A novel Halo Tag-based photo-crosslinking system for efficient drug target identification via chemoproteomic...

Target discovery of natural products is critical for novel drug development. However, it remains challenging due to the risk of disrupting bioactive molecule integrity during chemical modification. Moreover, many current methods for identifying drug targets rely on chemical synthesis to design the probes and enrichment systems. This is not friendly to researchers lacking experience in chemical synthesis, which limits their large-scale applications. To address these limitations, we present a novel chemoproteomic strategy to identify drug targets by integrating Halo Tag technology with photo-crosslinking chemistry.

A bifunctional probe was synthesized by conjugating the Halo Tag ligand with a photo-crosslinker, 3-Phenyl-3-(trifluoromethyl)-3H-diazirine (TAD). TAD induced UV-triggered covalent binding with drug molecules. And Halo Tag ligand was enriched by Halo Protein magnetic beads, with less structural bias and reduced activity impairment. Taking sweroside (SWE) as an example, 159 target proteins were screened using this probe, and yin-yang 1 (YY1) was selected as the most potential target of SWE. Furtherly, in vitro small molecule-protein interaction analysis, including cell thermal shift assays (CETSA), surface plasmon resonance (SPR) and molecular docking were performed. These results demonstrated the strong binding affinity of YY1 and SWE. Moreover, SWE was demonstrated to relieve bile acid (BA) induced hepatocyte apoptosis by targeting YY1 to regulate its transcriptional activity to enhance Farnesoid X Receptor (FXR) expression. Furthermore, in vivo assays showed that SWE administration effectively ameliorated 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) induced cholestatic liver injury in mice. It was evidenced by markedly reducing the serum biochemical biomarkers and liver pathological change. Notably, SWE decreased YY1 expression and promoted FXR and BSEP expression. Thus, SWE exerted hepatoprotective effects by regulating YY1/FXR pathway.

In conclusion, our method without pre-derivatization simplifies workflows and preserves native pharmacophores. It is compatible with routine laboratory equipment and can be widely used in drug target discovery.