Identification of Hepatitis C Virus NS5A Inhibitors

Abstract

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ϳ5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.Hepatitis C virus (HCV) is the major causative agent for non-A, non-B hepatitis worldwide, which affects more than 3% of the world population. HCV establishes chronic infections in a large percentage of infected individuals, increasing the risk for developing liver cirrhosis and, in some cases, hepatocellular carcinoma. Although the current standard of care for HCV infection involves the use of PEGylated interferon and ribavirin, a large proportion of patients fail to respond to this therapy, and treatment is associated with frequent and sometimes serious side effects (9). Given the limited efficacy of the current therapy, the development of safer and more effective therapies is of tremendous importance.HCV is a positive-strand RNA virus belonging to the family Flaviviridae. The HCV genome consists of a ϳ9.6-kb RNA with a large open reading frame encoding a polyprotein of ϳ3,010 amino acids. The polyprotein is cleaved co-and posttranslationally by both cellular and viral proteases into at least 10 different products (10, 11). The viral proteins required for RNA replication include NS3, NS4A, NS4B, NS5A, and NS5B (4, 19). NS3 consists of an amino-terminal protease domain required for the cleavage of the remaining nonstructural proteins and a carboxyl-terminal helicase/NTPase domain (8,11,30). NS4A serves as a cofactor for NS3 protease and helicase activities (8). NS4B is a...

Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

“…Because DGAT2 activity is sufficient to maintain steady LD levels in hepatoma cells treated with DGAT1 inhibitors (11), these inhibitors, some in clinical trials for treating obesity-associated diseases (32), may represent unique LD-targeting therapeutics that suppress HCV particle production without compromising overall lipid metabolism. As NS5A itself has become a focus of drug discovery in HCV infection (33,34), our findings that DGAT1 inhibitors suppress NS5A localization to LDs reveal a novel mechanism to explain how DGAT1 inhibitors interfere with HCV infection.…”

Hepatitis C Virus NS5A Activates the Mammalian Target of Rapamycin (mTOR) Pathway, Contributing to Cell Survival by Disrupting the Interaction between FK506-binding Protein 38 (FKBP38) and mTOR

“…Most importantly, anti-HCV drugs targeting NS5A are currently undergoing investigation (59). One such drug has a potent inhibitory effect on viral replication in a phase I clinical trial of chronically HCV-infected patients (60).…”

Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in Humans: In Vitro and In Vivo Correlations

“…6 Informed written consent was obtained from all patients. HCV RNA levels were determined using the Roche Cobas TaqMan HCV Test, v2.0 (lower limit of quantification, 25 IU/mL; lower limit of detection, 10 IU/mL; Roche, Pleasanton, CA) at baseline and days 1 (2,4,6,8,12,16, and 20 hours post-first dose), 2,3,4,5,7,9,11,14,15,16,17,21, and 28. Viral breakthrough was defined as an HCV RNA increase by at least 0.5 log 10 after HCV RNA nadir while receiving BMS-790052.…”

“…Many of these substitutions were previously identified during in vitro replicon studies, and others are novel substitutions. 4,5,11 Values for previously described substitutions (Tables 1 and 2) have been updated to reflect additional test occasions.…”

“…5 All amino acid substitutions associated with resistance to this class of inhibitors have mapped to the N-terminal region of NS5A. 4,5,11 Patients receiving BMS-790052 monotherapy in the MAD study generally experienced rapid, marked viral load declines at early time points. However, viral breakthrough, especially in patients infected with genotype 1a virus, was observed at later time points and was associated with the emergence of resistance.…”