In Vitro and Computational Evaluation of 1-O-Benzoylkarakoline on Vascular Calcium Transport
Аннотация
We aimed to investigate the vasorelaxant properties of 1-O-benzoylkarakoline, a semi-synthetic diterpenoid alkaloid, and to elucidate its interactions with calcium transport proteins in vascular smooth muscle. Isolated rat aortic rings were exposed to depolarization with 50 mM KCl and receptor-mediated contraction with 1 µM phenylephrine. Intracellular calcium mobilization was assessed in Ca2+-free medium using IP3R- and RyR-mediated contractions. NCX reverse mode was studied in Na+-free solution, and Na+/K+-ATPase involvement was tested in ouabain-induced contraction. Endothelium dependency was examined using mechanical removal and pharmacological inhibition (L-NAME, indomethacin). Molecular docking was conducted with L-type Ca2+ channels, RyR, SERCA, NCX1, and Na+/K+-ATPase. 1-O-benzoylkarakoline induced potent relaxation against KCl contraction (91.8 ± 3.7%, IC50 = 2.2 µM) and phenylephrine contraction (92.1 ± 3.1%, IC50 = 5.1 µM). It attenuated IP3R-mediated contraction by 13.1 ± 2.6% and RyR-mediated contraction by 27.6 ± 4.3%. Under Na+-free conditions, NCX reverse mode contraction was reduced by 22.3 ± 4.3%, while ouabain-induced contraction decreased by 28.1 ± 4.0%. Vasorelaxant effects were endothelium-independent. Docking analysis showed strong affinities with L-type Ca2+ channels (−8.4 kcal/mol), RyR (−7.7), SERCA (−8.1), NCX1 (−8.2), and Na+/K+-ATPase (−11.8), involving hydrogen bonds, π-π stacking, π-alkyl, van der Waals, and salt bridges. 1-O-benzoylkarakoline exerts strong vasorelaxant activity by modulating calcium transport across multiple membrane and intracellular targets. Its endothelium-independent mechanism and multi-target interactions suggest therapeutic potential for vascular disorders associated with calcium overload and hypercontractility. HIGHLIGHTS 1-O-benzoylkarakoline produced strong vasorelaxation against both depolarization (KCl) and receptor-mediated (phenylephrine) contractions in isolated rat aorta. Calcium transport was modulated at multiple levels, including L-type Ca²⁺ entry, IP₃R- and RyR-mediated intracellular release, SERCA handling, NCX reverse mode, and Na⁺/K⁺-ATPase activity. Endothelium-independent mechanism was confirmed through mechanical denudation and pharmacological inhibition. Molecular docking revealed high affinities for L-type Ca²⁺ channels, SERCA, NCX1, RyR, and Na⁺/K⁺-ATPase, involving hydrogen bonding, π-interactions, and van der Waals forces. Multi-target modulation of calcium homeostasis positions 1-O-benzoylkarakoline as a promising candidate for treating vascular disorders associated with calcium overload and hypercontractility. GRAPHICAL ABSTRACT