Abstract

KW-6356 is a novel adenosine A_2A (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 cocrystal 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 minute⁻¹ for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas 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, whereas 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 differences in vivo and help predict better clinical performance.