Probing the binding site of novel selective positive allosteric modulators at the M1 muscarinic acetylcholine receptor

Subtype-selective allosteric modulation of the M-1 muscarinic acetylcholine (ACh) receptor (M-1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M-1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M-1 PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 (N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl) benzyl)pyndine-2-carboxamide), VU6004256 (4,6-difluoro-N-(1S,2S)-2-hydroxycyclohexyl-1-((6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-yl)methyl)-1H-indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M-1 mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W400(7.35) in transmembrane domain (TM)7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M-1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.