A challenge to the treatment of chronic hepatitis C with direct-acting antivirals is the emergence of drug-resistant hepatitis C virus (HCV) variants. HCV with preexisting polymorphisms that are associated with resistance to NS3/4A protease inhibitors have been detected in patients with chronic hepatitis C. We performed a comprehensive pooled analysis from phase 1b and phase 2 clinical studies of the HCV protease inhibitor faldaprevir to assess the population frequency of baseline protease inhibitor resistance-associated NS3 polymorphisms and their impact on response to faldaprevir treatment. A total of 980 baseline NS3 sequences were obtained (543 genotype 1b and 437 genotype 1a sequences). Substitutions associated with faldaprevir resistance (at amino acid positions 155 and 168) were rare (<1% of sequences) and did not compromise treatment response: in a phase 2 study in treatment-naive patients, six patients had faldaprevir resistance-associated polymorphisms at baseline, of whom five completed faldaprevir-based treatment and all five achieved a sustained virologic response 24 weeks after the end of treatment (SVR24). Among 13 clinically relevant amino acid positions associated with HCV protease resistance, the greatest heterogeneity was seen at NS3 codons 132 and 170 in genotype 1b, and the most common baseline substitution in genotype 1a was Q80K (99/437 [23%]). The presence of the Q80K variant did not reduce response rates to faldaprevir-based treatment. Across the three phase 2 studies, there was no significant difference in SVR24 rates between patients with genotype 1a Q80K HCV and those without Q80K HCV, whether treatment experienced (17% compared to 26%; P = 0.47) or treatment naive (62% compared to 66%; P = 0.72).
ABSTRACT Background and aims Capsid assembly (CA) is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. CA is the target for various new anti-viral candidate therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and lead to hepatocyte cell death. The aim of the studies was to identify the mechanism of action of the CAM-A modulators leading to HBV infected hepatocyte elimination. Methods The CAM-A mediated mechanism of HBsAg reduction was evaluated in vitro in a stable HBV replicating cell line and in vivo in AAV-HBV transduced C57BL/6, C57BL/6 SCID and HBV-infected chimeric mice with humanized livers. Results In vivo treatment with CAM-A modulators induced pronounced reductions in HBe- and HBsAg which were associated with a transient increase in ALT. Both HBs- and HBeAg reduction and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute to this phenotype. However, depletion of CD8+ T-cells in transduced wild-type mice did not have a negative impact on antigen reduction, indicating that CD8+ T-cell responses are not essential. Coinciding with the transient ALT elevation in AAV-HBV transduced mice, we observed a transient increase in markers related to endoplasmic reticulum stress and apoptosis as well as cytokines related to apoptosis pathways, followed by the detection of a proliferation marker. Pathway enrichment analysis of microarray data revealed that antigen presentation pathway (MHC-I) was upregulated, overlapping with observed apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A mediated HBsAg reduction is dependent on de novo core protein translation and that the effect is dependent on high levels of core protein expression, which will likely focus the CHB sub-population that could respond. Conclusion CAM-A treatment eradicates HBV infected hepatocytes with high core protein levels through the induction of apoptosis a promising approach as part of a regimen to achieve functional cure. Lay summary Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.
