Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Abstract

Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

“…5F, 8.6-fold). Consistent with a previous report (15), HCV-796 displayed slow kinetics of binding to the genotype 1b enzymes, with slow association and dissociation rates. However, we found that the rate of association of HCV-796 with NS5B was faster in genotype 1a but that the overall binding affinity remained similar because of the concomitant increase in the dissociation rate.…”

“…A recent study has shown that the IC 50 s of NNI-3 and -4 binding derivatives correlate well with their respective K d values when tested against the NS5B⌬C21 Con1 enzyme (15). Therefore, we used the Con 1 strain as a 1b reference to compare to the 1a/1b subgenotypic profile of our NNI analogs.…”

“…In any case, the HCV NS5B chimeric replicon panel and SPR method described here are complementary tools for the genotypic and/or phenotypic assessment of NNI-3 analogs against clinical isolates. Alternatively, the tryptophan fluorescence quenching assay can perhaps also be used as an alternative to SPR studies (15).…”

A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

“…5A and B) very similar to that seen in the J6/JFH1 comparison. This is consistent with our proposed mechanism, although we cannot rule out that this is due in part to this Con1 NS5B being in complex with a nonnucleoside inhibitor of initiation (19). Although the replication levels of the V405I mutant did not reach those previously published for adapted Con1 replicons, this mutation remains a promising candidate for combination with other mutations to generate genotype 1b isolates with high replication levels and the capability of producing infectious virus.…”

Microwave assisted synthesis of some novel Flurbiprofen hydrazide hydrazones as anti-HCV NS5B and anticancer agents

“…Towards this end, we first examined the binding scores of compound 3m in the five reported NS5B allosteric sites, such as Thumb pocket(TP)-I (PDB ID: 2XWY) (26), TP-II (PDB ID: 3FRZ) (27), Palm pocket (PP)-I (PDB ID: 2JC1) (28), PP-II (PDB ID: 3FQL) (29), and PP-III, that significantly overlaps with PP-II (large grid box created around HCV-796 coordinates, with the objective of identifying the NS5B allosteric pocket to which compound 3m potentially binds. The binding energy (XP-Glide score) of (S)-isomer of compound 3m was found to be more negative than the corresponding (R)-isomer and moreover the relatively more negative XP-Glidescore in AP-B versus other pockets indicated a better fit of (S)-compound 3m in AP-B, thus suggesting that AP-B may be the potential binding site for flurbiprofen-hydrazide derivatives.…”