Characterization of the Inhibition of Hepatitis C Virus RNA Replication by Nonnucleosides

Abstract

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.Hepatitis C virus (HCV) chronically infects about 3% of the human population, causing a slowly evolving liver disease that leads to cirrhosis, liver failure, and occasionally hepatocellular carcinoma (39). Given the size of the HCV epidemic and the limited efficacy of the present therapy based on alpha interferon (16), the development of new, safer, and more effective drugs is of paramount importance and is presently an area of intensive research. The strategy most widely applied for developing novel anti-HCV therapeutics aims at identifying small molecule inhibitors of viral enzymes. The nonstructural protein 5B RNA-dependent RNA polymerase (NS5B RdRp) is an important target of drug discovery activities largely because it is essential for viral replication and also due to the clinical successes of inhibitors of other viral polymerases. In addition, the extensive structural and biochemical characterization of this enzyme provides the basis for drug design efforts as well as for elucidating the mechanism of action of inhibitors and for rapidly optimizing their potency.The NS5B protein was initially identified as an RdRp based on the presence of the signature GDD (Gly-Asp-Asp) motif characteristic for this class of enzymes (11). Its function was confirmed when an active form of the full-length protein was purified from baculovirus-infected insect cells (5). Subsequently, attempts to improve solubility, stability, and activity lead to the expression of C-terminal-truncated forms lacking the hydrophobic membrane anchor contained within the last 21 amino acids (1,17,25,33,35). In vitro, the enzyme shows little, if any, specificity for the HCV genome and can catalyze the synthesis of RNA by using a variety of homo-or heteropolymeric RNA templates both with and without a primer. In the absence of primer, NS5B can initiate RNA synthesis either by using the 3Ј-termi...

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

“…A K d of ϳ250 nM for a 16-mer single-stranded RNA and a 13/16-mer double-stranded RNA substrate was measured by equilibrium fluorescence titration (43). In addition, a K d of 420 nM was determined for a 14-mer RNA using fluorescence polarization (14,44). These previous data are in agreement with our results.…”

“…For instance, the single mutation H95R has been reported as a resistance mutation of benzothiadiazine (14), but this mutation did not affect compound binding to the NS5B-Con1 protein. H95R has been implicated in binding to the RNA template (6), and it has been proposed to affect the ability of the polymerase to recognize the viral genome.…”

“…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).…”

“…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.…”

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

“…Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanced to clinical trials (for a recent review, see reference 7). Nonnucleoside inhibitors of NS5B bind reversibly to distinct allosteric sites of the enzyme (4,9,15,41).…”