The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors. The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; NS, nonstructural; NOESY, nuclear Overhauser effect spectroscopy; HPLC, high-performance liquid chromatography; 1H NMR, proton nuclear magnetic resonance; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; ROESY, rotating frame Overhauser effect spectroscopy; T 1, longitudinal (spin-lattice) relaxation time; NT 1, spin-lattice relaxation multiplied by the number of covalently attached hydrogen atoms.infection is an important cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure worldwide (1Hoofnagle J.H. Di Bisceglie A.M. N. Engl. J. Med. 1997; 336: 347-356Crossref PubMed Scopus (996) Google Scholar). Approved therapies with proven benefit for patients with chronic hepatitis C include various drug regimens of interferon-α. These therapies have limited efficacy with a low sustained response rate and frequent side effects (1Hoofnagle J.H. Di Bisceglie A.M. N. Engl. J. Med. 1997; 336: 347-356Crossref PubMed Scopus (996) Google Scholar). Therefore, there is an urgent need for the development of new therapies for the treatment of HCV infections. HCV is a small enveloped virus containing a single-stranded RNA genome of positive polarity, which encodes a unique polyprotein of approximately 3000 amino acids (for reviews see Refs. 2Bartenschlager R. Antiviral Chem. Chemother. 1997; 8: 281-301Crossref Scopus (62) Google Scholar and 3Reed K.E. Rice C.M. Curr. Stud. Hematol. Blood Transfus. 1998; 62: 1-37Crossref PubMed Scopus (62) Google Scholar). This polyprotein is the precursor of four structural and six nonstructural (NS) proteins (4Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (582) Google Scholar, 5Hijikata M. Mizushima H. Agaki T. Mori S. Kakiuchi N. Kato N. Tanaka T. Kimura K. Shimotohno K. J. Virol. 1993; 67: 4665-4675Crossref PubMed Google Scholar, 6Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 7Bartenschlager R. Ahlborn-Laake L. Mous J. Jacobsen H. J. Virol. 1993; 67: 3835-3844Crossref PubMed Google Scholar, 8Tomei L. Failla C. Santolini E. De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar, 9Eckart M.R. Selby M. Masiarz F. Lee C. Berger K. Crawford C. Kuo C. Kuo G. Houghton M. Choo Q.-L. Biochem. Biophys. Res. Commun. 1993; 192: 399-406Crossref PubMed Scopus (171) Google Scholar, 10Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar). The structural proteins are proteolytically processed by host signal peptidases, whereas two virally encoded proteases within the NS2 and NS3 regions process the remaining nonstructural proteins. The NS3 serine protease domain (20 kDa), located within the N-terminal portion of the NS3 protein, mediates the proteolysis at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions (6Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 7Bartenschlager R. Ahlborn-Laake L. Mous J. Jacobsen H. J. Virol. 1993; 67: 3835-3844Crossref PubMed Google Scholar, 8Tomei L. Failla C. Santolini E. De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar, 9Eckart M.R. Selby M. Masiarz F. Lee C. Berger K. Crawford C. Kuo C. Kuo G. Houghton M. Choo Q.-L. Biochem. Biophys. Res. Commun. 1993; 192: 399-406Crossref PubMed Scopus (171) Google Scholar, 10Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar, 11Kwong A.D. Kim J.L. Rao G. Liposvek D. Raybuck S.A. Antiviral Res. 1998; 40: 1-18Crossref PubMed Scopus (92) Google Scholar). We and others have recently reported that N-terminal cleavage products of peptide substrates are competitive inhibitors of NS3 protease activity (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar,13Steinkühler C. Biasiol G. Brunetti M. Urbani A. Koch U. Cortese R. Pessi A. De Francesco R. Biochemistry. 1998; 37: 8899-8905Crossref PubMed Scopus (220) Google Scholar), which has served as the basis for designing substrate-based inhibitors (14Llinàs-Brunet M. Bailey M. Déziel R. Fazal G. Gorys V. Goulet S. Halmos T. Maurice R. Poirier M. Poupart M.-A. Rancourt J. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 2719-2724Crossref PubMed Scopus (99) Google Scholar, 15Ingallinella P. Altamura S. Bianci E. Taliani M. Ingenito R. Cortese R. De Francesco R. Steinkühler C. Pessi A. Biochemistry. 1998; 37: 8906-8914Crossref PubMed Scopus (164) Google Scholar). To date, there have been no reports in the literature on the structure of substrates or inhibitors when bound to NS3 protease, which would certainly be valuable for inhibitor design efforts. However, x-ray crystal structures have been determined for NS3 protease alone (16Love 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 (502) Google Scholar) and for NS3 protease in the presence of an NS4A peptide cofactor (17Kim 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 (677) Google Scholar, 18Yan Y. Li Y. Munshi S. Sardana V. Cole J.L. Sardana M. Steinkühler C. Tomei L. De Francesco R. Kuo L. Chen Z. Protein Sci. 1998; 7: 837-847Crossref PubMed Scopus (266) Google Scholar). These structures show that NS3 protease adopts a chymotrypsin/trypsin-like fold. In this report we applied NMR methods to study the structure of peptides and inhibitors, based on N-terminal cleavage products of peptide substrates when bound to the NS3 protease domain of HCV. Transferred NOESY experiments were used to determine the conformation of ligands when bound to the protease, and differential line-broadening experiments were used to identify which segments of the ligands contact the protease. A modification of a previously published procedure (19Steinkühler Urbani A. Tomei L. Biasiol G. Sardana M. Bianci E. Pessi A. De Francesco R. J. Virol. 1996; 70: 6694-6700Crossref PubMed Google Scholar) was used to purify the NS3 protease.Escherichia coli BL21(DE3) pLysS, transformed with a pET11d vector expressing amino acids 1–180 of HCV NS3 type 1b, was grown at 37 °C in CircleGrow (BIO 101, Inc., Vista, CA) medium supplemented with 200 μg/ml ampicillin and 34 μg/ml chloramphenicol. At mid-log phase the culture was cooled to 24 °C and induced with 1 mm isopropyl-β-d-thiogalactoside. Three hours post-induction, cells were harvested by centrifugation, and the cell paste was frozen at −80 °C. Following two freeze-thaw cycles the cell paste was resuspended in 3 ml of lysis buffer (25 mmNaPO4, pH 7.5, 5 mm dithiothreitol, 10% glycerol (v/v), 1 mm EDTA, 0.1% octyl-β-d-glucoside, 15 mm NaCl) per gram of cells. The suspension was processed in a Dounce homogenizer, supplemented with 20 mm MgCl2 and 10 μg/ml Dnase I (bovine pancreatic, Amersham Pharmacia Biotech), and incubated for 20 min on ice. Following a brief sonication, the extract was clarified by a 30-min centrifugation at 14,500 × g. The supernatant was applied to a SP-Sepharose column (equilibrated with 50 mm NaPO4, pH 6.5, 5 mmdithiothreitol, 10% glycerol, 1 mm EDTA, and 0.1% octyl-β-d-glucoside) and eluted with a 150–1000 mm NaCl gradient. NS3 protease enriched fractions were pooled and diluted with Buffer A (25 mm NaPO4, pH 7.5, 5 mm dithiothreitol, 10% glycerol, and 0.1% octyl-β-d-glucoside) to decrease the NaCl concentration to 100 mm. The NS3 protease pool was applied to a heparin-Sepharose column and eluted with 0.3 m NaCl in Buffer A. The eluted protein was concentrated and applied to a Superdex 75 column (in Buffer A with 350 mm NaCl) and eluted as a single major peak of homogeneous NS3 protease (Fig. 1). Purified protein preparations were checked by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and the enzyme concentration was quantified by PTH-derivatization of a total amino acid hydrolysate of the purified protein. A conversion factor of 1.22 was implemented to correct the protein concentration as determined from a Bio-Rad protein assay using a bovine serum albumin standard. The proportion of enzyme in active form was estimated to be greater than 95% based on a comparison of previously published activities (19Steinkühler Urbani A. Tomei L. Biasiol G. Sardana M. Bianci E. Pessi A. De Francesco R. J. Virol. 1996; 70: 6694-6700Crossref PubMed Google Scholar,20Urbani A. Bianci E. Narjes F. Tramontano A. De Francesco R. Steinkühler C. Pessi A. J. Biol. Chem. 1997; 272: 9204-9209Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Peptides 1, 2,and 3 (Table I shows the compounds used in this study) were synthesized using standard solid-phase methodology (21Stewart J.M. Young J.D. Gross Meienhofer Udenfriend Solid Phase Peptide Synthesis. 2nd Ed. Pierce Chemical Co., Rockford, IL1984: 149-170Google Scholar, 22Bodanszky M. Peptide Chemistry. 2nd Revised Ed. Springer-Verlag, Berlin1993Crossref Google Scholar). Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids and resins were obtained from Nova Biochem, Bachem, or Advanced ChemTech. The synthesis of 4 was carried out in solution using standard peptide chemistry (22Bodanszky M. Peptide Chemistry. 