title: Fluorescent photoaffinity probes for mitotic protein kinase Aurora A.
abstract: We combined the advantages of the selective inhibitor VX689, the bisubstrate-analogue conjugate approach, and photoreactive amino acids to develop 8 photoaffinity probes for Aurora A. The most efficient compounds possessed one-digit nanomolar KD values in the equilibrium binding assay, inhibited Aurora A at elevated concentrations of ATP in the phosphorylation assay in the presence of TPX2, and formed covalent complexes with the recombinant kinase or Aurora A in HeLa cells upon UV-irradiation. The recognition of the correct target by the probes during formation of the covalent complex in the biochemical assay and in situ was demonstrated by competition experiments using the non-labelled inhibitors VX689 and MLN8237.
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia. Electronic address: darja.lavogina@ut.ee.
authors.forename: Darja
authors.initials: D
authors.lastname: Lavogina
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia.
authors.forename: Katariina
authors.initials: K
authors.lastname: Kisand
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia.
authors.forename: Gerda
authors.initials: G
authors.lastname: Raidaru
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia.
authors.forename: Asko
authors.initials: A
authors.lastname: Uri
mesh_terms: D064096:Aurora Kinase A / Q000378:metabolism*; D001681:Biological Assay; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D019920:Photoaffinity Labels; D064096:Aurora Kinase A
keywords: Aurora A; Covalent irreversible inhibitor; Fluorescent probe; Photoaffinity labelling; Protein kinase; VX689 (MK-5108)