Abstract
Point mutations and molecular modeling have been used to study the activation of the M1 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 M1 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 M1 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 M1 selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.
Footnotes
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This work was supported by GlaxoSmithKline and by the Medical Research Council (United Kingdom) [Grant in Aid U.1175.03.003.00008.01].
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ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; NSA, novel selective agonist; ACh, acetylcholine; TM, transmembrane domain; NMS, (-)-N-methyl scopolamine; PhI, phosphoinositide; NDMC, N-desmethylclozapine; AC-42, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine; 77-LH-28-1, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide.
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↵1 Current affiliation: Division of Structural Studies, Laboratory of Molecular Biology, Cambridge, United Kingdom.
- Received July 31, 2008.
- Accepted November 11, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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