title: An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors.
abstract: N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.
authors.affiliation: LRL, Alcobendas, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Jambrina
authors.affiliation: LRL, Indianapolis, IN, USA.
authors.forename: Rok
authors.initials: R
authors.lastname: Cerne
authors.affiliation: Scripps Research Institute Molecular Screening Center, Jupiter, Florida, USA.
authors.forename: Emery
authors.initials: E
authors.lastname: Smith
authors.affiliation: Scripps Research Institute Molecular Screening Center, Jupiter, Florida, USA.
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.affiliation: LRL, Alcobendas, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Cuadrado
authors.affiliation: Eli Lilly & Company Ltd., Windlesham, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Findlay
authors.affiliation: LRL, Indianapolis, IN, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Krambis
authors.affiliation: LRL, Indianapolis, IN, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Wakulchik
authors.affiliation: Scripps Research Institute Molecular Screening Center, Jupiter, Florida, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Chase
authors.affiliation: Eli Lilly & Company Ltd., Windlesham, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Brunavs
authors.affiliation: LRL, Indianapolis, IN, USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Burris
authors.affiliation: Eli Lilly & Company Ltd., Windlesham, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Gallagher
authors.affiliation: Scripps Research Institute Molecular Screening Center, Jupiter, Florida, USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Spicer
authors.affiliation: Eli Lilly & Company Ltd., Windlesham, UK Ursu_Daniel@lilly.com.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ursu
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000544:Alzheimer Disease / Q000188:drug therapy; D002118:Calcium / Q000737:chemistry; D055808:Drug Discovery / Q000379:methods*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009473:Neuronal Plasticity / Q000187:drug effects; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D012559:Schizophrenia / Q000188:drug therapy; D054852:Small Molecule Libraries / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D054852:Small Molecule Libraries; D018698:Glutamic Acid; D002118:Calcium
keywords: NMDA receptor; PAM; automated patch-clamp; calcium flux; ionotropic glutamate receptors