Negative allosteric modulators (NAMs) are of great interest in drug development because they offer improved scope for the production of receptor antagonists with enhanced subtype-selectivity. Indeed, many NAMs are already on the market or undergoing clinical trials. NAMs act by binding to sites within receptors that are distinct from the primary, orthosteric ligand binding site and can inhibit the structural rearrangements of a receptor that are induced by orthosteric agonist binding.
P2X receptors are ligand-gated cation channels for which ATP is the endogenous orthosteric agonist. They are expressed throughout the body and the evidence indicates that they have numerous functions, including in sympathetic and parasympathetic neurotransmission, perception of sound, taste and pain, and immune regulation. Seven P2X subunits have been identified, which form trimers, to produce at least twelve different receptor subtypes. A major issue within the field has been a lack of selective antagonists for most P2X subtypes. This is unsurprising given the amino acid sequence similarity within the ATP binding site. Several selective NAMs have now been developed, but little is known about where in receptors they act and how exactly they inhibit receptor activation.
AF-219 is small molecule NAM at P2X3 receptors that was reported to be effective in a phase II clinical trial for treatment of refractory chronic cough. Wang et al., (1) combined X-ray crystallography, molecular modelling, and mutagenesis, to identify the site and mode of action of AF-219. P2X3 receptors are composed of three subunits, each of which adopts a conformation that could be likened to the shape of a leaping dolphin. The tail represents the transmembrane-spanning regions, the upper body the bulk of the extracellular loop and the head the most distal part of the extracellular loop. Also attached to the body are three structurally-distinct elements: the dorsal fin, the right flipper, and the left flipper. As a trimer, the subunits wrap round each other to produce a structure that resembles a chalice.
The AF-219 binding site is formed by the lower body and dorsal fin of one subunit and the lower body and left flipper of an adjacent subunit. Mutational analysis identified which amino acid residues within this pocket are essential for AF-219 binding, whilst in silico modelling showed that the small molecule P2X3 NAMS, AF-353, RO-51, RO-3 and TCP 262, but not the large NAMS suramin and PPADS, also bind to the same site. Activation of P2X3 receptors by ATP closes the binding cavity, so by occupying it, AF-219 prevents the protein structural rearrangements that lead to opening of the P2X3 receptor ion pore.
This identification of the AF-219 NAM binding site in P2X3 receptors is an opportunity for rational, intelligent drug design. It enables virtual screening of compound libraries, with the aim of identifying potential new molecular core structures, which can then be modified in order to optimise the structure of a novel NAM. In addition, this site differs among P2X receptor subtypes, so it is highly possible that drugs with greatly enhanced subtype-selectivity can be developed.
Comments by Dr. Charles Kennedy, University of Strathclyde
(1) Wang J, Wang Y, Cui WW, Huang Y, Yang Y, Liu Y, Zhao WS, Cheng XY, Sun WS, Cao P, Zhu MX, Wang R, Hattori M, Yu Y. (2018). Druggable negative allosteric site of P2X3 receptors. Proc Natl Acad Sci U S A. 2018 pii: 201800907. doi: 10.1073/pnas.1800907115. [PMID: 29674445]