Low Agonism and Balanced Pathway Modulation Distinguish an M1 Muscarinic Receptor Positive Allosteric Modulator Lacking Cholinergic Adverse Effects

Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) have progressed into clinical trials for cognitive disorders, such as Alzheimer's disease and schizophrenia. However, their successful translation has been limited by on-target cholinergic adverse effects. These limitations have been attributed to excessive allosteric agonism, however, the contribution of allosteric cooperativity, efficacy modulation and receptor regulatory mechanisms remains poorly defined. Here, we performed a comparative pharmacological analysis of five structurally distinct M1 PAMs (BQCA, MK-7622, PF-06767832, MIPS1780 and VU0486846) in HEK293A cells expressing the human M1 mAChR (hM1-WT) or a phosphorylation site-deficient human M1 mAChR (hM1-PD). Radioligand binding and functional assays were used to quantify binding affinity and cooperativity, as well as allosteric agonism and efficacy modulation on G protein-dependent and β-arrestin-associated pathways. Uniquely, VU0486846, previously reported to lack cholinergic adverse effects, displayed consistently low allosteric agonism, weaker binding and functional cooperativity but comparable efficacy modulation across all signaling pathways. In contrast, PAMs associated with cholinergic adverse effects had higher allosteric agonism, stronger cooperativity and preferential enhancement of β-arrestin-associated signaling. Removing the M1 mAChR phosphorylation sites uncoupled allosteric agonism from efficacy modulation for most M1 PAMs, with the loss of phosphorylation acting as a key regulator of allosteric signaling at the M1 mAChR. Together these results suggest that low allosteric agonism and balanced efficacy modulation may be essential for improved tolerability of M1 PAMs. This work provides a mechanistic framework for differentiating M1 PAM pharmacology beyond intrinsic agonism alone and may inform the design of safer M1-targeted therapeutics.