Bioactive lipid modulation of glycine transporter 2 (GlyT2)

Chronic neuropathic pain is debilitating affecting the quality of life globally. There is also an economic cost associated with unrelieved chronic pain. Currently used pain relievers (analgesics) do not provide adequate pain relief in most chronic pain patients. They also show significant side effects. Inhibitory glycinergic neurotransmission is severely impaired in chronic pain. Glycine concentrations are controlled by two glycine transporters, GlyT1 and GlyT2. GlyT1 is predominantly expressed in astrocytes surrounding both inhibitory and excitatory synapses, whereas GlyT2 is predominantly expressed by inhibitory glycinergic neurons. Thus, inhibition of GlyT2 has the potential to selectively regulate glycine concentrations at only inhibitory glycinergic synapses. Several bioactive lipids have been identified as effective analgesics which inhibit glycine transporter 2 (GlyT2) with reduced side effects compared to other GlyT2 inhibitors. A limited structure-activity profile of bioactive lipid inhibitors of GlyT2 has been established. The crucial determinants include: (1) the lipid head-group, (2) the length of the acyl chain and (3) the number and position of double bonds within the acyl tail influencing potency, reversibility and maximal inhibition. The objective of this project is to elucidate the mechanism of bioactive lipid modulation of GlyT2. My working hypothesis states that, bioactive lipids are specific and partial, non-competitive inhibitors of GlyT2, providing analgesia without side effects. The specific aims of my project are: (1) to identify and characterize the bioactive lipid- GlyT2 interface, (2) to characterize azologized bioactive lipid interactions with GlyT2, and (3) to achieve precise temporal control of GlyT2 inhibition in the dorsal horn of the spinal cord using azologized photoswitchable bioactive lipids. I will carry out functional analysis of the novel bioactive lipid inhibitors of GlyT2 in the lab of my host abroad, Dr. Robert J. Vandenberg (The University of Sydney). In collaboration, with Dr. Megan OMara (Australian National University), potential residues in GlyT2 forming the bioactive lipid interface would be identified. I will characterize these putative interaction sites by systematic mutagenesis of GlyT2 and test function using electrophysiology. In collaboration with Dr. Tristan Rawling (University of Technology, Sydney), novel azo-derivatized bioactive lipids will be synthesized by incorporating an azobenzene photoswitch along the acyl chain. As photoswitchable selective ligands, they are inactive in the dark and activated upon irradiation with ultraviolet-A light. Activation is rapidly reversed by irradiation with blue light. I will use the azo-derivatized bioactive lipids to provide robust optical control of pain sensitive neurons with temporal precision to investigate GlyT2 inhibition kinetics. In collaboration with Dr. MacDonald J. Christie (The University of Sydney), these experiments will provide the cellular basis for the analgesic action of bioactive lipids. The application of photopharmacology in these experiments will break new ground in drug discovery for chronic pain treatment. Key words: neuropathic pain, analgesia, glycine transporter, bioactive lipids, dorsal horn, photopharmacology Running title: GlyT2 modulation alleviates pain 1

Chronic neuropathic pain is debilitating affecting the quality of life globally. There is also an economic cost associated with unrelieved chronic pain. Currently used pain relievers (analgesics) do not provide adequate pain relief in most chronic pain patients. They also show significant side effects. Inhibitory glycinergic neurotransmission is severely impaired in chronic pain. Glycine concentrations are controlled by two glycine transporters, GlyT1 and GlyT2. GlyT1 is predominantly expressed in astrocytes surrounding both inhibitory and excitatory synapses, whereas GlyT2 is predominantly expressed by inhibitory glycinergic neurons. Inhibition of GlyT2 has the potential to selectively regulate glycine concentrations at only inhibitory glycinergic synapses. Several bioactive lipids were identified as effective analgesics which inhibited glycine transporter 2 (GlyT2) with reduced side effects compared to other GlyT2 inhibitors. A limited structure-activity profile of bioactive lipid inhibitors of GlyT2 was established. The crucial determinants included: (1) the lipid head-group, (2) the length of the acyl chain and (3) the number and position of double bonds within the acyl tail influencing potency, reversibility and maximal inhibition. The objective of this project was to elucidate the mechanism of bioactive lipid modulation of GlyT2. My working hypothesis stated that, bioactive lipids are specific and partial, non-competitive inhibitors of GlyT2, providing analgesia without side effects. The specific aims of my project were: (1) to identify and characterize the bioactive lipid-GlyT2 interface, (2) to characterize azologized bioactive lipid interactions with GlyT2, and (3) to achieve precise temporal control of GlyT2 inhibition in the dorsal horn of the spinal cord using azologized photoswitchable bioactive lipids. I carried out functional analysis of the novel bioactive lipid and ORG 25543-based inhibitors of GlyT2 in the lab of my host abroad, Dr. Robert J. Vandenberg (The University of Sydney). In collaboration, with Dr. Megan OMara (Australian National University), potential residues in GlyT2 forming the bioactive lipid interface were identified. I characterized these putative interaction sites by systematic mutagenesis of GlyT2 and tested function using electrophysiology. In collaboration with Dr. Tristan Rawling (University of Technology, Sydney), and Dr. Christopher L. Cioffi (Albany College of Pharmacy and Health Sciences, USA) novel azo-derivatized bioactive lipids and ORG- 25543-based compounds were synthesized, respectively. As photoswitchable selective ligands, they were inactive in the dark and activated upon irradiation with ultraviolet-A light. Activation was rapidly reversed by irradiation with blue light. I obtained reversible optical control of GlyT2 inhibition in Xenopus laevis oocytes using azologized photoswitchable ORG 25543-based compounds. The application of photopharmacology in these experiments broke new ground in drug discovery for chronic pain treatment. Key words: neuropathic pain, analgesia, glycine transporter, bioactive lipids, dorsal horn, photopharmacology Running title: GlyT2 modulation alleviates pain