Abstract
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 and three mutant M1 mAChRs. Both agonists inhibited the binding of the orthosteric antagonist, [3H]N-methylscopolamine ([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 kinetic assays revealed that the agonists could bind allosterically when the orthosteric site was pre-labeled with [3H]NMS, and that 77-LH-28-1 competed with the prototypical allosteric modulator, heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide (C7/3-phth) 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 Ca++ elevation and phosphorylation of ERK1/2. Molecular modeling suggested a possible "bitopic" binding mode, whereby the agonists extend down into the orthosteric site as well as up towards extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.
- Muscarinic cholinergic
- Structure-activity relationships and modeling
- Thermodynamic and kinetic processes and modeling
Footnotes
- Received February 23, 2010.
- Revision received April 21, 2010.
- Accepted April 21, 2010.
- The American Society for Pharmacology and Experimental Therapeutics