Selection of Replicon Variants Resistant to ACH-806, a Novel Hepatitis C Virus Inhibitor with No Cross-Resistance to NS3 Protease and NS5B Polymerase Inhibitors

Abstract

We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. It is estimated that 170 million individuals are infected with HCV (56). The current therapeutic combination of pegylated alpha interferon (IFN-␣) and ribavirin has a sustained viral response rate of ϳ50% in genotype 1 HCV-infected patients and is limited by the adverse effects of both agents (8, 13). Therefore, the development of oral anti-HCV agents with improved efficacy and better tolerance is urgently needed.HCV is an enveloped virus with a positive-stranded RNA genome of 9.6 kb. The viral genome encodes a large polyprotein that is cleaved cotranslationally and/or posttranslationally into at least 10 mature viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3, 26). Specific functions have been attributed to most of these viral proteins. For example, NS5B protein has an RNA-dependent RNA polymerase activity, the amino-terminal domain of NS3 carries serine protease activity, and NS4A is a cofactor of NS3 which enhances NS3 protease activity by forming a stable heterodimeric complex with NS3 (4,5,24,26,28,30). Both NS5B polymerase and NS3 protease have been the prime targets for the development of HCV-specific agents. To date, multiple small molecules against the two targets have been reported (7,17,18,48), and some of them have shown antiviral activity in HCV-infected patients (9,20,42,43,44,54).Among HCV-specific inhibitors discovered so far, NS5B nucleoside analogs target the polymerase catalytic site of NS5B. T...

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

“…PSI-7977 (prodrug of 2=-F-2=-C-methyluridine monophosphate) (38), PSI-352938 (prodrug of 2=-F-2=-C-methylguanosine monophosphate) (34), PSI-6130 (2=-F-2=-C-methylcytidine) (40), R1479 (4=-azidocytidine) (13), IDX-184 and INX-189 (prodrugs of 2=-C-meth-ylguanosine monophosphate) (43,48), NS5B nonnucleoside inhibitors NNI-1 (an indole analog), NNI-2 (a thiophene analog), NNI-3 (a benzothiadiazine analog), and HCV-796 (a benzofuran analog or NNI-4) (39), NS3 protease inhibitors telaprevir and RG7227 (ITMN-191) (21,22), NS3/4a inhibitor ACH-806 (46), and NS5A inhibitor BMS-790052 (8) were synthesized at Pharmasset and shown to be Ͼ95 to 99% pure by proton nuclear magnetic resonance, mass spectroscopy, and highperformance liquid chromatography analysis. Ribavirin was obtained from Sigma-Aldrich.…”

ACH-806, an NS4A Antagonist, Inhibits Hepatitis C Virus Replication by Altering the Composition of Viral Replication Complexes

“…This ACH-806-resistant cell line harbors a replicon carrying a C16S substitution in the N-terminal region of NS3, where NS3 interacts with NS4A, and displays ϳ10-fold less susceptibility to ACH-806 (19). As shown in Fig.…”

“…It was discovered through compound library screening, hit/lead identification, and lead optimization using HCV subgenomic replicon-containing cells (hereafter HCV replicon cells). ACH-806 has exhibited potent activity against genotype 1 HCV replication in vitro (19) and also showed antiviral activity in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Resistance substitutions that emerged under ACH-806 selection in replicon cells were mapped to the N-terminal region of NS3 and were not crossresistant with NS3 protease inhibitors and NS5B polymerase inhibitors (19).…”

“…ACH-806 exhibits potent anti-HCV activities in both genotype 1a and 1b replicon-containing cells with EC 50 s between 0.01 and 0.05 M, depending on the replicon cells and the endpoints (19). To study whether ACH-806 blocks de novo initiation of HCV RNA synthesis, RCs were prepared as membrane fractions from genotype 1b replicon cells and then treated with ACH-806 in vitro.…”

Specifically targeted antiviral therapy for hepatitis C virus

“…Resistance induction studies in replicon cells have shown a lack of cross resistance between ACH-806 and protease or polymerase inhibitors; thus confirming its different mode of action. Interestingly, viral quasispecies resistant to protease or polymerase inhibitors remain sensitive to ACH-806 and vice versa [40] . In vitro studies of a combination of ACH-806 with telaprevir or valopicitabine or interferon have demonstrated potent antiviral effects, which are synergistic when cells are exposed to ACH-806 with telaprevir or ACH-806 with valopicitabine.…”