Glycine receptors (GlyRs) are members of the Cys-loop family of pentameric ion channels. GlyRa1 and GlyRa3 are expressed throughout the adult central nervous system (CNS) where they modulate inhibitory glycinergic neurotransmission. Previous studies with modestly potent pharmacological modulators of GlyRs have implicated these channels in the control of both inflammatory (GlyRa3) and neuropathic (GlyRa3, GlyRa1) pain processing. Thus far, the published small molecule tools interrogated in the context of preclinical pain models have lacked in both potency and pharmacokinetics. As such, new potent, CNS penetrant, positive modulators of GlyRs would serve as valuable proof-of-concept tools and may ultimately offer mechanistically differentiated therapies for the treatment of chronic inflammatory and neuropathic pain. Herein, we describe the identification of a novel class of allosteric GlyR potentiators, originating from a 3 mM hit derived from a functional cellular HTS assay. Property-guided optimization resulted in potent, selective, orally bioavailable, CNS penetrant tool molecules. These tools demonstrated (1) in vitro potentiation of Gly-evoked current in stably expressed cells, (2) ex vivo potentiation of Gly-evoked current in mouse spinal dorsal horn slices, and (3) in vivo proof-of-concept in a mouse model of neuropathic pain. Furthermore, a highly potent binder (K<sub>i</sub>: 0.011 mM; EC<sub>50</sub>: 0.066 mM) from this series was leveraged facilitated the solution of the first high resolution X-ray co-crystal structure of hGlyRa3 in complex with both glycine and an allosteric potentiator. The co-crystal structure reveals a novel binding pocket, which explains the high degree of selectivity over other Cys-loop family receptors and introduces opportunities for structure-based lead optimization and additional lead generation.

Discovery of potent, selective, CNS-penetrant potentiators of glycine receptors

252nd National Meeting (2016)

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