Revealing and Distinguishing the Binding Sites of Agonists/Antagonists on the MrgprX2 by CMC-MD Strategy

Ligand-receptor interactions are fundamental in pharmacology. Mas-related G protein-coupled receptor X2 (MrgprX2) has emerged as a promising therapeutic target for pseudoallergic reactions. Thus, investigating the ligands of MrgprX2 holds substantial potential for early warning of drug allergies and for the discovery of both agonists and antagonists of this receptor. At present, no antagonists specifically targeting MrgprX2 have been widely commercialized. Consequently, identifying and characterizing the binding sites for these antagonists is of great significance. Notably, whether antagonists and agonists vie for the identical binding site has long been a subject that has captivated the attention of researchers. In this study, a combined approach integrating cell membrane chromatography (CMC) and molecular dynamics (MD) simulations was adopted to pinpoint and systematically explore the binding sites of both agonists and antagonists on the MrgprX2. The results indicate that agonists and antagonists do not compete for an identical MrgprX2 binding site. Specifically, antagonists predominantly interact with the amino acid residues in transmembrane domain 2 (TM2) and transmembrane domain 7 (TM7), while agonists preferentially form favorable interactions with the residues in transmembrane domain 6 (TM6) and extracellular loop 3 (ECL3). Calcium imaging results confirmed that antagonists suppress mast cell activation by noncompetitive binding to MrgprX2, inhibiting agonist-induced calcium influx, which further elucidates the distinct binding behaviors of agonists and antagonists. These findings provide insights into the development of MrgprX2-targeted compounds and offer a theoretical foundation for the future design of antagonists.