2nd Revised Ed. Springer-Verlag, Berlin1993Crossref Google Scholar). Each compound was purified by preparative reversed-phase HPLC on a C18 column using an acetonitrile gradient. Satisfactory 1H NMR, mass spectrometry, amino acid analysis, and homogeneity data (> 90% HPLC) were obtained for all the compounds.Table ICompounds, sequences, and IC50 values of HCV NS3 protease inhibitorsNameP10P9P8P7P6P5P4P3P2P1P1′P2′P3′P4′P5′P6′IC50μm1AspAspIleValProCys682SerMetSerTyrThrTrp>25003ThrGluAlaGlyAspAspIleValProCys344AcAspAspIleValHbpaHbp represents 4-trans-benzyloxyproline.NvabNva represents norvaline.12Residue positions are labeled according to the nomenclature described in Ref. 30Schechter J. Berger A. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1970; 257: 249-264Crossref PubMed Scopus (379) Google Scholar.a Hbp represents 4-trans-benzyloxyproline.b Nva represents norvaline. Open table in a new tab Residue positions are labeled according to the nomenclature described in Ref. 30Schechter J. Berger A. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1970; 257: 249-264Crossref PubMed Scopus (379) Google Scholar. The enzymatic assay was performed in 50 mm Tris-HCl, pH 7.5, 30% (w/v) glycerol, 2% (w/v) CHAPS, 1 mg/ml bovine serum albumin, and 1 mm TCEP. 25 μm of the substrate DDIVPC-SMSY/TW, ∼1 nmbiotin-DDIVPC-SMSY-125I-labeled TW, and various concentrations of inhibitor were incubated with 11 nmprotease for 60 min at 23 °C. These conditions were chosen to obtain < 20% substrate conversion to minimize the effect of product inhibition (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar, 13Steinkühler C. Biasiol G. Brunetti M. Urbani A. Koch U. Cortese R. Pessi A. De Francesco R. Biochemistry. 1998; 37: 8899-8905Crossref PubMed Scopus (220) Google Scholar). The final Me2SO concentration did not exceed 6.4%. The reaction was terminated with the addition of 0.025 N NaOH. The separation of substrate from products was performed by adding avidin-coated agarose beads to the assay mixture followed by filtration. A nonlinear curve fit using the Hill model was then applied to the percent inhibition-concentration data, and 50% effective concentration (IC50) was calculated through the use of SAS (Statistical Software System, SAS Institute Inc., Cary, NC). The initial velocities were determined at multiple inhibitor and substrate concentrations under the assay conditions described above in the presence of 22 nmprotease. To minimize the effect of product inhibition, the cleavage rates were only measured during the initial phase of substrate conversion (<20%). Accordingly, the initial rates were linear and displayed Michaelis-Menten kinetics. K i calculations were performed by nonlinear regression analysis of the velocity data using the GraFit software (version 3.0, Erithacus Software Ltd., Staines, UK) and Equation 1 for competitive inhibition.V=VmaxS(Km(1+I/Ki)+S)Equation 1 Two identical samples were initially prepared for each one-dimensional 1H NMR experiment. Sample tubes (5 mm) containing 2 mm inhibitor were prepared by adding 10 μl of concentrated solutions in Me2SO-d 6 to an aqueous buffer composed of 25 mm Na2PO4, 300 mm NaCl, 5 mmdithiothreitol-d 10, 10% (v/v) glycerol-d 8, and 10% (v/v) D2O spiked with 3-(trimethylsilyl)-proprionic 2,2,3,3-d 4. The final pH values of the solutions were adjusted to 7.0, and buffer was added to a final volume of 600 μl. To one of the samples described above was added a concentrated stock solution of NS3 protease (depending on the experiment, the concentration used ranged between 2.1 to 3.6 mg/ml) in a buffer identical to that employed above, with the exception of 0.01% Nonidet P-40 detergent, such that an inhibitor/protease ratio of 30:1 was typically achieved. All spectra were acquired on a Bruker DRX 600-MHz NMR spectrometer at 30 °C with the exception of the spectra shown in Fig. 2 B, which were acquired on a Bruker AMX 400-MHz NMR. Suppression of the solvent signal was achieved by the use of presaturation or by inserting a 3–9-19 WATERGATE module prior to data acquisition (23Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3539) Google Scholar, 24Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar). Phase-sensitive NOESY, total correlation spectroscopy (TOCSY), and ROESY experiments were acquired using the time-proportional phase incrementation method. NOESY experiments were recorded with mixing times of 100, 200, and 250 ms and a water-selective flip-back pulse prior to the readout pulse (25Lippens G. Dhalliun C. Wieruszeski J.