Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4)

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 M4 (M4), we developed a homogenous, multiparametric, 1536-well assay to measure M4 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 M1 and M5 subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M4 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.

[1]  Susan R. George,et al.  G-Protein-coupled receptor oligomerization and its potential for drug discovery , 2002, Nature Reviews Drug Discovery.

[2]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[3]  C. Lindsley,et al.  Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity , 2010 .

[4]  Christopher J Langmead,et al.  Muscarinic acetylcholine receptors as CNS drug targets. , 2008, Pharmacology & therapeutics.

[5]  A. Lees,et al.  Anticholinergics for symptomatic management of Parkinson's disease. , 2002, The Cochrane database of systematic reviews.

[6]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. , 1993, Nature.

[7]  M. Ishii,et al.  Muscarinic acetylcholine receptors. , 2006, Current pharmaceutical design.

[8]  P. Gentry.  Discovery, optimization, and characterization of novel subtype-selective M5 muscarinic acetylcholine receptor ligands , 2014 .

[9]  P. Insel,et al.  Genetic variations and polymorphisms of G protein-coupled receptors: functional and therapeutic implications. , 2001, Annual review of pharmacology and toxicology.

[10]  H. Lavreysen,et al.  Functional Impact of Allosteric Agonist Activity of Selective Positive Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 in Regulating Central Nervous System Function , 2012, Molecular Pharmacology.

[11]  B. Strulovici,et al.  Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system. , 2003, Analytical biochemistry.

[12]  D. Sheffler,et al.  Discovery and optimization of a novel, selective and brain penetrant M1 positive allosteric modulator (PAM): the development of ML169, an MLPCN probe. , 2011, Bioorganic & medicinal chemistry letters.

[13]  J. Dalton,et al.  Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening , 2014, Journal of biomolecular screening.

[14]  Craig W Lindsley,et al.  Activation of metabotropic glutamate receptors as a novel approach for the treatment of schizophrenia. , 2009, Trends in pharmacological sciences.

[15]  Noel Southall,et al.  A multiplex calcium assay for identification of GPCR agonists and antagonists. , 2010, Assay and drug development technologies.

[16]  G. Mcallister,et al.  Orphan G-protein-coupled receptors and natural ligand discovery. , 2001, Trends in pharmacological sciences.

[17]  M. Banks,et al.  Identification of Selective Agonists and Positive Allosteric Modulators for µ- and δ-Opioid Receptors from a Single High-Throughput Screen , 2014, Journal of biomolecular screening.

[18]  C. Lindsley,et al.  Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders. , 2009, Trends in pharmacological sciences.

[19]  C. Lindsley,et al.  Discovery, Synthesis and Characterization of a Highly Muscarinic Acetylcholine Receptor (mAChR)‐Selective M5‐Orthosteric Antagonist, VU0488130 (ML381): A Novel Molecular Probe , 2014, ChemMedChem.

[20]  Arthur Christopoulos,et al.  Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders , 2009, Nature Reviews Drug Discovery.

[21]  M. Banks,et al.  Development of a high-throughput calcium flux assay for identification of all ligand types including positive, negative, and silent allosteric modulators for G protein-coupled receptors. , 2012, Assay and drug development technologies.

[22]  P Jeffrey Conn,et al.  Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Reveals Chemical and Functional Diversity and In Vivo Activity in Rat Behavioral Models of Anxiolytic and Antipsychotic Activity , 2010, Molecular Pharmacology.

[23]  Kari A. Johnson,et al.  A Novel Assay of Gi/o-Linked G Protein-Coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors , 2008, Molecular Pharmacology.

[24]  Edgar Jacoby,et al.  The 7 TM G‐Protein‐Coupled Receptor Target Family , 2006, ChemMedChem.

[25]  Bernd Rinn,et al.  Quantifying Bioactivity on a Large Scale: Quality Assurance and Analysis of Multiparametric Ultra-HTS Data , 2005 .

[26]  C. Wahlestedt,et al.  Selective and Brain Penetrant Neuropeptide Y Y2 Receptor Antagonists Discovered by Whole-Cell High-Throughput Screening , 2010, Molecular Pharmacology.

[27]  Peter Hodder,et al.  Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators. , 2015, Bioorganic & medicinal chemistry letters.

[28]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gqα to that of Giα , 1993, Nature.