HCV NS5A Inhibitor Research Articles

Sub-stoichiometric Modulation of Viral Targets-Potent Antiviral Agents That Exploit Target Vulnerability.

The modulation of oligomeric viral targets at sub-stoichiometric ratios of drug to target has been advocated for its efficacy and potency, but there are only a limited number of documented examples. In this Viewpoint, we summarize the invention of the HIV-1 maturation inhibitor fipravirimat and discuss the emerging details around the mode of action of this class of drug that reflects inhibition of a protein composed of 1,300-1,600 monomers that interact in a cooperative fashion. Similarly, the HCV NS5A inhibitor daclatasvir has been shown to act in a highly sub-stoichiometric fashion, inhibiting viral replication at concentrations that are ∼23,500 lower than that of the protein target.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir’s apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Sofosbuvir-velpatasvir-voxilaprevir in adolescents 12 to 17 years old with HCV infection.

Sofosbuvir-velpatasvir-voxilaprevir is a pangenotypic regimen for chronic HCV infection. In the USA and Europe, sofosbuvir-velpatasvir-voxilaprevir once daily for 12weeks is indicated for adults who previously received an HCV NS5A inhibitor. In Europe, sofosbuvir-velpatasvir-voxilaprevir is also indicated in the absence of prior HCV direct-acting antiviral (DAA) therapy as an 8-week or 12-week regimen. In an open-label study, we evaluated the safety, efficacy, and pharmacokinetics of sofosbuvir-velpatasvir-voxilaprevir in adolescents 12 to 17years with chronic HCV of any genotype. In this Phase 2, multicenter study, sofosbuvir-velpatasvir-voxilaprevir 400/100/100mg daily was administered to adolescents for 8weeks if DAA-naïve or for 12weeks for cirrhosis or prior DAA failure. The key efficacy endpoint was sustained virologic response 12weeks after therapy (SVR12). Intensive pharmacokinetic sampling was done in 14 patients at week 2 or 4, and samples for population pharmacokinetics were collected in all patients. All patients (n=21) were naïve to HCV DAAs, and none had cirrhosis. HCV genotype 3a infection was most common, occurring in 43% of patients. Overall, 100% of patients (21 of 21) reached SVR12. The most common adverse events were abdominal pain and headache (24% each) and nausea (19%); no adverse events led to discontinuation. The only serious adverse event, hypotension, was considered related to study drug and resolved the same day without interruption of treatment. Sofosbuvir-velpatasvir-voxilaprevir exposures were similar to those observed in adults. The pangenotypic regimen of sofosbuvir-velpatasvir-voxilaprevir is highly efficacious and well-tolerated in treating chronic HCV infection in adolescents.

An Improved Process for the Enantioselective Synthesis of HCV NS5A Inhibitor Elbasvir (MK-8742) Chiral Amine Intermediate

An Improved Process for the Enantioselective Synthesis of HCV NS5A Inhibitor Elbasvir (MK-8742) Chiral Amine Intermediate

Ravidasvir hydrochloride for genotype 1 hepatitis C treatment.

Effective oral combination regimens have been approved for treatment of hepatitis C virus (HCV) infection and demonstrated high cure rates in different HCV genotypes. These direct-acting agents target a variety of HCV proteins, including HCV-NS5A (nonstructural protein 5A). Ravidasvir hydrochloride, a potent pan-genotypic HCV-NS5A inhibitor approved in Egypt for treatment of HCV genotype 4 (G4), demonstrated impressive efficacy, safety profiles and a high barrier to resistance in multiple clinical trials when used as a key component in combination with other direct-acting agents in treating patients with HCV-G1.

Prodrug Strategies to Improve the Solubility of the HCV NS5A Inhibitor Pibrentasvir (ABT-530).

A research program to discover solubilizing prodrugs of the HCV NS5A inhibitor pibrentasvir (PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety is attached to a benzimidazole nitrogen atom via an oxymethyl linkage to allow for rapid and complete release of the drug for absorption following phosphate removal by intestinal alkaline phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided superior in vivo performance, delivering high plasma concentrations of PIB in PK studies conducted in mice, dogs, and monkeys. The improved dissolution properties of these phosphate prodrugs provide them the potential to simplify drug dosage forms for PIB-containing HCV therapy.

