Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M 2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [ 3 H]Nmethylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M 2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M 1 and M 3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.GPCR | allosteric modulators | ensemble docking | affinity | cooperativity

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SignificanceThe orthosteric binding sites of the five muscarinic acetylcholine receptor (mAChR) subtypes are highly conserved, making the development of selective antagonists challenging. The allosteric sites of these receptors are more variable, allowing one to imagine allosteric modulators that confer subtype selectivity, which would reduce the major off-target effects of muscarinic antagonists. Accordingly, a large library docking campaign was prosecuted seeking unique positive allosteric modulators (PAMs) for antagonists, ultimately revealing a PAM that substantially potentiates antagonist binding leading to subtype selectivity at the M2 mAChR. This study supports the feasibility of discovering PAMs that can convert an armamentarium of potent but nonselective G-protein–coupled receptor (GPCR) antagonist drugs into subtype-selective reagents.

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