Complexities of measuring antagonist potency at P2X7 receptor orthologs

Abstract

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X7 receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS), which was previously thought to be a weak P2X7 receptor antagonist, possessed high potency (nanomolar IC50) at human and rat P2X7 receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5′-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X7 receptors. HMA (5-(N,N-hexamethylene)amiloride) was also selective for human over rat P2X7 receptors but potentiated responses at mouse P2X7 receptors. Coomassie Brilliant Blue G (CBB) was a nonselective antagonist with high potency at mouse P2X7 receptors (IC50 sim; 100 nM). All compounds were noncompetitive antagonists, and potency could only be quantified by measuring IC50 values. These values were similar when determined against EC50 concentrations of ATP or 2′- and 3′-O-4(-benzoylbenzoyl)-ATP and, for most compounds, only slightly (3- to 5-fold) affected by agonist concentration. However, IC50 values for KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) and suramin, varied up to 25-fold depending upon agonist concentration. Furthermore, IC50 values for KN62 and OxATP were 10-fold lower at 22°C than at 37°C, whereas IC50 values for PPADS, P5P, suramin, and OxATP were up to 20-fold lower in NaCl than in sucrose buffer. Potency estimates for CBB and PPADS decreased 5-fold in the presence of bovine serum albumin, possibly due to protein binding. Given the species differences, and the effects of assay conditions on antagonist potency, caution must be exercised when interpreting results obtained with the available antagonists.