Multiple Mutations in Hepatitis C Virus NS5A Domain II Are Required To Confer a Significant Level of Resistance to Alisporivir

Abstract

Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance. Hepatitis C virus (HCV) is the major causative agent of acute and chronic liver diseases (9). Nearly 200 million people worldwide (3% of the population), including 4 to 5 million in the United States, are chronically infected with HCV, and 4 million new infections occur every year (2, 48). In the developed world, HCV accounts for 2/3 of all cases of liver cancer and transplants (45), and in the United States, ϳ12,000 people are estimated to die from HCV each year (3).The introduction of alpha interferon (IFN-␣) and the nucleoside analog ribavirin (RBV) greatly increased the percentage of chronically HCV-infected patients able to reach a sustained antiviral response (SVR) (49, 51). However, the current standard PEGylated IFN-␣-plus-RBV (pIFN␣/RBV) therapy has a success rate of ϳ80% in patients with genotypes 2 (GT2) and 3 (GT3) and only ϳ50% in patients with GT1 (8, 47), and it causes severe side effects (35). Not only is GT1 the most prevalent HCV genotype in Europe, North and South America, China, and Japan, it is also the most difficult to treat (56). Although the recent approval of the protease inhibitors boceprevir and telaprevir should improve the efficacy of the previous standard of care, there is an urgent need for the development of additional anti-HCV agents with novel mechanisms of action (MOA) in order to provide alternative treatments for the increasing numbe...

Characterization of the Anti-HCV Activities of the New Cyclophilin Inhibitor STG-175

“…This is consistent with the fact that CypI have a unique MoA, which is the prevention of contacts between the host protein CypA and the viral protein NS5A [38,[43][44][45][46][47][51][52][53]. We showed that CypI such as ALV and SCY-635 block interactions between CypA and NS5A derived from various GTs [43][44].…”

“…Two mutations-D320E and Y321N -were identified in the NS5A region of the STG-175-resistant replicon colonies. The D320E and Y321N mutations have been previously identified in HCV variants resistant to other CypI including CsA, ALV, SCY-635 and NIM811 [43][44][45][46][47][48][49]. Thus, it is likely that the double E320/N321 substitutions represent a common motif of partial resistance to CypI including STG-175.…”

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

“…Targeting a cellular process also has the potential advantage of presenting a higher barrier for the development of resistance, a concept with substantial experimental and clinical support. For example, alisporivir (Debio 025) is another HCV inhibitor in advanced clinical development that targets host cyclophilin A, where the emergence of resistance is low and requires multiple viral mutations (59)(60)(61). However, cell-targeted antiviral compounds also have potential disadvantages, including toxicity and the potential for limited in vivo activity due to the redundancy of physiologic systems, such as exogenous dietary pyrimidine uptake mitigating the antiviral activity of dihydroorotate dehydrogenase inhibitors in mice (51).…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…The most advanced congeners are alisporivir (or DEB025) (7) and SCY-635 (8) 116 . Although virus mutations that confer resistance to alisporivir have been reported 109,110 , a synergistic effect when used in combination with anti-HCV DAAs has been noted 156 and the drug is in clinical trials for the treatment of HCV infection 69 . Additional in vivo studies with other relevant viruses will determine whether cyclophilin inhibitors will be of broad applicability.…”

“…CypA interacts with viral proteins supporting viral replication 69 . CypA inhibitors such as cyclosporine A (CsA) have been shown to inhibit the replication of HIV, HCV, influenza virus, CoV, HBV, HSV, human cytomegalovirus (HCMV), VSV, vaccinia virus (VV) and human papillomavirus (HPV) [105][106][107][108][109][110][111][112][113][114] . Alisporivir (Debio-025) and SCY-635, both CsA analogues, have shown antiviral activity against HCV in vivo and are currently in combination with other anti-HCV compounds in various clinical trials 115,116 .…”