Mapping the Allosteric Action of Antagonists A740003 and A438079 Reveals a Role for the Left Flipper in Ligand Sensitivity at P2X7 Receptors

Abstract

P2X7 receptor (P2X7R) activation requires ∼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site. Within the allosteric site large effects on antagonist action were found for point mutants of residues F88A, D92A, F95A, and F103A that were conserved or similar between sensitive/insensitive P2XR subtypes, suggesting that these side-chain interactions were not solely responsible for high-affinity antagonist binding. Antagonist sensitivity was increased with mutations that remove the bulk of side chains around the center of the binding pocket, suggesting that the dimensions of the pocket make a significant contribution to selectivity. Chimeric receptors swapping the left flipper (around the orthosteric site) reduced both ATP and antagonist sensitivity. Point mutations within this region highlighted the contribution of a P2X7R-specific aspartic acid residue (D280) that modeling suggests forms a salt bridge with the lower body region of the receptor. The D280A mutant removing this charge increased ATP potency 15-fold providing a new insight into the low ATP sensitivity of the P2X7R. The ortho- and allosteric binding sites form either side of the β-strand Y291-E301 adjacent to the left flipper. This structural linking may explain the contribution of the left flipper to both agonist and antagonist action.

Dissection of P2X4 and P2X7 Receptor Current Components in BV-2 Microglia

“…A438079 had a strong blocking effect at low ATP concentrations but at >0.3 mM ATP 4− , the currents that are mainly carried by P2X7 receptors were only partially blocked by A438079. Although A438079 binds to P2X7 receptors at an allosteric binding site [ 65 ], indications for a competitive blocking effect were found [ 66 , 67 ]. The partial block can therefore be explained by a competitive displacement of A438079 from its blocking site by ATP.…”

P2X7 in Cancer: From Molecular Mechanisms to Therapeutics

“…However, the lack of specificity of these inhibitors led to the identification and development of several classes of inhibitors that bind to the inter-subunit allosteric pocket preventing ATP induced rotation of each subunit and closure of the turret (Karasawa and Kawate, 2016). Within this inter-subunit allosteric pocket, several point mutants including but not limited to F88A, D92A, F95A, and F103A were identified to play an important role in the mode of action of these inhibitors (Allsopp et al, 2017;Allsopp et al, 2018;Bin Dayel et al, 2019). Engagement of this allosteric pocket allowed progressive development of antagonists in the low nanomolar range ( Table 2) while providing better selectivity for P2X7 against other P2X family members (Donnelly-Roberts et al, 2009a;Allsopp et al, 2017;Allsopp et al, 2018;Bin Dayel et al, 2019).…”

“…Within this inter-subunit allosteric pocket, several point mutants including but not limited to F88A, D92A, F95A, and F103A were identified to play an important role in the mode of action of these inhibitors (Allsopp et al, 2017;Allsopp et al, 2018;Bin Dayel et al, 2019). Engagement of this allosteric pocket allowed progressive development of antagonists in the low nanomolar range ( Table 2) while providing better selectivity for P2X7 against other P2X family members (Donnelly-Roberts et al, 2009a;Allsopp et al, 2017;Allsopp et al, 2018;Bin Dayel et al, 2019). Initial identification of lead candidates against the intersubunit allosteric pocket revealed compounds with good activity against human P2X7 but inactive against the rat isoform making pharmacology studies difficult (Michel et al, 2008a;Michel et al, 2008b;Caseley et al, 2015).…”

“…Initial identification of lead candidates against the intersubunit allosteric pocket revealed compounds with good activity against human P2X7 but inactive against the rat isoform making pharmacology studies difficult (Michel et al, 2008a;Michel et al, 2008b;Caseley et al, 2015). However, docking of these candidates in the inter-subunit allosteric pocket highlighted the importance of human F95 (L95 in rat P2X7) in developing pi-stacking interactions with inhibitors (Caseley et al, 2015;Allsopp et al, 2017;Allsopp et al, 2018).…”

Mapping the Site of Action of Human P2X7 Receptor Antagonists AZ11645373, Brilliant Blue G, KN-62, Calmidazolium, and ZINC58368839 to the Intersubunit Allosteric Pocket

“…A wide range of chemically distinct, highly selective P2X7R antagonists have been reported (Young and Gorecki, 2018). Crystallization and mutagenesis studies have identified an allosteric binding site for six of these P2X7R antagonists (Karasawa and Kawate, 2016; Allsopp et al, 2017, 2018). This allosteric site is at the subunit interface at the apex of the receptor (and referred to in this paper as the “intersubunit allosteric site”).…”