Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

Abstract

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC 50 ) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC 50 s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC 50 , 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC 50 s of >4 M) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC 50 . Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6-to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were >10-fold above the inhibitor EC 50 s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

Synergistic Activity of Combined NS5A Inhibitors

“…Thus, we expect the impact on authentic gt 3a virus to be similar to the impact on the hybrid virus used in the experiment (33,34).…”

“…NS3 protease inhibitors bind the catalytic site or the NS4A activator site, defining 2 targets that inactivate protease function to inhibit replication (47). NS5B inhibitors bind to 1 of 4 allosteric inhibitor binding sites or the catalytic site, defining 5 targets that inactivate the polymerase to inhibit virus replication (33,48,49). Displacement of bound radiolabeled compound suggests the less wellcharacterized inhibitors of NS4B bind at least one site that inhibits replication (50).…”

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

“…The activity of the P495L variant is ϳ2-fold lower than WT and is highly resistant to BMS-791325 inhibition (EC 50 Ͼ500 nM and IC 50 431 Ϯ 110 nM; Table 2) (10, 11). It was observed in replicons after selection in vitro and in clinical specimens of GT-1 patients treated with BMS-791325 (7,11). The P495L and L30S variants were used in this study to confirm that the inhibition and binding observed with WT NS5B is specific and to probe the impact of the weakened interaction between the thumb and finger on the inhibitor-enzyme interactions.…”

“…In a more recent Phase 2 study, 63 of 66 HCV-infected GT-1 patients treated for 12 or 24 weeks with a combination of BMS-791325 (75 or 150 mg twice daily), daclatasvir (60 mg once daily), and asunaprevir (200 mg twice daily) achieved sustained virologic response (9). The preclinical profile of BMS-791325, including potent activity in GT-1a and -1b enzyme and replicon assays (IC 50 and EC 50 values of 0.7-4 nM), selection of significant resistance at a single substitution site, and a robust pharmacokinetic profile in animal models, anticipated the strong antiviral effect observed in patients (10,11).…”

“…It is also highly selective for NS5B, showing no activity against the closely related RNA-dependent RNA polymerase from the pestivirus, bovine viral diarrhea virus, or human DNA polymerases ␣, ␤, and ␥ (IC 50 values Ͼ25 M) (11).…”