title: Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor.
abstract: Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.
authors.affiliation: From the Departments of Drug Discovery Biology and.
authors.forename: Alaa
authors.initials: A
authors.lastname: Abdul-Ridha
authors.affiliation: From the Departments of Drug Discovery Biology and.
authors.forename: J Robert
authors.initials: JR
authors.lastname: Lane
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Shailesh N
authors.initials: SN
authors.lastname: Mistry
authors.affiliation: From the Departments of Drug Discovery Biology and.
authors.forename: Laura
authors.initials: L
authors.lastname: López
authors.affiliation: From the Departments of Drug Discovery Biology and.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Scammells
authors.affiliation: From the Departments of Drug Discovery Biology and arthur.christopoulos@monash.edu.
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: From the Departments of Drug Discovery Biology and meri.canals@monash.edu.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
mesh_terms: D000109:Acetylcholine / Q000737:chemistry*; D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006912:Hydroxyquinolines / Q000737:chemistry*; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D006912:Hydroxyquinolines; D043643:Receptor, Muscarinic M1; D000109:Acetylcholine
keywords: Allosteric Regulation; Cell Signaling; Drug Discovery; G Protein-coupled Receptor (GPCR); Muscarinic Acetylcholine Receptor; Mutagenesis