In Vitro Resistance Studies of Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313020200
ISSN1083-351X
AutoresChao Lin, Kai Lin, Yu-Ping Luong, B. Govinda Rao, Yunyi Wei, Debra Brennan, John R. Fulghum, Hsun-Mei Hsiao, Sue Ma, John P. Maxwell, Kevin M. Cottrell, Robert B. Perni, Cynthia A. Gates, Ann D. Kwong,
Tópico(s)Hepatitis B Virus Studies
ResumoWe have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3·4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3·4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3·4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp168 are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala156 remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061. We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3·4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3·4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3·4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp168 are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala156 remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; PI, protease inhibitor; DMEM, Dulbecco's modified minimal essential medium; FBS, fetal bovine serum; RT, reverse transcriptase; FRET, fluorescence resonance energy transfer; HIV, human immunodeficiency virus. 1The abbreviations used are: HCV, hepatitis C virus; PI, protease inhibitor; DMEM, Dulbecco's modified minimal essential medium; FBS, fetal bovine serum; RT, reverse transcriptase; FRET, fluorescence resonance energy transfer; HIV, human immunodeficiency virus. (1Wasley A. Alter M.J. Semin. Liver Dis. 2000; 20: 1-16Crossref PubMed Google Scholar). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2Kenny-Walsh E. Clin. 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It has been shown that the central region (amino acids 21–30) of the 54-residue NS4A protein is essential and sufficient for the enhancement of proteolytic activity of the NS3 serine protease (18Bartenschlager R. Lohmann V. Wilkinson T. Koch J.O. J. Virol. 1995; 69: 7519-7528Crossref PubMed Google Scholar, 19Failla C. Tomei L. De Francesco R. J. Virol. 1995; 69: 1769-1777Crossref PubMed Google Scholar, 20Lin C. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7622-7626Crossref PubMed Scopus (84) Google Scholar, 21Lin C. Thomson J.A. Rice C.M. J. Virol. 1995; 69: 4373-4380Crossref PubMed Google Scholar, 22Tanji Y. Hijikata M. Satoh S. Kaneko T. Shimotohno K. J. Virol. 1995; 69: 1575-1581Crossref PubMed Google Scholar). The central region of NS4A forms a tight heterodimer with the NS3 protein (21Lin C. Thomson J.A. Rice C.M. J. Virol. 1995; 69: 4373-4380Crossref PubMed Google Scholar), for which the first x-ray crystal structure was solved in 1996 (23Kim 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 (675) Google Scholar). BILN 2061 is the first HCV serine protease inhibitor (PI) in clinical trials for hepatitis C (24Lamarre D. Anderson P.C. Bailey M. Beaulieu P. Bolger G. Bonneau P. Bos M. Cameron D.R. Cartier M. Cordingley M.G. Faucher A.M. Goudreau N. Kawai S.H. Kukolj G. Lagace L. LaPlante S.R. Narjes H. Poupart M.A. Rancourt J. Sentjens R.E. St George R. Simoneau B. Steinmann G. Thibeault D. Tsantrizos Y.S. Weldon S.M. Yong C.L. Llinas-Brunet M. Nature. 2003; 426: 186-189Crossref PubMed Scopus (847) Google Scholar). In phase I trials, a 2–3-log reduction of HCV viral load was observed after a 2-day treatment, which provided the first proof-of-concept evidence that HCV NS3·4A protease inhibitors could be a new therapeutic option for hepatitis C patients (24Lamarre D. Anderson P.C. Bailey M. Beaulieu P. Bolger G. Bonneau P. Bos M. Cameron D.R. Cartier M. Cordingley M.G. Faucher A.M. Goudreau N. Kawai S.H. Kukolj G. Lagace L. LaPlante S.R. Narjes H. Poupart M.A. Rancourt J. Sentjens R.E. St George R. Simoneau B. Steinmann G. Thibeault D. Tsantrizos Y.S. Weldon S.M. Yong C.L. Llinas-Brunet M. Nature. 2003; 426: 186-189Crossref PubMed Scopus (847) Google Scholar). Recently, another HCV NS3·4A protease inhibitor, VX-950 (25Babine, R. E., Chen, S.