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ACS Med Chem Lett. 2014 Jul 11;5(9):1043-8. doi: 10.1021/ml5002486. eCollection 2014 Sep 11.

Structure-Based Design of Reactive Nucleosides for Site-Specific Modification of the A2A Adenosine Receptor.

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Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0810, United States.


Adenosine receptors (ARs) are members of the G protein-coupled receptor (GPCR) superfamily and have shown much promise as therapeutic targets. We have used an agonist-bound A2AAR X-ray crystallographic structure to design a chemically reactive agonist for site-specific chemical modification of the receptor. To further explore and chemically engineer its binding cavity, a 2-nitrophenyl active ester was attached through an elongated chain at adenine C2 position. This general structure was designed for irreversible transfer of a terminal acyl group to a nucleophilic amino group on the A2AAR. Preincubation with several O-acyl derivatives prevented radioligand binding that was not regenerated upon extensive washing. In silico receptor docking suggested two lysine residues (second extracellular loop) as potential target sites for an O-acetyl derivative (MRS5854, 3a), and site-directed mutagenesis indicated that K153 but not K150 is essential. Similarly, a butyl azide for click reaction was incorporated in the active ester moiety (3b). These promising results indicate a stable, covalent modification of the receptor by several reactive adenosine derivatives, which could be chemical tools for future imaging, structural probing, and drug discovery. Thus, structure-based ligand design has guided the site-specific modification of a GPCR.


G protein-coupled receptor; adenosine receptor; affinity labeling; covalent modification; nucleoside

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