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

Abstract

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-ofconcept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication. C hronic hepatitis C virus (HCV) infection is a major cause of liver diseases worldwide. It is estimated that 170 million individuals are infected with HCV (1-4). A significant portion of these infected people will develop liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma (5). Treatment with pegylated alpha interferon (IFN-␣) and ribavirin has a sustained virologic response or cure rate of ϳ45% in genotype 1 HCV-infected patients (6, 7), and the addition of boceprevir or telaprevir, HCV NS3 protease inhibitors newly approved by the U.S. Food and Drug Administration, increases the cure rate to ϳ70% (8). The new standard care of the triple combination, however, also leads to more toxic effects (9). Hence, development of new treatment regimens with higher efficacy, as well as better tolerability is urgently needed (10).HCV, a member of the Flaviviridae family, is an enveloped virus with a positive-stranded RNA genome of 9.6 kb. The viral genome encodes a large polyprotein that is cleaved co-and/or posttranslationally into at least 10 mature viral proteins: structural proteins, including C, E1, E2, and p7, and nonstructural (NS) proteins, including NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The functions of these viral proteins in the HCV life cycle have been extensively studied and mostly clarified (11). For example, NS5B has an RNA-dependent RNA polymerase activity, NS3 possesses a serine protease activity in its N-terminal domain and a helicase activity in the C-terminal domain, and NS4A is a cofactor of NS3...

Adapted J6/JFH1-Based Hepatitis C Virus Recombinants with Genotype-Specific NS4A Show Similar Efficacies against Lead Protease Inhibitors, Alpha Interferon, and a Putative NS4A Inhibitor

“…The C1042S aa change was also suggested to confer resistance of genotype 1b replicons to ACH-806 (11,13). J6/JFH1-based NS4A recombinants (Fig.…”

“…The A1065V amino acid change (aa 39 of NS3P) was suggested to confer resistance of genotype 1b replicons to ACH-806 (11,13). To exclude the possibility that the limited efficacy of ACH-806 against the developed J6/JFH1-based NS4A recombinants was caused by the genotype 2a-specific valine at aa position 1065, we constructed 2a(JFH1)V1065A.…”

“…In 2011, the first two directly acting antivirals targeting HCV NS3P, telaprevir and boceprevir, were licensed for treatment of chronic genotype 1 infection (10). Several other protease inhibitors are advanced in clinical trials, while inhibitors of HCV NS4A are in preclinical development (10)(11)(12)(13). We and others have reported that HCV recombinants with genotype-specific NS3P/NS4A show differential responses to protease inhibitors (14)(15)(16).…”

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

“…Yang et al recently detected small quantities of p14, a putative homodimer of NS4A, in genotype 1b replicon-bearing cells (83). The p14 form of NS4A was apparent only in the presence of ACH-806, an inhibitor of NS3-NS4A interaction (84), or when NS4A was overexpressed in the absence of NS3.…”

Synergistic Activity of Combined NS5A Inhibitors

“…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).…”