NATAP: HCV Protease Inhibitors & Drug Resistance
- Subject: NATAP: HCV Protease Inhibitors & Drug Resistance
HCV Protease inhibitors & Drug resistance
Note from Jules Levin: two HCV protease inhibitors are currently being studied in the early phases of research in HCV+ patients, the Schering drug 503034 (studied in treatment-experienced patients) & Vertex's VX-950 (studies in treatment-naive patients). Both show potency from 10-14 day studies as monotherapy and added potency when combined with peginterferon. Phase II studies with these drugs are in progress. SCH-503034 is starting dose-ranging studies in treatment-experienced patients with several different dose regimens. Vertex has selected a dose. Over the next several years development studies for these drugs and additional HCV drugs in early development will take place, as there are several other types of HCV drugs starting studies in patients at this time. Study for NM283, a polymerase inhibitor, is entering phase III now. In HIV we learned that drug resistance is crucial in understanding how to use ART medications. Resistance to one drug in a class usually results in some
degree of cross-resistance to other drugs in the same class. Early studies of HCV protease inhibitors have obserbed resistance mutations emerging. The following report contains a published article on resistance mutations discovered associated with HCV protease inhibitors, and several interesting slides presented at CROI by presenters talking about HCV pathogenesis, and treatment, and HCV protease inhibitor drug resistance.
ââ¦..From these examples, it is clear that, as new HCV-specific inhibitors enter clinical trials, resistance could become a major problem in patients treated with drugs targeting the HCV enzymes, especially in monotherapy. The replication rate of HCV in patients was reported to be in the range of 1010 to 1012 viral particles/day, higher than the replication rate of HIV in patients (38). In vitro resistance mutations against the HCV protease or polymerase inhibitors have also been identified in the replicon system (25, 34, 35, 39, 40). These studies suggest that future hepatitis C therapy involving small molecule inhibitors of HCV enzymes might require multidrug combination, as in the case of the current HIV treatmentsâ¦..â you can read the full portion of this discussion in the author's discussion at the end of this report.
as you can see in this chart just below up to 61% of HCV-monoinfed patients in studies with peginterferon plus ribavirin can achieve a sustained viral response which can translate into virus eradication and an effectual 'cure'. However, the therapy is difficult to tolerate and more effective results are the goal, and results are less in certain patient populations: African-Americans, HIV-coinfected, and patients with genotype 1 achieve much less viral clearance rates. Therefore, we are now entering a new era of the development of more effective HCV drugs and orally administered drugs which include HCV protease inhibitors and ploymerase inhibiitors.
This is a schematic view of the various HCV associated extra-hepatic conditions that can be caused by having HCV including diabetes and fatty liver.
The course of HCV disease is affected by two factors, the host or patient response to the invasion of the body by a virus, HCV, and the characteristics of the virus. The host may try to fight the virus with its immune system but usually the virus, HCV, can evade the immune response.
The immune system's response to HCV is complicated but ultimately tries to fight the virus & usually fails as chronic infection becomes established & HCV invades the liver cell, the hepatocyte.
HCV Protease Inhibitors
Activity & Resistance
Oral Presentation at 13th CROI Feb 2006
By Ann Kwong, Vertex Pharmaceuticals
In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: Structural analysis indicates different resistance mechanisms
Chao Lin, Kai Lin, Yu-Ping Luong, B. Govinda Rao, Yun-Yi Wei, Debra L. Brennan, John R. Fulghum, Hsun-Mei Hsiao, Sue Ma, John P. Maxwell, Kevin M. Cottrell, Robert B. Perni, Cynthia A. Gates, and Ann D. Kwong
Biology, Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139
We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3o4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3o4A 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 sub-genomic replicon system to compare VX-950 to another HCV NS3o4A 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.
Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease
Antimicrobial Agents and Chemotherapy, March 2006, p. 899-909, Vol. 50, No. 3
Robert B. Perni,* Susan J. Almquist, Randal A. Byrn, Gurudatt Chandorkar, Pravin R. Chaturvedi, Lawrence F. Courtney, Caroline J. Decker, Kirk Dinehart, Cynthia A. Gates, Scott L. Harbeson, Angela Heiser, Gururaj Kalkeri, Elaine Kolaczkowski, Kai Lin, Yu-Ping Luong, B. Govinda Rao, William P. Taylor, John A. Thomson, Roger D. Tung, Yunyi Wei, Ann D. Kwong, and Chao Lin*
ABSTRACT: VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (Ki*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse
model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.
