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Site‐directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non‐Nucleoside Inhibitors
Author(s) -
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
Publication year - 2016
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/cbdd.12630
Subject(s) - allosteric regulation , nucleoside , chemistry , adenosine , mutagenesis , adenosine kinase , site directed mutagenesis , biochemistry , binding site , computational biology , enzyme , stereochemistry , biology , mutation , adenosine deaminase , gene , mutant
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 ( h AK ), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on h AK , 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 h AK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of h AK (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 h AK and may pave the way to the development of novel selective and potent non‐nucleoside inhibitors of h AK endowed with therapeutic potential.