Screening licorice for H2 receptor-binding anti-ulcer components using affinity chromatography

Recent studies have confirmed the significant potential of the histamine H2 receptor (H2R) as a therapeutic target for multiple diseases, creating an urgent demand for efficient drug screening methodologies. To address this need, we developed an innovative technique utilizing immobilized H2R for the rapid identification and characterization of bioactive compounds from complex matrices. Compared with the previous screening methods, this approach can detect the binding mode of ligands on the basis of screening. In this study, the prepared H2R chromatographic column was successfully applied for kinetic interaction analysis between two specific ligands and the receptor. The bioactive compound screened and collected via the H2R column was identified as liquiritin using high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-IT-MS). Competitive displacement assays revealed that liquiritin competes with nizatidine and ranitidine for the same binding sites. Notably, as the concentration of famotidine in the mobile phase increased, an inverse shift in the retention time of liquiritin was observed. To elucidate this phenomenon, molecular docking simulations were performed. The results suggest that nizatidine and ranitidine compete with liquiritin for the same binding sites, reducing liquiritin-protein binding and increasing the proportion of free liquiritin, thereby causing a forward shift in the retention peak. In contrast, the acidic residues (ASP186, ASP98) of famotidine may reorganize the protein's surface charge through salt bridges or hydrogen bonds, altering the electrostatic interactions of liquiritin and leading to the observed peak shift. Additionally, at higher concentrations, famotidine may form multimolecular complexes that indirectly stabilize liquiritin-protein binding. Literature review revealed that, compared to nizatidine and ranitidine, famotidine exhibits a positive regulatory effect on β-arrestin-mediated receptor desensitization and internalization. These findings further demonstrate that the established H2R receptor chromatography model can effectively screen bioactive components and determine their binding modes. This study lays the foundation for screening bioactive compounds from traditional Chinese medicines using receptor chromatography and provides preliminary insights into their mechanisms of action.