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Human Adenosine Kinase: Characterization of a Novel Inhibitor-Enzyme Interaction through Mutagenesis Studies
Adenosine Kinase (hAK) converts Adenosine (Ado) into AMP. Ado acts as a homeostatic modulator of cellular activity and has several important roles in central and peripheral nervous system (CNS, PNS).
Recently, in collaboration with Dott. Campiani (University of Siena), we have developed a new series of non-nucleoside noncompetitive allosteric inhibitors of hAK that were found to inhibit the enzyme in the µM range. Computational analysis of hAK suggested us the existence of a novel putative allosteric site where specific amino-acid residues were identified as relevant for the interaction of our inhibitors in the proposed binding mode. In order to validate our original hypothesis we have cloned and expressed single and double mutants of hAK in which the identified amino acids were substituted with Ala. We determined kinetic parameters of each hAK mutant and performed inhibition studies to validate our model.
The biochemical characterization of the recombinant enzymes showed that every mutation, present in the putative allosteric site, changes the affinity of the enzyme for the natural substrate and some non-nucleoside inhibitors. Our findings represent an intriguing starting point for further structure-activity relationship studies between non-nucleoside inhibitors and hAK, with the aim to identify new essential insights in the enzyme-inhibitors interaction, which may be useful for the synthesis of novel and more potent non-nucleoside inhibitors of hAK with therapeutic purposes.
Adenosine Kinase (hAK) converts Adenosine (Ado) into AMP. Ado acts as a homeostatic modulator of cellular activity and has several important roles in central and peripheral nervous system (CNS, PNS).
Recently, in collaboration with Dott. Campiani (University of Siena), we have developed a new series of non-nucleoside noncompetitive allosteric inhibitors of hAK that were found to inhibit the enzyme in the µM range. Computational analysis of hAK suggested us the existence of a novel putative allosteric site where specific amino-acid residues were identified as relevant for the interaction of our inhibitors in the proposed binding mode. In order to validate our original hypothesis we have cloned and expressed single and double mutants of hAK in which the identified amino acids were substituted with Ala. We determined kinetic parameters of each hAK mutant and performed inhibition studies to validate our model.
The biochemical characterization of the recombinant enzymes showed that every mutation, present in the putative allosteric site, changes the affinity of the enzyme for the natural substrate and some non-nucleoside inhibitors. Our findings represent an intriguing starting point for further structure-activity relationship studies between non-nucleoside inhibitors and hAK, with the aim to identify new essential insights in the enzyme-inhibitors interaction, which may be useful for the synthesis of novel and more potent non-nucleoside inhibitors of hAK with therapeutic purposes.
