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.
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 Pharmacol 61:1297-1302, 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.
Jacobson
Marlene A
MA
Merck Research Labs, Schizophrenia Research, WP26-265, West Point, PA 19486, USA. marlene_jacobson@merck.com
Kreatsoulas
Constantine
C
Pascarella
Danette M
DM
O'Brien
Julie A
JA
Sur
Cyrille
C
D000109:Acetylcholine / Q000378:metabolism*; D000494:Allosteric Regulation / Q000502:physiology; D000495:Allosteric Site / Q000502:physiology; D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010880:Piperidines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000378:metabolism*
C532626:1-(1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo(d)imidazol-2-(3H)-one; C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; D001562:Benzimidazoles; D010880:Piperidines; D043643:Receptor, Muscarinic M1; D000109:Acetylcholine