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. © 2015 John Wiley & Sons A/S.

Savi, L., Brindisi, M., Alfano, G., Butini, S., La Pietra, V., Novellino, E., et al. (2016). Site-directed mutagenesis of key residues unveiled a novel allosteric site on human adenosine kinase for Pyrrolobenzoxa(thia)zepinone non-nucleoside inhibitors. CHEMICAL BIOLOGY & DRUG DESIGN, 87(1), 112-120 [10.1111/cbdd.12630].

Site-directed mutagenesis of key residues unveiled a novel allosteric site on human adenosine kinase for Pyrrolobenzoxa(thia)zepinone non-nucleoside inhibitors

BRINDISI, MARGHERITA;ALFANO, GLORIA;BUTINI, STEFANIA;CAVELLA, CATERINA;CAMPIANI, GIUSEPPE;GEMMA, SANDRA
2016-01-01

Abstract

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. © 2015 John Wiley & Sons A/S.
2016
Savi, L., Brindisi, M., Alfano, G., Butini, S., La Pietra, V., Novellino, E., et al. (2016). Site-directed mutagenesis of key residues unveiled a novel allosteric site on human adenosine kinase for Pyrrolobenzoxa(thia)zepinone non-nucleoside inhibitors. CHEMICAL BIOLOGY & DRUG DESIGN, 87(1), 112-120 [10.1111/cbdd.12630].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/981875