The genetic diversity of HCV, which classifies the virus into six major genotypes, multiple subtypes, and numerous quasispecies, presents the virus with opportunities to naturally select for the genetic isolate that is best fit for replication. With the development of antiviral compounds and the monitoring of resistance, it has become apparent that mutations which would allow the virus to escape inhibition by drugs will be enriched over the baseline genomic sequences. These studies have also led to the identification of subtype-dependent resistant mutations for NS3 protease, NS5A, and NS5B nonnucleoside inhibitors in both replicons and clinical isolates (7
). Compared to these three classes of HCV inhibitors, relatively less is known about genotype- and subtype-dependent resistance for nucleoside/tide analogs targeting the active site of NS5B. Herein we present anti-HCV results, cross-resistance analysis, and resistance selection studies of the phosphoramidate nucleotide prodrug PSI-7977 using replicons from various genotypes and subtypes.
Results from studies using GT 1a (H77)-, 1b (Con1)-, and 2a (JFH-1)-derived replicons and chimeric replicons with the NS5B region from GT 2a (J6), 2b, and 3a clearly showed that PSI-7977 is a potent HCV inhibitor across NS5B proteins from different isolates. Our previous work with PSI-7409, the active 5′-triphosphate metabolite of PSI-7977, also showed that it inhibited the enzymatic activity of NS5B polymerase from GTs 1 to 4 with similar 50% inhibitory concentrations (18
). In comparison, nonnucleoside inhibitors are more selective in their targets and exhibit a genotype- and/or subtype-dependent range of activities. Currently, we are working on generating replicative chimeric replicons with NS5B from GTs 4 to 6, but as the 5′-triphosphate metabolite of PSI-7977 targets the active conserved site of the polymerase, we expect that PSI-7977 would retain its activity in these constructs.
Cross-resistance studies using the panels of replicons with mutations in NS3/4A protease, NS5A, and NS5B showed that with the exception of GT 1b S282T, these replicon variants remained fully susceptible to PSI-7977. An efficacious antiviral therapy for hepatitis C patients will likely require the combination of two or more HCV inhibitors to prevent the emergence of resistant variants. Our data therefore indicate that PSI-7977 can be combined with different classes of HCV inhibitors, including ribavirin and the 2′-F-2′-C
-methylguanosine analogs, which exhibit a different resistance profile. It was interesting to note that the F415Y change, which was previously observed in GT 1a-infected patients treated with ribavirin (47
), did not confer resistance to ribavirin in our hands. As GT 1a replicons were not available at that time, the cross-resistance data from the Young et al. study were generated using GT 1b replicons (which naturally contained a Tyr at position 415) and reverse substitution to generate the Y415F change (47
). It is possible that the F415Y change alone in GT 1a replicons is not sufficient to reduce the activity of ribavirin.
Unlike the NS3 protease, NS5A, and NS5B nonnucleoside inhibitors, PSI-7977 did not appear to exhibit genotype- or subtype-dependent resistance, as it selected the S282T change in replicons from H77 GT 1a, Con1 GT 1b, and JFH-1 GT 2a. While selection was not performed with the chimeric replicons, variants containing NS5B from the J6 GT 2a and GT 2b isolates confirmed that S282T did confer resistance to PSI-7977, comparable to the effect observed in GT 1b and 1a S282T replicons. Interestingly, analysis of the PSI-7977-selected JFH-1 GT 2a replicons indicates that JFH-1 was capable of enriching for amino acid changes in addition to S282T and that reduction of PSI-7977 activity was associated with additional mutations in combination with S282T. In particular, S282T together with mutations from both the finger (T179A) and palm (M289L and I293L) domains was essential to conferring resistance to PSI-7977. The two changes on the surface of the thumb domain, M434T and H479P, each showed a compensatory effect that improved the fitness of the S282T variants. Using the JFH-1 infectious virus system, Cheng et al. have recently reported that PSI-7851 (a diastereoisomer mixture that contains PSI-7977), but not 2′-C
-methyladenosine or 2′-C
-methylcytidine analogs, selected the M289L, S282T, and R543H changes (5
). M289L and S282T were the dominant amino acid changes according to our genotypic analysis. Together the data support the suggestion that similar amino acid changes which at least include S282T and M289L could be selected by PSI-7851 and its pure diastereoisomer PSI-7977 using both the JFH-1 replicon and infectious virus systems.
The locations of these residues on the polymerase suggest that they could be involved in regulating the conformation of NS5B. Residues 179, 289, and 293 are within two alpha helices that are across from each other in the finger and palm domains. In particular, T179 is on top of M289, which is in turn stacked directly above I293. S282 is an extension from the helix containing residues 289 and 293. Mutations in 179, 289, and 293 could affect the interaction between the two helices, which might in turn impact the strand containing S282. Residues 434 and 479 are located on the surface of the thumb domain far away from the active site of RNA synthesis and thus are unlikely to directly interfere with the nucleotide substrates. Schmitt et al. have suggested that extensive hydrophobic interaction within the thumb domain of JFH-1 NS5B could provide stabilization of this enzyme (36
). It is possible that altering amino acid 434 or 479 could compensate for conformation changes induced by residue 282, which is close to the active site, and residues 179, 289, and 293, which are along the alpha helices within the finger and palm domains.
The susceptibility of the JFH-1 S282T replicons appeared to vary among the different classes of nucleoside/tide inhibitors (). JFH-1 replicons with the single S282T mutation showed 4- and 7-fold increases in EC50s for IDX-184 and INX-189, respectively, but were less resistant to PSI-7977 and PSI-6130. Among JFH-1 S282T replicons with multiple mutations, PSI-6130 (a 2′-F-2′-C-methylcytidine) showed the lowest EC50 fold changes (~5-fold). PSI-7977 (a 2′-F-2′-C-methyluridine prodrug), which shares a similar modified sugar as PSI-6130 but a different base, showed ~9-fold increases in EC50s. INX-189 and IDX-184 (prodrugs of 2′-C-methylguanosine), which contain a different modified sugar and a different base, had the highest increases in EC50s (19- and 15-fold, respectively). In contrast, PSI-352938 (a prodrug of 2′-F-2′-C-methylguanosine monophosphate), which shares the same 2′-F-2′-C-methyl-modified sugar as PSI-7977 and PSI-6130 but carries a different base, remained active against the S282T replicon variants. These data suggest that the Thr substitution at residue 282 and possibly its nearby amino acids could affect the interaction with the sugar and base moieties of the nucleotides. Work is under way using molecular modeling, crystallization, and mutagenesis of residue 282 within both the replicons and NS5B polymerase to determine how this would affect nucleotide interaction.
In conclusion, PSI-7977 is a potent HCV inhibitor with broad genotype coverage. Cross-resistance and selection studies showed that S282T is likely the amino acid change that will be selected by PSI-7977 across various genotypes and subtypes. JFH-1 is a highly unique strain capable of efficient replication and infection (11
), and this particular isolate appeared to require additional amino acid changes together with S282T to reduce the activity of PSI-7977. Our studies presented here further suggest that it is possible for the diverse genome of HCV to evolve a mechanism to compensate for the poor fitness as a result of the S282T amino acid alteration.