Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase

Abstract

1. The haeme-containing soluble guanylyl cyclase (α₁β₁-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle.

2. Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC₅₀ 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for K_i, the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min⁻¹ for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm.

3. S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC₅₀ 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH₂- decreased the inhibition.

4. In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC₅₀ 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC₅₀ 30 nM).

5. In prostaglandin F_2α-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC₅₀ 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 μM) suppressed the relaxant response to nitroglycerin from 88.3±2.1 to 26.8±6.4% and induced a 9 fold rightward shift (EC₅₀ 15 μM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 μM), but did not affect the vasorelaxation to the K_ATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028.

6. In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 μM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase.

7. NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 μM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 μM).

8. NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism.

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