Unique residues in the ATP gated human P2X7 receptor define a novel allosteric binding pocket for the selective antagonist AZ10606120

Abstract

The P2X7 receptor (P2X7R) for ATP is a therapeutic target for pathophysiological states including inflammation, pain management and epilepsy. This is facilitated by the predicted low side effect profile as the high concentrations of ATP required to activate the receptor are usually only found following cell damage/disease and so P2X7Rs respond to a “danger” signal and are not normally active. AZ10606120 is a selective antagonist for P2X7Rs (IC50 of ~10 nM) and ineffective at the P2X1R (at 10 μM). To determine the molecular basis of selectivity we generated a series of P2X7/1R chimeras and mutants. Two regions that are unique to the P2X7R, a loop insertion (residues 73–79) and threonine residues T90 and T94, are required for high affinity antagonist action. Point mutations ruled out an orthosteric antagonist site. Mutations and molecular modelling identified an allosteric binding site that forms at the subunit interface at the apex of the receptor. Molecular dynamics simulations indicated that unique P2X7R features regulate access of AZ10606120 to the allosteric site. The characterisation of the allosteric pocket provides a new and novel target for rational P2X7R drug development.

P2X7 as a scavenger receptor for innate phagocytosis in the brain

“…These data show that the selective and potent P2X7 antagonists currently available are effective at blocking P2X7 pore formation but do not affect P2X7 phagocytosis in monocytes expressing wild‐type P2X7 receptors or the two common P2X7 functional variants. This divergence of features between pore formation and phagocytosis is consistent with recent data showing antagonists such as AZ10606120 bind to an allosteric site, which is separate from the ATP‐binding site that controls pore opening (Karasawa and Kawate, ; Allsopp et al ., ).…”

“…While the open state mediates the pro‐inflammatory downstream effects, it is likely that the closed state is responsible for P2X7 scavenger activity. These structural data have identified the inhibitory binding sites separate from previously identified ATP binding sites (Karasawa and Kawate, ; Allsopp et al ., ; Karasawa and Kawate, ). Thus, selective P2X7 antagonists may block the open pore state without affecting P2X7 scavenger activity, although oxidized ATP is an exception as it blocks both pore and scavenger function (Ou et al ., ).…”

“…The crystal structure of the vertebrate P2X7 receptor has been published showing clear differences between the open and closed pore state of this transmembrane molecule (Karasawa and Kawate, 2016;Allsopp et al, 2017;Karasawa and Kawate, 2017). While the open state mediates the proinflammatory downstream effects, it is likely that the closed state is responsible for P2X7 scavenger activity.…”

Purinergic drug targets for gastrointestinal disorders

“…Examples of such heterotrimers are P2X2/3, P2X2/4 and P2X4/6, (Figure 1). X-ray structures of the zebrafish P2X4R, human P2X3R and panda P2X7R are reported and are being utilized for the homology modeling of binding of various ligands at related P2X1R, P2X3R and P2X7R subtypes (Allsopp et al, 2017; Mansoor et al, 2016; Fryatt et al, 2016). …”

“…AZD9056 32 was the first P2X7R antagonist to be tested clinically, significantly improving in rheumatoid arthritis scores (Keystone et al, 2012). AZ10606120 33 binding at the P2X7R was probed structurally (Allsopp et al, 2017). Compounds 38 and 40 are PET ligands for imaging of P2X7R in the brain.…”