-H., Lamar, J. E., Snyder, N. J., Sun, X. D., Tebbe, M. J., Victor, F., Wang, Q. M., Yip, Y. Y. M., Collado, I., Garcia-Paredes, C., Parker, R. S. I., Jin, L., Guo, D., and Glass, J. I. (March 7, 2002), International Patent WO 0218369, Eli Lilly and Co.Google Scholar), was selected as a clinical candidate for hepatitis C (26Perni R.B. Chandorkar G. Chaturvedi P.R. Courtney L.F. Decker C.J. Gates C.A. Harbeson S.L. Kwong A.D. Lin C. Lin K. Luong Y.P. Markland W. Rao B.G. Tung R.D. Thomson J.A. Hepatology. 2003; 38 (Abstr. 972)Crossref Google Scholar). Resistance to specific antiviral drugs is a major factor limiting the efficacy of therapies against many retroviruses or RNA viruses, due to the error-prone nature of the viral reverse transcriptases or RNA-dependent RNA polymerases. As new HCV-specific inhibitors enter clinical trials, resistance could become a major problem in patients treated with drugs targeting HCV NS3·4A serine protease or NS5B RNA polymerase. In this report, we used the HCV subgenomic replicon system to identify resistance mutations against two HCV protease inhibitor clinical candidates, BILN 2061 and VX-950. The in vitro resistance mutations selected against either inhibitor resulted in a significant reduction in susceptibility to the inhibitor itself. However, the primary resistance mutations against BILN 2061 were fully susceptible to VX-950, and the major resistance mutation against VX-950 remained sensitive to BILN 2061. Plasmid Construction—A DNA fragment encoding residues Ala1–Ser181 of the HCV NS3 protease (GenBank™ CAB46913) was obtained by PCR from the HCV Con1 replicon plasmid, I377neo/NS3–3′/wt (renamed as pBR322-HCV-Neo in this study) (10Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2490) Google Scholar) and inserted into pBEV11 2S. Chamber, personal communication. for expression of the HCV proteins with a C-terminal hexa-histidine tag in Escherichia coli. Resistance mutations against the HCV NS3·4A PI were introduced into this construct by PCR-based, site-directed mutagenesis. To generate the HCV replicon containing the PI-resistant mutations, a 1.2-kb HindIII/BstXI fragment derived from the HCV Con1 replicon was subcloned into a TA cloning vector, pCR2.1 (Invitrogen). The PI-resistant mutations in the NS3 serine protease domain were introduced into the pCR2.1 vector containing the HindIII/BstXI HCV fragment by PCR, and a 579-bp BsrGI/BstXI fragment containing the mutated residue was subcloned back into a second generation Con1 replicon plasmid containing three adaptive mutations, pBR322-HCV-Neo-mADE (see below). All constructs were confirmed by sequencing. Generation of HCV Replicon Cells—The Con1 subgenomic replicon plasmid, pBR322-HCV-Neo, was digested with ScaI (New England Biolabs). Full-length HCV subgenomic replicon RNA was generated from the linearized DNA template using a T7 Mega-script kit (Ambion) and treated with DNase to remove the template DNA. The run-off RNA transcripts were electroporated into Huh-7 cells, and stable HCV replicon cell lines were selected with 0.25 or 1 mg/ml G418 (Geneticin) in Dulbecco's modified minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). HCV replicon-stable cells were maintained in DMEM, 10% FBS, and 0.25 mg/ml G418. During the course of generation of the HCV subgenomic replicon stable cell lines, several different patterns of adaptive mutations were identified. One of these patterns contains three substitutions in the HCV nonstructural proteins, 3C. Lin, unpublished results. which were introduced into the original pBR322-HCV-Neo plasmid by site-directed mutagenesis to generate the second generation subgenomic replicon plasmid, pBR322-HCV-Neo-mADE. When the T7 run-off RNA transcripts from the ScaI-linearized pBR322-HCV-Neo-mADE plasmid were electroporated into Huh7 cells, stable replicon cell colonies were formed at a much higher efficiency than the original Con1 replicon RNA. The resistance