-M. J. Biomol. NMR. 1995; 5: 327-331Crossref PubMed Scopus (182) Google Scholar). The ROESY experiment was recorded with a 250-ms spin-lock period. Two-dimensional data sets were typically acquired with 2048 points in t 2 and 400 points in t 1. 128 scans were averaged for each NOESY t1 increment and 64 scans for each TOCSYt1 increment. The data were processed and analyzed using XWinNMR and WinNMR software (Bruker Canada, Milton, Ontario) and TRIAD software (Tripos, St. Louis, MO). Data sets were typically zero-filled to yield 2048 × 1024 real points after transformation using a phase-shifted sine bell window function. One-dimensional 13C spectra for T 1relaxation measurements for 4 (36 mm) were acquired at 150 MHz and 27 °C in Me2SO solvent. Inversion recovery experiments were run with power-gated proton decoupling during acquisition. Twelve spectra were acquired corresponding to the τ delays (0.01, 0.1, 0.2, 0.37, 0.65, 1.0, 1.5, 2.2, 3.0, 5.0, 6.0, and 7.5 s). Each spectrum was acquired by adding 1600 transients and using a relaxation delay of 7.54 s. ROESY and J-coupling data collected in both Me2SO solvent and Buffer A, described above, were found to be similar, suggesting that 4 assumes similar conformational properties in both solvents. The structure of 4 was modeled by a simulated annealing protocol using Discover 95.0 and the CFF95 force field (Molecular Simulations Inc., San Diego, CA). All calculations were performed without nonbonded or coulombic cut-offs and a dielectric constant of 1.0. NMR-derived distance restraints were generated from the NMR data using a method similar to that of Sykes (26Baleja J.D. Moult J. Syrosces B.D. J. Magn. Reson. 1990; 87: 375-384Google Scholar). The 27 restraints derived from NOESY distance data were applied as strong (1.8–2.7 Å), medium (1.8–3.5 Å), or weak (1.8–5.0 Å) flat-bottomed potentials having force constants of 50 kcal/mol·Å2. A single, high temperature, unrestrained dynamics run was performed at 1000 K using a time step of 1 fs, with 50 structures collected at 10-ps intervals to generate a starting set of conformations. Each structure was cooled and minimized using the following simulated annealing protocol. The temperature was lowered to 500 K at a rate of 50 K/ps−1 where strong restraints were applied, followed by additional cooling to 250 K (5 K/ ps−1). The remaining restraints were added and cooling to 50 K (0.5 K/ ps−1) was performed followed by restrained minimization to a final gradient of 0.01 kcal/mol−1Å−1. Sixteen low energy, NMR-consistent structures were isolated and are shown superimposed based on the P1–P4 backbone in Fig. 5 A. The root mean square deviation for the backbone heavy atoms of P1–P4 (excluding the acid oxygens of P1) is 0.25 Å. The average total restraint violation energy is 0.14 kcal/mol with a S.D. = 0.13 kcal/mol. The differential line-broadening experiment is a powerful NMR method for detecting protein-ligand interactions of moderate affinity (K d = 10−3 to 10−6m) and for identifying the contact sites on ligands that interact with a target protein (27Ni F. Prog. NMR Spectrosc. 1994; 26: 517-606Abstract Full Text PDF Scopus (327) Google Scholar). The relative ease and simplicity of the method makes it particularly attractive. For a protein-ligand complex in fast exchange on the NMR time scale, one can observe changes in the predominant ligand resonances upon addition of less than stoichiometric amounts of protein (e.g. 30:1 ligand to protein ratio). The observed protein-induced changes in ligand resonances are due to the different environments of the ligand hydrogen nuclei in the free and the bound states. We have previously described differential line-broadening and transferred NOESY NMR methods that were successful for determining the structures of inhibitors (28LaPlante S.R. Cameron D.R. Aubry N. Bonneau P.R. Déziel R. Grand-Maı̂tre C. Ogilvie W.W. Kawai S.H. Angew. Chem. Int. Ed. 1998; 37: 2729-2732Crossref PubMed Scopus (12) Google Scholar) and peptides (29LaPlante S.R. Aubry N. Bonneau P.R. Cameron D.R. Lagacé L. Massariol M.