P2RX7 Purinoceptor as a Therapeutic Target-The Second Coming?

Abstract

The P2RX7 receptor is a unique member of a family of extracellular ATP (eATP)-gated ion channels expressed in immune cells, where its activation triggers the inflammatory cascade. Therefore, P2RX7 has been long investigated as a target in the treatment of infectious and inflammatory diseases. Subsequently, P2RX7 signaling has been documented in other physiological and pathological processes including pain, CNS and psychiatric disorders and cancer. As a result, a range of P2RX7 antagonists have been developed and trialed. Interestingly, the recent crystallization of mammalian and chicken receptors revealed that most widely-used antagonists may bind a unique allosteric site. The availability of crystal structures allows rational design of improved antagonists and modeling of binding sites of the known or presumed inhibitors. However, several unanswered questions limit the cogent development of P2RX7 therapies. Firstly, this receptor functions as an ion channel, but its chronic stimulation by high eATP causes opening of the non-selective large pore (LP), which can trigger cell death. Not only the molecular mechanism of LP opening is still not fully understood but its function(s) are also unclear. Furthermore, how can tumor cells take advantage of P2RX7 for growth and spread and yet survive overexpression of potentially cytotoxic LP in the eATP-rich environment? The recent discovery of the feedback loop, wherein the LP-evoked release of active MMP-2 triggers the receptor cleavage, provided one explanation. Another mechanism might be that of cancer cells expressing a structurally altered P2RX7 receptor, devoid of the LP function. Exploiting such mechanisms should lead to the development of new, less toxic anticancer treatments. Notably, targeted inhibition of P2RX7 is crucial as its global blockade reduces the immune and inflammatory responses, which have important anti-tumor effects in some types of malignancies. Therefore, another novel approach is the synthesis of tissue/cell specific P2RX7 antagonists. Progress has been aided by the development of p2rx7 knockout mice and new conditional knock-in and knock-out models are being created. In this review, we seek to summarize the recent advances in our understanding of molecular mechanisms of receptor activation and inhibition, which cause its re-emergence as an important therapeutic target. We also highlight the key difficulties affecting this development.

Keywords: ATP; DMD; P2RX7 KO mouse; P2X7; cancer; inflammation; large pore.

Figure 1

Recent P2RX7 structural developments: the ATP binding pocket and the allosteric groove. (A) PyMOL (pymol.org) generated surface plot of P2RX7 trimer using RCSB PDB data file (rcsb.org) for chicken variant bound to the competitive antagonist TNP-ATP (structure 5XW6, 3.1A). ROI shows the ATP binding pocket (RHS, upper), PyMOL Video file gives structural overview of the trimer with detailed orientations of the ATP binding pocket and the central ion channel (see Supplementary Material Video S1). RCSB PDB generated images show location of the ATP binding pocket in relation to the newly discovered allosteric site on the monomer (B) and at the subunit interface of the trimer, forming an allosteric groove—here shown occupied by A740003 antagonist (C). When imaged together from intracellular projection, the three allosteric grooves appear as a Shuriken, whose rotation is impeded by the allosteric antagonist A740003 (D). (E) Cartoon representation of the relationship between the agonist binding site and the allosteric antagonist-binding groove. Adapted from Karasawa and Kawate (2016), with permission.

Figure 2

Selective regulation of cationic and anionic dye permeation through the P2RX7 LP. Example of differences in P2RX7 LP permeation preferences to EtBr (cationic) and Lucifer Yellow (anionic) dyes in individual cells of a clonal population (Young and Gorecki, 2016): Compared to WT myoblasts (Left), the dystrophic mdx myoblasts (Center) display upregulated P2RX7 expression and increased LP permeability in response to eATP (Young et al., 2015, 2017). The mdx/P2RX7^−/− myoblasts (Right) show no dye uptake under these conditions, as expected. Note that the majority of the seemingly identical cells in this clonal population shows a clear preferential uptake of EtBr over LY upon the LP opening, which is considered to be a non-selective pore. The molecular mechanisms underlying this phenomenon are currently under investigation as this experiment illustrates the potential for diverse LP responses in heterogeneous and functionally plastic populations of e.g., immune cells in vivo.

Figure 3

P2RX7 expression is elevated in human cancers. cBioPortal (cbioportal.org) derived expression levels for P2RX7 multiple human cancer patient biopsies from TCGA data (cancergenome.nih.gov). Noticeable, across almost all tumor types, is the significant overexpression of P2RX7 and a paucity of detected mutations. This indicates that malignant cells express high levels of fully-functional receptors, which is an important consideration for drug treatment.