One of the major factors limiting the efficacy of virus-specific therapies against many retroviruses and RNA viruses is the development of resistance to antiviral drugs. Resistance to many antiviral drugs, including HIV protease inhibitors, HIV or hepatitis B virus reverse transcriptase inhibitors, or influenza neuraminidase, is caused by specific mutations in the viral enzymes. In vitro resistance mutations against HCV protease inhibitors have been identified using replicon cells (14, 15, 20, 39). The major in vitro resistance mutation against VX-950 is a substitution of Ala156 of the NS3 protease with Ser (A156S), which remained sensitive to BILN 2061 (15). Reciprocally, the dominant in vitro resistance mutations against BILN 2061, substitutions of Asp168 with Ala (D168A) or Val (D168V), conferred several-hundredfold-reduced sensitivity to BILN 2061 while remaining fully sensitive to VX-950 (15). Not surprisingly, none of these 3 mutations appeared under the selective pressure of
both inhibitors. Instead, substitutions of Ala156 by either Val (A156V) or Thr (A156T) emerged to confer cross-resistance to both VX-950 and BILN 2061 (14). It should be noted that many, if not all, of these in vitro resistance mutants remain susceptible to IFN- and/or ribavirin and have significantly decreased replication capacity or fitness in viral RNA replication in cell culture (14, 20). It remains to be seen whether and, if so, which, resistance mutation(s), if any, will appear in HCV patients treated with new antiviral drugs. In this study, we demonstrated that a prolonged incubation with VX-950 resulted in a >4-log10 reduction in the HCV RNA levels in the replicon cells and no HCV replicon-containing cells remained after selection with G418 upon the withdrawal of VX-950. However, the replication rate of HCV in patients has been reported to be in the range of 1010 to 1012 viral particles per day, which is higher than the viral replication rate in HIV-infected patients (29).
Further studies are needed to determine whether a single antiviral agent, such as VX-950, or a combination of two or more antiviral drugs will be needed (as in the case of HIV) to suppress the emergence of resistant variants in patients with hepatitis C.
In Vitro Studies of Cross-resistance Mutations against Two Hepatitis C Virus Serine Protease Inhibitors, VX-950 and BILN 2061*
J. Biol. Chem., Vol. 280, Issue 44, 36784-36791, November 4, 2005
Chao Lin1, Cynthia A. Gates, B. Govinda Rao, Debra L. Brennan, John R. Fulghum, Yu-Ping Luong, J. Daniel Frantz, Kai Lin, Sue Ma, Yun-Yi Wei, Robert B. Perni, and Ann D. Kwong
VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3Â·4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3Â·4A protease inhibitor, BILN 2061 (Lin, C., Lin, K., Luong, Y.-P., Rao, B. G., Wei, Y.-Y., Brennan, D. L., Fulghum, J. R., Hsiao, H.-M., Ma, S., Maxwell, J. P., Cottrell, K. M., Perni, R. B., Gates, C. A., and Kwong, A. D. (2004) J. Biol. Chem. 279, 17508-17514). Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of
resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala156 with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.
Chronic hepatitis C has become one of the most common liver diseases and is estimated to affect 170 million patients worldwide and 1% of the population in developed countries (1). In many patients, hepatitis C virus (HCV)2 infection leads to liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care, a 48-week treatment with pegylated interferon (IFN)- in combination with ribavirin, has a sustained viral response rate of 40-50% in the difficult-to-treat genotype 1 HCV-infected patients (Refs. 4 and 5; for a review, see Refs. 6 and 7), which accounts for the majority of the hepatitis C patient population in the developed countries. A more effective treatment with fewer side effects and shorter treatment durations is urgently needed for HCV-infected patients.