-J. Montpetit H. Plouffe C. Kawai S.H. Fulton B.D. Chen Z.G. Ni F. Biochemistry. 1998; 37: 9793-9801Crossref PubMed Scopus (21) Google Scholar) (based on N-terminal cleavage products of peptide substrates) when bound to human cytomegalovirus serine protease. Based on this experience, our initial aim was to identify whether the N-terminal cleavage products of peptide substrates of NS3 protease also exhibited fast exchange appropriate for line-broadening experiments. The N-terminal cleavage product of an NS5A/5B-derived peptide substrate (1, TableI) was tested for inhibitory activity. The moderate affinity of 1 (IC50 = 68 μm) for NS3 protease suggested that this competitive inhibitor (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar) whould be a good candidate for line-broadening and transferred NOESY studies. A differential line-broadening study began by acquiring 1H NMR spectra of 1 (Fig.2 A) in the absence (blue) and presence of the protease (red). A comparison of these spectra shows changes to specific resonances of1 (Fig. 2 A) that results from fast-exchange binding to the protease (on the NMR time scale). The specific nature of the protease-induced perturbations shown in Fig. 2 A suggests that these segments of 1 likely contact the enzyme (e.g. see the resonances of P4 αH, P1 βCH2, and P4 γCH3/δCH3; residue positions are labeled according to the nomenclature described in Ref. 30Schechter J. Berger A. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1970; 257: 249-264Crossref PubMed Scopus (379) Google Scholar). Other resonances of nonexchangeable hydrogens did not exhibit significant perturbations upon addition of the protease (e.g. P6 αH and P3 γCH3 in Fig. 2 A). These hydrogen atoms do not experience significantly different environments in their free and bound states and may not directly contact the enzyme. In contrast to the observation of N-terminal product inhibition, peptide 2, which is based on the C-terminal cleavage product of the NS5A/5B-derived peptide substrate, does not bind to the protease. Fig. 2 B shows no difference between the spectrum of free 2 (blue) and the spectrum of 2in the presence of protease (red). Moreover, peptide2 does not inhibit NS3 protease activity (IC50> 2500 μm). To investigate other ligand contact points with NS3 protease, a line-broadening study was performed on 3 (IC50 = 34 μm). This compound is based largely on the P10–P1 sequence of the NS5A/5B cleavage site. A comparison of transferred NOESY data (not shown) and line-broadening patterns for 1and 3 in the presence of the protease shows that the P1–P6 residues bind the protease in a similar mode (e.g. 1 and 3 have similar broadening patterns for P1 βCH2, P3 γCH3, and P4 γCH3/δCH3, shown in Fig. 2, Aand C). However, the lack of protease-induced resonance perturbations for the residues comprising P7–P10 indicate the lack of significant contacts between these residues and the protease (e.g. see the resonances labeled P8 βCH3 and P10 δCH3 in Fig. 2 C). These results are consistent with the observation that extension of the hexapeptide1 N terminus does not increase inhibitory potency (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar). Further along in our efforts to design potent inhibitors, we found that a (4R)-benzyloxy substituent on the P2 proline improved inhibitory potency by 7–10-fold. The question arose as to whether or not the benzyl group improves potency by interacting directly with the protease. A line-broadening study was undertaken to help answer this question using 4, which has a (4R)-benzyloxy substituent on the P2 proline and in which the P1 cysteine was replaced by the chemically more stable norvaline (14Llinàs-Brunet M. Bailey M. Déziel R. Fazal G. Gorys V. Goulet S. Halmos T. Maurice R. Poirier M. Poupart M.