Pubblicazioni IGM

Site-directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone-Non-Nucleoside Inhibitors.


Savi L, Brindisi M, Alfano G, Butini S, La Pietra V, Novellino E, Marinelli L, Lossani A, Focher F, Cavella C, Campiani G, Gemma S.


CHEMICAL BIOLOGY & DRUG DESIGN 87(1) 112-120, 2016

Autori CNR




Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non-substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non-nucleoside non-competitive inhibitors of human Adenosine Kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies and site-directed mutagenesis to validate our hypothesis. Based on a three-dimensional model of interaction between hAK and our molecules, we designed, cloned and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A and Q74A-F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non-nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK, and may pave the way to the development of novel selective and potent non-nucleoside inhibitors of hAK endowed with therapeutic potential. This article is protected by copyright. All rights reserved.

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