The M1 Muscarinic Receptor Allosteric Agonists AC-42 and 1-[1′-(2-Methylbenzyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one Bind to a Unique Site Distinct from the Acetylcholine Orthosteric Site

• Published in: Molecular pharmacology Vol. 78; no. 4; pp. 648 - 657
• Main Authors: Jacobson, Marlene A., Kreatsoulas, Constantine, Pascarella, Danette M., O’Brien, Julie A., Sur, Cyrille
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2010
• Subjects: Acetylcholine - metabolism; Allosteric Regulation - physiology; Allosteric Site - physiology; Amino Acid Sequence; Benzimidazoles - chemistry; Benzimidazoles - metabolism; Benzimidazoles - pharmacology; Binding Sites - physiology; Humans; Molecular Sequence Data; Piperidines - chemistry; Piperidines - metabolism; Piperidines - pharmacology; Protein Structure, Secondary; Receptor, Muscarinic M1 - agonists; Receptor, Muscarinic M1 - metabolism; QNB; NMS; TBPB; TM; CHONFAT; AC-42; FLIPR
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.110.065771

Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1′-(2-methylbenzyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol61:1297–1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.