Further exploration of M1 allosteric agonists: Subtle structural changes abolish M1 allosteric agonism and result in pan-mAChR orthosteric antagonism

Abstract

This letter describes the further exploration of two series of M1allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M1 allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M1 allosteric agonist family (VU0357017) identified similar, subtle ‘molecular switches’ that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M1 activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.

Reverse Engineering of the Selective Agonist TBPB Unveils Both Orthosteric and Allosteric Modes of Action at the M1 Muscarinic Acetylcholine Receptor

“…The apparently competitive behavior of TBPB argues against a purely allosteric mechanism of agonism at the M 1 mAChR. Indeed, consideration of the evidence for a purely allosteric mechanism of action by TBPB is somewhat inconclusive, with recent exploration of TBPB's structural activity relationships also uncovering competitive orthosteric pharmacology (Sheffler et al, 2013). In contrast to initial observations of noncompetitive antagonism of TBPB by atropine , we observed competitive antagonism.…”

“…Recent studies by Digby et al (2012) and Sheffler et al (2013) have made similar proposals, but remained unclear as to whether this mechanism involved concomitant binding to both orthosteric and allosteric sites, or acting as pure allosteric ligands at low concentrations and recognizing the orthosteric site only at higher concentrations. Such distinctions are important, because ascertaining differences between classes of ligands at the preclinical stage of discovery is vital in improving the likelihood of clinical translation of such molecules.…”

“…Notably, the concentration of TBPB required to observe the allosteric effect on radioligand dissociation would likely saturate the receptor population in the absence of other ligands, based on our binding data. Additionally, a lower concentration of TBPB (50 mM) has been reported to have no effect on [ 3 H]NMS dissociation (Sheffler et al, 2013), suggesting that a higher-affinity orthosteric/ bitopic interaction predominates over a much lower-affinity (purely) allosteric interaction. In support of this notion, the retardation of [ 3 H]NMS dissociation by the bitopic ligand McN-A-343, at the M 2 mAChR, has been defined at concentrations that are greater than its equilibrium dissociation constant (May et al, 2007;Valant et al, 2008).…”

Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

“…Interestingly, a recent study identified "molecular switches" within the structure of TBPB that convert the ligand from a selective allosteric agonist to a pan-mAChR orthosteric antagonist. 132 Surprisingly, addition of a single fluorine atom to the central piperidine ring was sufficient to disrupt an interaction critical for allosteric binding. This result, combined with the finding that TBPB behaves as an orthosteric antagonist at the other mAChR subtypes (in Ca 2+ mobilization assays), led to the first definitive classification of TBPB as a bitopic ligand; though additional supportive pharmacological data will add further weight to this classification.…”

Discovery and optimization of a novel series of highly CNS penetrant M 4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3- d ]pyrimidine core

“…4). The majority of analogs were inactive, but once the alkyl chain reached 3-carbons in length, a ‘molecular switch’ was noted (rare amongst muscarinic allosteric modulators 20–22 and the first report for M 4 ), wherein the ligands became M 4 antagonists. For example, 19 (human M 4 IC 50 = 2.4 μM, 9.3% ACh min) and 20 (human M 4 IC 50 = 1.5 μM, 8.7% ACh min) were robust M 4 antagonists.…”