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Long Residence Times Revealed by Aurora A Kinase‐Targeting Fluorescent Probes Derived from Inhibitors MLN8237 and VX‐689
Author(s) -
Lavogina Darja,
Enkvist Erki,
Viht Kaido,
Uri Asko
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300613
Subject(s) - fluorescence , chemistry , biophysics , kinase , activator (genetics) , aurora kinase , substrate (aquarium) , fluorescence anisotropy , biochemistry , stereochemistry , biology , physics , receptor , ecology , quantum mechanics , cell cycle , cell , membrane
We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well‐known inhibitors MLN8237 and VX‐689 (MK‐5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes ( k on 10 5 –10 7 M −1 s −1 , k off 10 −3 –10 −4 s −1 ; residence time 500–3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on‐rate and increase in the off‐rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX‐689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.