mutations identified in this study were introduced into the pBR322-HCV-Neo-mADE replicon plasmid by site-directed mutagenesis. Stable replicon cell lines were generated using the T7 transcripts derived from either wild type pBR322-HCV-Neo-mADE or the ones with the resistance mutations. IC50 Determination of HCV PIs in the HCV Replicon Cell Assay— HCV Con1 subgenomic replicon cells were maintained in DMEM containing 10% FBS and 0.25 mg/ml G418. On the day prior to the assay, 10,000 HCV replicon cells/well were plated in a 96-well plate in DMEM plus 10% FBS. The next day, the medium was removed, and a compound serially diluted in DMEM, 2% FBS, and 0.5% Me2SO was added. The replicon cells were incubated with the compounds for 48 h. Total cellular RNA was extracted using RNeasy-96 (Qiagen), and the copy number of the HCV RNA was determined by a quantitative, real time RT-PCR (Taqman) assay. The cytotoxicity of the compounds was measured using a mitochondrial enzyme-based cell viability assay, CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). The IC50 and CC50 values of the compounds were calculated using four-parameter curve fitting (SoftMax Pro). Selection of HCV PI-resistant Replicon Cells—The HCV Con1 subgenomic replicon stable cells were serially passed in the presence of 0.25 mg/ml G418 and slowly increasing concentrations of VX-950 (series A) or BILN 2061 (series B). The concentrations of VX-950 ranged from 3.5 μm (or 10× IC50) in the 48-h assay (see above), to 28 μm (80× IC50). For BILN 2061, the starting concentration was 80 nm (80× IC50), and the final concentration was 12.5 μm (12,500× IC50). During the course of selection, replicon cells were split twice per week when a 70–90% confluence was reached. Fresh HCV PI was added every 3–4 days regardless of whether the cell culture was split. Identification of HCV PI Resistance Mutations—During the selection of HCV PI-resistant replicon cells, cell pellets were collected every time the cell culture was split. Total cellular RNA was extracted using the RNeasy miniprep kit (Qiagen). A 1.7-kb-long cDNA fragment encompassing the HCV NS3 serine protease region was amplified with a pair of HCV-specific oligonucleotides (5′-CCTTCTATCGCCTTCTTG-3′ and 5′-CTTGATGGTCTCGATGG-3′) using the Titan One-Step RT-PCR kit (Roche Applied Science). The amplified products were purified using the QIA-quick PCR purification kit (Qiagen). To monitor the emergence of the HCV PI-related mutations in the HCV NS3 serine protease domain during the selection, the purified 1.7-kb RT-PCR products of PI-treated replicons from several different culture time points were subjected to sequence determination. To determine the frequency of PI-resistant mutations, the 1.7-kb RT-PCR products of HCV RNA of the VX-950 or BILN 2061-resistant replicon cells were ligated into the TA cloning vector pCR2.1 (Invitrogen). For each time point, multiple individual bacterial colonies were isolated, and the HCV NS3 protease coding region of the purified plasmid DNA was sequenced. Expression and Purification of the HCV NS3 Serine Protease Domain—Each of the expression constructs for the HCV NS3 serine protease domain containing the wild type sequence or the resistance mutations (A156S, D168V, or D168A) were transformed into BL21/DE3 pLysS E. coli cells (Stratagene). Freshly transformed cells were grown at 37 °C in a BHI medium (Difco) supplemented with 100 μg/ml carbenicillin and 35 μg/ml chloramphenicol to an optical density of 0.75 at 600 nm. Induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside was performed for 4 h at 24 °C. Cell pastes were harvested by centrifugation and flash frozen at -80 °C prior to protein purification. All purification steps were performed at 4 °C. For each of the HCV NS3 proteases, 100 g of cell paste was lysed in 1.5 liters of buffer A (50 mm HEPES (pH 8.0), 300 mm NaCl, 0.1% n-octyl-β-d-glucopyranoside, 5 mm β-mercaptoethanol, 10% (v/v) glycerol) and stirred for 30 min. The lysates were