Mechanismof Action and Antiviral Activity of Benzimidazole-Based AllostericInhibitors of the Hepatitis C Virus RNA-Dependent RNAPolymerase

Abstract

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.Hepatitis C virus (HCV) is the causative agent of the majority of chronic liver disease throughout the world. More than 170 million individuals are estimated to be infected with this virus (27). The size of the HCV epidemic and the limited efficacy of current therapy (based on the use of alpha interferon) have stimulated intense research efforts towards the development of antiviral drugs that are both better tolerated and more effective. The most widely established strategy for developing novel anti-HCV therapeutics aims at the identification of low-molecular-weight inhibitors of essential HCV enzymes.RNA-dependent RNA polymerase (RdRP) activity, carried out by the NS5B protein, is essential for virus replication (13) and has no functional equivalent in uninfected mammalian cells. It is thus likely that specific inhibitors of this enzyme can be found that block HCV replication with negligible associated toxicity. The NS5B RdRP has been expressed in a variety of recombinant forms (2, 4). The production of highly soluble forms of the enzyme (12, 24), devoid of the C-terminal membrane anchoring domain (23), has allowed considerable progress toward the determination of the enzyme's three-dimensional structure and mechanism of action. The crystal structure of NS5B revealed a classical "right hand" shape, showing the characteristic fingers, palm, and thumb subdomains (1,7,14). More recently, the three-dimensional structure of the HCV polymerase was solved in complex with RNA (20) as well as in a complex with nucleoside triphosphates (6). Three distinct nucleotide-binding sites were observed in the catalytic center of HCV RdRP whose geometry was remarkably similar to that observed in the initiation complex of the RNA phage ⌽6 RdRP (8), strengthening the proposal that the two enzymes initiate replication de novo by similar ...

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

“…These results confirm that BMS-791325, like the previous thumb site 1 inhibitors tested, has no impact on elongation (16,18). The inhibition in systems that model elongation suggests that BMS-791325 blocks a step before the incorporation of nucleotides.…”

“…Mode of Inhibition-Previous reports showed that compounds binding at thumb site 1 inhibit the initiation of RNA replication (16,18) and not elongation; however, BMS-791325 appears to be 5-75 times more potent than previously studied compounds and inhibits equally in model systems of de novo and primer-dependent synthesis. Because two of the four replication systems are assumed to model elongation (poly(A)⅐dT 12 and HCV RNA), a trapping experiment was performed to confirm that BMS-791325 has no impact on the elongation phase of replication.…”

“…Primer-dependent replication model systems were used to characterize 2 thumb site 1 inhibitors with K i values of 120 -200 nM. The inhibitors were shown to be non-competitive with primer⅐template and NTP and unable to inhibit preformed replication complexes (18). Resistance selection in the replicon system identified substitutions at proline 495, an amino acid 30 Å from the active site, as responsible for resistance.…”

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

“…Activity after hybridization to the DNA strand used in single molecule experiments was tested using the R (lanes 4 and 8). Lanes 2,4,10,12,14, and 16 show full-length products in addition to shorter products representing pausing sites. Lanes 6 and 8 show a truncated product.…”

“…Thumb pocket 1 non-nucleoside inhibitors (NNIs) or finger loop NNIs bind to a region of the thumb that is otherwise occupied by the flexible ⌬1 finger loop, which seems to impede productive RNA binding (Fig. 1b) (11)(12)(13)(14).…”