A high-throughput radiometric assay for hepatitis C virus NS3 protease

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• Hepatitis C virus (HCV) nonstructural protein 3 (NS3) contains a serine protease essential for viral polyprotein processing.
• HCV NS4A protein acts as a cofactor for NS3 protease activity.
• Developing effective inhibitors requires robust assays for screening and characterization.

Purpose of the Study:

• To develop a novel radiometric in vitro assay for the discovery of inhibitors targeting HCV protease activity.
• To establish a high-throughput screening (HTS) compatible assay for HCV NS3 serine protease.
• To characterize the influence of physicochemical conditions on NS3 protease activity.

Main Methods:

• Purification of NS3 proteases from different HCV strains.
• Use of a synthetic radiolabeled peptide substrate mimicking the NS4A-NS4B junction.
• Separation of substrate and product using ion exchange resin in a microtiter plate format.
• Optimization of reaction conditions by varying buffer components.

Main Results:

• A novel radiometric in vitro assay for HCV protease was successfully developed.
• The assay is suitable for high-throughput screening and mechanistic characterization of inhibitors.
• Physicochemical conditions were found to impact NS3 protease activity in a strain-specific manner.
• The assay demonstrated sensitivity for detecting low-nanomolar affinity inhibitors.

Conclusions:

• The developed radiometric assay is a valuable tool for identifying and characterizing inhibitors of the hepatitis C virus NS3 serine protease.
• The assay's HTS compatibility and sensitivity facilitate drug discovery efforts against HCV.
• Understanding strain-specific activity is crucial for developing broad-spectrum HCV protease inhibitors.