Site-directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non-Nucleoside Inhibitors
| Autor: | Lida Savi, Federico Focher, Ettore Novellino, Valeria La Pietra, Caterina Cavella, Luciana Marinelli, Margherita Brindisi, Gloria Alfano, Stefania Butini, Andrea Lossani, Sandra Gemma, Giuseppe Campiani |
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| Přispěvatelé: | 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 |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
non-nucleoside human AK inhibitor Allosteric regulation Adenosine kinase Ala-scan mutagenesis Allosteric binding site Allosteric inhibitors Drug discovery Non-nucleoside human AK inhibitors Small molecule Biochemistry Molecular Medicine 03 medical and health sciences 0302 clinical medicine Humans Site-directed mutagenesis Pharmacology chemistry.chemical_classification allosteric inhibitor biology Organic Chemistry Nucleosides Azepines Ala-scan mutagenesi 030104 developmental biology Enzyme Allosteric enzyme chemistry biology.protein Mutagenesis Site-Directed Nucleoside 030217 neurology & neurosurgery Allosteric Site |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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