Muscarinic receptors as model targets and antitargets for structure-based ligand discovery

Abstract

G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

Fig. 1.

Docking poses for selected M₂ muscarinic receptor hits. (A) The overall structure of the M₂ receptor (Haga et al., 2012) with the orthosteric site outlined. (B) The chemical structure of the cocrystallized antagonist QNB, its crystallographic geometry, and key interactions (dashed lines). (C) Docking-discovered ligands (carbons in cyan) are superimposed in their docked poses on the crystallographic structure of QNB (carbons in yellow).

Fig. 2.

Docking for selective M₃ receptor ligands. (A) The M₃ (green) and M₂ receptor (orange) binding pockets are superimposed and rendered as solvent-accessible surfaces, highlighting the enlarged binding pocket in the M₃ subtype (Kruse et al., 2012). (B) Specific interactions with the cocrystallized M₃ antagonist tiotropium are shown. (C) Docking poses for select new ligands.

Fig. 3.

Compound 16 activates M₃ but not M₂ receptors. (A) Compound 16 showed partial agonism at the M₃ subtype, but not at the M₂ receptor in a calcium mobilization assay using CHO cells stably expressing M₂ or M₃ receptors (see Materials and Methods for details). This effect was blocked by the muscarinic antagonist atropine (Atr), consistent with direct activity at the M₃ receptor. (B) In a fluorescence resonance energy transfer–based cAMP assay (see Materials and Methods for details), compound 16 did not lead to changes in intracellular cAMP levels in CHO-M₂ cells, confirming that this agent lacks efficacy at M₂ receptors. In this assay, an elevated 665 nm/620 nm ratio corresponds to decreased cAMP levels. The curves shown in A and B are representative of three independent experiments. (C) The unique structure and predicted binding mode of compound 16 may account for its novel activity profile. ACh, acetylcholine; Cmpd, compound.

Fig. 4.

Ligand-stimulated insulin release in MIN6 cells. (A) MIN6 cells, which express endogenous M₃ receptors, were incubated with increasing concentrations of OXO-M and compound 16, and ligand-induced insulin release was measured. (B) The responses to both agonists were sensitive to blockade by atropine, indicating that the observed effects result from direct M₃ receptor activation. Data (mean ± S.E.) are from three independent experiments: OXO-M pEC₅₀ = 5.75 ± 0.17; E_max = 453 ± 21; compound 16 pEC₅₀ = 4.21 ± 0.18; E_max = 261 ± 21. Atr, atropine; comp, compound. **P < 0.0092; ***P < 0.0001.