Reverse engineering of the selective agonist TBPB unveils both orthosteric and allosteric modes of action at the M₁ muscarinic acetylcholine receptor.

Recent interest in the M₁ muscarinic acetylcholine (ACh) receptor (mAChR) has led to the discovery of various selective agonists for the receptor. The novel selective agonist 1-(1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (TBPB) displays unprecedented functional selectivity at the M₁ mAChR. This functional selectivity has been described to stem from sole interaction with an allosteric site, although the evidence for such a mechanism is equivocal. To delineate TBPB's mechanism of action, several truncated variants of TBPB were synthesized and characterized. Binding experiments with [³H]N-methylscopolamine at the M₁, M₂, M₃, and M₄ mAChRs revealed radioligand displacement in a manner consistent with a competitive binding mode at the orthosteric site by TBPB and fragment derivatives. Cell-based functional assays of fragment derivatives of TBPB identified both agonistic and antagonistic moieties, one of which, 1-(1-cyclohexylpiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (VCP794), lost agonistic selectivity for the M₁ mAChR. Further interaction experiments between TBPB or its antagonist fragments with ACh also indicated a mechanism consistent with competitive binding at mAChRs. However, interaction with an allosteric site by an antagonist fragment of TBPB was demonstrated via its ability to retard radioligand dissociation. To reconcile this dual orthosteric/allosteric pharmacological behavior, we propose that TBPB is a bitopic ligand, interacting with both the orthosteric site and an allosteric site, at the M₁ mAChR. This mechanism may also be the case for other selective agonists for mAChRs, and should be taken into consideration in the profiling and classification of new novel selective agonists for this receptor family.