Structure of the Adenosine A2A Receptor in Complex with ZM241385 and the Xanthines XAC and Caffeine

Abstract

Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

Adenosine Signaling in Striatal Circuits and Alcohol Use Disorders

“…Furthermore, specific actions of caffeine, including increased arousal (Huang et al, 2005), increased locomotor activity (El Yacoubi et al, 2000) and increased ethanol tolerance can be attributed to its inhibition of A2AR. In addition, a recent co-crystal structural study has revealed that ZM241385 (A2AR specific antagonist) and caffeine have similar pharmacological binding properties to A2AR (Dore et al, 2011), suggesting that inhibition of A2AR by either ZM241385 or caffeine might enhance goal-directed behavior and promote ethanol drinking (Fig. 2).…”

Ice breaking in GPCR structural biology

“…To date, most crystallization studies are focused on indirect uses, such as expression or stability screening, to facilitate higher throughput in a short period of time. The best example of this was provided by the Tate group in solving the crystal structures of the β1 adrenergic receptor and the A 2A adenosine receptor in the active state [16,37] . All of the mutants were expressed in E coli using a procedure that is similar to Grisshammer's, and the construct optimization was performed accordingly, before the final transfer to insect cells for large-scale preparations.…”

“…The most promising mutations were then combined and further screened for the highest stability to tolerate the harsh environment during extraction, purification and crystalliza tion [38,45] . Guided by this method, they solved the crystal structures of the β1 adrenergic receptor with different ligands and the A 2A adenosine receptor structure in an active state [4,16] . Similarly, PLÜCKTHUN et al used error-prone PCR (epPCR) to construct a library of mutations of target gene which were expressed and sorted by flow cytometry to screen for higher expression or stability.…”

“…To date, all the receptor analogs achieved from mutation screening had a modified binding preference depending on the type of ligands used [4,16] . For example, in the structure of the A 2A adenosine receptor bound to agonists adenosine and NECA, four mutations in the receptor greatly reduced the affinity with the antagonists, while the agonist binding was unaltered [16] . The shift in binding affinity indicates that the receptor could only adopt restrained conformations, thus its stability and homogeneity were improved, which would further allow crystallization.…”

“…Despite the current low success rates for achieving high-resolution structures, years of technology developments and studies of this protein family have shed light on this challenging project. Recent breakthroughs include the structures of novel GPCRs [7] , discovery of new activity states of GPCRs [10,[14][15][16] and a structure of GPCR in complex with other proteins [11] . This review will focus on new…”