Safety and efficacy of daclatasvir at doses other than 60 mg daily in HIV/HCV co-infected subjects: Data from the ICONA/HepaICONA foundation cohorts

BackgroundDaclatasvir (DCV) is a HCV NS5A inhibitor whose plasma exposure may be influenced by co-administration with inducers or inhibitors of CYP3A4 such as many antiretrovirals. AimsDescribe the use of different DCV dosages; assess if dose prescription complies with Summaries of Product Characteristics (SmPC); evaluate safety and efficacy of 60 versus 30/90 mg and adequate (i.e. concordant with SmPC) versus incorrect prescriptions. MethodsRetrospective analysis of patients included in ICONA/HepaICONA starting a DCV-including treatment. Incidence rates of liver adverse events (LAE) were calculated; Poisson regression model was used to identify factors associated with LAE. Results311 patients were included: 250 (80.4%) received DCV at a dosage of 60 mg, 52 (16.7%) 30 mg and 9 (2.9%) 90 mg. An inadequate dosage was used in 18 individuals (5.8%). No difference in SVR was observed (93.8% with 60 mg and 94.2% with 30/90 mg, p = 0.910; 93.5% with adequate and 100% with incorrect dosage, p = 0.277). There were 36 LAE with no differences in the two-paired groups. Decompensated liver disease was a risk factor for LAE (aRR = 2.37; p = 0.034), while HIV RNA < 50 copies/ml resulted protective (aRR = 0.22; p = 0.003). ConclusionsDCV use resulted in high SVR rate regardless of dosage and correctness of prescription.

Discovery of a novel unsymmetrical structural class of HCV NS5A inhibitors with low picomolar antiviral activity

A novel unsymmetrical structural class of HCV NS5A inhibitors showing picomolar range antiviral activity has been identified. An unsymmetrical lead compound 2, generated from a substructure of a known symmetrical inhibitor 1, was optimized by extension of its substituents to interact with the hitherto unexplored site of the target protein. This approach afforded novel highly potent unsymmetrical inhibitor 20, which not only equally inhibited HCV genotypes1a, 1b, and 2a with EC50 values in the picomolar range, but also inhibited the 1a Q30K mutant induced by a launched symmetrical inhibitor daclatasvir with an EC50 in the low nanomolar range.

Discovery and Characterization of Potent Pan-Genotypic HCV NS5A Inhibitors Containing Novel Tricyclic Central Core Leading to Clinical Candidate.

The identification of a novel class of potent pan-genotypic NS5A inhibitors with good pharmacokinetic profile suitable for potential use in treating HCV infections is disclosed here. The present series of compounds are with less complex tricyclic central core, identified through a systematic SAR study carried out on biphenyl moiety. The SAR outcome has confirmed the requirement of near planar and linear conformation of the molecule to achieve the best pan-genotypic activity. In addition, SAR with substituted imidazoles on improvement of antiviral activity is disclosed. The newly identified compounds 12, 16, 19-21 have shown desirable pharmacokinetic profiles with a favorable uptake of compounds in liver and maintained a significant concentration for up to 8 h in the liver. In addition, compounds 20 and 21 have shown superior pan-genotypic anti-HCV activity compared to ledipasvir and daclatasvir. Additional characterization and preliminary safety assessment resulted in the identification of compound 20 as a potential clinical candidate.

A novel, potent, and orally bioavailable thiazole HCV NS5A inhibitor for the treatment of hepatitis C virus

A medicinal chemistry program based on the small-molecule HCV NS5A inhibitor daclatasvir has led to the discovery of dimeric phenylthiazole compound 8, a novel and potent HCV NS5A inhibitor. The subsequent SAR studies and optimization revealed that the cycloalkyl amide derivatives 27a-29a exhibited superior potency against GT1b with GT1b EC50 values at picomolar concentration. Interestingly, high diastereospecificity for HCV inhibition was observed in this class with the (1R,2S,1′R,2′S) diastereomer displaying the highest GT1b inhibitory activity. The best inhibitor 27a was found to be 3-fold more potent (GT1b EC50 = 0.003 nM) than daclatasvir (GT1b EC50 = 0.009 nM) against GT1b, and no detectable in vitro cytotoxicity was observed (CC50 > 50 μM). Pharmacokinetic studies demonstrated that compound 27a had an excellent pharmacokinetic profiles with a superior oral exposure and desired bioavailability after oral administration in both rats and dogs, and therefore it was selected as a developmental candidate for the treatment of HCV infection.

Discovery of novel pan-genotypic HCV NS5A inhibitors containing a novel tetracyclic core

A series of novel tetracyclic core-containing HCV NS5A inhibitors has been discovered. Incorporation of tetrahydropyran-substituted amino acid moiety improved their potency and yielded HCV NS5A inhibitors with a minimum potency shift from the GT1a strain compared to other genotypes and mutants. Compounds 53 and 54 showed the best potency profile and had reasonable half-times in rat PK studies. However, further optimization of their oral bioavailability is still needed in order to advance them for further development. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.

