Shared binding modes of M3 muscarinic agonists and antagonists may incentivize development of novel IOP-reducing drugs: findings from in-silico assays

M3 muscarinic acetylcholine receptors (M3R) are G-protein-coupled receptors expressed in the ciliary muscle. Pilocarpine, an M3R agonist, reduces intraocular pressure (IOP) by causing contraction of the ciliary muscle, which leads to widening of the spaces in the trabecular meshwork and facilitates aqueous humor flow out of the eye. Despite their IOP-reducing potentials, there are only four M3R agonists that have been FDA-approved, and the chemical features that differentiate agonists from antagonists are poorly understood. In this study, we created a homology model of human M3R (hM3R) to analyze the binding modes that differentiate M3R agonists from antagonists, using such in-silico assays as high-throughput virtual screening and molecular dynamics. Our in-silico results suggest that a nitrogen ring with a positive charge is crucial for forming cation-π exchange with hM3R, regardless of agonism or antagonism. However, we discovered π-π stacking exchange that is exclusively found among M3R antagonists. Structural analysis suggests that this π-π stacking exchange leads to structural changes in one of the α-helices and causes instability in the cytoplasmic domain, where G protein interactions occur. Together, these finding may prove useful in developing drugs that not only target but also selectively agonize hM3R to reduce IOP in glaucoma patients.