homogenized using a microfluidizer (Microfluidics, Newton, MA), followed by ultracentrifugation at 54,000 × g for 45 min. Imidazole was added to the supernatants to a final concentration of 5 mm along with 2 ml of Ni2+-nitrilotriacetic acid resin pre-equilibrated with buffer A containing 5 mm imidazole. The mixtures were rocked for 3 h and washed with 20 column volumes of buffer A plus 5 mm imidazole. The HCV NS3 proteins were eluted in buffer A containing 300 mm imidazole. The eluates were concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, pre-equilibrated with buffer A. The appropriate fractions of the purified HCV proteins were pooled and stored at -80 °C. Enzymatic Assays for the HCV NS3 Serine Protease Domain—Enzymatic activity was determined using a modification of the assay described by Taliani et al. (27Taliani M. Bianchi E. Narjes F. Fossatelli M. Rubani A. Steinkuhler C. De Francesco R. Pessi A. Anal. Biochem. 1997; 240: 60-67Crossref Scopus (105) Google Scholar). An internally quenched fluorogenic depsipeptide (FRET substrate), Ac-DED(EDANS)EEαAbuΨ[COO]ASK (DABCYL)-NH2, was purchased from AnaSpec Inc. (San Jose, CA). The assay was run in a continuous mode in a 96-well microtiter plate format. The buffer was composed of 50 mm HEPES (pH 7.8), 100 mm NaCl, 20% glycerol, 5 mm dithiothreitol, and 25 μm KK4A peptide (KKGSVVIVGRIVLSGK). The KK4A peptide represents the central region of the NS4A cofactor from genotype 1a with lysine residues added for improved solubility (28Landro J.A. Raybuck S.A. Luong Y.-C. O'Malley E.T. Harbeson S.L. Morgenstern K.A. Rao G. Livingston D.J. Biochemistry. 1997; 36: 9340-9348Crossref PubMed Scopus (105) Google Scholar). The reaction was initiated by the addition of the FRET substrate after a 10-min preincubation of the buffer components with a 2 nm concentration of the NS3 protease at room temperature. The reaction was monitored at 30 °C for 20 min using a Molecular Devices fmax fluorometric plate reader. The filters for excitation and emission wavelengths were 355 and 495 nm, respectively. For determination of substrate kinetic parameters, concentrations of the FRET peptide were varied from 0.5 to 7.0 μm. Intermolecular quenching was not observed in this range. The substrate kinetic parameters, Km and Vmax, were determined by fitting the data to the Michaelis-Menten equation. Inhibition constants (Ki) were determined by titration of enzyme activity using the assay described above, except that compound dissolved in Me2SO (no greater than 2% (v/v) Me2SO; solvent only was used as control) was added to the buffer components and enzyme after the initial 10-min preincubation as described above. This mixture was incubated for an additional 15 min at room temperature prior to an incubation with the FRET substrate for another 20 min at 30 °C. Seven or eight concentrations of compound were assayed, and the resulting data were fitted to the integrated form of Morrison's equation for tight binding inhibition (29Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (720) Google Scholar). All substrate and inhibitor data were fitted using Marquardt-Levenberg nonlinear regression with GraphPad Prism software. Modeling—VX-950 and BILN 2061 were modeled into the active site of the NS3 serine protease domain using the crystal structure of a full-length HCV NS3 protein fused with a NS4A polypeptide, which was published by Yao et al. (30Yao N. Reichert P. Taremi S.S. Prosise W.W. Weber P.C. Struct. Fold Des. 1999; 7: 1353-1363Abstract Full Text Full Text PDF Scopus (367) Google Scholar) (Protein Data Bank code 1CU1). The coordinates of the protease domain of the A segment in this structure showed that the C-terminal strand of the NS3 protein binds in the substrate-binding site of the protease. The terminal carboxyl group of this strand is located near active site residues (His57, Asp81, and Ser139) such that it forms hydrogen bonds with the side chains of His57 and Ser139 as well as the backbone amides