Identification and characterization of agnuside, a natural proangiogenic small molecule

Due to its important role in regulating angiogenesis, vascular homeostasis and remodeling, and arteriogenesis in blood vascular and lymphatic endothelial cells, VEGFR2/KDR/Flk-1 stimulation has demonstrated promise in preclinical studies as an endovascular treatment for ischemic myocardial and peripheral disease. However, the short half-life of protein- and cytokine-based strategies and transduction inefficiency of vector-based modalities have hindered its clinical therapeutic applications. In the present study, we used a streamlined bioinformatics strategy combining ligand-based pharmacophore development and validation, virtual screening, and molecular docking to identify agnuside, a non-toxic, natural small molecule extract of Vitex agnus-castus possessing strong binding affinity, druggable physiochemical properties, and conformationally stable hydrogen bond and hydrophobic interactions with catalytically important residues within VEGFR2's active and allosteric sites. In-vitro proliferation, tube formation, and scratch wound migration assays provide evidence that agnuside promotes endothelial cell angiogenesis. Agnuside increases HUVEC proliferation with an EC₅₀ of 1.376 μg/mL, stimulates tubulogenesis dose-dependently, and increases scratch wound migration rate. An additional angiogenesis assay suggests that agnuside may actively compete with a VEGFR2/KDR/Flk-1 inhibitor for VEGFR2/KDR/Flk-1 binding site occupancy to increase total length and branching length of HUVEC tubular networks. Chemometric analysis of molecular interaction fields (MIFs) by partial least squares (PLS)-derived quantitative structure activity relationship (QSAR) analysis and MIF contours provides the framework for the formulation of agnuside analogues possessing greater potency. Our research supports that agnuside may be a lead molecule for therapeutic angiogenesis.

Agnuside; Agonist; Drug discovery; Molecular modeling; QSAR; Therapeutic angiogenesis.