KW-6356 is a novel adenosine A 2A receptor (A 2A receptor) antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A 2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing OFF episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A 2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined co-crystal structures of A 2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A 2A receptor (the −log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 min -1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, while istradefylline exhibits surmountable antagonism. Crystallography of KW-6356-and istradefylline-bound A 2A receptor have indicated that interactions with His250 6.52 and Trp246 6.48 are essential for the inverse agonism, while the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important difference in vivo and help predict better clinical performance.

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