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.
Nipah virus (NiV), a highly pathogenic paramyxovirus, causes respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. On the basis of the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we designed and tested 18 quinolone derivatives in a NiV and measles virus (MV) envelope protein-based fusion assay beside evaluation of cytotoxicity. We found five compounds successfully inhibiting NiV envelope protein-induced cell fusion. The most active molecules (19 and 20), which also inhibit the syncytium formation induced by infectious NiV and show a low cytotoxicity in Vero cells, represent a promising lead quinolone-type compound structure. Molecular modeling indicated that compound 19 fits well into a particular protein cavity present on the NiV F protein that is important for the fusion process.
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