Peripheral Administration of Selective Glycine Transporter-2 Inhibitor, Oleoyl-D-Lysine, Reverses Chronic Neuropathic Pain but Not Acute or Inflammatory Pain in Male Mice.

Aberrations in spinal glycinergic signaling are a feature of pain chronification. Normalizing these changes by inhibiting glycine transporter (GlyT)-2 is a promising treatment strategy. However, existing GlyT2 inhibitors (e.g., ORG25543) are limited by narrow therapeutic windows and severe dose-limiting side effects, such as convulsions, and are therefore poor candidates for clinical development. Here, intraperitoneally administered oleoyl-D-lysine, a lipid-based GlyT2 inhibitor, was characterized in mouse models of acute (hot plate), inflammatory (complete Freund's adjuvant), and chronic neuropathic (chronic constriction injury) pain. Side effects were also assessed on a numerical rating score, convulsions score, for motor incoordination (rotarod), and for respiratory depression (whole body plethysmography). Oleoyl-D-lysine produced near complete antiallodynia for chronic neuropathic pain, but no antiallodynia/analgesia in inflammatory or acute pain. No side effects were seen at the peak analgesic dose, 30 mg/kg. Mild side effects were observed at the highest dose, 100 mg/kg, on the numerical rating score, but no convulsions. These results contrasted markedly with ORG25543, which reached less than 50% reduction in allodynia score only at the lethal/near-lethal dose of 50 mg/kg. At this dose, ORG25543 caused maximal side effects on the numerical rating score and severe convulsions. Oleoyl-D-lysine (30 mg/kg) did not cause any respiratory depression, a problematic side effect of opiates. These results show the safe and effective reversal of neuropathic pain in mice by oleoyl-D-lysine and provide evidence for a distinct role of glycine in chronic pain over acute or short-term pain conditions. SIGNIFICANCE STATEMENT: Partially inhibiting glycine transporter (GlyT)-2 can alleviate chronic pain by restoring lost glycinergic function. Novel lipid-based GlyT2 inhibitor ol-D-lys is safe and effective in alleviating neuropathic pain, but not inflammatory or acute pain. Clinical application of GlyT2 inhibitors may be better suited to chronic neuropathic pain over other pain aetiologies.

The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity.

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric-binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here, we use a combination of medicinal chemistry, electrophysiology, and computational modeling to develop a rational structure-activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length, and double-bond position promote enhanced inhibition. Overall, the l-stereoisomer is generally a better inhibitor than the d-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric-binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment.

Photoswitchable ORG25543 Congener Enables Optical Control of Glycine Transporter 2.

Glycine neurotransmission in the dorsal horn of the spinal cord plays a key role in regulating nociceptive signaling, but in chronic pain states reduced glycine neurotransmission is associated with the development of allodynia and hypersensitivity to painful stimuli. This suggests that restoration of glycine neurotransmission may be therapeutic for the treatment of chronic pain. Glycine transporter 2 inhibitors have been demonstrated to enhance glycine neurotransmission and provide relief from allodynia in rodent models of chronic pain. In recent years, photoswitchable compounds have been developed to provide the possibility of controlling the activity of target proteins using light. In this study we have developed a photoswitchable noncompetitive inhibitor of glycine transporter 2 that has different affinities for the transporter at 365 nm compared to 470 nm light.

Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics.

The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.

Development of an N-Acyl Amino Acid That Selectively Inhibits the Glycine Transporter 2 To Produce Analgesia in a Rat Model of Chronic Pain.

Inhibitors that target the glycine transporter 2, GlyT2, show promise as analgesics, but may be limited by their toxicity through complete or irreversible binding. Acyl-glycine inhibitors, however, are selective for GlyT2 and have been shown to provide analgesia in animal models of pain with minimal side effects, but are comparatively weak GlyT2 inhibitors. Here, we modify the simple acyl-glycine by synthesizing lipid analogues with a range of amino acid head groups in both l- and d-configurations, to produce nanomolar affinity, selective GlyT2 inhibitors. The potent inhibitor oleoyl-d-lysine (33) is also resistant to degradation in both human and rat plasma and liver microsomes, and is rapidly absorbed following an intraperitoneal injection to rats and readily crosses the blood-brain barrier. We demonstrate that 33 provides greater analgesia at lower doses, and does not possess the severe side effects of the very slowly reversible GlyT2 inhibitor, ORG25543 (2).

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuTAa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuTAa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC50 of 9 µM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 µM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Glycine transporter2 inhibitors: Getting the balance right.

Neurotransmitter transporters are targets for a wide range of therapeutically useful drugs. This is because they have the capacity to selectively manipulate the dynamics of neurotransmitter concentrations and thereby enhance or diminish signalling through particular brain pathways. High affinity glycine transporters (GlyTs) regulate extracellular concentrations of glycine and provide novel therapeutic targets for neurological disorders.