CHARACTERIZATION OF NS-2028 AS A SPECIFIC INHIBITOR OF SOLUBLE GUANYLYL CYCLASE

1 The haeme-containing soluble guanylyl cyclase (alpha(1) beta(1)-hete rodimer) is a major intracellular receptor and effector for nitric oxi de (NO) and carbon monoxide (GO) 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 cy clase activity in vitro, on the formation of cyclic GMP in cultured ce lls and on the NO-dependent relaxation of vascular and non-vascular sm ooth muscle. 2 Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by mo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)ox azin-1-one (NS 2028) in a concentration-dependent and irreversible man ner (IC50 30 nM for basal and 200 nM for NO-stimulated enzyme activity ). Evaluation of the inhibition kinetics according to Kitz & Wilson yi elded 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 inhibitio n. Inhibition was accompanied by a shift in the soret absorption maxim um of the enzyme's haem cofactor from 430 to 390 nm. 3 S-nitroso-gluta thione-enhanced soluble guanylyl cyclase activity in homogenates of mo use cerebellum was inhibited by NS 2028 (IC50 17 nM) and by 17 structu ral analogues in a similar manner, albeit with different potency, depe nding on the type of substitution at positions 1, 7 and 8 of the benzo xazin structure. Small electronegative ligands such as Br and Cl at po sition 7 or 8 increased and substitution of the oxygen at position 1 b y -S-,- NH- or -CH2- decreased the inhibition. 4 In tissue slices prep ared 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 a nalogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formati on of cyclic GMP in human cultured umbilical vein endothelial cells wa s inhibited by NS 2028 (IC50 30 nM). 5 In prostaglandin F-2 alpha-cons tricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC50 17 0 nM), which was abolished by removal of the endothelium. NS 2028 (1 m u M) 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 mu M) of the concentration-relaxation response curve to nitroglycerin. It abol ished the relaxation to sodium nitroprusside (1 mu M), bur did not aff ect the vasorelaxation to the K-ATP channel opener cromakalim. Approxi mately 50% of the relaxant response to sodium nitroprusside was recove red after 2 h washout of NS 2028. 6 In phenylephrine-preconstricted, e ndothelium-denuded aorta of the rabbit NS 2028 (1 mu M) did not affect relaxant responses to atrial natriuretic factor, an activator of part iculate guanylyl cyclase, or forskolin, an activator of adenylyl cycla se. 7 NO-dependent relaxant responses in non-vascular smooth muscle we re also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 mu M), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The re laxant responses to electrical field stimulation of non-adrenergic, no n-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 mu M) 8 NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3, a]quinoxali n-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a cha nge in the coordination of the haeme-iron of guanylyl cyclase is a lik ely inhibitory mechanism.