An Exosome-Based Affinity Screening Platform for Identifying Bioactive Compounds against Colon Cancer

Abstract Exosomes, nanoscale extracellular vesicles carrying native membrane proteins, play crucial roles in intercellular communication and are critically involved in colorectal cancer progression and drug resistance, thereby offering a biologically relevant platform for targeted drug discovery. In this study, we developed an innovative immobilized exosome affinity screening platform (Exo@SiO2–NH2) using HT-29 cell-derived exosomes to selectively capture antitumor bioactive compounds from Sophora flavescens. Through UHPLC-MS/MS analysis, four compounds─Kushenol I, Kurarinone, 2′-O-MethylKurarinone, and Isoxanthohumol were successfully enriched and identified. To explore potential exosome-associated targets, systematic molecular docking and molecular dynamics simulations were conducted on representative exosome-enriched proteins against these four compounds. Further combinatorial alanine scanning mutagenesis within the predicted binding pockets (<5.0 Å) suggested CD81 and HSP90 as probable protein targets. Subsequent functional validation via siRNA-mediated knockdown of CD81 and HSP90, combined with IC50 determination via CCK-8 assays, confirmed target-dependent alterations in drug responsiveness, supporting their role as key functional mediators. Together, this integrated strategy provides a practical and novel approach for discovering anti-colorectal cancer molecules targeting exosome-associated protein interfaces.