Deciphering the Mechanism and Impact of NS 2028 on Soluble Guanylyl Cyclase Inhibition

The study investigates the effects of newly synthesized oxadiazolo-benz-oxazins, particularly NS 2028, on soluble guanylyl cyclase, a key intracellular receptor for nitric oxide (NO) and carbon monoxide (CO), which mediates their biological actions by increasing cyclic GMP levels.

NS 2028 was found to inhibit soluble guanylyl cyclase purified from bovine lung in a concentration-dependent and irreversible manner, with distinct IC50 values for basal and NO-stimulated enzyme activity. The inhibition kinetics revealed a Ki value and an irreversible inhibition rate constant. The inhibition was associated with a shift in the enzyme's heme cofactor absorption maximum.

In mouse cerebellum homogenates, NS 2028 and its analogues inhibited S-nitrosoglutathione-enhanced soluble guanylyl cyclase activity, with the potency varying based on the structural modifications. Small electronegative ligands increased the inhibition, while substitution of oxygen at position 1 decreased it.

NS 2028 also inhibited neuronal NO synthase-dependent activation of soluble guanylyl cyclase in mouse cerebellum tissue slices and the formation of cyclic GMP in human endothelial cells induced by SIN-1.

In endothelium-intact porcine coronary arteries, NS 2028 increased contractile tone in a concentration-dependent manner, an effect abolished by endothelium removal. It suppressed the relaxant response to nitroglycerin and shifted the concentration-relaxation response curve. NS 2028 did not affect vasorelaxation to cromakalim or forskolin, indicating selectivity.

In rabbit aorta, NS 2028 did not influence relaxant responses to atrial natriuretic factor or forskolin. Furthermore, NS 2028 inhibited NO-dependent relaxant responses in non-vascular smooth muscle, such as guinea-pig trachea preconstricted with histamine.

The findings suggest that NS 2028 and its analogues are potent and specific inhibitors of soluble guanylyl cyclase in various cell types, with a possible inhibitory mechanism involving oxidation and/or changes in the coordination of the heme-iron of guanylyl cyclase.