Vasodilation Elicited by Isoxsuprine, Identified by High-Throughput Virtual Screening of Compound Libraries, Involves Activation of the NO/cGMP and H₂S/K Pathways and Blockade of α₁-Adrenoceptors and Calcium Channels.

Recently, our research group demonstrated that uvaol and ursolic acid increase NO and H₂S production in aortic tissue. Molecular docking studies showed that both compounds bind with high affinity to endothelial NO synthase (eNOS) and cystathionine gamma-lyase (CSE). The aim of this study was to identify hits with high binding affinity for the triterpene binding-allosteric sites of eNOS and CSE and to evaluate their vasodilator effect. Additionally, the mechanism of action of the most potent compound was explored. A high-throughput virtual screening (HTVS) of 107,373 compounds, obtained from four ZINC database libraries, was performed employing the crystallographic structures of eNOS and CSE. Among the nine top-scoring ligands, isoxsuprine showed the most potent vasodilator effect. Pharmacological evaluation, employing the rat aorta model, indicated that the vasodilation produced by this compound involved activation of the NO/cGMP and H₂S/K signaling pathways and blockade of α₁-adrenoceptors and L-type voltage-dependent Ca channels. Incubation of aorta homogenates in the presence of isoxsuprine caused 2-fold greater levels of H₂S, which supported our preliminary in silico data. This study provides evidence to propose that the vasodilator effect of isoxsuprine involves various mechanisms, which highlights its potential to treat a wide variety of cardiovascular diseases.