Membrane Lipids and Water Shape the Unbinding Kinetics of ZM241385 from Human Adenosine A2A Receptor Variants

Abstract

Ligand residence time is increasingly recognized as an important determinant of drug efficacy. In the human adenosine A2A receptor (hA2AR) 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 E169ECL2-H7.29 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 E169ECL2–H7.29 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. Significance Drug efficacy depends not only on how tightly a ligand binds its target, but also on how long it remains bound. Why mutations can dramatically alter residence time without substantially changing binding affinity remains poorly understood. Here, we uncover a solvent-mediated mechanism of ligand dissociation from an important pharmaceutical target, the adenosine receptor A2A. For this system, the bound-state hydrogen-bond network is replaced by water-mediated interactions during unbinding, while lipid interactions emerge specifically in the receptor variants. These findings identify receptor–lipid interactions along the dissociation pathway as a potential target for rationally tuning drug kinetics.