title: Site-directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non-Nucleoside Inhibitors.
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.
authors.affiliation: Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100, Pavia, Italy.
authors.forename: Lida
authors.initials: L
authors.lastname: Savi
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Brindisi
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Alfano
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Butini
authors.affiliation: European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|Dipartimento di Farmacia, Università di Napoli Federico II, via D. Montesano 49, 80131, Napoli, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: La Pietra
authors.affiliation: European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|Dipartimento di Farmacia, Università di Napoli Federico II, via D. Montesano 49, 80131, Napoli, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.affiliation: European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|Dipartimento di Farmacia, Università di Napoli Federico II, via D. Montesano 49, 80131, Napoli, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Marinelli
authors.affiliation: Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100, Pavia, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Lossani
authors.affiliation: Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100, Pavia, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Focher
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Cavella
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Campiani
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.|European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena, Italy.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gemma
mesh_terms: D000248:Adenosine Kinase / Q000378:metabolism*; D000495:Allosteric Site; D001381:Azepines / Q000494:pharmacology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000037:antagonists & inhibitors*
chemical_list: D001381:Azepines; D009705:Nucleosides; D000248:Adenosine Kinase
keywords: Ala-scan mutagenesis; adenosine kinase; allosteric binding site; allosteric inhibitors; drug discovery; non-nucleoside human AK inhibitors; small molecule