An online rapid screening HPLC system establishing and applying discovered a new type of natural chemical ligand of the BRD4-BD1 from Hedyotis diffusa

Herein, we report an online high performance liquid chromatography (HPLC) for screening of the bromodomain-containing protein 4-first bromodomain (BRD4-BD1) ligands, leading to screen a new type of natural chemical ligand of the BRD4-BD1 from Hedyotis diffusa. The system was first established, validated, and then applied for rapid screening the ligands of BRD4-BD1 from Hedyotis diffusa. It was investigated using the positive ligand JQ1 in five aspects including the suitability, the specificity, the reproductivity, the limit of detection (LOD), and the saturability of this system. When it was been applied to screen BRD4-BD1 ligands from Hedyotis diffusa, one candidate ligand 1 was fished out. Then the ligand 1 was isolated and identified. And their interaction between the screened ligand 1 and the BRD4-BD1 was assay on the online system again and followed verified by the surface plasmon resonance (SPR) technique. The molecular docking was performed to the binding mode of ligand 1 with BRD4-BD1. The ligand 1 was found as a new type of natural ligand for BRD4-BD1 protein. In a conclusion, we have systemically demonstrated the feasibility of the online HPLC system screening method applying to screen the chemical ligand of the BRD4-BD1 in complex substance systems. Besides, the ligand 1 provided a potential new type of scaffold for chemical modification for BRD4-BD1 inhibitors in future.

BRD4-BD1; Hedyotis diffusa; Natural chemical ligand; Online HPLC system.