Investigation of the Binding Characteristics of Agonists and Various Antagonists Targeting Histamine 1 Receptor.

Objective: The histamine H1 receptor (H1R) plays a central role in mediating allergic responses, making it a critical target for therapeutic intervention. However, the molecular mechanisms underlying drug binding to H1R remain incompletely elucidated.

Methods: We employed an integrated approach combining site-directed mutagenesis, cell membrane chromatography (CMC) and pharmacological activity assays to systematically characterize the binding mechanisms of H1R agonists and antagonists. We constructed various H1R/CMC systems using high-expression H1R cells (wild type, TM3, TM5, TM6, and ECL2 mutants) and evaluated the binding affinities of three agonists (histamine, HTMT, betahistine) and three classes of antagonists (ethylenediamine/propanamine, tricyclic, piperidine derivatives).

Results: Our findings reveal distinct agonist binding preferences: histamine primarily targets TM3, HTMT interacts with TM5, and betahistine shows a strong preference for TM6. Among antagonists, ethylenediamine/propanamine and piperidine classes predominantly block TM3 and TM6 regions, while tricyclic antagonists additionally depend on TM5 region for their inhibitory effects. Pharmacological validation through phospholipase C (PLC) activity assays corroborated these results, demonstrating that mutations in specific transmembrane domains significantly alter agonist-induced signaling and antagonist-mediated efficacy.

Conclusion: These mechanistic insights into H1R ligand binding provide a structural foundation for the rational design of targeted therapies with improved selectivity and efficacy against allergic disorders.