A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase.
Discovery and development of effective antiviral agents to combat hepatitis C virus (HCV) is the focus of intensive research both in academia and in pharmaceutical companies. One of the HCV nonstructural proteins, NS5B (an RNA-dependent RNA polymerase), represents an attractive target in light of the clinical success of human immunodeficiency virus reverse transcriptase inhibitors. To identify and evaluate NS5B inhibitors, we developed a homogeneous, solid-phase, high-throughput biochemical assay for detecting NS5B enzymatic activity. In this assay, a biotinylated oligo(dT(12)) primer was immobilized onto streptavidin-coated scintillation proximity assay (SPA) beads, and after addition of homopolymeric A template, NS5B enzyme, and radiolabeled uridine 5'-triphosphate, the primer-dependent RNA synthesis occurred on beads. Optimization of the on-bead reaction resulted in the use of significantly less RNA template and NS5B enzyme while producing a faster steady state reaction rate compared to the solution-phase or off-bead SPA. The newly developed solid-phase assay is functionally comparable to the solution-phase assay as similar potencies of HCV NS5B inhibitors tested were obtained with the two assays. Furthermore, the solid-phase assay offers the advantage of delaying initiation, mimicking a physical preincubation step required for evaluating time-dependent inhibitors.
Wang
Ying-Kai
YK
Department of Virology, Bristol Myers Squibb Co. Pharmaceutical Research Institute, Wallingford, CT 06067, USA. ying-kai.wang@bms.com
Rigat
Karen L
KL
Roberts
Susan B
SB
Gao
Min
M
D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001681:Biological Assay; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004800:Enzymes, Immobilized; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D020128:Inhibitory Concentration 50; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase