Enzyme Kinetics and Hit Validation in Fluorimetric Protease Assays

Title: Enzyme Kinetics and Hit Validation in Fluorimetric Protease Assays

Volume: 10 Issue: 3

Author(s): Stephanie Ludewig, Markus Kossner, Markus Schiller, Knut Baumann and Tanja Schirmeister

Affiliation:

Keywords: Protease inhibitor, fluorimetric assay, enzyme kinetics, false positive hit

Abstract: Fluorimetric assays are convenient and efficient to determine the inhibitory potency of enzyme inhibitors. Since enzyme activity can be blocked in a number of ways, it is important to determine the exact mode of inhibition. The first part of the review deals with kinetic methods to distinguish among the different modes of inhibition. In addition to that, pitfalls are discussed that can be encountered if the mode of inhibition was not thoroughly investigated. The second part of the review deals with some basic techniques of hit validation. Specifically, three error sources that may result in misleadingly strong inhibitors are scrutinized and exemplified for two different typical protease assays (cathepsin B, chymotrypsin). The studied error sources are attenuation of the fluorescence signal, aggregation of the analysed molecules, and irreversible binding of the inhibitor to the enzyme. A simple experimental protocol to detect the aforementioned problems is proposed.

Cite this article as:

Ludewig Stephanie, Kossner Markus, Schiller Markus, Baumann Knut and Schirmeister Tanja, Enzyme Kinetics and Hit Validation in Fluorimetric Protease Assays, Current Topics in Medicinal Chemistry 2010; 10 (3) . https://dx.doi.org/10.2174/156802610790725498