The combining of multiple direct-acting antiviral agents with diverse resistance profiles will be the key to a truly effective direct antiviral therapy. Here we have reported on a novel HCV-specific inhibitor, GS-9190 (Tegobuvir), that is highly potent against genotype 1 HCV, the most prevalent genotype in the Western world and one that is significantly refractory to the current standard of care. Although clearly possessing antiviral activity as demonstrated in vitro
and in HCV-infected patients (1
), little was known about the exact mechanism of action of GS-9190 and other imidazopyridine analogues. Like most other anti-HCV compounds that have been optimized specifically for activity against genotype 1, GS-9190 exhibits reduced potency against genotype 2a. Here we were able to take advantage of this genotype-dependent difference in potency to explore the mechanism by which GS-9190 inhibits HCV replication.
As mentioned, previous analyses of GS-9190 in in vitro assays for effects on the NS3 serine protease, NS3 RNA helicase, IRES-directed translation, and NS5B polymerase showed no inhibition, suggesting that GS-9190 may be dependent on the cellular context of HCV replication for activity. We therefore created a series of chimeric replicons carrying various combinations of nonstructural proteins derived from genotypes 1b and 2a and assessed their antiviral phenotypes in a transient cell-based replicon assay. Using the combined data from the chimeras, we found that the NS5B genotype defined sensitivity to GS-9190, identifying the NS5B polymerase as a likely target of activity. This conclusion was further corroborated by a kinetics experiment using real-time quantitative PCR to measure replicon RNA inhibition, in which GS-9190 behaved in nearly an identical manner to the NS5B NNI HCV-796 and faster than the NS3 protease inhibitor BILN-2061, suggesting GS-9190 inhibits replication.
To further identify the mechanism of action of GS-9190, we performed resistance selections in genotype 1b replicon cells. Replicon sequences were obtained from phenotypically resistant colonies, and individual mutations were reengineered into wild-type 1b replicons. Using this method, we uncovered that the only mutations conferring GS-9190 resistance were those found in NS5B. Specifically, C316Y, C445F, and Y452H individually induced 7- to 10-fold reductions in susceptibility to GS-9190. When a closely related analogue of GS-9190 was used to select for drug resistance, an additional mutation, Y448H, was identified. This particular mutation resulted in 36-fold resistance to GS-9190. Furthermore, combining these mutations had an even greater effect on GS-9190 potency. Taken together, these data pinpoint NS5B as the target by which GS-9190 enacts antiviral activity. In addition, by using a β-hairpin chimeric replicon, we found specifically that the 20-amino-acid of the β-hairpin region of the polymerase thumb subdomain played a significant role in establishing GS-9190 potency. The involvement of this region as a site for inhibition of HCV replication is not unprecedented, as a secondary resistance mutation for the benzothiadiazine NNI A-782759 has previously been reported to reside in the β-hairpin (Y448H) (24
). We confirmed this finding in our study, as A-782759 was markedly less active against the β-hairpin chimeric replicon. Interestingly, the amino acid Y448 was not changed in this chimera, suggesting that resistance arising from sequence modification within the β-hairpin may be due to gross conformational change versus alterations of compound interactions with specific residues. Cross-resistance analyses of the site III inhibitor A-782759 and site IV inhibitor HCV-796 with GS-9190 revealed that GS-9190 has a different drug resistance profile from that of the known site III and site IV inhibitors, further implying that GS-9190 has an NS5B interaction mechanism distinct from other NNIs.
Using the NS5B crystal structure, we were able to map these mutations and found that their locations centered on the β-hairpin and near the catalytic active site. Interestingly, although the Y448H mutation was previously shown to be involved in resistance to benzothiadiazines (NNI site III), the primary site of resistance for that class is M414 (25
). We observed no M414 mutations in GS-9190-resistant replicons. Furthermore, additional studies evaluating GS-9190 cross-resistance showed no alteration of the GS-9190 EC50
in replicons harboring the NS5B M414T mutation. These data, along with a model overlapping GS-9190, HCV-796, and A-782759 in a putative NS5B pocket, suggest that the mode by which GS-9190 interacts with NS5B is different from other palm-site NNIs.
Given the preponderance of evidence obtained in cell-based assays that NS5B is the target of GS-9190, it is bewildering that the compound is inactive in in vitro
NS5B polymerase assays. There are several possible hypotheses that may explain this apparent discrepancy: (i) GS-9190 may only interact with the NS5B protein in the context of the intact replication complexes. It is conceivable that the conformation of NS5B in the context of the replication complexes is different from that of the C-terminally truncated recombinant NS5B protein used in the polymerase assays and that the precise conformation needed to allow binding with GS-9190 may not be adopted in the purified recombinant enzyme preparation. (ii) To address this possibility, we evaluated the effect of GS-9190 on the polymerization activity of endogenous replication complexes by employing membrane fractions isolated from replicon cells (15
). However, GS-9190 failed to show inhibitory activity in this assay (data not shown), suggesting that it is unlikely that the lack of direct interaction between GS-9190 and purified NS5B protein can be ascribed to the absence of other viral or cellular proteins in the replication complexes or due to certain conformational differences. (iii) The interaction of GS-9190 and NS5B may occur during protein translation and before the enzyme adopts its final conformation. It is possible that the binding pocket for GS-9190 is formed transiently during the translation process. GS-9190 may require metabolic activation before it can interact with the enzyme and exert antiviral activity. To explore this hypothesis, we tested the effects of several known cytochrome P450 (CYP) inhibitors on the antiviral activity of GS-9190 in a replicon assay. These inhibitors include 1-benzylimidazole, α-naphthoflavone, and tetramethoxystilbene, which have been shown to be either nonselective CYP inhibitors or selective for certain CYP isoforms (5
). To our surprise, GS-9190 showed significantly reduced antiviral activity (up to 50- to 90-fold) in the replicon assay when tested in combination with the CYP inhibitors (). Note that the concentrations of the CYP inhibitors used in this experiment were chosen based on their lack of cytotoxic or antireplicon effect when tested individually in the replicon assay. Furthermore, these CYP inhibitors had no effect on the antiviral activity of 2′CMeA, suggesting that this detrimental effect is specific for GS-9190. These data suggest that GS-9190 may undergo an intracellular activation step, likely through a CYP-mediated oxidative reaction, to produce the active metabolite and subsequently interact with and inhibit the NS5B polymerase function. This hypothesis could explain the lack of inhibitory activity of GS-9190 in NS5B biochemical and biophysical assays in vitro
. Follow-up studies are under way to further investigate the molecular mechanism of this effect and how it relates to the GS-9190 antiviral mechanism of action. In conclusion, given the data from this current study, it is clear that GS-9190 exerts its antiviral activity through targeting the NS5B polymerase via a mechanism different from other NNIs. The locations of the primary resistance mutations further suggest that inhibition of NS5B by GS-9190, or most likely by an intracellular metabolite from GS-9190, is via a potentially new mode of binding.
GS-9190 replicon EC50s in the presence of cytochrome P450 inhibitors