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

Abstract

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, 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.

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

“…Consistent with previous reports (15,22,23), improved binding of TBPB and 77-LH-28-1 was detected at the W101 3.28 A receptor. This was also observed for the agonist fragment derivative of TBPB (VCP794), and all three ligands exhibited improved binding at the Y381 6.51 A receptor (Fig.…”

“…In comparison, the efficacy of 77-LH-28-1-activated pERK1/2 was reduced at the F77 2.56 I mutant (Fig. 10C), consistent with previous reports (15,22,23). However, its efficacy in [Ca 2ϩ ] i mobilization experiments was unchanged (Fig.…”

“…3.28 A and F77 2.56 I because of their unique discriminatory effects on the function of TBPB and 77-LH-28-1 relative to other prototypical ligands (15,22,23).…”

“…In addition to previous investigation with 77-LH-28-1 (15), our predicted poses of TBPB and VCP794 at the M 1 mAChR suggest that the flexibility of amino acid residues in the aromatic cage can accommodate bitopic ligands. Furthermore, previous reports have indicated that residues of the orthosteric site aromatic cage are required for receptor activation (23,24,61). Hence, the lower efficacies of bitopic agonists may be due to the disruption of the aromatic cage as they extend up toward an allosteric site and, consequently, the disruption of key activation networks.…”

“…Prior mutational studies have provided some insight into bitopic mechanisms of action and the engagement of mAChRs by the structural congeners AC-42 and 77-LH-28-1 (10, 15, 20 -22). A more recent mutagenesis investigation focuses on the pharmacology of TBPB and AC-42 (23). However, this latter study does not differentiate among the effects of mutations on the affinity, efficacy, and signaling bias of the selective agonists.…”