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

Abstract

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.

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).…”

“…Preclinical N/A (Stokes et al, 2006;Caseley et al, 2015;Bin Dayel et al, Pain from inflammatory disease such as arthritis Han et al, 2017) GW791343 Small molecule (positive modulator)…”

The purinergic P2X7 receptor as a potential drug target to combat neuroinflammation in neurodegenerative diseases

“…A structural characteristic of the P2X7R is an extracellular pocket located internally at the top of the UB, which is wider than in other family subtypes. This is the allosteric binding site for several antagonists, as demonstrated by the crystallized panda P2X7R and in subsequent mutagenesis and molecular docking studies 49,63 . This information could help design and develop new selective ligands.…”

“…This is the allosteric binding site for several antagonists, as demonstrated by the crystallized panda P2X7R and in subsequent mutagenesis and molecular docking studies. 49,63 This information could help design and develop new selective ligands.…”