of residues 137 and 139, which form the oxyanion hole. Additionally, the last six residues (residues 626–631) of the NS3 protein form an extended, antiparallel β strand along the edge of the E2 strand of the protease β barrel (31Love 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 (500) Google Scholar) and makes 12 backbone-to-backbone hydrogen bonds. A product-based inhibitor like BILN 2061 is expected to bind to the NS3 protease in a similar fashion. Therefore, we utilized the coordinates of this crystal structure to build our models of inhibitor-protease co-complexes. BILN 2061 molecule was built using QUANTA molecular modeling software (Accelrys Inc., San Diego, CA), and manually docked into the active site such that its carboxyl group overlays with the C-terminal carboxylate of the full-length NS3 protein. The inhibitor molecule was then rotated such that it makes all of the following backbone hydrogen bonds: P1 NH with Arg155 carbonyl, P3 carbonyl with Ala157 NH, and P3 NH with Ala157 carbonyl. This mode of binding placed the large P2 group of the BILN 2061 in direct clash with the Arg155 side chain. To avoid the clash, the Arg155 side chain was modeled in an extended conformation, which was observed in a crystal structure of NS3 protease complexed with a close analogue of BILN 2061 (32Tsantrizos Y.S. Bolger G. Bonneau P. Cameron D.R. Goudreau N. Kukolj G. LaPlante S.R. Llinas-Brunet M. Nar H. Lamarre D. Angew. Chem. Int. Ed. Engl. 2003; 42: 1356-1360Crossref PubMed Scopus (157) Google Scholar). The inhibitor was energy-minimized in two stages. In the first stage, only the inhibitor and the side-chain atoms of Arg123, Arg155, and Asp168 of the protease were allowed to move during energy minimization for 1000 steps. In the second stage, all of the side-chain atoms of the protease were allowed to move along with the inhibitor for 1000 additional steps. This modeled structure closely mimics the published structure of the BILN 2061 analog (32Tsantrizos Y.S. Bolger G. Bonneau P. Cameron D.R. Goudreau N. Kukolj G. LaPlante S.R. Llinas-Brunet M. Nar H. Lamarre D. Angew. Chem. Int. Ed. Engl. 2003; 42: 1356-1360Crossref PubMed Scopus (157) Google Scholar). A similar procedure was adopted for modeling VX-950 into the NS3 protease active site. VX-950 was modeled as a covalent adduct with si-face attachment of the Ser139 side chain to the keto carbonyl of the inhibitor. This binding mode was observed for analogous ketoamide inhibitors (33Perni R.B. Pitlik J. Britt S.D. Court J.C. Courtney L.F. Deininger D.D. Farmer L.J. Gates C.A. Harbeson S.L. Levin R.B. Lin C. Lin K. Moon Y.-C. Luong Y.P. O'Malley E.T. Rao B.G. Thomson J.A. Tung R.D. Van Drie J.H. Wei Y. Bioorg. Med. Chem. Lett. 2004; 14: 1441-1446Crossref PubMed Scopus (66) Google Scholar) and ketoacid inhibitors (34Di Marco S. Rizzi M. Volpari C. Walsh M.A. Narjes F. Colarusso S. De Francesco R. Matassa V.G. Sollazzo M. J. Biol. Chem. 2000; 275: 7152-7157Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The main chain of the inhibitor was overlaid with residues 626–631 of the C-terminal strand of the full-length NS3 protein such that it makes all of the following backbone hydrogen bonds: P1 NH with Arg155 carbonyl, P3 carbonyl with Ala157 NH, P3 NH with Ala157 carbonyl, and P4 cap carbonyl with NH of Cys159. In this binding mode, the P2 group of VX-950 was placed in the S2 pocket without any need to move the Arg155 side chain. The t-butyl and the cyclohexyl groups were placed in S3 and S4 pockets, respectively. To be consistent, we used the same two-stage energy minimization protocol used for the BILN 2061 model. The side chain of Asp168 is exposed to solvent. The valine side chain of the D168V mutant can adopt three canonical conformations with χ1 = 60, -60, or 180°. All three orientations of the Val168 side chain were modeled. The interaction energy of the D168V mutant enzyme and the inhibitor was minimized by allowing the inhibitor and Val168 atoms to move while