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38287) Google Scholar) and with the CCP4 suite (9.Collaborative Computational Project, Number 4Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19676) Google Scholar). Crystals belong to the space group P61 with two molecules in the asymmetric unit. A summary of the diffraction data is presented in TableI. The noninhibited structure was solved by molecular replacement with AMoRe (10.Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar), using the NS3BK/4A coordinates as a search model, Protein Data Bank code 1JXP. The starting models of the inhibited structures were obtained by rigid body refinement of the refined native coordinates within AMoRe. In each case, the unique inhibitor was built into the initial, clearly interpretable 2F o − F cand F o − F c density maps. Refinement, using a maximum likelihood target function, was performed with REFMAC (11.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13735) Google Scholar). All data were used (no ς cutoff) from 20 Å to the high resolution limit of each data set (Table I). Modeling of solvent sites was executed with an automatic refinement program, ARP (12.Lamzin V.S. Wilson K.S. Methods Enzymol. 1997; 277: 269-305Crossref PubMed Scopus (278) Google Scholar). In the refinement, 5% of the data was set aside for use as a cross-validation set (13.Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3839) Google Scholar). Refinement was continued interspersed with manual model building with the program O (14.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13002) Google Scholar). The final statistics are given on Table I. The refined 2F o −F c electron density maps for the inhibitors I and II and for the glycerol molecule are shown in Fig.2.Table IData collection and refinement statisticsNative+Inhibitor I+Inhibitor IIData setaλ, data set 1, 0.91160 Å; data set 2, 0.9360 Å; data set 3, 1.1938 Å. a, b (Å)92.98094.35293.820 c (Å)81.80582.37480.950 Resolution (Å)bHighest resolution of data set with highest resolution bin in parentheses.2.4 (2.53–2.4)2.1 (2.14–2.1)2.4 (2.53–2.4) R merge(%)cR merge = ΣhklΣi=1N‖< I hkl > − I hkl‖ / ΣhklΣi=1N I ihkl.7.1 (35.5)3.5 (15.0)13.7 (42.5) <I/ςI>9.0 (2.2)32 (3.2)4.7 (1.7) Number of measurements126,23866,92455,295 Number of unique observations15,78623,96015,872 Completeness (%)100 (99.9)98.2 (98.4)99.8 (99.8)Refinement statistics Resolution (Å)20–2.420–2.120–2.4 R-factordR-factor = Σh ‖Fo − F c‖ / Σh ‖F o‖.0.1950.2070.215 R freeeR free is calculated from 5% of the data that were omitted during the course of the refinement.0.2980.2750.317 r.m.s.d. bond lengths (Å)fr.m.s.d. is the root mean square deviation from ideal geometry.0.0100.0100.009 r.m.s.d. bond angles (Å)0.0370.0300.032 Overall B-factor (Å2)46.7843.545.4 φψ angle distributiongAs defined by PROCHECK (25), the percentage distribution is given in parentheses.in core region261 (88.6)272 (91.3)266 (89.3)in additionally allowed region34 (11.4)25 (8.4)32 (10.7)in generously allowed region0 (0)0 (0)0 (0)in disallowed region0 (0)1 (0.3)0 (0) Number of atomsin structure2,9563,0222,908in NS3/4A2,7382,7382,738in inhibitor07074 Solvent21021396 Other (zinc ion and glycerol)2 (Zn ions), 6 (glycerol)1 (Zn ion)0a λ, data set 1, 0.91160 Å; data set 2, 0.9360 Å; data set 3, 1.1938 Å.b Highest resolution of data set with highest resolution bin in parentheses.c R merge = ΣhklΣi=1N‖< I hkl > − I hkl‖ / ΣhklΣi=1N I ihkl.d R-factor = Σh ‖Fo − F c‖ / Σh ‖F o‖.e R free is calculated from 5% of the data that were omitted during the course of the refinement.f r.m.s.d. is the root mean square deviation from ideal geometry.g As defined by PROCHECK (25.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), the percentage distribution is given in parentheses. Open table in a new tab The coordinates of the three structures have been deposited in the Protein Data Bank (accession codes 1dxp, 1dy8, and 1dyg). Two crystal forms of the NS3/4A complex (H and BK strains), have been reported (3.Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 4.Yan Y. Li Y. Munshi S. Sardana V. Cole J.L. Sardana M. Steinkuehler C. Tomei L. De Francesco R. Kuo L.C. Chen Z. Protein Sci. 1998; 7: 837-847Crossref PubMed Scopus (263) Google Scholar). Likewise, the NS3J/4A complex contains a C-terminal domain (residues 94–175) with a six-stranded β-barrel that ends with a C-terminal helix. The N-terminal domain (NS3 residues 1–93 and NS4A residues 21–34), also a β-barrel, contains eight β-strands, with one of the two additional strands contributed by the N-terminal region of the protease and the other by the NS4A cofactor (Fig. 1 B). The cofactor assumes an extended conformation forming main chain hydrogen bonds with the protease in an anti-parallel fashion. The N terminus is well ordered in both molecules of the asymmetric unit, and, as such, it is similar to the NS3BK/4A crystal structure (4.Yan Y. Li Y. Munshi S. Sardana V. Cole J.L. Sardana M. Steinkuehler C. Tomei L. De Francesco R. Kuo L.C. Chen Z. Protein Sci. 1998; 7: 837-847Crossref PubMed Scopus (263) Google Scholar). Conversely, in the NS3H/4A structure (3.Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar), only one N terminus is ordered. Both inhibitors bind at the active site (His57, Asp81, and Ser139) located at the crevice between the two domains and are rather solvent exposed (Fig. 1 B). In one of the two molecules of the asymmetric unit of the noninhibited enzyme, a glycerol molecule is bound at the oxyanion hole through a hydrogen bonding system (Fig. 3 A). This finding correlates with biochemical data showing glycerol-induced stabilization of NS3/4A complex (15.Urbani A. Biasol G. Brunetti M. Volpari C. Di Marco S. Sollazzo M. Orru‘ S. Dal Piaz F. Casbarra A. Pucci P. Nardi C. Gallinari P. De Francesco R. Steinkühler C. Biochemistry. 1999; 38: 5206-5215Crossref PubMed Scopus (32) Google Scholar). At the metal binding site, the Zn2+ion is present in the noninhibited structure (Fig. 3 B) and is coordinated to Cys97, Cys99, Cys145 and, via a water molecule, to the imidazole ring of His149. The electron density at this site is very well defined. On the contrary, in the inhibited structures, the metal ion is absent or present only with a low occupancy, and this site is quite disordered. The absence of Zn2+ is likely due to the presence of dimethyl sulfoxide (∼5%), necessary to dissolve the inhibitors. This is the only region where there is a large root mean square deviation (r.m.s.d.) for the C-α distance (∼0.6–1.0 Å) between the noninhibited and inhibited structures. Otherwise the r.m.s.d. is within the coordinate experimental error. Perturbations at the Zn2+ site do not affect the NS3/4A complex and the active site conformation in our crystal structures. This appears to be in conflict with folding studies (6.De Francesco R. Urbani A. Nardi M.C. Tomei L. Steinkühler C. Tramontano A. Biochemistry. 1996; 35: 13282-13287Crossref PubMed Scopus (85) Google Scholar). Although, as of today, it has not been formally shown that the removal of Zn2+ from the folded NS3 protease has an effect on the activity or the structure. The binding mode of the two inhibitors is equivalent, forming an antiparallel β-sheet with the protease, with one strand contributed by the inhibitor and one by the protease (Fig.1, B and C). Two pathways leading to different conformations (Fig. 4) are possible, depending on the stereochemistry of the nucleophilic attack at the carbonyl carbon of the ketoacid by the serine OH group. In the x-ray crystal structures of thrombin and trypsin complexed withp-amidinophenylpyruvate (16.Chen Z. Li Y. Mulichak A.M. Lewis S. Shafer J.A. Arch. Biochem. Biophys. 1995; 322: 198-203Crossref PubMed Scopus (46) Google Scholar, 17.Walter J. Bode W. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 949-959Crossref PubMed Scopus (65) Google Scholar) the attack is from there-side (Fig. 4, complex I). In the case of HCV NS3 protease, the carbonyl carbon of the inhibitor is attacked from thesi-side by the catalytic serine, forming a covalent bond (1.44 Å) with the inhibitor via the Oγ, resulting in the tetrahedral intermediate shown in Fig. 4 (complex II). The carbonyl oxygen does not point into the oxyanion hole, as observed for thrombin and trypsin (16.Chen Z. Li Y. Mulichak A.M. Lewis S. Shafer J.A. Arch. Biochem. Biophys. 