Prevalence of relevant NS5A resistance-associated substitutions to elbasvir in genotype 1a hepatitis C virus patients in Spain

Resistance-associated substitutions (RASs) to the new HCV NS5A inhibitor elbasvir may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are no data outside clinical trials evaluating their prevalence and impact in grazoprevir/elbasvir in GT1a-infected patients in Spain. A multicentre cross-sectional study of 632 initial patients was conducted. In 13 of these patients, the sample could not be amplified or a consensus sequence by Sanger sequencing could not be performed. Ultimately, 617 HCV-G1a-infected individuals treated at 84 Spanish hospitals from the 17 autonomous communities plus the 2 autonomous cities of Spain were analysed. HCV population sequencing was used to identify RAS to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-G1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%, respectively). Only 3.4% of the identified RASs to elbasvir conferred reduced susceptibility to elbasvir by geno2pheno[HCV], which exclusively identified the positions Q30H/R (n=7) and Y93C/H/N (n=8) as single mutations and Q30H+Y93H (n=4) and Q30R+Y93H (n=2) as double mutations as the major RASs to elbasvir. A lower prevalence of RASs to elbasvir was observed in our HCV-G1a Spanish cohort than reported previously in clinical trials evaluating patients from the USA. This information may be essential to guide the implementation of grazoprevir/elbasvir in Spain and to manage G1a-infected patients.

MK-8325: A silyl proline-containing NS5A inhibitor with pan-genotype activity for treatment of HCV

HCV NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays thus making them attractive components for inclusion in an all oral fixed dose combination regimen. Herein, we describe the discovery and characterization of silyl proline-containing HCV NS5A inhibitor MK-8325 with good pan-genotype activity and acceptable pharmacokinetic properties.

Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with pan-genotype activity

Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d with improved pan-genotype NS5A inhibition and better pharmacokinetic properties. The key structural changes to ombitasvir include bioisosteric replacement of carbon with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT 1 to 6) of 10d is increased to some extent, especially the inhibitory activity against genotype 3a and 6a is increased by more than seven times, and the dog's in vivo pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation showed that 10d was similar to ombitasvir on plasma protein binding and liver distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450 and hERG ligand binding. However, permeability assay results indicated that 10d was not the substrate of P-gp or BCRP transporter, which is different from that of ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity with 10d. Our findings in preclinical tests suggest that the silicon-containing compound 10d could be worthy of continued study as a potential drug candidate.

Synthesis of BMS-986097

Key words BMS-986097 - HCV NS5A inhibitor - diastereoselective Michael addition

Development of the Large-Scale Synthesis of Tetrahydropyran Glycine, a Precursor to the HCV NS5A Inhibitor BMS-986097

An efficient large-scale synthesis of acid 1, a penultimate precursor to the HCV NS5A inhibitor BMS-986097, along with the final API step are described. Three routes were devised for the synthesis of 1 at the various stages of the program. The third generation route, the one that proved scalable and is the main subject of this paper, features a one-step Michael addition of t-butyl 2-((diphenylmethylene)amino)acetate (24) to (E)-benzyl 4-(1-hydroxycyclopropyl)but-2-enoate (28) followed by cyclization and chiral separation to form 27c, the core skeleton of cap piece 1. The epimerization and chiral resolution of 27c followed by further synthetic manipulations involving the carbamate formation, lactone reduction and cyclization, afforded cyclopropyl pyran 1. A detailed study of diphenylmethane deprotection via acid hydrolysis as well as a key lactone to tetrahydropyran conversion, in order to avoid a side reaction that afforded an alternative cyclization product, are discussed. This synthesis was applied to the preparation of more than 100 g of the final API BMS-986097 for toxicology studies.

Efficacy of 12 or 18weeks of elbasvir plus grazoprevir with ribavirin in treatment-naïve, noncirrhotic HCV genotype 3-infected patients.

Elbasvir (EBR; HCV NS5A inhibitor) and grazoprevir (GZR; HCV NS3/4A protease inhibitor) are approved as a fixed-dose combination to treat patients chronically infected with HCV genotypes 1 and 4. During the development programme and supported by in vitro potency, the efficacy of EBR+GZR was assessed in HCV GT3-infected patients. This study's aim was to determine the efficacy and tolerability of 12 or 18weeks of EBR+GZR with ribavirin (RBV) in treatment-naïve, noncirrhotic HCV GT3-infected patients. Randomized patients received open-label EBR (50mg once daily) + GZR (100mg once daily) + RBV. The primary efficacy objective was to evaluate the sustained virologic response rates 12weeks after the end of all study therapy (SVR12). SVR12 rates (95% confidence interval) were 45.0% (23.1, 68.5) and 57.1% (34.0, 78.2) after treatment with EBR+GZR+RBV for 12weeks or 18weeks, respectively. On-treatment virologic failure was observed in 41% (17 of 41) of patients. At virologic failure, resistance-associated substitutions (RASs) with a >five-fold shift in potency occurred in the NS3 region in six (35%) patients and in the NS5A region in 16 (94%) patients. The most common RAS at virologic failure was Y93H in NS5A which was identified in 13 of 17 (76%) patients. The efficacy of EBR+GZR+RBV was suboptimal in HCV GT3-infected patients due to a high rate of on-treatment virologic failure and treatment-emergent RASs which demonstrates an inadequate barrier to the development of GT3 resistance. However, rapid viral clearance demonstrated the antiviral activity of EBR+GZR+RBV in GT3-infected patients.clinicaltrials.gov: NCT01717326.

