Mechanism of action of species‐selective P2X7 receptor antagonists

Background and purpose: AZ11645373 and N‐{2‐methyl‐5‐[(1R, 5S)‐9‐oxa‐3,7‐diazabicyclo[3.3.1]non‐3‐ylcarbonyl]phenyl}‐2‐tricyclo[3.3.1.13,7]dec‐1‐ylacetamide hydrochloride (compound‐22) are recently described P2X7 receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. Experimental approach: Antagonist effects at recombinant and chimeric P2X7 receptors were assessed by ethidium accumulation and radioligand‐binding studies. Key results: AZ11645373 and compound‐22 were confirmed as selective non‐competitive antagonists of human or rat P2X7 receptors respectively. Both compounds were weak antagonists of the mouse and guinea‐pig P2X7 receptors and, for each compound, their potency estimates at human and dog P2X7 receptors were similar. The potency of compound‐22 was moderately temperature‐dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio‐labelled P2X7 receptor antagonist. The species selectivity of AZ11645373, but not compound‐22, was influenced by the nature of the amino acid at position 95 of the P2X7 receptor. N2‐(3,4‐difluorophenyl)‐N1‐[2‐methyl‐5‐(1‐piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound‐22 at the rat receptor but had little effect on the actions of AZ11645373. Conclusions: AZ11645373 and compound‐22 are allosteric antagonists of human and rat P2X7 receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X7 receptor at which antagonists can bind and affect receptor function.