Relaxant Activity of KC-4 Associated with Modulation of Calcium Transport and Calcium Homeostasis Pathways

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Calcium dysregulation plays a critical role in vascular dysfunction and the development of cardiovascular diseases. The present study investigated the relaxant activity of KC-4 and its interaction with calcium transport-associated proteins using integrated in vitro and in silico approaches. Vasorelaxant activity was evaluated in isolated rat aortic rings precontracted with 50 mM KCl or 1 μM phenylephrine. The involvement of calcium transport pathways was further assessed using verapamil and phentolamine. Molecular docking studies were performed against major calcium transport proteins, including Ca²⁺-ATPase, SERCA, NCX1, RyR, and voltage-gated calcium channels. KC-4 produced concentration-dependent relaxation of both KCl- and phenylephrine-induced contractions, with IC₅₀ values of 23.15 μM and 18.21 μM, respectively. The greater inhibition of phenylephrine-induced contraction suggests that KC-4 modulates both voltage-dependent and receptor-operated calcium signaling pathways. Comparative experiments with verapamil and phentolamine further supported the involvement of calcium transport mechanisms in the relaxant activity of KC-4. Molecular docking analysis revealed favorable binding affinities of KC-4 toward Ca²⁺-ATPase (−11.7 kcal/mol), L-type calcium channels (−10.8 kcal/mol), SERCA (−10.2 kcal/mol), and R-type calcium channels (−10.2 kcal/mol), whereas weaker interactions were observed with NCX1 and RyR. These findings indicate that KC-4 may regulate vascular smooth muscle contraction through modulation of multiple calcium transport systems. The combined experimental and computational results identify KC-4 as a promising calcium transport-modulating compound with potential relevance for the development of novel agents targeting vascular disorders.