Nonstructural Protein 5A Replication Complex Research Articles

Association between interleukin 28B polymorphism and sustained virological response to sofosbuvir plus daclatasvir in chronic hepatitis C genotype 4 Egyptian patients

Sofosbuvir has been approved as the first nonstructural protein 5Bpolymerase inhibitor with pan-genotypic activity against the hepatitis C (HCV) virus. Daclatasvir is a first-in-class hepatitis C virus nonstructural protein 5A replication complex inhibitor. We aimed to evaluate the usefulness of the reference single nucleotide polymorphism (rs12979860) interleukin 28B (CC genotype) for predicting sustained virological response to sofosbuvir plus daclatasvir in Egyptian patients infected with HCV-4. Samples were collected at week zero. One hundred and thirty-one patients who reached the end of treatment (at week 12) were divided into three groups, according to their interleukin 28B genotype: Group A included 31 patients (CC genotype), group B included 79 patients (CT genotype) and group C had 21 patients (TT genotype). All patients received treatment for 3months in the form of sofosbuvir plus daclatasvir with ribavirin (in case of cirrhotic patients) or without ribavirin (in case of non-cirrhotic patients). Sustained virological response rate was significantly higher in patients with IL28B (CC genotype) vs. (non-CC genotype) (100 vs.88%) (p<0.0001).These patients also showed lower rates of post-treatment relapse and non-response, compared with the CT and TT patients (0% vs. (7.59% and 28.5%, respectively) (p<0.0001). Also, patients with CC genotype showed higher sustained virological response than non-CC genotypes on both cirrhotic (100% vs. 68.75%) and non-cirrhotic patients (100% vs. 91.66%) (p≤0.0001). Our results suggest that IL28B genotype contributes to the prediction of response to sofosbuvir plus daclatasvir.

GSK2818713, a Novel Biphenylene Scaffold-Based Hepatitis C NS5A Replication Complex Inhibitor with Broad Genotype Coverage.

Pan-genotype NS5A inhibitors underpin hugely successful hepatitis C virus (HCV) therapy. The discovery of GSK2818713 (13), a nonstructural protein 5A (NS5A) HCV inhibitor characterized by a significantly improved genotype coverage relative to first-generation NS5A inhibitor daclatasvir (DCV), is detailed herein. The SAR analysis revealed cooperative potency effects of the biphenylene, bicyclic pyrrolidine (Aoc), and methyl-threonine structural motifs. Relative to DCV, 13 improved activity against genotype 1a (gt1a) and gt1b NS5A variants as well as HCV chimeric replicons containing NS5A fragments from genotypes 2-6. Long-term treatment of subgenomic replicons with 13 potently and durably decreased HCV RNA levels for gt1a, gt2a, and gt3a. These properties, suitable pharmacokinetics, and the lack of cross-resistance resulted in the selection of 13 as a preclinical candidate.

Population Pharmacokinetic Analysis for Daclatasvir and Asunaprevir in Japanese Subjects With Chronic Hepatitis C Virus Infection.

Daclatasvir is a nonstructural protein 5A replication complex inhibitor, and asunaprevir is a nonstructural protein 3 protease inhibitor for hepatitis C virus (HCV). In 2014, the combination therapy of daclatasvir and asunaprevir received the first global approval in Japan as the first nonribavirin, all‐oral therapy for HCV treatment. The population pharmacokinetics (popPK) of daclatasvir and asunaprevir were characterized by nonlinear mixed‐effects modeling using 3801 and 2626 concentration data from 336 and 265 Japanese HCV subjects, respectively. The plasma pharmacokinetic profiles of daclatasvir and asunaprevir were described by a 1‐compartment model. Parameter estimates (interindividual variability) of daclatasvir apparent clearance (CL/F) and apparent volume of the central compartment (V/F) were 5.29 L/h (39.4%) and 64.2 L (38.1%). The effects of all statistically significant covariates on daclatasvir PK parameters were within or overlapped the 80% to 125% boundaries, suggesting a lack of clinical relevance. Parameter estimates (interindividual variability) of asunaprevir CL/F and V/F were 52.1 L/h (41.5%) and 75.1 L (93.4%), respectively. Baseline and time‐varying aspartate aminotransferase (AST) and cirrhosis on CL/F and formulation (soft‐gel capsule or tablet) on F were included as significant covariates in the asunaprevir popPK model. The effects of all covariates exceeded the 80% to 125% boundaries, indicating that the asunaprevir soft‐gel capsule had higher bioavailability than the tablet and that asunaprevir exposure increased with cirrhosis and increasing baseline and time‐varying AST values. The popPK models adequately described the PK profiles of daclatasvir and asunaprevir in Japanese HCV subjects.

