摘要The vasopressin V2 receptor(V2R)is a validated therapeutic target for autosomal dominant polycystic kidney disease(ADPKD),with tolvaptan being the first FDA-approved antagonist.Herein,we used Gaussian accelerated molecular dynamics simulations to investigate the spontaneous binding of tolvaptan to both active and inactive V2R conformations at the atomic-level.Overall,the binding process consists of two stages.Tolvaptan binds initially to extracellular loops 2 and 3(ECL2/3)before overcoming an energy barrier to enter the pocket.Our simulations result highlighted key residues(e.g.,R181,Y205,F287,F178)involved in this process,which were experimentally confirmed by site-directed mutagenesis.This work provides structural insights into tolvaptan-V2R interactions,potentially aiding the design of novel antagonists for V2R and other G protein-coupled receptors.