Synergy of a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors

Abstract

Rapid emergence of resistance to monotherapy with virus-specific inhibitors necessitates combination therapy. ACH-806 is a hepatitis C virus NS4A inhibitor with a novel mechanism of action and resistance pathway. This compound was synergistic with NS3 protease inhibitors and NS5B nucleoside and nonnucleoside polymerase inhibitors.Significant progress has been made in the discovery and testing of novel inhibitors of hepatitis C virus (HCV) replication (4). The majority of the compounds evaluated in vitro and in early clinical trials have belonged to one of three classes of HCV inhibitors: NS3 protease inhibitors (PI) (11,17,29), NS5B nucleoside polymerase inhibitors (21,22,24), and NS5B nonnucleoside (NNI) polymerase inhibitors (2,7,8,12). Importantly, resistance to each of these compound classes has been described with some resistance mutations conveying cross-resistance to several inhibitors within a given class (e.g., A156T in HCV protease) (15,18,20,28). Analogous to the experience with human immunodeficiency virus type 1 therapy, combinations of several classes of viral inhibitors with unique mechanisms of action and resistance pathways will be integral to the success of small-molecule-based antiviral therapy for chronic HCV infection.ACH-806 [1-(4-pentyloxy-3-trifluoromethylphenyl)-3-(pyridine-3-carbonyl)thiourea] is a novel acylthiourea compound with a 50% effective concentration (EC 50 ) of 14 nM in the genotype 1b replicon system and 30 nM in a genotype 1a replicon system (13). A phase 1b proof-of-concept study showed significant antiviral activity at the lowest dose tested (23). ACH-806 possesses a unique mechanism of action. It selectively binds to the NS4A protein, resulting in altered protein composition and inactivation of the replicase complex (13). Given its unique mechanism of action, we sought to evaluate ACH-806 in combination with other small-molecule inhibitors of HCV replication, as well as alpha interferon, in a genotype 1b luciferase reporter replicon system.(A portion of the results described here was presented as an abstract at the XVI International HIV Drug Resistance Workshop, Hilton Barbados, Bridgetown, Barbados, 12 to 16 June 2007.)Replicon constructs. The BM4-5 replicon is a subgenomic HCV genotype 1b replicon which contains a deletion of a serine in NS5A (10). The firefly luciferase gene was inserted into the BM4-5 replicon, in a manner we and others have previously described (26,27), to generate the BM4-5 FEO replicon. The sequence of the replicon was verified by DNA sequencing.Cell culture and luciferase compound assays. Cell culture and luciferase compound assays were performed as previously described in detail (9, 27). Briefly, 10,000 BM4-5 FEO cells/ well were seeded into 96-well plates and incubated for 4 h. Medium was then aspirated and replaced with 100 l of complete medium containing a single compound or combinations at the desired concentration(s). Plates with compounds were incubated for 48 h and then assayed for luciferase expression (Bright-Glo; Promega). All conditio...

Resistance Analysis and Characterization of a Thiazole Analogue, BP008, as a Potent Hepatitis C Virus NS5A Inhibitor

“…More importantly, no antagonistic antiviral effects or enhanced cytotoxicity was observed in any of these combinations. Although the EC 50 (55.2 IU/ml) of IFN-␣ for stable HCV1b replicon cells was higher than the values reported by other groups (Table 4) (34,35,39,59), the EC 50 of IFN-␣ for our parental HCV1b replicon measured by transient HCV1b replicon assay was in the region of 3 IU/ml (data not shown), comparable to most of the values reported by others. Moreover, several mutations within the C terminus of NS5A were identified by sequencing analysis of cDNAs derived from viral RNAs of our stable HCV replicon cells compared to the sequences of parental HCV1b replicon plasmid for transient replicon assay (data not shown).…”

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

“…Given the limited efficacy of ACH-806 in the applied assay, calculation of the EC 50 was not possible for all treatments done; overall determinable EC 50 s were in the range of 1,000 to 10,000 nM, thus being several orders of magnitude higher than EC 50 values determined in genotype 1 replicon systems (11)(12)(13). EC 50 values strongly depend on the assay used.…”

“…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).…”

MK-7009, a Potent and Selective Inhibitor of Hepatitis C Virus NS3/4A Protease

“…NS3/4A protease inhibitors have previously been shown to demonstrate synergy with agents acting via multiple other mechanisms, including peginterferon alfa 2a (20,45), NS5B polymerase inhibitors (7,14), NS4A antagonists (53), and cyclosporine analogs (3,30). In the short term, they may allow a reduced duration of therapy when they are used in combination with interferon and ribavirin, coupled with improved SVR rates, particularly in difficult-to-treat genotype 1-infected patients.…”