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Biophysical mapping of the adenosine A2A receptor.

ABSTRACT: A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (K(D), k(on), and k(off)) of receptor-ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein-ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A(2A) receptor are presented.

SUBMITTER: Zhukov A

PROVIDER: S-EPMC3308198 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Biophysical mapping of the adenosine A2A receptor.

Zhukov Andrei A Andrews Stephen P SP Errey James C JC Robertson Nathan N Tehan Benjamin B Mason Jonathan S JS Marshall Fiona H FH Weir Malcolm M Congreve Miles M

Journal of medicinal chemistry 20110610 13

A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding ...[more]

PMID: 21661720

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Project description:ObjectivePharmacological activation of adenosine signaling has been shown to increase ?-cell proliferation and thereby ?-cell regeneration in zebrafish and rodent models of diabetes. However, whether adenosine has an endogenous role in regulating ?-cell proliferation is unknown. The objective of this study was to determine whether endogenous adenosine regulates ?-cell proliferation-either in the basal state or states of increased demand for insulin-and to delineate the mechanisms involved.MethodsWe analyzed the effect of pharmacological adenosine agonists on ?-cell proliferation in in vitro cultures of mouse islets and in zebrafish models with ?- or ?-cell ablation. In addition, we performed physiological and histological characterization of wild-type mice and mutant mice with pancreas- or ?-cell-specific deficiency in Adora2a (the gene encoding adenosine receptor A2a). The mutant mice were used for in vivo studies on the role of adenosine in the basal state and during pregnancy (a state of increased demand for insulin), as well as for in vitro studies of cultured islets.ResultsPharmacological adenosine signaling in zebrafish had a stronger effect on ?-cell proliferation during ?-cell regeneration than in the basal state, an effect that was independent of the apoptotic microenvironment of the regeneration model. In mice, deficiency in Adora2a impaired glucose control and diminished compensatory ?-cell proliferation during pregnancy but did not have any overt phenotype in the basal state. Islets isolated from Adora2a-deficient mice had a reduced baseline level of ?-cell proliferation in vitro, consistent with our finding that UK432097, an A2a-specific agonist, promotes the proliferation of mouse ?-cells in vitro.ConclusionsThis is the first study linking endogenously produced adenosine to ?-cell proliferation. Moreover, we show that adenosine signaling via the A2a receptor has an important role in compensatory ?-cell proliferation, a feature that could be harnessed pharmacologically for ?-cell expansion and future therapeutic development for diabetes.

Dataset Information

Biophysical mapping of the adenosine A2A receptor.

Publications

Biophysical mapping of the adenosine A2A receptor.

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