HEADS OR TAILS: LIPID INHIBITORS OF THE GLYCINE TRANSPORTER, GLYT2

Abstract

Membrane proteins are influenced by the dynamic lipid membrane environment, which can impart stability, mediate protein interactions, and provide highly selective contacts essential for function. Membrane proteins can also bind endogenous lipid ligands or are able to be allosterically modulated by lipids, many of which are thought to access their specific binding sites via the cell membrane. N-arachidonyl glycine (NAGly) is a bioactive lipid that is found in its highest concentrations within the spinal cord and may play an important role in endogenous regulation of glycinergic neurotransmission and pain perception through inhibition of the glycine transporter, GlyT2. In addition to NAGly, a number of lipid inhibitors of GlyT2 have been identified. These compounds are comprised of a long flexible unsaturated acyl tail conjugated to an amino acid or amino acid derivative head group. The aims of my study were two-fold; first to identify new, more potent, lipid inhibitors and develop a structure activity relationship for these compounds; and second, to elucidate the molecular mechanisms of inhibition. Wild type and mutant GlyT2 transporters were expressed in Xenopus laevis oocytes with glycine transport and the subsequent inhibition of transport measured using two-electrode voltage clamp electrophysiology, and radiolabelled uptake of glycine. A library of 55 N-acyl amino acids with varying head and tail groups were synthesised and tested at both GlyT2 and the closely related glycine transporter, GlyT1. Two distinct groups of compounds were tested: the first group maintaining a glycine head group and altering the lipid tail; and the second conjugating the [C18 ω9] oleoyl tail to amino acids with varying properties. I found the lipid constituent of the acyl-glycine analogues is essential for specific interactions and the mechanism of inhibition and is not merely a non-selective, sticky adjunct. There was an ideal chain length, with an order of potency C18 > C16 > C14, and stringently defined double bond conformation and position. Conservative differences between compounds are sufficient to impart or remove inhibitory activity which validates highly specific binding to a subtype specific, allosteric pocket. While changing the tail did not greatly alter potency, analogues where the head group was altered significantly influenced apparent affinity. Acyl amino acids containing an aromatic or positively charged side chain conferred the highest apparent affinity, with C16 ω3 L-Lys possessing the highest potency (10.7 nM). 12 compounds inhibited GlyT2 < 100nM, and one of these inhibitors, oleoyl D-Lys, is also metabolically stable and produces analgesia in a rat model of neuropathic pain. Mutagenesis of extracellular loop 4 (EL4), and transmembrane helices TM5 and TM8 suggest that the allosteric binding site is comprised of a cluster of aromatic residues which may strongly coordinate aromatic or positively charged head groups of the most potent analogues. Additionally, changing the properties of a membrane facing residue alters the otherwise slow washout of lipids. From these results, in addition to dynamic docking studies, it is proposed that acyl amino acids may first diffuse into the lipid bilayer and interact with regions of GlyT2 at the protein-membrane interface. Acyl amino acids then access their final binding site formed by aliphatic and aromatic residues from TM5, TM8, and EL4. It has previously been shown that EL4 undergoes important conformational changes in this family of transporters, where EL4 shifts into the outward facing vestibule to occlude the extracellular side and continue the transport cycle. Acyl amino acids may therefore inhibit GlyT2 by stabilising EL4 in a conformation that does not favour transport. The combination of structure-activity studies with molecular insights provides key information on the mechanism of inhibition which will drive further generation of GlyT2 inhibitors for the treatment of neuropathic pain.