1995; 322: 198-203Crossref PubMed Scopus (46) Google Scholar, 17.Walter J. Bode W. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 949-959Crossref PubMed Scopus (65) Google Scholar), instead it is hydrogen bonded to the Nε2 of His57. The two carboxyl oxygens point into the oxyanion hole: one forms a hydrogen bond with Ser139nitrogen, the other is hydrogen bonded with Gly137nitrogen. This different binding mode may be influenced by the soaking conditions, although it explains the remarkable discrepancy between the kinetics of hemiketal formation in thep-amidinophenylpyruvate/trypsin complex (18.Tanizawa K. Kanaoka Y. Wos J.D. Lawson W. Biol. Chem. Hoppe-Seyler. 1985; 366: 871-878Crossref PubMed Scopus (13) Google Scholar) compared with the kinetics of covalent bond formation in the α-ketoacid/NS3 complexes (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar). A biphasic kinetic behavior, analogous to that reported for our inhibitors (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar), was observed for the reaction between trypsin and p-amidinophenylpyruvate. In the latter case, the rate constants describing the equilibrium between the initial complex and the covalent complex were determined to be 3.15 and 0.1 s−1, respectively (18.Tanizawa K. Kanaoka Y. Wos J.D. Lawson W. Biol. Chem. Hoppe-Seyler. 1985; 366: 871-878Crossref PubMed Scopus (13) Google Scholar). These data should be compared with 7.5 × 10−3 s−1 and 1.2 × 10−5 s−1, for the NS3-inhibitor I and to 5 × 10−3 and 1.8 × 10−5s−1, for the NS3-inhibitor II complexes, respectively. Therefore, these results suggest a significantly larger energy barrier for both the formation and the dissociation of the covalent complex, in the case of the NS3 protease, and correlate with the structural differences of the final covalent complexes (Fig. 4). Another characteristic of our structures is that His57makes two strong hydrogen bonds (ranging from 2.33 to 2.75 Å for all three structures; Fig. 1 C); one with its Nδ1 to the Oδ2 of Asp81, another with its main chain nitrogen to the Oδ1 of the same aspartate. The presence of these particularly short hydrogen bonds is consistent with the structure of the subtilisin protease from Bacillus lentus, solved at 0.78 Å (19.Kuhn P. Knapp M. Soltis S.M. Ganshaw G. Thoene M. Bott R. Biochemistry. 1998; 37: 13446-13452Crossref PubMed Scopus (176) Google Scholar). More generally, the existence of hydrogen bonds with a partially covalent character has been demonstrated by a recent Compton scattering study (20.Martin T.W. Derewenda Z.S. Nat. Struct. Biol. 1999; 6: 403-406Crossref PubMed Scopus (105) Google Scholar). The His57-Asp81 interaction is missing in the structures of the noninhibited NS3 without the NS4A cofactor (21.Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomaw E.W. Adachi T. Hostomska Z. Cell. 1996; 87: 331-342Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar,22.Barbato G. Cicero D.O. Nardi M.C. Steinkühler C. Cortese R. De Francesco R. Bazzo R. J. Mol. Biol. 1999; 289: 371-384Crossref PubMed Scopus (103) Google Scholar), whereas it is present in our noninhibited NS3/4A complex (Fig.3 A). The solvent exposed residue, Lys136, close to the active site likely stabilizes the carboxylate bound at the oxyanion hole (Fig. 1, C and D). The P1 and P2 sites for both inhibitors are the same, (difluoro)aminobutyric acid and Leu, respectively (Fig. 1, Band C). The S1 pocket constitutes a small nonpolar depression on the protease surface and is formed by Val132, Leu135, and Phe154, which, together with the aliphatic part of Lys136, can make lipophilic interactions, thus explaining the preference for a Cys residue in the P1 position of substrates. The P1 residue of both inhibitors, a chemically inert fluorocarbon group, mimics the cysteine thiol of natural substrates (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar) and makes hydrophobic interactions with Val132, Leu135, the