A piperamide natural compound is compatible with an inactive DFG-out conformation of vascular endothelial growth...

Vascular endothelial growth factor receptor-2 (VEGFR2) is a well-known key regulator of angiogenic cancers. In a structure-based virtual screening of Indonesian natural compounds, dehydropipernonaline (DPN), a piperamide from Piper species, emerged as a promising VEGFR2-binding candidate. However, its inhibitory activity against VEGFR2 has not been experimentally validated. This study combined molecular simulations and cellular assays to investigate the potential interaction between DPN and VEGFR2. Molecular docking predicted that DPN occupies the regulatory domain pocket (RDP) spanning the catalytic and juxtamembrane (JM) domains, overlapping the binding region of the type II inhibitor tivozanib. All-atom simulations revealed that DPN remains stably associated with the DFG loop. Free-energy surface analysis identified two dominant minima corresponding to a DFG-out/JM-out conformation resembling the inactive, tivozanib-bound VEGFR2 structure rather than the active DFG-in state. Binding free-energy calculations further supported favourable, predominantly hydrophobic stabilization of the DPN-VEGFR2 complex. Experiments in human umbilical vein endothelial cells (HUVECs) were consistent with these predictions. At 10 μM, DPN reduced VEGFR2, PLCG1 (phospholipase C gamma 1) and PRKCE (protein kinase C epsilon) transcripts, total and phosphorylated VEGFR2 (Tyr1054/1059), and VEGF-induced proliferation, but this concentration also reduced cell viability by ~24%, so non-specific cytotoxicity cannot be excluded. At 1 μM, which did not significantly reduce viability, DPN reduced proliferation relative to VEGF-stimulated cells but had no significant effect on the molecular readouts. Collectively, these findings indicate that DPN is compatible with, and remains stably bound within, an inactive type II-like DFG-out conformation of VEGFR2, consistent with (though not proof of) a stabilizing effect on this state; this is accompanied by reduced VEGFR2 signalling readouts at a partially cytotoxic concentration. DPN is therefore highlighted as a candidate natural lead compound for further evaluation in anti-angiogenic drug development.