Biochemical evaluation of HCV NS3 protease inhibitors.

This unit describes assays for characterizing the potency and mechanism of action of NS3 protease inhibitors. Determination of IC(50) values is described using in vitro expressed and purified NS3 protease. This assay can also be used for the rapid exploration of structure-activity relationships. Another protocol describes using the full-length NS3/4A complexes expressed in HCV replicon cell lines for a rapid alternative method for assessing protease activity without requiring conventional protein expression and purification. A method is then provided for determination of inhibitor K(i), which more accurately assesses the potency of inhibitors compared to the IC(50) assay, particularly for potent inhibitors that reach the sensitivity limit for the basic IC(50) assay. The final protocol describes how to determine the reversibility of inhibitor binding to the enzyme, an important parameter that can affect the pharmacodynamic properties of a compound.