title: Long residence times revealed by Aurora A kinase-targeting fluorescent probes derived from inhibitors MLN8237 and VX-689.
abstract: 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 (kon 10(5)-10(7) M(-1) s(-1), koff 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.
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu (Estonia). darja.lavogina@ut.ee.
authors.forename: Darja
authors.initials: D
authors.lastname: Lavogina
authors.forename: Erki
authors.initials: E
authors.lastname: Enkvist
authors.forename: Kaido
authors.initials: K
authors.lastname: Viht
authors.forename: Asko
authors.initials: A
authors.lastname: Uri
mesh_terms: D064096:Aurora Kinase A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001381:Azepines / Q000737:chemistry* / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism
chemical_list: C547876:4-(3-chloro-2-fluorophenoxy)-1-((6-(1,3-thiazol-2-ylamino)pyridin to 2-yl)methyl) cyclohexanecarboxylic acid; D001381:Azepines; D003509:Cyclohexanecarboxylic Acids; D005456:Fluorescent Dyes; C550258:MLN 8237; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D064096:Aurora Kinase A
keywords: Aurora A; fluorescent probes; inhibitors; kinetics; protein kinases; residence time