Biotransformation of Daclatasvir In Vitro and in Nonclinical Species: Formation of the Main Metabolite by Pyrrolidine δ-Oxidation and Rearrangement.

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.

In Silico Approaches to the Design of NS5A Inhibitors.

In recent years, nonstructural protein 5A (NS5A) has rapidly emerged as a promising therapeutic target for Hepatitis C (HCV) virus therapy. It is involved in both viral RNA replication and virus assembly and NS5A plays a critical role in the regulation of HCV life cycle. NS5A replication complex inhibitors (NS5A RCIs) have demonstrated strong antiviral activity in vitro and in vivo. However, wild-type resistance mutations and a wide range of genotypes significantly reduce their clinical efficacy. The exact mechanism of NS5A action still remains elusive, therefore several in silico models have been constructed to gain insight into the drug binding and subsequent structural optimization to overcome resistance. This paper provides a comprehensive overview of the computational studies towards NS5A mechanism of action and the design of novel small-molecule inhibitors.

Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A.

It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.

The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile-Part 2.

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability. The novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A.

Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5a replication complex inhibitor, BMS-790052

The influence of naturally occurring polymorphisms on the potency of the HCV nonstructural protein 5A (NS5A) replication complex inhibitor, BMS-790052, was investigated by evaluating hybrid replicons in which the entire NS5A coding region of genotype (GT) la and 1b laboratory (lab) strains (H77c and Con1) were replaced with the corresponding regions of specimens collected from 10 GT-1a- and 6 GT-1b-infected subjects. For baseline (BL) specimens, with no previously observed resistance variants identified by population sequencing, the median 50% effective concentration (EC(50) ) values for BMS-790052 were similar for the clinically derived and lab strains. A Q30R variant was observed at viral breakthrough (VBT) in one of the GT-1a-infected subjects. Because the lowest plasma exposure of BMS-790052 observed in this subject was 117 nM and the median 50% effective concentration value for a GT-1a H77c replicon containing a Q30R substitution is ~7 nM, a rigorous investigation was initiated to determine the basis for resistance. Three approaches were used: (1) replacement of the entire H77c NS5A or (2) replacement of the N-terminal region of NS5A, with sequence from BL and day 14, and (3) substitution of specific amino acids. A BL polymorphism (E62D) did not contribute resistance to BMS-790052; however, the linked variant, Q30R-E62D, conferred high-level resistance in vitro and is likely responsible for VBT in vivo. Our data show that a BL polymorphism with minimal effect on the anti-HCV effect of BMS-790052 can affect the emergence of resistance and significantly affect clinical outcome. This work establishes a clear, systematic approach to monitor resistance to NS5A inhibitors in the clinic.

Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b–infected null responders

Patients with chronic hepatitis C virus (HCV) infection and previous null response to pegylated interferon (Peg-IFN) and ribavirin (RBV) have limited therapeutic options. HCV genotype 1 is the most common worldwide and the most difficult to treat; genotype 1b is the most common subtype of genotype 1 outside North America. The enhanced antiviral activity achieved by combining two direct-acting antiviral (DAA) agents may improve clinical outcomes. This open-label, phase IIa study included 10 patients with chronic HCV genotype 1b infection and previous null response (<2 log(10) reduction in HCV RNA after 12 weeks) to Peg-IFN and RBV. Patients received dual DAA treatment for 24 weeks with the nonstructural protein 5A replication complex inhibitor, daclatasvir (60 mg once-daily), and the nonstructural protein 3 protease inhibitor, asunaprevir (initially 600 mg twice-daily, then subsequently reduced to 200 mg twice-daily). The primary efficacy endpoint was the proportion of patients with sustained virologic response (SVR) at 12 weeks post-treatment (SVR(12) ). Nine patients completed 24 weeks of treatment; 1 patient discontinued treatment after 2 weeks. In the 9 patients who completed the full course of treatment, HCV RNA was undetectable at week 8 and remained undetectable through the end of treatment; all 9 patients achieved SVR(12) and SVR(24) . HCV RNA also remained undetectable post-treatment in the patient who discontinued after 2 weeks. There was no viral breakthrough. Diarrhea and headache, generally mild, were the most common adverse events; transaminase elevations were reported in 3 patients, but did not result in discontinuation. Dual therapy with daclatasvir and asunaprevir, without Peg-IFN and RBV, can achieve high SVR rates in difficult-to-treat patients with HCV genotype 1b infection and previous null response to Peg-IFN and RBV.

Multiple ascending dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1

The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of BMS-790052, a nonstructural protein 5A (NS5A) replication complex inhibitor, were evaluated in a double-blind, placebo-controlled, sequential panel, multiple ascending dose study. Thirty patients with chronic hepatitis C virus (HCV) genotype 1 infection were randomized to receive a 14-day course of BMS-790052 (1, 10, 30, 60, or 100 mg once daily or 30 mg twice daily) or placebo in a ratio of 4:1. The mean maximum decline from baseline in HCV RNA ranged from 2.8 to 4.1 log(10) IU/mL; the placebo group showed no evidence of antiviral activity. Most patients experienced viral rebound on or before day 7 of treatment with BMS-790052 monotherapy; viral rebound was associated with viral variants that had been previously implicated in resistance development in the in vitro replicon system. The PK profile was supportive of once-daily dosing with median peak plasma concentrations at 1-2 hours postdose and mean terminal half-life of 12-15 hours. Steady state was achieved following 3-4 days of daily dosing. BMS-790052 was well tolerated in all dose groups, with adverse events occurring with a similar frequency in BMS-790052- and placebo-treated groups. There were no clinically relevant changes in vital signs, laboratory, or electrocardiogram parameters. BMS-7590052 is the first NS5A replication complex inhibitor with multiple dose proof-of-concept in clinic. At doses of 1-100 mg daily, BMS-790052 was well tolerated, had a PK profile supportive of once-daily dosing, and produced a rapid and substantial decrease in HCV-RNA levels in patients chronically infected with HCV genotype 1.

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

The NS5A replication complex inhibitor, BMS-790052, inhibits hepatitis C virus (HCV) replication with picomolar potency in preclinical assays. This potency translated in vivo to a substantial antiviral effect in a single-ascending dose study and a 14-day multiple-ascending dose (MAD) monotherapy study. However, HCV RNA remained detectable in genotype 1a-infected patients at the end of the MAD study. In contrast, viral breakthrough was observed less often in patients infected with genotype 1b, and, in several patients, HCV RNA declined and remained below the level of quantitation (<25 IU/mL) through the duration of treatment. Here, we report on the results of the genotypic and phenotypic analyses of resistant variants in 24 genotype 1-infected patients who received BMS-790052 (1, 10, 30, 60, and 100 mg, once-daily or 30 mg twice-daily) in the 14-day MAD study. Sequence analysis was performed on viral complementary DNA isolated from serum specimens collected at baseline and days 1 (4, 8, and 12 hours), 2, 4, 7, and 14 postdosing. Analyses of the sequence variants (1) established a correlation between resistant variants emerging in vivo with BMS-790052 treatment and those observed in the in vitro replicon system (major substitutions at residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b); (2) determined the prevalence of variants at baseline and the emergence of resistance at different times during dosing; and (3) revealed the resistance profile and replicative ability (i.e., fitness) of the variants. Although resistance emerged during monotherapy with BMS-790052, the substantial anti-HCV effect of this compound makes it an excellent candidate for effective combination therapy.