Metal complexes with N-heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry and are now increasingly considered for the development of new chemical tools and metal based drugs. Ruthenium complexes of the type (p-cymene)(NHC)RuCl(2) interacted with biologically relevant thiols and selenols, which resulted in the inhibition of enzymes such as thioredoxin reductase or cathepsin B. Pronounced antiproliferative effects could be obtained provided that an appropriate cellular uptake was achieved. Inhibition of tumor cell growth was accompanied by a perturbation of metabolic parameters such as cellular respiration.

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.

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