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

Abstract

1 The haeme-containing soluble guanylyl cyclase (a 1 b 1 -heterodimer) is a major intracellular receptor and e ector 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, puri®ed 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 (IC 50 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 71 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 (IC 50 17 nM) and by 17 structural analogues in a similar manner, albeit with di erent 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 -CH 2 -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 (IC 50 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 (IC 50 30 nM). 5 In prostaglandin F 2a -constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC 50 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 mM) suppressed the relaxant response to nitroglycerin from 88.3+2.1 to 26.8+6.4% and induced a 9 fold rightward shift (EC 50 15 mM) of the concentrationrelaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 mM), but did not a ect 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 mM) did not a ect 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 a...

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

“…These data indicate that an agonist-induced rise in cGMP levels does not result from modulation of PDE activity but represents de novo cGMP production. To identify which guanylyl cyclase, particulate or soluble, is responsible for agonist-induced cGMP production, cells were incubated in the presence of ODQ and NS 2028, two inhibitors of sGC (37,38). As shown in Fig.…”

Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells

“…To determine whether sGC is an important regulator of cGMP content in human ovarian cancer cells, OV2008 cells were cultured for 48 h, and then treated 20 min with ODQ (0-100 mM), a potent and specific sGC inhibitor (Garthwaite et al, 1995;Moro et al, 1996;Mulsch et al, 1997;Olesen et al, 1998). Cells were harvested and cGMP assays were performed as detailed in Materials and methods.…”

“…Furthermore, these findings suggest that sGC/cGMP-mediated survival of these cells proceeds, in part, by lowering basal p53 protein content and phosphorylation and p53-induced apoptosis, and that the basal activity of this pathway may alter p53 content and function by promoting p53 degradation. [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one) depletes cellular cGMP levels To determine whether sGC is an important regulator of cGMP content in human ovarian cancer cells, OV2008 cells were cultured for 48 h, and then treated 20 min with ODQ (0-100 mM), a potent and specific sGC inhibitor (Garthwaite et al, 1995;Moro et al, 1996;Mulsch et al, 1997;Olesen et al, 1998). Cells were harvested and cGMP assays were performed as detailed in Materials and methods.…”

Activators of soluble guanylate cyclase for the treatment of male erectile dysfunction

“…14 The ODQ analog, NS 2028, was described as another potent inhibitor of sGC (IC 50 ¼ 30 nM). 15 Methylene blue 16 and LY-83583 17 were reported as inhibitors of sGC, but they are less potent than ODQ and NS 2028. Furthermore, they inhibit not only sGC activity but they affect the biological activity of NO synthase and glucose transporters.…”