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

Abstract

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor-and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

“…With the development of antiviral compounds and the monitoring of resistance, it has become apparent that mutations which would allow the virus to escape inhibition by drugs will be enriched over the baseline genomic sequences. These studies have also led to the identification of subtype-dependent resistant mutations for NS3 protease, NS5A, and NS5B nonnucleoside inhibitors in both replicons and clinical isolates (7,21,27,32,42). Compared to these three classes of HCV inhibitors, relatively less is known about genotype-and subtype-dependent resistance for nucleoside/tide analogs targeting the active site of NS5B.…”

Antiviral Activity and Mode of Action of TMC647078, a Novel Nucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

“…The transient replicon assay, replicon mutants, and chimeras were described in detail elsewhere (20,32). In brief, replicon plasmid DNA was prepared and in vitro transcribed to yield replicon RNA.…”

“…The kinetic parameters of the phosphorylation of TMC647078 were determined by using phosphoryl transfer assays as described previously (15), using a mixture containing 0.05 M [␥- 32 P]ATP (10 Ci/l) (Perkin-Elmer Sweden AB), 100 M ATP, 20 mM Tris-HCl (pH 7.6), 5 mM MgCl 2 , 100 mM KCl, 0.5 mg/ml bovine serum albumin (BSA), 5 mM dithiothreitol (DTT), and different concentrations of the nucleoside analog. The reaction was initiated by the addition of the enzyme to the mixture followed by incubation at 37°C and was terminated after 25 min by heating to 100°C.…”

Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

“…There is evidence that the selection of resistant variants and virus breakthrough is more frequent in patients infected with subtype 1b than in those harboring subtype 1a (13,25,40). The antiviral activities of nucleoside analogs of polymerase inhibitors are similar regardless of the HCV subtype, while nonnucleoside inhibitors are more active against subtype 1b than against subtype 1a (18,31,41). These findings suggest that the antiviral activity of new anti-HCV agents may also vary with the subtypes of genotypes other than 1.…”

“…One study of 597 difficult-to-treat patients found that subtypes 1b, 4a, and 4d were independently associated with SVR (16). The virological response to new anti-HCV agents could also be influenced by the HCV subtype (31,42).…”