Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors

Abstract

A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

“…Potency of Selected Compounds in a Cell-Based Replicon Assay . To further evaluate the potency and the “in vivo” characteristics of these inhibitors, a few compunds were tested in a replicon-based cellular assay, and the results are included in Table . IC 50 , the concentration required for inhibition of 50% of virus replication, was recorded as a measure of cell-based replicon potency, which is a more accurate reflection of the potency of the inhibitors in a “real” living environment.…”

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation

“…avibactam, have been developed for clinical use as reversibly reacting covalent inhibitors of nucleophilic serine blactamases. [80][81][82] g-Lactams have been developed as inhibitors of both human and viral serine proteases including e.g., human neutrophil elastase, [31][32][33] the hepatitis C virus (HCV) ns3/4a serine protease, [34][35][36][37] and the human cytomegalovirus (HCMV) serine protease. 38,39 They inhibit via acylation of the nucleophilic serine residue, at least in some cases in a reversible manner.…”

“…29 At least in part, this gap may reect the reduced intrinsic reactivity of g-lactams compared to more strained b-lactams based on (non-enzymatic) ring closure rates. 30 The covalent reaction of g-lactams with nucleophilic serine enzymes is reported; [31][32][33][34][35][36][37][38][39] however, their analogous reactivity with nucleophilic cysteine enzymes, many of which are contemporary drug targets, 25,26 is, to our knowledge, unknown. SARS-CoV-2 M pro appears to be a suitable target to investigate the reactivity of g-lactams with nucleophilic cysteine enzymes, because many small-molecule inhibitors are reported which employ an electrophilic group for reversible or irreversible covalent reaction with the nucleophilic thiolate of the catalytic cysteine residue of M pro (i.e., Cys145, Fig.…”

Issues in designing and interpreting clinical trials of treatments for chronic hepatitis C

“…This, combined with a lack of small animal models for the disease and the expense of chimpanzees, has hindered the development of HCV interventions [2]. Although this situation has been alleviated by the introduction of HCV replicons and newer cell‐based assay systems, the major lessons in identifying the best treatment, dose, and duration have come from randomized clinical trials (RCTs) of patients with the disease, and this will most likely continue long into the future with the evaluation of new, small molecular therapies as well as the next generation of antiviral compounds [1–3]. This article reviews the history, general design, and conduct of RCTs in medicine and then focuses on the specific elements in the design of clinical trials of interventions for chronic hepatitis C that can influence the outcomes and interpretation of the studies.…”