The diterpenoids derived from Daphne genkwa (Thymelaeaceae) and Wikstroemia chamaedaphne (Thymelaeaceae) have been regarded as important subjects in natural product research [1]. Among them, daphnane- and tigliane-type diterpenoids have attracted significant attention due to their notable pharmacological activities, including antitumor and activation of latent HIV effects [2,3]. However, their practical application has been hindered by three major bottlenecks: (1) difficulties in isolation caused by low natural abundance, (2) inefficiency of traditional activity screening methods, and (3) synthetic challenges arising from complex chiral centers. Notably, structure-based drug design has been demonstrated to facilitate the rapid identification of potential target-binding compounds, effectively narrowing down compound libraries and providing candidate molecules for subsequent experimental validation [4]. By establishing structure-activity prediction models, the efficiency of targeted bioactive screening can be significantly enhanced.

In the field of computer-aided drug design, molecular docking technology has emerged as a key method for bioactive compound screening by calculating ligand-receptor binding free energy to accurately predict their spatial binding modes and interaction strengths [5]. This technique constructs three-dimensional binding models between ligand molecules and target proteins, enabling the analysis of intermolecular interactions such as hydrogen bonds and van der Waals forces, thereby providing visual evidence for structure-activity relationship studies of natural products. For example, through molecular docking and molecular dynamics simulation, the binding modes of the isolated novel daphnane-type diterpenoids from W. chamaedaphne were revealed with PCAF and GLP, revealing the potential medicinal value and mechanism of action of these compounds [6]. By forming a closed-loop system with subsequent experimental validation, this method effectively avoids the blind spots of traditional isolation and enables the efficient screening of bioactive diterpenoids.

As a critical member of the Rho family GTPases, cell division cycle protein 42 (Cdc42) maintains cellular homeostasis by regulating processes such as cell proliferation, polarity establishment, and migration. During tumorigenesis, aberrant activation of Cdc42 has been shown to promote epithelial-mesenchymal transition (EMT) and cancer metastasis, while its overexpression in various malignant tissues suggests its potential as a novel therapeutic target [7]. Structural biology studies have revealed that Cdc42 contains two key functional domains [8]: (1) the GTPase domain (mediating GTP/GDP binding and hydrolysis to regulate protein activity switching) and (2) the C-terminal domain (facilitating membrane localization and downstream signaling via the CAAX box). Notably, the daphnane diterpenoid daphnepedunin A (DA), isolated from W. chamaedaphne, has been confirmed to specifically bind to the GTPase domain of Cdc42 [9]. By inhibiting its GTPase activity and downregulating the PKCβ/GSK-3β/β-catenin signaling pathway, DA not only exhibits anti-renal fibrosis effects but also demonstrates significant inhibitory activity against prostate cancer cells.

Based on the above background, this study aimed to establish a systematic screening system for bioactive diterpenoids from D. genkwa and W. chamaedaphne, with a focus on elucidating the interaction mechanisms between trace components and Cdc42. A multi-dimensional integrated strategy was employed: first, a targeted isolation method guided by characteristic core structures was developed using traditional extraction and separation techniques. Second, molecular docking was utilized to predict the binding modes and free energies between diterpenoids and Cdc42, enabling the screening of potential bioactive molecules. Subsequently, compound-protein interactions were validated through UV/fluorescence spectroscopy, cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) technology. Finally, the antiproliferative activities of diterpenoids against 4T1 breast cancer cells, RAW264.7 macrophages, and HepG2 liver cancer cells were systematically evaluated using the CCK-8 assay, with IC50 values calculated via concentration-gradient experiments to construct an antitumor activity profile of diterpenoids from D. genkwa and W. chamaedaphne. This research framework is expected to provide methodological insights for the efficient discovery of trace bioactive diterpenoids in natural products.