Identification and characterisation of lipids that are positive allosteric modulators of glycine receptors

Glycine receptors are ligand-gated ion channels that mediate fast inhibitory neurotransmission within the nervous system. They have recently emerged as targets for chronic pain therapies due to their role in nociceptive signalling. N-arachidonyl glycine is an endogenous lipid that modulates glycine receptors, however its mechanism of action remains unclear. We have identified a series of N-acyl amino acids that positively modulate glycine receptors. From this, a second series of lipids containing a phenylene-moiety within the lipid tail were synthesised. This improved their efficacy by up to 6-fold, and increased the potency of receptor activation by over 10-fold. (8-2-octylphneyl)octanoyl)glycine (OPOG) had the greatest activity - potentiating the glycine EC5 by 1500% with an EC50 of 664 nM at the α1 receptor. OPOG was also found to be analgesic and produces a dose-dependent reversal of allodynia in rodent models of neuropathic pain. The second aim of this study was to identify lipid binding sites on the glycine receptor. Molecular dynamic simulations of OPOG at the α1 receptor were conducted and indicated consistent binding within a cavity at the intracellular portion of transmembrane domains (TM) 1 and 4. This site has previously been suggested to bind neurosteroids. A cryoEM structure of OPOG bound to the zebrafish glycine receptor was also obtained and identified binding to an inter-subunit cavity between the extracellular portions of TM1(-), TM2(+) and TM3(+). This cavity has previously been shown to bind the positive allosteric modulator ivermectin. Mutagenesis of the residues identified within the two sites was indicated potentiation occurs primarily through the neurosteroid binding site involving aromatic and positively charged residues. Further studies are required to explore binding within the ivermectin cavity and its relative contribution to lipid modulation.