A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• Hepatitis C virus (HCV) infection remains a significant global health concern.
• Developing effective antiviral therapies is crucial for combating HCV.
• The HCV NS5B RNA-dependent RNA polymerase is a validated target for antiviral drug development.

Purpose of the Study:

• To develop a novel, homogeneous, solid-phase, high-throughput biochemical assay for detecting HCV NS5B enzymatic activity.
• To optimize the assay for efficiency and comparability with existing methods.
• To facilitate the identification and evaluation of potential NS5B inhibitors.

Main Methods:

• Development of a solid-phase assay using biotinylated oligo(dT(12)) primer immobilized on streptavidin-coated scintillation proximity assay (SPA) beads.
• Detection of primer-dependent RNA synthesis catalyzed by NS5B enzyme using radiolabeled uridine 5'-triphosphate.
• Optimization of on-bead reaction conditions to reduce reagent usage and increase reaction rate.

Main Results:

• The optimized solid-phase SPA assay demonstrated comparable potency of HCV NS5B inhibitors to solution-phase assays.
• The assay requires significantly less RNA template and NS5B enzyme.
• The solid-phase format allows for delayed initiation, enabling evaluation of time-dependent inhibitors.

Conclusions:

• A novel, efficient, and robust solid-phase biochemical assay for HCV NS5B activity has been successfully developed.
• This assay is suitable for high-throughput screening and drug discovery efforts targeting HCV.
• The assay offers advantages over traditional methods, including reduced reagent consumption and the ability to assess time-dependent inhibition.