HCV is an enveloped virus containing a single-stranded, positive polarity RNA that encodes a polyprotein precursor of 3000 amino acids. The HCV polyprotein is proteolytically processed by cellular and viral proteases into at least 10 distinct products in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 8). The structural proteins are processed by host signal peptidases, whereas the nonstructural (NS) proteins are processed by two virally encoded proteases, the NS2Â·3 and NS3Â·4A proteases. The NS2Â·3 protease is responsible for the cleavage between the NS2 and NS3 proteins, whereas the NS3Â·4A serine protease is responsible for the release of the remaining four nonstructural proteins, NS4A, NS4B, NS5A, and NS5B (9-13). The essentiality of the NS3Â·4A serine protease for viral replication has been demonstrated by the nonproductive infection following liver inoculation of chimpanzees with a genomic HCV RNA containing a mutation in the NS3 protease
active site (14). 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 the proteolytic activity of the NS3 serine protease (15-19). The central region of NS4A forms a tight heterodimer with the NS3 protein (18), for which the first x-ray crystal structure was solved in 1996 (20). The NS3Â·4A serine protease has been one of the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 21). VX-950, a potent, small molecule, selective inhibitor of the HCV NS3Â·4A serine protease, was discovered using structure-based drug design techniques (22). Clinical proof of concept for HCV protease inhibitors (PIs) has been demonstrated by Boehringer Ingelheim and Vertex Pharmaceuticals Inc. using BILN 2061 (23) and VX-950,3 respectively. Both compounds reduced HCV viral load in patients by 2-3 log10 in the first 3 days of dosing. In some patients treated
with VX-950, the HCV viral load dropped by >4 log10 to below the limit of detection (<10 IU/ml) during 14 days of dosing.3
Because of the error-prone nature of the viral reverse transcriptase of retroviruses or the RNA-dependent RNA polymerase of RNA viruses, drug resistance frequently emerges in patients treated with antiviral drugs and therefore limits the efficacy of these therapies. For these new HCV NS3Â·4A serine protease inhibitors, resistance could become a major issue in treated patients. In our previous study, we used the HCV subgenomic replicon system to identify resistance mutations against two HCV PI clinical candidates, BILN 2061 and VX-950 (25). The in vitro resistance mutations selective against either inhibitor result in a significant reduction in susceptibility to the same inhibitor. However, the primary BILN 2061-resistant mutants are fully susceptible to VX-950, and the major VX-950-resistant mutant remains sensitive to BILN 2061. In this study, we identified mutants that are cross-resistant to both PIs. Analysis of structural models of these mutants indicated that steric hindrance is
the primary reason for the resistance of these mutants against both HCV PIs. HCV replicon cells containing either cross-resistance mutation A156T or A156V displayed significantly reduced fitness (or replication capacity) and remained as sensitive to IFN- or ribavirin as the wild-type replicon in cell assays.
HCV NS3Â·4A Serine Protease Inhibitors VX-950 and BILN 2061- VX-950 (Fig. 1) has recently been shown to possess antiviral activity in chronic hepatitis C patients in a phase I trial.3 VX-950 is a reversible covalent inhibitor of the HCV NS3Â·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.4 Incubation of the HCV Con1 subgenomic replicon cells with VX-950 resulted in a concentration-dependent decline in the HCV RNA level as measured by real-time RT-PCR (TaqMan), with an average IC50 of 354 nM in the 48-h assay (22, 25). Another HCV NS3Â·4A protease inhibitor, BILN 2061 (Fig. 1), is the first PI to demonstrate proof of concept in hepatitis C patients (23). Its average IC50 value in the 48-h replicon cell assay is in the low single digit nanomolar range.
