From cells to mice and back again: Designing a series of GlyT2 inhibitors for the treatment of neuropathic pain

Abstract

Impaired glycinergic neurotransmission has been implicated in the development of neuropathic pain. With a widespread interest in non-opioid analgesics, there has been a concerted effort to develop glycine transporter 2 (GlyT2) inhibitors to restore normal glycinergic and thus pain signalling. The overall aim of this thesis was to elucidate the optimal characteristics and the structure-activity relationships (SAR) of varying classes of GlyT2 inhibitors for analgesia in neuropathic pain.

The first aim of this thesis was to develop a SAR scaffold based primarily on ORG25543. 92 compounds were first screened for affinity on GlyT2 expressed in Xenopus laevis oocytes measured by TEVC. These results were used to develop SAR profiles for structural modifications made to the candidate 29. Analogues determined to have optimal GlyT2 affinities, transport recovery washout half-lives, and other CNS-desirable pharmacokinetic characteristics were designated as candidates for further experimentation, including ADME profiling assessing liver metabolism, off-target selectivity, and mouse CNS PK studies. SAR rules for indoline and ORG25543-derived compounds were established.

The second aim of this thesis was to characterise a competitive inhibitor, ALX3255, in GlyT2 WT oocytes. Using site-directed mutagenesis, the binding site of ALX3255 was determined to be within primary substrate and vestibule allosteric sites.

The final aim of this thesis was to evaluate the analgesic efficacy of these candidate compounds in behavioural mouse models. These candidates were initially administered to neuropathic pain mice which were tested for allodynia via the von Frey test. Promising compounds were then assessed in further models. Compound 82 provided dose-dependent reductions in allodynia without side effects and was also used to compare differing models of neuropathic pain. Additionally, for the first time in GlyT2 inhibitor studies, a compound was assessed in both male and female mice.