Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

Abstract

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV)polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb-and a palmbinding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.Hepatitis C virus (HCV), a positive-strand RNA virus, is a member of the genus Hepacivirus in the Flaviviridae family and is the leading cause of liver disease worldwide. It is estimated that over 170 million individuals are infected with HCV (43). The current standard of care provides good clinical efficacy for patients infected with genotype 2 and 3 but is less efficacious for patients infected with the most prevalent genotype, genotype 1, thereby emphasizing the urgent need for more effective HCV-specific antiviral therapies (15,27).The HCV RNA-dependent RNA polymerase is an essential enzyme for viral RNA replication and represents an attractive therapeutic target. HCV polymerase has the "right-hand" polymerase fold with finger, thumb, and palm domains (22). As with other RNA-dependent RNA polymerases, the extended "fingertips" contact a thicker thumb domain to create an encircled active site constituting the closed, active conformation of the enzyme (7,16,22,32). With the advent of the HCV replicon system there have been extensive developments supporting the discovery of new HCV polymerase nonnucleoside inhibitors (1-3, 5, 6, 11, 36). Several chemical classes of nonnucleoside inhibitors that inhibit the isolated enzyme and replication in the replicon system have been shown to bind at distinct sites on HCV polymerase. These polymerase inhibitors include benzothiadiazines, binding to the palm domain near the active site (38, 40), thiophene carboxylic acids which bind at the...

Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

“…The cyclopropyl group of NNI-3 extends deeper into this lipophilic pocket [15]. The 4-CF 3 group in the benzamide of NNI-4 locates also in this lipophilic pocket [16].…”

“…2, NNI-1) , proline sulfonamide derivative [14] (Fig. 2, NNI-2), thiadiazine derivative [15] (Fig. 2, NNI-3), acyl pyrrolidine derivative [16] (Fig.…”

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

“…Expression and Purification of HCV NS5B Proteins-The expression and purification of wild-type and mutant NS5B570 proteins were as described (18,20). Briefly, protein expression was induced in Escherichia coli strain M15 harboring an inducible NS5B expression vector by the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside when optical density at 600 nm had reached 1.5-3.5, after which the induced cultures were incubated for 16 -18 h at 22°C.…”

“…Mapping of replicon cells resistant to HCV-796 identified specific mutations in NS5B involving changes at residues Cys 316 and Ser 365 at the palm binding site (8,24). Viral resistance selection against thiophene-based carboxylic acid derivatives NNI-3 identified major substitutions at Leu 419 and Met 423 at the base of one of the thumb domain binding pockets (20), whereas the selection of resistance mutations against the benzimidazole 5Ј-carboxylic acid series localized at the top of the thumb domain with Pro 495 as a key residue (12,37). For the present study, resistance mutations in binding sites in palm 1 (M414T), thumb 2 (L419M), and thumb 1 (P495L) were selected in order to determine NNI binding specificity (Table 3 and Fig.…”

Discovery of Novel Allosteric HCV NS5B Inhibitors. 2. Lactam-Containing Thiophene Carboxylates

“…Based on the X-ray crystal structure of compound 4 bound to the thumb pocket 2 allosteric site of NS5B (PDB: 2GIR), 13 compound 1 was docked, maintaining the same key contacts. The model illustrated in Figure 1 shows the trans-4-hydroxycyclohexyl group protruding from the binding site and in proximity to the surface residue Arg501 of the long helix forming the side of the binding pocket.…”