-A. Rancourt J. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 2719-2724Crossref PubMed Scopus (99) Google Scholar). Although the latter replacement typically reduces the potency of inhibitors (14Llinàs-Brunet M. Bailey M. Déziel R. Fazal G. Gorys V. Goulet S. Halmos T. Maurice R. Poirier M. Poupart M.-A. Rancourt J. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 2719-2724Crossref PubMed Scopus (99) Google Scholar), it does favor fast-exchange binding, which is required for line-broadening and transferred NOESY experiments. A rapid, reversible binding of4 to the protease was then confirmed by comparing the1H spectra in Fig. 2 D of free 4(blue) to that of 4 in the presence of protease (red). Specific binding to the active site of the protease was also demonstrated by kinetics and NMR studies. The Dixon and the Cornish-Bowden plots (31Cornish-Bowden Biochem. J. 1974; 137: 143-144Crossref PubMed Scopus (788) Google Scholar) obtained with 4 (Fig.3) are characteristic of a competitive mode of inhibition. Furthermore, the best fit observed by nonlinear regression analysis of these data also shows that 4competitively inhibits the NS3 protease with a K i of 5.4 μm. Also, NMR data illustrate that the protease-induced line broadening observed in Fig. 2 D is lost (black spectrum in Fig. 2 E) upon addition of a more potent inhibitor (IC50 = 0.041 μm). The fortuitous resonance dispersion in the 1H spectrum of4 allowed us to identify most of the hydrogen resonances that are perturbed by the protease. For example, Fig. 2 Dshows that the P2 aromatic ring resonances of free 4 were significantly altered with the addition of the protease, suggesting that this group contacts the protease. Many of the resonances of the side chains of P1, P2, and P4 also exhibited protease-induced line broadening, which helps explain their role in the binding of4 to the protease. A summary of the line-broadening data for resonances of P1 to P4 is illustrated in Fig. 5 C and discussed later. Interestingly, little or no broadening was observed for the side chains of P3 (e.g. see Fig. 2 D), P5, and P6. The lack of protease-induced broadening for the P3 side chain resonances suggests a minimal role in direct binding by this group. It also highlights the conformational role that P3 likely plays in orienting the main chain in the free state for optimal binding to the protease (see below). The exact role of P5 and P6 in binding to the protease is still unclear. The resonances of these residues lack significant protease-induced broadening for the nonexchangeable hydrogen atoms, yet these residues are important for optimal inhibitor potency (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar). This would suggest that the acids of the side chains may have an important binding role. Unfortunately, this can not be tested using these NMR methods because of the lack of observable1H resonances for rapidly exchanging hydrogen atoms. However, we have shown elsewhere that the aspartic acid at P5 can be replaced by tert-butylglycine with no loss in inhibitory potency (12Llinàs-Brunet M. Bailey M. Fazal G. Goulet S. Halmos T. LaPlante S. Maurice R. Poirier M. Poupart M.-A. Thibeault D. Wernic D. Lamarre D. Bioorg. & Med. Chem. Lett. 1998; 8: 1713-1718Crossref PubMed Scopus (172) Google Scholar). On the other hand, replacement of the aspartic acid at P6 by alanine results in a significant loss in inhibitory potency. The importance of this acid group is highlighted by the fact that sequence comparisons among isolated HCV strains have revealed a structurally conserved acid residue at the P6 position (10Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar). We applied transferred NOESY methods to determine the structure of4 when bound to NS3 protease. As a control experiment, a NOESY spectrum of free 4 was acquired. Fig.4 B shows a subregion that contains only a few distance-related cross-peaks, as expected for a small molecule of this size. A subsequent NOESY spectrum (Fig.4 A) of the same sample, following addition of NS3 protease (30:1 inhibitor to protease ratio), exhibited many new negative cross-peaks, as