title: Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4).
abstract: Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M(4) (M(4)), we developed a homogenous, multiparametric, 1536-well assay to measure M(4) receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z' of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M(1) and M(5) subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M(4) selective primary agonists, 1450 PAMs, and 2389 antagonist hits. Concentration-response analysis identified 25 selective agonists, 4 PAMs, and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors: Emery
authors: E
authors: Smith
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors: Peter
authors: P
authors: Chase
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Colleen M
authors: CM
authors: Niswender
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Thomas J
authors: TJ
authors: Utley
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA Cell Death and Survival Networks Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors: Douglas J
authors: DJ
authors: Sheffler
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Meredith J
authors: MJ
authors: Noetzel
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Atin
authors: A
authors: Lamsal
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Michael R
authors: MR
authors: Wood
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: P Jeffrey
authors: PJ
authors: Conn
authors: Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Medical Center, Nashville, TN, USA.
authors: Craig W
authors: CW
authors: Lindsley
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors: Franck
authors: F
authors: Madoux
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors: Mary
authors: M
authors: Acosta
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors: Louis
authors: L
authors: Scampavia
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA spicert@scripps.edu.
authors: Timothy
authors: T
authors: Spicer
authors: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA Amgen Inc., Thousand Oaks, CA, USA.
authors: Peter
authors: P
authors: Hodder
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D018721:Muscarinic Agonists / Q000737:chemistry / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000737:chemistry / Q000494:pharmacology*; D043588:Receptor, Muscarinic M4 / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries
chemical_list: D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043588:Receptor, Muscarinic M4; D054852:Small Molecule Libraries
keywords: GPCR; muscarinic; parallel screening; positive allosteric modulators