Abstract

Activators of M₁ muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M₁-active compounds has resulted in nonselective activation of the highly related M₂ to M₅ mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M₁ with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N′-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M₁ that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M₁ activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M₁ receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M₁ agonist, 1-[1′-(2-tolyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M₁-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M₁ is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M₁ potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.