Lipid modulators of glycine receptors and their potential use for pain therapies

Glycine receptors are pentameric ligand-gated ion channels that mediate fast inhibitory neurotransmission within the nervous system. Compounds which positively modulate receptor activity have been shown to produce antinociceptive effects and provide analgesia in rodent models of chronic pain. One example of this is N-arachidonyl glycine, which is a lipid that occurs endogenously within the spinal cord and modulates glycine receptors in a subtype-specific manner. Whilst lipid modulation of other pentameric ligand-gated ion channels has been extensively characterised, it is yet to be fully explored for glycine receptors. Developing lipids which positively modulate glycine receptors may provide a promising avenue for the development of novel pain therapies. To characterise lipid modulation of glycine receptors, a series of N-acyl amino acid which were structurally similar to N-arachidonyl glycine were screened at human GlyRs expressed in Xenopus laevis oocytes using, two-electrode voltage-clamp electrophysiology. This highlighted key structure activity relationships that govern receptor potentiation. Lipids with smaller amino acid head groups such as glycine are favourable for potentiation, whilst bulkier head groups cause significant inhibition. Lipids also require a long 18-carbon tail with a cis double bond in the centre region to be active. This pharmacophore was used to optimise lipid modulators and develop a second series of lipids which contained a phenylene moiety. This improved their efficacy by up to 6-fold, and increased the potency of receptor activation by over 10-fold. 8-8 Ortho-phenylene glycine was found to have the greatest activity. This lipid contains a glycine head group conjugated to an 18-carbon tail that contains an ortho-phenylene moiety in the centre. At the α1 receptor this lipid potentiates the glycine EC5 by 1500 % with an EC50 of 664 nM. 8-8 Ortho-phenylene glycine was also found to be analgesic and produces a dose-dependent reversal of allodynia in rodent models of neuropathic pain Molecular dynamics simulations of 8-8 ortho-phenylene glycine at the α1 receptor were conducted to determine lipid binding cavities and help elucidate their mechanism of action. These simulations indicated consistent binding within a cavity at the intracellular portion of TM1 and TM4, in a site that has previously been suggested to bind neurosteroids. Mutagenesis of this site suggests that the 8-8 ortho-phenylene glycine elicits potentiation by forming π-stacking interactions with the aromatic residue W239 on TM1, and electrostatic interactions with the positively charged residue K397 on TM4. These studies also suggested the presence of additional binding sites that produce inhibitory activity. CryoEM of the zebrafish glycine receptor was used to identify this secondary site. A structure of the receptor bound to 8-8 ortho-phenylene glycine revealed binding within an inter-subunit cavity between the extracellular portions of TM1 (-), TM2 (+) and TM3 (+), in a cavity that has previously been shown to bind the positive allosteric modulator ivermectin. Competition assays conducted between 8-8 ortho-phenylene glycine and ivermectin unveiled a complex interaction between the two sites which has not previously been shown in literature. These sites can positively and negatively modulate each other which is likely to have significant impacts in vivo, as endogenous lipids have also been suggested to occupy these cavities. Glycine receptors are also endogenously modulated by zinc, which is a common contaminant in functional studies and can impact the activity of other receptor modulators. This study has developed a system to determine the activity of positive modulators at the α1 glycine receptor in the absence zinc. This was used to validate that lipid modulation is independent from zinc modulation and was not previously impacted by contaminating zinc concentrations. 8-8 ortho-phenylene glycine was also assessed in the presence of a physiologically relevant concentration of zinc that occurs at nociceptive synapses. This was found to enhance the degree of potentiation and indicated a synergistic interaction between the two modulators which may enhance lipid modulation in vivo, and therefore help achieve greater antinociceptive activity. The findings presented in this thesis have contributed significantly to our understanding of how lipids interaction with glycine receptors. This study has identified numerous lipids that positively modulate glycine receptors and illuminated how they mechanistically elicit potentiation through multiple binding cavities. Further development of these compounds may provide a novel approach that leads to the development of clinically relevant therapeutics for the treatment of chronic pain.