aliphatic part of Lys136, and Phe154 (Fig. 1 D). In addition, attractive interactions between the fluorine lone pairs and the aromatic C-H of Phe154 are possible. In the S2 region, Ala156side chain together with His57 and the aliphatic part of Arg155 provide a small hydrophobic patch on the protein surface (Fig. 1 D). The side chains of His57, Arg155, and Ala156 sterically limit the space available to the P2 residue. Because of this steric hindrance, hydrophobic β-branched amino acids cannot be accommodated in S2, hence explaining the lower activity of inhibitors with Val or Ile in P2 (23.Ingallinella P. Altamura S. Bianchi E. Taliani M. Ingenito R. Cortese R. De Francesco R. Steinkühler C. Pessi A. Biochemistry. 1998; 37: 8906-8914Crossref PubMed Scopus (164) Google Scholar), whereas Leu in P2 fits well. Inhibitor binding is further reinforced through its main chain N1 and O3, which form hydrogen bonds with the main chain carbonyl of Arg155 and the main chain amide of Ala157, respectively (Fig. 1 C). The S3 surface, an extension of the S1 pocket, is formed by the hydrophobic side chains of Ala157 and Cys159and makes lipophilic interactions with the nonpolar part of the P3 side chain of the inhibitors (Fig. 1 D; Glu for inhibitor I and Ile for inhibitor II). In the structure of NS3/4A in complex with inhibitor I, the carboxylate of Glu forms a salt bridge with the NZ of Lys136 (Fig. 1 C). Because of the bulky nature of the P3 side chain of inhibitor II, Lys136 is sterically restricted and also visible, whereas it is disordered in the noninhibited structure. In addition, at the P3 position, a carbon-sulfur interaction with the enzyme residue Cys159 is possible, thereby helping to stabilize the inhibitor in an extended conformation. A hydrogen bond is formed between the backbone N2 of the inhibitors and the main chain carbonyl of Ala157 (Fig.1 C). The S4 site consists of a solvent exposed hydrophobic patch, created by Arg123 and Val158 (Fig.1 D). This small hydrophobic area should favor hydrophobic amino acids. The preference for bulky hydrophobic amino acids in P4, shown previously (23.Ingallinella P. Altamura S. Bianchi E. Taliani M. Ingenito R. Cortese R. De Francesco R. Steinkühler C. Pessi A. Biochemistry. 1998; 37: 8906-8914Crossref PubMed Scopus (164) Google Scholar), can be explained by the hydrophobic interaction of the P4 residue (tert-butyl in inhibitor I and benzyl capping group in inhibitor II) with Arg123 and Val158 of the enzyme and by the intramolecular hydrophobic contact with Leu in P2 (Fig. 1 D). To our knowledge, this is the first report of the crystal structure of inhibitors bound to the HCV NS3/4A complex and permits the interpretation of structure/activity relationship data (5.Narjes F. Brunetti M. Colarusso S. Gerlach B. Koch U. Biasol G. Fattori D. De Francesco R. Matassa V.G. Steinkühler C. Biochemistry. 2000; 39: 1849-1861Crossref PubMed Scopus (73) Google Scholar). These structures show that even in the presence of an inhibitor, the substrate-binding region remains largely featureless and solvent-exposed and underscores the likely difficulties of developing small drug-like inhibitors. We thank C. Steinkühler and R. Cortese for helpful discussions and critical reading of the manuscript; B. Gerlach for contributing to the synthesis of one compound; U. Koch for the initial modeled coordinates of the inhibitors; F. Bonelli for mass spectrometry and C. Capo for N-terminal sequencing; and M. Emili for help with the art work. We especially thank the staff of European Synchrotron Radiation Facility (Grenoble, France) and of EMBL (Deutsches Elektronen Synchrotron, Hamburg, Germany) for assistance in data collection." @default.
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W2108093903 date "2000-03-01" @default.
W2108093903 modified "2023-10-03" @default.
W2108093903 title "Inhibition of the Hepatitis C Virus NS3/4A Protease" @default.
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W2108093903 doi "https://doi.org/10.1074/jbc.275.10.7152" @default.
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