fixing positions of all of the other atoms of the protein molecule. In all cases, the Val168 side chain does not cause any steric clash with the inhibitor atoms. The serine mutation at Ala156 was modeled by the following procedure. The Ala156 side chain is in van der Waals contact with the P2 group of both of the inhibitors. The serine side chain of the A156S mutant was modeled at three canonical conformations of χ1 = 60, -60, and 180°, and the energy was minimized by holding the conformation of the rest of the protein fixed. These models were used to examine the effects of this mutation on inhibitor binding. The -60° conformation was found to have the lowest energy as it forms a hydrogen bond with the neighboring Arg155 carbonyl, but it causes the maximal number of unfavorable contacts with both inhibitors. The 60 and 180° conformations are energetically equivalent, but the 60° conformation has fewer unfavorable contacts and was used in our analysis. Development of Resistance to VX-950 in HCV Replicon Cells—VX-950 (Fig. 1) (25Babine, R. E., Chen, S.-H., Lamar, J. E., Snyder, N. J., Sun, X. D., Tebbe, M. J., Victor, F., Wang, Q. M., Yip, Y. Y. M., Collado, I., Garcia-Paredes, C., Parker, R. S. I., Jin, L., Guo, D., and Glass, J. I. (March 7, 2002), International Patent WO 0218369, Eli Lilly and Co.Google Scholar) was recently selected as a clinical candidate for hepatitis C treatment (26Perni R.B. Chandorkar G. Chaturvedi P.R. Courtney L.F. Decker C.J. Gates C.A. Harbeson S.L. Kwong A.D. Lin C. Lin K. Luong Y.P. Markland W. Rao B.G. Tung R.D. Thomson J.A. Hepatology. 2003; 38 (Abstr. 972)Crossref Google Scholar). VX-950 is a reversible, covalent inhibitor of the HCV NS3·4A serine protease. Although competitive with the peptide substrate in the active site, it exhibits apparent noncompetitive inhibition as a result of its tight binding properties and time-dependent inhibition mechanism. 4C. A. Gate and Y.-P. Luong, unpublished data. Incubation of the HCV Con1 subgenomic replicon cells with VX-950 resulted in a concentration-dependent decline of the HCV RNA level, as measured by the real time RT-PCR (Taqman) method (Fig. 2B). The IC50 value of VX-950 is 354 nm in the 48-h assay.Fig. 2Development of VX-950-resistant replicon cells. A, HCV Con1 subgenomic replicon cells were serially passed in the presence of G418 and increasing concentrations of VX-950. Replicon cells were split, and fresh VX-950 was added to medium twice a week, as indicated by filled diamonds. The open rectangle indicates the time period in which the replicon cells had little or no overall growth accompanied by a concurrent massive cell death. Total cellular RNA of replicon cells at various time points (indicated by open arrows) during the resistance selection was extracted and the RT-PCR product covering the HCV NS3 serine protease was sequenced either directly or after being subcloned into the TA vector. B, dose-dependent inhibition of the wild type (filled triangle) or the series A (VX-950-resistant) (open circle) replicon cells at day 56 by VX-950 was shown. HCV RNA level was determined after 48-h incubation with VX-950.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify VX-950 resistance mutations, the Con1 subgenomic replicon cells were serially passed in the presence of 0.25 mg/ml G418 and slowly increasing concentrations of VX-950 (series A) (Fig. 2A). The starting concentration of VX-950 was 3.5 μm, or 10 times the IC50, and the highest concentration was 28 μm, or 80 times the IC50. Replicon cells were split, or the medium was replenished every 3 or 4 days, and fresh VX-950 was added. Since a HCV NS3 serine protease inhibitor, such as VX-950, inhibits the HCV polyprotein processing and consequently blocks replication of HCV RNA, the steady state level of HCV proteins and neomycin transferase protein gradually declined and eventually became undetectable in the presence of high concentrations of VX
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