Natural diterpenoids isolated-interaction-phenotypic target characterization: An idea performed with Daphne genkwa...

In this study, twenty-two diterpenoids (including two unreported structures) were isolated and identified from the buds of Daphne genkwa and Wikstroemia chamaedaphne. Molecular docking analysis revealed that this series of compounds could stably bind to the active site of Cdc42 protein (-6.47 to -10.39 kcal/mol), with their binding energies showing significant correlation to structural features. Spectroscopic experiments confirmed that the compounds could bind to Cdc42 and alter its microenvironment. Cellular assays demonstrated that some compounds exhibited selective cytotoxicity against RAW264.7/HepG2/4T1 cells (lowest IC₅₀ 2.25 μM). CETSA verification showed that compound 6 could enhance the thermal stability of Cdc42 (∆Tm >5 °C), suggesting Cdc42 as a potential antitumor target for this compound. In conclusion, the interactions between these 22 compounds and Cdc42 protein were systematically investigated by using an "isolated-interaction-phenotypic target characterization" strategy. These findings not only established a crucial foundation for elucidating the antitumor mechanisms mediated by diterpenoid-Cdc42 interactions but also provided a practical methodological framework for natural product-based drug discovery.