Discovery of VEGFR-2 Inhibitors From Leonurus japonicus via Integrated Genetic Authentication, Molecular Networking, and In Silico Analysis.

Angiogenesis, regulated by vascular endothelial growth factor (VEGF), is crucial in tumor growth, metastasis, and inflammation. Leonurus japonicus Houtt., a traditional Chinese herb, was investigated for its anti-angiogenic potential. DNA sequencing confirmed its identity, distinguishing it from the common morphological misidentification with Leonurus sibiricus by the public. Using UHPLC-MS/MS and GNPS (Global Natural Products Social) molecular networking on the active methanol partition, over 6000 nodes were grouped into 273 clusters. Diterpenoids and flavonoids were targeted. Six compounds were successfully isolated from L. japonicus, including four labdane-type diterpenoids (1-4) and two flavonoids (5, 6) using column chromatography. Compound 1, (-)-8S-acetoxy-15,16-epoxy-8,9-seco-13(16),14-labdadiene, was a new stereoisomer, while Compound 6, nevadensin, was reported for the first time from this species. Compounds were characterized by NMR, ESI-MS, and ECD. At 50 μM, Compounds 1, 4, and 5 exhibited inhibitory effects in endothelial progenitor cell (EPC) tube formation. Molecular docking and dynamics simulations supported this anti-angiogenic activity. Compound 1 showed the highest VEGFR-2 binding affinity (-9.24 kcal/mol), comparable to sunitinib (-9.52 kcal/mol), further verified by an immunoblotting assay. This study demonstrated the successful integration of GNPS and bioactivity-guided isolation for identifying natural VEGFR-2 inhibitors. Our findings highlight Compounds 1 and 4 as promising lead candidates for anti-angiogenic therapy.