title: Enzyme kinetics and hit validation in fluorimetric protease assays.
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.
authors.affiliation: Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, D 97074 Wuerzburg, Germany. s.ludewig@tu-bs.de
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ludewig
authors.forename: Markus
authors.initials: M
authors.lastname: Kossner
authors.forename: Markus
authors.initials: M
authors.lastname: Schiller
authors.forename: Knut
authors.initials: K
authors.lastname: Baumann
authors.forename: Tanja
authors.initials: T
authors.lastname: Schirmeister
mesh_terms: D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D015203:Reproducibility of Results; D012107:Research Design; D013329:Structure-Activity Relationship
chemical_list: D011480:Protease Inhibitors; D002918:Chymotrypsin; D002401:Cathepsin B