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 K_i 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.