title: Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay.
abstract: Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Clara
authors.initials: C
authors.lastname: Stead
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Adam
authors.initials: A
authors.lastname: Brown
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Cathryn
authors.initials: C
authors.lastname: Adams
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Nickolls
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Gareth
authors.initials: G
authors.lastname: Young
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Juha
authors.initials: J
authors.lastname: Kammonen
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: David
authors.initials: D
authors.lastname: Pryde
authors.affiliation: 1 Neusentis (Pfizer Ltd.), Granta Park, Great Abington, Cambridgeshire, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Cawkill
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000700:Analgesics / Q000302:isolation & purification* / Q000627:therapeutic use; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D010146:Pain / Q000188:drug therapy* / Q000235:genetics; D018408:Patch-Clamp Techniques / Q000379:methods; D018009:Receptors, Glycine / Q000187:drug effects / Q000235:genetics*; D054852:Small Molecule Libraries / Q000032:analysis; D009435:Synaptic Transmission / Q000187:drug effects / Q000235:genetics
chemical_list: D000700:Analgesics; D005456:Fluorescent Dyes; D018009:Receptors, Glycine; D054852:Small Molecule Libraries; C113965:glycine receptor alpha3 subunit
keywords: glycine receptor; pain; positive allosteric modulator