RT Journal Article SR Electronic T1 Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 94 OP 104 DO 10.1124/mol.110.064345 VO 78 IS 1 A1 Vimesh A. Avlani A1 Christopher J. Langmead A1 Elizabeth Guida A1 Martyn D. Wood A1 Ben G. Tehan A1 Hugh J. Herdon A1 Jeannette M. Watson A1 Patrick M. Sexton A1 Arthur Christopoulos YR 2010 UL http://molpharm.aspetjournals.org/content/78/1/94.abstract AB Recent years have witnessed the discovery of novel selective agonists of the M1 muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M1 mAChR and three mutant M1 mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [3H]N-methyl scopolamine ([3H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [3H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y381A (transmembrane helix 6) and W101A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F77I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca2+ elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible “bitopic” binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics