Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor

Magdalena Korczynska, Mary J. Clark, Celine Valant, Jun Xu, Ee Von Moo, Sabine Albold, Dahlia R. Weiss, Hayarpi Torosyan, Weijiao Huang, Andrew C. Kruse, Brent R. Lyda, Lauren T. May, Jo Anne Baltos, Patrick M. Sexton, Brian K. Kobilka, Arthur Christopoulos, Brian K. Shoichet, Roger K. Sunahara

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49 Citations (Scopus)


Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound’628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover,’628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of’628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between’628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like’628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

Original languageEnglish
Pages (from-to)E2419-E2428
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number10
Publication statusPublished - 6 Mar 2018


  • Docking
  • GPCR
  • PAM antagonist
  • Structure-based ligand discovery
  • Subtype selectivity

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