expected for a small molecule when bound to a large, slow tumbling macromolecule. Also, the negative cross-peaks present in the spectrum of free 4 (Fig. 4 B) were more intense in the transferred NOESY spectrum (Fig. 4 A). These results indicate a well defined and rapidly reversible binding of4 to the protease. Other changes included the loss of artifact zero quantum cross-peaks (e.g. P1-NH/P1-αH, P3-NH/P3-αH, and P4-NH/P4-αH) observed for free 4 (Fig.4 B) and their replacement with negative NOESY cross-peaks (Fig. 4 A) in the presence of the protease. Upon addition of a more potent inhibitor (IC50 = 0.041 μm) that blocks the active site, the transferred NOESY data of 4in the bound state is lost (Fig. 4 C), and the resulting spectrum resembles that of free 4 (compare Fig. 4,C and B). Having demonstrated that the NOESY cross-peaks in Fig. 4 Aarise from 4 when bound to the protease, an NMR-derived structure of enzyme-bound 4 was obtained through restrained simulated annealing techniques. The NOESY cross-peak volumes were scaled and converted to interproton distance restraints (similar to a method described in Ref. 26Baleja J.D. Moult J. Syrosces B.D. J. Magn. Reson. 1990; 87: 375-38
BackgroundBILB 1941 is a potent and specific non-nucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase in vitro.MethodsIn a double-blind sequential group comparison, 96 male HCV genotype 1 patients with minimal to mild liver fibrosis (Ishak or Metavir score 0–2) were randomized (8 to active treatment and 2 to placebo per dose group) and treated with 10–450 mg BILB 1941 every 8 h over 5 days. Viral load (VL) was measured using Roche Cobas TaqMan® assays.ResultsVL decreased by ≥1 log10 IU/ml in 2/8, 2/8, 1/8, 2/7, 0/8, 2/8 and 4/5 patients on 60, 80, 100, 150, 200, 300 and 450 mg, respectively. No response was seen with placebo. HCV subtype 1b showed better response than 1a, the effect of other covariables including prior interferon treatment was not significant. NS5B population sequencing and phenotyping identified baseline samples with reduced BILB 1941 susceptibility, but did not detect an on-treatment emergence of resistant mutants. Plasma drug levels were linear until 300 mg. No serious adverse events (AEs) were reported. AEs were mainly gastrointestinal-related (most frequent diarrhoea) and frequency increased with dose. On 450 mg, all five active-treated patients discontinued (four for gastrointestinal intolerance and one for increased aspartate aminotransferase and alanine aminotransferase levels) and the trial was discontinued.ConclusionsBILB 1941 monotherapy demonstrated antiviral activity against HCV genotype 1, but gastrointestinal intolerance precluded testing of higher doses.
Combinations of direct acting antivirals (DAAs) that have the potential to suppress emergence of resistant virus and that can be used in interferon-sparing regimens represent a preferred option for the treatment of chronic HCV infection. We have discovered allosteric (thumb pocket 1) non-nucleoside inhibitors of HCV NS5B polymerase that inhibit replication in replicon systems. Herein, we report the late-stage optimization of indole-based inhibitors, which began with the identification of a metabolic liability common to many previously reported inhibitors in this series. By use of parallel synthesis techniques, a sparse matrix of inhibitors was generated that provided a collection of inhibitors satisfying potency criteria and displaying improved in vitro ADME profiles. "Cassette" screening for oral absorption in rat provided a short list of potential development candidates. Further evaluation led to the discovery of the first thumb pocket 1 NS5B inhibitor (BILB 1941) that demonstrated antiviral activity in patients chronically infected with genotype 1 HCV.
The Ner protein of bacteriophage Mu acts as a λ cro‐like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner ‐operator‐specific DNA‐binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5′‐ANPyTAPuCTAAGT‐3′, separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar λ cro protein, gel filtration experiments show the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.
