Development of cell-based assays for in vitro characterization of hepatitis C virus NS3/4A protease inhibitors.
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.
Chung
Victoria
V
Department of Virology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom. Vicky.2.chung@gsk.com
Carroll
Anthony R
AR
Gray
Norman M
NM
Parry
Nigel R
NR
Thommes
Pia A
PA
Viner
K Claire
KC
D'Souza
Eric A
EA
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007769:Lactams / Q000737:chemistry / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests / Q000379:methods; D011480:Protease Inhibitors / Q000494:pharmacology*; D012093:Replicon; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D007769:Lactams; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus