Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M₁Muscarinic Acetylcholine Receptor

Abstract

Recent years have witnessed the discovery of novel selective agonists of the M(1) 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 M(1) mAChR and three mutant M(1) mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [(3)H]N-methyl scopolamine ([(3)H]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 [(3)H]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 Y(381)A (transmembrane helix 6) and W(101)A (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, F(77)I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca(2+) 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.

Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

“…115 Notably, single point mutations of key orthosteric site residues Y381A and W101A, 114,115,117 while reducing the affinity of prototypical orthosteric muscarinic agonists ACh and pilocarpine, actually led to increases in the affinity of AC-42 from that recorded at the wild-type M 1 mAChR. 115 Furthermore, AC-42 exhibited a divergent signaling and regulatory profile to that of other orthosteric muscarinic agonists such as oxotremorine-M. AC-42 was unable to stimulate M 1 mAChR-Gα i1/2 coupling 119 and did not result in M 1 mAChR internalization after prolonged (24 h) exposure. Rather, it actually upregulated total receptor expression.…”

“…Ligand docking studies performed on an M 1 mAChR homology model further support a bitopic binding mode for 77-LH-28-1. 115 N-Desmethylclozapine. Clozapine is a second-generation antipsychotic known for its unrivaled efficacy as a dual-action treatment for the positive and negative symptoms of schizophrenia, 126 and notorious for promiscuous off-target activity that has rendered it a risky therapeutic option.…”

A Monod-Wyman-Changeux Mechanism Can Explain G Protein-coupled Receptor (GPCR) Allosteric Modulation

“…Cell Culture and Transfections-FlpIn CHO cells stably expressing wild type (WT) or DREADD (Y106C/A196G) hM 1 mAChR were generated and cultured as described previously (11). Transient transfections were performed using linear polyethyleneimine (molecular mass 25 kDa) (12).…”

“…Membrane Preparation and Radioligand Binding-Membrane preparation of CHO FlpIn hM 1 cells was performed as described previously (11). Competition binding assays were performed in membranes derived from CHO FlpIn hM 1 WT cells or Y106C/A196G hM 1 .…”

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

“…Cell Culture and Transfections-FlpIn CHO cells stably expressing the WT, ⌬i3, or R123L hM 1 mAChRs were generated and cultured as described previously (16). Transient transfections of adherent HEK293 cells were performed using linear polyethyleneimine (molecular mass, 25 kDa) in a 1:6 ratio (8).…”