Development of PI-cross-resistant HCV Replicons from VX-950-resistant Cells-To identify mutants that are cross-resistant to both VX-950 and BILN 2061, several selection schemes were employed. First, a VX-950-resistant replicon cell line (series A in Ref. 25) was initially developed by serial passage of HCV subgenomic replicon cells in the presence of 0.25 mg/ml G418 and increasing concentrations of VX-950. After the series A replicon cells became resistant to 14 ÂµM VX-950, the cells were then serially passaged in the presence of slowly increasing concentrations of BILN 2061 in addition to 0.25 mg/ml G418 and 14 ÂµM VX-950 (designated series C) (Fig. 2A). For BILN 2061, the starting and final concentrations were 40 nM and 6.4 ÂµM, respectively. Every 3 or 4 days, replicon cells were split; the medium was replenished; and fresh VX-950 and BILN 2061 were added. Because HCV PIs inhibit NS3Â·4A serine protease activity and consequently block replication of HCV RNA, the steady-state
levels of HCV proteins and neomycin phosphotransferase protein gradually declined over time and eventually became undetectable in the presence of high concentrations of HCV PI (data not shown). Cells with low or no neomycin phosphotransferase protein proliferated at a gradually decreasing rate and eventually died in the presence of G418. Replicon cells with the dominant VX-950 resistance mutation (A156S) were expected to die in the presence of increasing concentrations of BILN 2061 because they have been shown to be susceptible to inhibition by BILN 2061 (25). Only HCV RNA with mutations that were cross-resistant to both VX-950 and BILN 2061 could replicate in the presence of high concentrations of both HCV PIs and support the growth of the replicon cells harboring them. During the development of series A (VX-950-resistant) or series B (BILN 2061-resistant) replicon cell lines, the cell growth became stalled for a period of 7-10 days, concurrent with massive cell death. However,
replicon cells in series C grew normally for the entire selection process, which lasted for 2 months. The IC50 of BILN 2061 for the series C replicon cells at day 52 was determined to be 3.9 ÂµM in the 48-h assay, which is 390-fold higher than the IC50 for the series A (VX-950-resistant) replicon cells (10 nM) (Fig. 2C). The series C replicon cells at day 52 remained resistant to VX-950, with IC50 > 30 ÂµM (Fig. 2B). Therefore, the series C replicon cells at day 52 were cross-resistant to both VX-950 and BILN 2061.
Total cellular RNA was extracted from the series C replicon cells (which had been cultured in the presence of 14 ÂµM VX-950 and 0.32 ÂµM BILN 2061) at day 32 and subjected to RT-PCR to amplify the coding region of the HCV NS3 serine protease domain. The RT-PCR product was bulk-sequenced to identify the position(s) of potential mutations that could be responsible for the observed reduction in sensitivity to both HCV PIs. Substitutions at Ala156 in the protease domain were observed, suggesting that mutations at residue 156 might be critical for the reduced sensitivity to both PIs. No amino acid substitution was observed in the NS4A coding sequences or at any of the four proteolytic sites in the HCV nonstructural protein region that are cleaved by the NS3Â·4A serine protease. To delineate the identity and frequency of the substitutions, a 1.7-kb RT-PCR product of the series C replicon RNA at day 32 was subcloned into the TA vector, and 10 individual colonies were subjected to
sequencing. Six clones had the A156T substitution; three clones had the A156V substitution; and the last clone retained the A156S mutation.
Development of PI-cross-resistant HCV Replicons from BILN 2061-resistant Cells-The second selection scheme was to grow BILN 2061-resistant replicon cells in the presence of both BILN 2061 and VX-950. In this case, a BILN 2061-resistant replicon cell line (series B in Ref. 25) was initially developed by serial passage of the HCV subgenomic replicon cells in the presence of 0.25 mg/ml G418 and increasing concentrations of BILN 2061. The BILN 2061-resistant series B replicon cells were then subsequently serially passaged in the presence of 0.25 mg/ml G418 and slowly increasing concentrations of VX-950 and BILN 2061 (designated series D) (Fig. 3A). For BILN 2061, the concentrations ranged from 160 nM to 6.4 ÂµM. Only two concentrations of VX-950 were used: 7 and 14 ÂµM. Replicon cells containing the major BILN 2061 resistance mutations (D168V and D168A) were expected to die in the presence of high concentrations of VX-950 because they have been shown to be susceptible to inhibition by
VX-950 (25). Again, only HCV RNA with mutations that were cross-resistant to both VX-950 and BILN 2061 could replicate in the presence of high concentrations of both HCV PIs and support the growth of the replicon cells harboring them. However, the replicon cells in series D grew normally for most of the selection process, which lasted for 2 months. Because 30 ÂµM VX-950 did not result in a >50% reduction in HCV RNA in the series D replicon cells at day 52, the actual IC50 values of VX-950 could not be determined, but would be >100-fold higher than the IC50 (0.26 ÂµM) for the series B (BILN 2061-resistant) replicon cells (Fig. 3B). The IC50 values of BILN 2061 for the series D replicon cells at day 52 were determined to be 2.3 ÂµM, indicating that the series D replicon cells at day 52 remained resistant to BILN 2061 (Fig. 3C). Therefore, the series D replicon cells at day 52 were cross-resistant to both VX-950 and BILN 2061.
Total cellular RNA was extracted from the series D replicon cells (which had also been cultured in the presence of 14 ÂµM VX-950 and 0.32 ÂµM BILN 2061) at day 32 and subjected to RT-PCR to amplify the coding region of the HCV NS3 serine protease domain. The RT-PCR product was bulk-sequenced to identify the position(s) of potential mutations that could be responsible for the observed reduction in sensitivity to both HCV PIs. Again, substitutions at Ala156 in the protease domain were observed, confirming that mutations at residue 156 might be critical for the reduced sensitivity to both PIs. No amino acid substitution was observed in the NS4A coding sequences or at any of the four proteolytic sites in the HCV nonstructural protein region that are cleaved by the NS3Â·4A serine protease. To delineate the identity and frequency of the substitutions, a 1.7-kb RT-PCR product from the series D replicon RNA at day 32 was subcloned into the TA vector, and 14 individual colonies were subjected
to sequencing. 12 clones had the A156V substitution; one clone had the A156T mutation; and the last clone had two mutations (A156S and D168V).
Development of PI-cross-resistant HCV Replicons from NaÃ¯ve Replicon Cells-In our previous study of resistance mutations against a single HCV PI (VX-950 or BILN 2061), cell growth became stalled for several days, during which time massive cell death was observed (25), signaling the emergence of resistant mutant replicon cells and concurrent death of nonresistant replicon cells. However, no such cell death or slow down in cell growth was observed during selection of the cross-resistant series C and D replicon cells as described above. It is possible that the cross-resistance mutations A156T and A156V may have already existed in VX-950-resistant (series A) or BILN 2061-resistant (series B) replicon cells as a minor population. If so, these two selection schemes could provide bias toward the A156T or A156V mutation over other potential cross-resistance mutations. Thus, a third selection scheme was performed using the naÃ¯ve HCV replicon cells that had not been exposed to either
The Con1 subgenomic replicon cells derived from pBR322-HCV-Neo-mADE (25) were serially passaged in the presence of 0.25 mg/ml G418 and slowly increasing concentrations of both VX-950 and BILN 2061 (designated series E) (Fig. 4). The starting and final concentrations of VX-950 were 3.5 and 14 ÂµM, respectively. For BILN 2061, the starting and final concentrations were 80 nM and 1.6 ÂµM, respectively. Every 3-4 days, replicon cells were split if they were confluent, the medium was replenished, and fresh VX-950 and BILN 2061 were added. Replicon cells in series E grew normally for the first 10 days in the presence of 3.5 ÂµM VX-950 and 160 nM BILN 2061. After 10 days, the series E cells grew significantly slower, and massive cell death was observed between days 10 and 21 (Fig. 4). Normal growth did not resume until day 21. Total cellular RNA was extracted from the series E cells at days 10, 21, and 48 and subjected to RT-PCR to amplify the coding region of the HCV NS3 serine protease
domain. No HCV PI-related mutation was observed in the NS3 serine protease domain of the series E replicon cells at day 10 compared with the wild-type Con1 replicon cells cultured in the absence of both HCV PIs. To delineate the identity and frequency of the substitutions, a 1.7-kb RT-PCR product from the series E replicon RNA at day 21 or 48 was subcloned into the TA vector, and multiple clones were sequenced for both samples. In the day 21 sample of the series E replicon cells (which had been cultured in the presence of 3.5 ÂµM VX-950 and 0.32 ÂµM BILN 2061 for 14 days), 65% or 30 of 46 clones had the A156T substitution, whereas 35% or 16 of 46 clones had the A156V substitution. In the day 48 sample of the series E (which had been cultured in the presence of 14 ÂµM VX-950 and 1.6 ÂµM BILN 2061 for 14 days), 80% or 35 of 44 clones had the A156T substitution, whereas 20% or 9 of 44 clones had the A156V substitution. In either case, no other mutation in the NS3 serine protease domain
was found in >10% of the TA plasmid clones, indicating that A156T and A156V are the only two mutations that confer cross-resistance to both VX-950 and BILN 2061.
Either Mutation A156V or A156T Is Sufficient to Confer Cross-resistance to both VX-950 and BILN 2061-To confirm that the observed mutations at Ala156 are sufficient to confer cross-resistance to both VX-950 and BILN 2061, site-directed mutagenesis was used to replace Ala156 with either Val or Thr in the wild-type NS3 protease domain. The NS3 serine protease domain containing either mutation was expressed in E. coli and purified for enzyme characterization. These mutations were also introduced into high efficiency subgenomic replicon plasmids for characterization in the HCV replicon system.
The catalytic efficiency (kcat/Km) of the A156T or A156V mutant protease for the FRET substrate was 7- or 4-fold lower than that of the wild-type protease, respectively (TABLE ONE). The Ki values of VX-950 were 9.9 and 33 ÂµM for the A156T and A156V mutant proteases, respectively, which are 99- or 330-fold higher than that for the wild-type protease (0.1 ÂµM), respectively (TABLE TWO). Both mutant proteases were apparently unaffected by up to 1.2 ÂµM BILN 2061 (TABLE TWO). These data indicate that either mutant protease is at least 63-fold less susceptible to BILN 2061 compared with the wild-type protease. The actual magnitude of resistance could not be determined because the solubility of BILN 2061 was limited at concentrations >1.2 ÂµM in the assay buffer as measured by the absorbance at 650 nm (data not shown).
The fitness or replication capacity of the PI cross-resistance mutations was determined in a transient transfection system using the luciferase activity as the surrogate readout. Because the luciferase mRNA is part of the HCV replicon RNA, the amount of luciferase protein or its activity (as a direct consequence of its mRNA translation) can be used as an indirect readout of the HCV replicon RNA levels in the transiently transfected cell. The normalized replication capacity or fitness of the HCV replicon containing the A156T or A156V mutation was 5 or 3%, respectively, of that of the wild-type replicon in the luciferase transient transfection assay. These results are consistent with the lower catalytic efficiency of the two mutants compared with that of the wild-type HCV NS3 serine protease.
The HCV RNA level in the stable replicon cells containing the A156T or A156V substitution was also lower than that in the stable wild-type replicon cells (data not shown), which was not unexpected given the reduced replication capacity or fitness of the mutant replicons and the lower catalytic efficiency of the mutant proteases. No significant reduction in HCV replicon RNA by up to 30 ÂµM VX-950 was observed in either mutant replicon cell line, indicating at least a 75-fold decrease in sensitivity conferred by either mutation (TABLE THREE). The IC50 of BILN 2061 for the A156T replicon cells was 1.09 ÂµM, which is 272-fold higher than that for the wild-type replicon cells (4 nM). The IC50 of BILN 2061 for the A156V mutant replicons was 10 ÂµM, indicating a >2500-fold decrease in sensitivity conferred by the A156V mutation (TABLE THREE).
Because the current standard of care is a combination of pegylated IFN- and ribavirin, we wanted to determine whether these PI-cross-resistant HCV replicon cells remain sensitive to either IFN- or ribavirin. As shown in TABLE THREE, the IC50 of either IFN- or ribavirin remained virtually the same for HCV replicon cells containing A156T or A156V compared with the wild-type replicon cells. These results suggest that combination with IFN- or even ribavirin could be a potential therapeutic strategy to suppress the emergence of HCV PI-resistant mutants.
We have previously shown that the A156S mutant is resistant to VX-950, but not to BILN 2061 (25). Substitution of Asp168 with Val or Ala causes resistance to BILN 2061, but susceptibility to VX-950 remains. Because there is no apparent overlap between the in vitro dominant resistance mutation profiles of VX-950 and BILN 2061, it is likely that a combination of VX-950 and BILN 2061 would suppress the appearance of their dominant resistance mutations. Indeed, not one of these three single residue substitutions was observed when the HCV replicon cells were treated with both HCV PIs, as we reported in this study. Instead, we found that substitution of Ala156 with either Val or Thr in the HCV serine protease domain conferred cross-resistance to both inhibitors. Ala156 is located on the E2 -strand of the HCV NS3Â·4A protease, which is involved in backbone-to-backbone hydrogen bond interactions with the inhibitor, and its side chain divides the S2 and S4 subsites of the substrate-binding
site of the protease. The Ala156 side chain is in van der Waals contact with the P2 group of the two inhibitors (Fig. 5). The A156S substitution puts the Ser side chain too close to the P4 group of the two inhibitors. Because the P4 group of BILN 2061 is a terminal group, it could avoid the repulsive interaction by moving out without losing any other interactions between BILN 2061 and the HCV serine protease. On the other hand, any movement of the P4 group of VX-950 would cause loss of the hydrogen bond with the cap carbonyl as well as hydrophobic interactions of the P4 group of the inhibitor (25).
Of the three possible conformations of the Ser side chain at position 156, the conformation of 1 = -60Â° (Fig. 6) has the least number of unfavorable contacts with VX-950 and BILN 2061. The other two conformations (1 = 180Â° and 60Â°) (Fig. 6) have unfavorable contacts with both inhibitors either at the P2 side chain or at the P3 carbonyl group. In the A156T or A156V mutant, the additional hydroxyl or methyl group, respectively, at the C- atom of the residue 156 side chain is forced to occupy one of these two positions with 1 = 180Â° or 60Â°, which has unfavorable interactions with the inhibitors. The three possible conformations of Thr156 are shown schematically in Fig. 6. In all cases, the additional methyl and hydroxyl groups of Thr156 have a repulsive interaction with the inhibitor and/or enzyme backbone atoms. By energy minimization, we found that the 180Â°/-60Â° conformation (Fig. 6) has the least repulsive interaction and that the main cause of the repulsion is the close clash
of the terminal hydroxyl or methyl group of the Thr156 or Val156 side chain, respectively, against the P3 carbonyl group of the inhibitors. Therefore, the A156T and A156V mutants are resistant to both inhibitors.
A BLAST search of the GenBankTM Data Bank was conducted using the amino acid sequences of the HCV NS3 protease domain from the Con1 replicon. A total of 437 HCV isolates from all six major genotypes were identified, and Ala156 is absolutely conserved in all of the isolates. The lack of polymorphism at amino acid 156 of the NS3 serine protease suggests that substitution at this position might be unfavorable for viral replication. It remains to be seen if substitution at Ala156 has a deleterious effect on the virus life cycle in vivo. However, several lines of evidence suggest that the HCV protease with either cross-resistance mutation A156V or A156T may have lower fitness and be more compromised in its ability to support viral replication than the wild-type protease or other PI-resistant mutant proteases containing the A156S or D168V/D168A mutations. First, the catalytic efficiency of the A156V and A156T mutant proteases was 4- and 7-fold lower, respectively, than that of the
wild-type protease. Second, A156S was found to be the dominant resistance mutation against VX-950, even though A156T and A156V were at least as resistant to VX-950 as A156S. The same applies to BILN 2061 resistance mutations: D168V and D168A were found to be the dominant mutations even though A156T and A156V were as resistant to BILN 2061 as the substitutions at Asp168. Finally, both cross-resistance mutations were shown to have a significantly reduced replication capacity or fitness in transiently transfected replicon cells.
Several in vitro studies suggest that the HCV NS3Â·4A serine protease may block the IFN signal transduction pathway (31-33) and therefore interfere with host innate immune responses, which could be one of the reasons that HCV escapes immune clearance and maintains chronic infection. One may expect that the HCV PI-resistant mutants, with a significantly diminished fitness and a less efficient NS3Â·4A protease, may be less able to interfere with the IFN pathway. If so, the HCV replicon cells containing these PI resistance mutations may become more sensitive to inhibition by IFN-. However, no change in IC50 values was observed for A156T or A156V replicon cells compared with wild-type replicon cells in our study, which is similar to what has been previously reported for other HCV PI resistance mutations (34, 35).
