Roles of Lys191 and Lys179 in regulating thermodynamic binding forces of ligands to determine their binding affinity...

Docking simulations based on the crystal structure of human histamine H₁ receptors have predicted crucial roles of Lys191^5.39 and Lys179^ECL2, which exist at the entrance of the ligand-binding pocket, in increasing the H₁-receptor selectivity for carboxylated second-generation antihistamines via electrostatic interaction. In this study, we evaluated the roles of Lys191^5.39 and Lys179^ECL2 in regulating the thermodynamic binding forces of non-carboxylated and carboxylated antihistamines that determine their binding affinity for human H₁ receptors. The binding enthalpy and entropy of the 3 sets of non-carboxylated and corresponding carboxylated antihistamines (doxepin and olopatadine, desloratadine and loratadine, and terfenadine and fexofenadine, respectively) were estimated using the van't Hoff equation with the dissociation constants obtained from the displacement curves of the non-carboxylated and carboxylated antihistamines against the binding of [³H]mepyramine to the membrane preparations of Chinese hamster ovary cells expressing human H₁ receptors at various temperatures, ranging from 4 °C to 37 °C. We found that the affinity for carboxylated antihistamines was lower than that for the corresponding non-carboxylated compounds due to lower enthalpy-dependent electrostatic binding forces and/or entropy-dependent hydrophobic binding forces. Mutations of Lys191^5.39 and/or Lys179^ECL2 to alanine mostly increased the binding affinity for antihistamines due to a variety of changes in both enthalpy- and entropy-dependent binding forces. These results suggest that Lys191^5.39 and Lys179^ECL2 may not contribute to selectively increasing the binding affinity for carboxylated antihistamines via electrostatic interaction, but that they can negatively modulate the binding affinity for non-carboxylated and carboxylated antihistamines non-selectively by affecting their electrostatic as well as hydrophobic binding forces.