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(dT12) 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.