Preparation and characterization of immobilized 5-HT(1A) receptor stationary phase for high throughput screening of the receptor-binding ligands from complex systems like Curcuma wenyujin Y. H. Chen et C. Ling extract

The incidence of depression has increased significantly during the COVID-19 pandemic. This disease is closely associated with serotonin (1A) (5-HT(1A)) receptor and often treated by complex prescription containing Curcuma wenyujin Y. H. Chen et C. Ling. Therefore, we hypothesized that this herb contains bioactive compounds specially binding to the receptor. However, the rapid discovery of new ligands of 5-HT(1A) receptor is still challenging due to the lack of efficient screening methods. To address this problem, we developed and characterized a novel approach for the rapid screening of ligands by using immobilized 5-HT(1A) receptor as the chromatographic stationary phase. Briefly, haloalkane dehalogenase was fused at the C-terminal of 5-HT(1A) receptor, and the modified 5-HT(1A) receptor was immobilized on amino-microspheres by the reaction between haloalkane dehalogenase and 6-chlorohexanoic acid linker. Scanning electron microscope and X-ray photo-electron were used to characterize the morphology and element of the immobilized receptor. The binding of three specific ligands to 5-HT(1A) receptor was investigated by two different methods. Moreover, we examined the feasibility of 5-HT(1A) receptor colume in high throughput screening of new ligands from complex systems as exemplified by Curcuma wenyujin Y. H. Chen et C. Ling. Gweicurculactone, 2-hydroxy-1-(3,4-dihydroxybenzene)-7-(4′-hydroxybezene)-heptane and curcuminol F were identified as the ligands of 5-HT(1A) receptor with the binding energies of −7.06 kcal/mol, −7.77 kcal/mol and −5.26 kcal/mol, respectively. Collectively, these results indicated that the immobilized 5-HT(1A) receptor was capable of screening bioactive compound from complex system, providing an effective methodology for high throughput screening.