Low frequency of NS5A relevant resistance-associated substitutions to Elbasvir among hepatitis C virus genotype 1a in Spain: a cross-sectional study

Relevant resistance-associated substitutions (RASs) to elbasvir, the new HCV NS5A inhibitor, may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are few data outside clinical trials evaluating their prevalence and impact of elbasvir/grazoprevir. A multicenter cross-sectional study of 617 HCV-GT1a-infected individuals attended in 84 Spanish hospitals from the 17 Autonomous Communities and two Autonomous cities was performed. HCV population sequencing was used to identify RASs to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-GT1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%; respectively). Only 3.4% of patients had viruses with RASs that confer reduced susceptibility to elbasvir by geno2pheno[HCV] that identified exclusively the positions Q30H/R (n = 7) and Y93C/H/N (n = 8) as single mutations and Q30H + Y93H (n = 4) and Q30R + Y93H (n = 2) as double mutations considered as RASs to elbasvir. Lower prevalence of RASs to elbasvir in our HCV-GT1a-Spanish cohort was observed than reported previously in clinical trials. This information may be essential to guiding the implementation of elbasvir/grazoprevir in Spain, expected at the beginning of 2017 and the management of GT1a-infected patients.

Frecuencia de sustituciones relevantes asociadas a resistencia en la región NS5A a elbasvir en el virus de la hepatitis C en pacientes con genotipo 1a en España

Resistance-associated substitutions (RASs) to the new HCV NS5A inhibitor elbasvir may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are no data outside clinical trials evaluating their prevalence and impact in grazoprevir/elbasvir in GT1a-infected patients in Spain. A multicentre cross-sectional study of 632 initial patients was conducted. In 13 of these patients, the sample could not be amplified or a consensus sequence by Sanger sequencing could not be performed. Ultimately, 617 HCV-G1a-infected individuals treated at 84 Spanish hospitals from the 17 autonomous communities plus the 2 autonomous cities of Spain were analysed. HCV population sequencing was used to identify RAS to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-G1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%, respectively). Only 3.4% of the identified RASs to elbasvir conferred reduced susceptibility to elbasvir by geno2pheno[HCV], which exclusively identified the positions Q30H/R (n=7) and Y93C/H/N (n=8) as single mutations and Q30H+Y93H (n=4) and Q30R+Y93H (n=2) as double mutations as the major RASs to elbasvir. A lower prevalence of RASs to elbasvir was observed in our HCV-G1a Spanish cohort than reported previously in clinical trials evaluating patients from the USA. This information may be essential to guide the implementation of grazoprevir/elbasvir in Spain and to manage G1a-infected patients.

Long-term persistence of HCV NS5A resistance-associated substitutions after treatment with the HCV NS5A inhibitor, ledipasvir, without sofosbuvir.

Data on persistence of NS5A resistance-associated substitutions (RASs) may have implications for resistance testing approaches and selection of initial and retreatment strategies. Long-term persistence of NS5A RASs in HCV genotype (GT) 1 infected subjects (n=76) who did not achieve sustained virological response after receiving ledipasvir (LDV) without sofosbuvir (SOF) and were subsequently enrolled in an ongoing 3-year follow-up registry study was investigated by population or deep sequencing. Of the 76 subjects enrolled, 67 and 9 subjects had GT1a and GT1b infection, respectively. At pretreatment, NS5A RASs were detected in 14% of subjects (11/76) by population sequencing, with three subjects having >1 RAS. All RASs that were detected at pretreatment persisted and were observed at the 96 week visit in the follow-up study (FU96). For the remaining subjects with no detectable RASs at pretreatment, RASs were detected in 98% (63/64) of subjects at virological failure in the parent study and persisted at detectable levels through FU96 in 86% of subjects by deep sequencing (1% cutoff). However, a decline in the quasispecies frequency of most RASs and the number of RASs per subject was observed over time. Phenotypic analysis demonstrated that the majority of NS5A RASs confer similar levels of resistance to LDV and daclatasvir. The majority of NS5A RASs can persist at detectable levels for >96 weeks post-treatment in subjects who failed treatment with regimens containing an NS5A inhibitor without SOF, suggesting relatively high fitness of NS5A RASs even in the absence of drug pressure.