Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

Abstract

A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique ␤-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8-to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 M. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.A wealth of literature has documented that RNA viruses or viruses that use RNA replicative intermediates have high degrees of genetic variability (5, 15). The high mutation rates found in these organisms are largely due to the lack of proofreading mechanisms and/or a high recombination frequency during cellular DNA replication. A DNA-dependent DNA polymerase complex allows an error rate of 10 Ϫ9 mutations/ nucleotide, whereas the error rates for an RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase (RdRp), such as those in human immunodeficiency virus (HIV) and hepatitis C virus (HCV), respectively, are on the order of 10 Ϫ4 to 10 Ϫ5 mutations/nucleotide (35, 37). The robust viral replication that leads to a large population size during viral infection in these RNA viruses and retroviruses contributes to the generation of quasispecies in infected patients (7,12,13).During chemotherapy, the high rates of viral replication and the high frequency of mutation lead to the rapid generation of drug-resistant mutants. In the case of HIV, numerous mutations have been identified in patients treated with protease inhibitors as well as nucleoside and nonnucleoside reverse transcriptase inhibitors. The emergence of resistant viruses becomes one of the biggest challenges in developing effective antiviral therapies to eradicate these ...

In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

“…A close examination of the binding pocket revealed that this substitution introduced a polar hydroxyl group in the otherwise hydrophobic pocket, thus significantly altering the hydrophobic interaction between the protein and the cyclopentyl group of AG-021541. Changes at residues M423, M426, and I482 were previously observed in in vitro resistance studies of compounds containing pyranoindole or thiophene-2-carboxylic acid cores, both of which bind to a similar region in the thumb domain of the polymerase as the dihydropyrone inhibitors analyzed in this study (10,17). The baseline prevalence of the resistance substitutions observed in this study in naturally occurring HCV isolates cannot be accurately determined due to the lack of adequate clonal sequences of the HCV genome.…”

“…In vitro resistance studies of various HCV inhibitors, including NS3 protease (20,21,24,41,44) and NS5B polymerase inhibitors (10,11,15,17,27,30,39,40,43), identified resistance mutations in the corresponding viral target regions, some of which have also been observed in subsequent clinical studies. A recent report indicated that resistance mutations observed in vitro were also developed in vivo after a 14-day monotherapy treatment with an NS3 protease inhibitor, VX-950, and correlated strongly with clinical outcome (33).…”

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

“…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).…”

Biochemical Study of the Comparative Inhibition of Hepatitis C Virus RNA Polymerase by VX-222 and Filibuvir

“…Although many small molecules have been reported to interact with the thumb II pocket, no specific analysis of the inhibition of de novo initiation has been reported (6,7,14,16,21,23,29,38,42). Filibuvir, also known as PF-00868554 (Fig.…”