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キーワード:
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要旨:
Analog-sensitive (as) kinases differ from their natural counterparts by a single amino-acid change in the ATP binding pocket. This so called gatekeeper mutation enlarges the ATP binding site and makes the kinase susceptible to specific inhibitors that typically do not target unmodified kinases. The high conservation of the kinase domain allows identification of the gatekeeper residue in most kinases, and as-kinases have been successfully created in organisms from yeast to human. Here we report on the introduction of a second mutation to successfully target a serine/threonine as-kinase with covalent inhibitors. Through structure-based design we identified a residue close to the ATP binding pocket, whose mutation to cysteine creates an anchor point for irreversible kinase inhibitors. We demonstrate the feasibility of this approach for an as-version of the conserved fission yeast (Schizosaccharomyces pombe) Aurora kinase Ark1. Aurora is essential for the proper execution of mitosis and small-molecule inhibitors selective for Aurora have been crucial to decipher its role during the cell cycle. We employed structure-guided chemical synthesis of a focused library of 4-anilino-quinazolines equipped with diverse reactive groups and show that some of the compounds specifically inhibit Ark1 carrying the gatekeeper mutation as well as the engineered cysteine, but not the kinase with only one of these mutations.
