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- Title
Site-directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non-Nucleoside Inhibitors.
- Authors
Savi, Lida; Brindisi, Margherita; Alfano, Gloria; Butini, Stefania; La Pietra, Valeria; Novellino, Ettore; Marinelli, Luciana; Lossani, Andrea; Focher, Federico; Cavella, Caterina; Campiani, Giuseppe; Gemma, Sandra
- 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 nonnucleoside 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.
- Subjects
MUTAGENESIS; RADIOGENETICS; GENETIC mutation; ADENOSINE kinase; RIBONUCLEOSIDES
- Publication
Chemical Biology & Drug Design, 2016, Vol 87, Issue 1, p112
- ISSN
1747-0277
- Publication type
Article
- DOI
10.1111/cbdd.12630