title: Mutagenic mapping suggests a novel binding mode for selective agonists of M1 muscarinic acetylcholine receptors.
abstract: Point mutations and molecular modeling have been used to study the activation of the M(1) muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M(1) mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M(1) mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M(1) selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.
authors.affiliation: Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lebon
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Langmead
authors.forename: Ben G
authors.initials: BG
authors.lastname: Tehan
authors.forename: Edward C
authors.initials: EC
authors.lastname: Hulme
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D010880:Piperidines / Q000494:pharmacology*; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; D010880:Piperidines; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic