Membrane Lipids and Water Shape the Unbinding Kinetics of ZM241385 from Human Adenosine A _2A Receptor Variants

Ligand residence time is increasingly recognized as an important determinant of drug efficacy. In the human adenosine A _2A receptor (hA _2A R) bound to the high-affinity antagonist ZMA, mutations at the extracellular entrance of the binding pocket markedly accelerate ZMA dissociation while largely preserving binding affinity. This effect has been attributed to disruption of the E169 ^ECL2 -H ^7.29 salt bridge that constrains the extracellular entrance in the wild-type receptor. However, how the interaction network is reorganized after salt-bridge disruption remains unknown. Here, we combine well-tempered metadynamics and infrequent metadynamics simulations to characterize the ZMA dissociation pathway and transition-state ensemble. The simulations reproduce the experimentally observed trend in dissociation rates and reveal that the hydrogen-bond network stabilizing ZMA in the bound state is largely replaced by water-mediated interactions during unbinding. In the mutants, disruption of the E169 ^ECL2 –H ^7.29 interaction expands the binding pocket and increases hydration, while additional ligand–lipid interactions emerge specifically at the transition state. These interactions are substantially less frequent in the wild type, indicating that membrane lipids can directly participate in ligand escape when the extracellular gate is disrupted. Thus, the mutations selectively reshape the transition-state interaction network – including recruitment of membrane lipids. Thus, the interaction of the latter with the ligand accompanies and may contribute to the mutants’ reduced dissociation barrier, thereby facilitating ligand release.