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

Elham Khajehali,Celine Valant,Manuela Jörg,Andrew B Tobin,P Jeffrey Conn,Craig W Lindsley,Patrick M Sexton,Peter J Scammells,Arthur Christopoulos

doi:10.1016/j.bcp.2018.05.009

Abstract

Subtype-selective allosteric modulation of the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M1 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)pyridine-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 M1 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 W4007.35 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 M1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.

Highlights

Muscarinic acetylcholine receptors are members of the Class A G protein-coupled receptor (GPCR) family [1] involved in central and peripheral biology [2]
Equilibrium [3H]NMS binding interaction studies between ACh and increasing concentrations of benzyl quinolone carboxylic acid (BQCA), PF-06767832, VU6004256 or MIPS1780 were performed at the M1 muscarinic acetylcholine receptor (mAChR) to determine the affinity (KB) of each positive allosteric modulator (PAM) and their binding cooperativity (α) with the endogenous agonist ACh or the radiolabelled antagonist [3H]NMS, using an allosteric ternary complex model (Eq (1))
Consistent with the results of previous studies [21,22,26], PF-06767832, VU6004256 and MIPS1780 have higher affinities at the M1 mAChR compared to BQCA (Table 1)

Summary

Introduction

Muscarinic acetylcholine receptors (mAChRs) are members of the Class A G protein-coupled receptor (GPCR) family [1] involved in central and peripheral biology [2]. Five mAChR subtypes (M1-M5), have been identified; M1, M3 and M5 mAChRs preferentially couple to Gq/11 proteins; M2 and M4 mAChRs preferentially couple to Gi/o proteins [3]. The M1 mAChRs are highly expressed in forebrain regions, including the cerebral cortex, hippocampus and striatum [4]. Transgenic M1 mAChR studies implicated roles for this receptor in neuronal excitability, locomotor activity and learning and memory [Reviewed in [5]]. Selective activation of the M1 mAChR has emerged as an approach for the treatment of cognitive deficits associated with disorders such as Alzheimer’s disease (AD) and schizophrenia [6,7]

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Conclusion