Enthalpy-Entropy Trade-Off Underlies Geometric Isomer Selectivity in Histamine H₁ Receptor-Doxepin Interaction

Understanding the thermodynamic basis of ligand recognition by G-protein-coupled receptors (GPCRs) is crucial for rational drug design. Here, we directly characterized the binding thermodynamics of the histamine H₁ receptor (H₁R) interacting with the geometric isomers of doxepin using isothermal titration calorimetry combined with molecular dynamics (MD) simulations. The Z-isomer binding to H₁R_WT exhibited a larger enthalpic gain but a greater entropic loss than the E-isomer, whereas these differences were diminished in the T112^3.37V mutant. Cluster analysis of MD trajectories revealed that Z-doxepin adopts a more restricted conformation upon binding, consistent with its enthalpy-driven interaction and reduced conformational entropy. These findings indicate that H₁R distinguishes between E- and Z-isomers not only by affinity but also through distinct thermodynamic fingerprints. This study provides mechanistic insight into the enthalpy-entropy trade-off in GPCR-ligand interactions, highlighting the importance of conformational restriction and flexibility in designing ligands with optimized thermodynamic and functional properties.