Differential Regulation of Bilastine Affinity for Human Histamine H1 Receptors by Lys 179 and Lys 191 via Its Binding Enthalpy and Entropy

Bilastine, a zwitterionic second-generation antihistamine containing a carboxyl group, has higher selectivity for H₁ receptors than first-generation antihistamines. Ligand-receptor docking simulations have suggested that the electrostatic interaction between the carboxyl group of second-generation antihistamines and the amino group of Lys179^ECL2 and Lys191^5.39 of human H₁ receptors might contribute to increased affinity of these antihistamines to H₁ receptors. In this study, we evaluated the roles of Lys179^ECL2 and Lys191^5.39 in regulating the electrostatic and hydrophobic binding of bilastine to H₁ receptors by thermodynamic analyses. The binding enthalpy and entropy of bilastine were estimated from the van ’t Hoff equation using the dissociation constants. These constants were obtained from the displacement curves against the binding of ³H] mepyramine to membrane preparations of Chinese hamster ovary cells expressing wild-type human H₁ receptors and their Lys179^ECL2 or Lys191^5.39 mutants to alanine at various temperatures. We found that the binding of bilastine to wild-type H₁ receptors occurred by enthalpy-dependent binding forces and, more dominantly, entropy-dependent binding forces. The mutation of Lys179^ECL2 and Lys191^5.39 to alanine reduced the affinity of bilastine to H₁ receptors by reducing enthalpy- and entropy-dependent binding forces, respectively. These results suggest that Lys179^ECL2 and Lys191^5.39 differentially contribute to the increased binding affinity to bilastine via electrostatic and hydrophobic binding forces.