One of the major factors limiting the efficacy of virus-specific therapies against many retroviruses and RNA viruses is the development of resistance to antiviral drugs. Resistance to inhibitors of human immunodeficiency virus (HIV) reverse transcriptase or protease is caused by specific mutations in the viral enzymes (for a review, see Ref. 36). Because of the error-prone nature of the HIV reverse transcriptase, resistance mutations emerge quickly in patients who are on monotherapy with HIV-specific inhibitors. It is estimated that all possible single mutations can be randomly generated within 1 day in an HIV-infected patient. Even though elimination or cure of HIV infection in patients remains an elusive goal, multidrug combination therapies have been shown to be more effective than monotherapy in reducing HIV viral load and suppressing the emergence of resistance mutants. Drug-resistant strains of hepatitis B virus containing specific mutations in the viral polymerase are the
primary cause of treatment failure of lamivudine, the first hepatitis B virus-specific drug. It has been reported that the frequency of resistance mutations against lamivudine increases from 24% in the first year to 67% in the fourth year in hepatitis B patients treated with lamivudine (37).
From these examples, it is clear that, as new HCV-specific inhibitors enter clinical trials, resistance could become a major problem in patients treated with drugs targeting the HCV enzymes, especially in monotherapy. The replication rate of HCV in patients was reported to be in the range of 1010 to 1012 viral particles/day, higher than the replication rate of HIV in patients (38). In vitro resistance mutations against the HCV protease or polymerase inhibitors have also been identified in the replicon system (25, 34, 35, 39, 40). These studies suggest that future hepatitis C therapy involving small molecule inhibitors of HCV enzymes might require multidrug combination, as in the case of the current HIV treatments. Because there is no overlap between the in vitro dominant resistance mutation profiles of VX-950 and BILN 2061 (25), it is possible that a combination of VX-950 and BILN 2061 would suppress the appearance of the dominant resistance mutations against each inhibitor. Indeed,
the dominant resistance mutations A156S, D168V, and D168A either did not emerge or disappeared when various types of HCV replicon cells were incubated with both VX-950 and BILN 2061. However, Thr or Val substitution at a single amino acid (Ala156)in the serine protease domain, which resulted in cross-resistance to both inhibitors, appeared in the replicon cell system under the selective pressure of both inhibitors.
It should be noted that all of the HCV PI-resistant replicon cells were selected under increasing concentrations of inhibitors, which would be ideal for the development of resistance. When a relatively high concentration of HCV PI was used at the beginning of selection, a 4-5 log10 reduction in HCV RNA levels was observed, and no replicon cells were recovered after 2 weeks of treatment (24). It remains to be seen whether treatment of hepatitis C patients with a single HCV PI or a combination of different PIs will be able to suppress the virus so that no resistant virus will appear. Finally, because all of these PI-resistant mutant replicons remained sensitive to IFN-, combinations of HCV PIs with IFN-, inhibitors targeting other host factors that support viral replication, or inhibitors against a different HCV protein or nucleic acid target could raise the barrier to the emergence of resistance against PI(s) and therefore increase the efficacy of anti-HCV therapy.
Lin, C., C. A. Gates, B. G. Rao, D. L. Brennan, J. R. Fulghum, Y.-P. Luong, J. D. Frantz, K. Lin, S. Ma, Y.-Y. Wei, R. B. Perni, and A. D. Kwong. 2005. In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061. J. Biol. Chem. 280:36784-36791
Lin, C., K. Lin, Y.-P. Luong, B. G. Rao, Y. Y. Wei, D. L. Brennan, J. R. Fulghum, H. M. Hsiao, S. Ma, J. P. Maxwell, K. M. Cottrell, R. B. Perni, C. A. Gates, and A. D. Kwong. 2004. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J. Biol. Chem. 279:17508-17514.
Trozzi, C., L. Bartholomew, A. Ceccacci, G. Biasiol, L. Pacini, S. Altamura, F. Narjes, E. Muraglia, G. Paonessa, U. Koch, R. De Francesco, C. Steinkuhler, and G. Migliaccio. 2003. In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor. J. Virol. 77:3669-3679
Lu, L., T. J. Pilot-Matias, K. D. Stewart, J. T. Randolph, R. Pithawalla, W. He, P. P. Huang, L. L. Klein, H. Mo, and A. Molla. 2004. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 48:2260-2266.
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