Mechanism of Resistance of Hepatitis C Virus Replicons to Structurally Distinct Cyclophilin Inhibitors

Abstract

The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.Hepatitis C virus (HCV) poses a serious medical problem, with more than 170 million people infected worldwide (27). Chronic HCV infection increases the risk of hepatocellular carcinoma and results in progressive liver disease and liver failure in approximately 30% of infected individuals (2, 13). HCV infection is the leading indication for liver transplantation in the United States, and HCV reinfection occurs in nearly all cases of chronically infected HCV patients receiving liver transplants. The effectiveness of the current standard therapy (pegylated alpha interferon [PEG-IFN-␣] and ribavirin) is genotype dependent. The response rate in genotype 1 patients, the most prevalent genotype in North America, Europe, and Japan, is only 48%, whereas in genotype 2 and 3 patients there is an 88% response rate (4). In view of these limitations to the current standard of care, the development of alternative, more effective treatment regimens is urgently needed.HCV is a positive-, single-stranded RNA virus with a genome approximately 9.6 kb in length that encodes a single polyprotein, which subsequently is cleaved into 10 distinct viral proteins. The NS3-4A serine protease and the NS5B RNAdependent RNA polymerase are the major foci of current anti-HCV drug discovery efforts. Both enzymes a...

Domain 3 of NS5A Protein from the Hepatitis C Virus Has Intrinsic α-Helical Propensity and Is a Substrate of Cyclophilin A

“…However, we showed in a recent study that NS5A-D2 from the JFH-1 strain (genotype 2a) interacts with, and is a substrate for, the PPIase of human cyclophilin A (35). In addition, we could correlate the CypA interaction sites with mutations in D2 that significantly influence viral RNA replication (27) and that confer resistance to cyclophilin inhibitors (14,22,37). Finally, D3 (residues 356 -447) has been shown, at least in the context of the JFH-1 strain (2a), to be involved in the production and assembly of viral particles (26,38).…”

“…The identification of mutations in NS3, a serine protease, NS5B, the viral RNA-dependent RNA polymerase, and NS5A that all confer in vitro resistance to CsA or non-immunosuppressive CsA analogs, suggested that these proteins are potential cyclophilin binding partners (21)(22)(23)(24). Recent studies also suggested that NS2, a cysteine protease, is an additional cyclophilin target (19,25).…”

“…Recent studies also suggested that NS2, a cysteine protease, is an additional cyclophilin target (19,25). NS5A mutations, mostly located in the C-terminal half of the protein, were shown to confer the highest resistance against cyclophilin inhibitors (21,22). NS5A is a 49-kDa phosphoprotein essential for RNA replication and production of HCV particles (26,27).…”

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…A-782759 selects highly fit mutations at NS5B residue M414 that confer a large decrease in drug susceptibility (17,18). In contrast, a single HCV mutation that confers a more than fivefold shift in potency has not been reported despite a large body of literature on CsA (19)(20)(21). When a large (10 5 ) population of 51C-RFP-1a replicon cells was treated with each of these drugs, a dramatic difference in the frequency of resistance foci was observed (Fig.…”

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

“…Debio 025 (alisporivir) and other structurally distinct cyclophilin inhibitors were shown to select for D320E in NS5A. Like S282T, this mutation emerges late in replicon-based selection experiments, and confers only low levels of resistance to the drug (13,14).Parameters that determine the outcome of a selection process are multifactorial and include the fitness of the mutant variant, the level of resistance that the specific mutation confers, and its availability within a given viral population. The HCV-associated RdRp is often described as an error-prone enzyme that is the source of genetic diversity, and, in turn, availability of mutant viruses.…”