Activation of the M2 Muscarinic Receptor and Computer-Aided Design of Receptor-Selective Allosteric Drugs

Abstract

G-protein coupled receptors (GPCRs) are important cell signaling membrane proteins that have been targeted by ∼30-40% of marketed drugs for treating many human diseases including cancer and heart failure. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined in an inactive antagonist-bound state. Through accelerated molecular dynamics (aMD) enhanced sampling simulation, we captured activation of the M2 receptor that occurs on the millisecond timescales(1). The receptor activation is characterized by large-scale structural rearrangements of the transmembrane helices via an intermediate state. With activation-associated conformers of the M2 receptor revealed from aMD simulation, a fragment-based site mapping program FTMAP is applied to explore the receptor surface. Seven allosteric sites are identified with two distributed in the solvent-exposed extracellular and intracellular mouth regions and five in the lipid-exposed pockets formed by transmembrane helices of the receptor(2). Virtual screening is then performed to select small-molecule drugs that bind these allosteric sites with differential affinity and thus stabilize the receptor in the corresponding conformational states. In contrast to highly conserved residues in the orthosteric site where endogenous ligands bind, residues in the allosteric sites exhibit large diversity across different GPCR subtypes. Therefore, ligands that bind GPCR allosteric sites are able to provide potential receptor-selective therapeutics.