Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.

1 Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (a/û) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2 Through a high-throughput screen we identi®ed BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem de®cient. 3 Binding studies with radiolabelled BAY 58-2667 show a high anity site on the enzyme. 4 Using photoanity labelling studies we identi®ed the amino acids 371 (a-subunit) and 231 ± 310 (û-subunit) as target regions for BAY 58-2667. 5 sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not in¯uenced by nitrate tolerance. 6 BAY 58-2667 shows a potent antihypertensive eect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic eects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7 This novel type of sGC activator is a valuable research tool and may oer a new approach for treating cardiovascular diseases.

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1 BAY 41-8543 is a novel, highly speci®c and so far the most potent NO-independent stimulator of sGC. Here we report the eects of BAY 41-8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendor heart preparation. 2 BAY 41-8543 stimulates the recombinant sGC concentration-dependently from 0.0001 mM to 100 mM up to 92-fold. In combination, BAY 41-8543 and NO have synergistic eects over a wide range of concentrations. Similar results are shown in implying that BAY 41-8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO. 3 In vitro, BAY 41-8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC 50 -values in the nM range. 4 In the rat heart Langendor preparation, BAY 41-8543 potently reduces coronary perfusion pressure from 10 79 to 10 76 g ml 71 without any eect on left ventricular pressure and heart rate. 5 BAY 41-8543 is eective even under nitrate tolerance conditions proved by the same vasorelaxing eect on aortic rings taken either from normal or nitrate-tolerant rats. 6 BAY 41-8543 is a potent inhibitor of collagen-mediated aggregation in washed human platelets (IC 50 =0.09 mM). In plasma, BAY 41-8543 inhibits collagen-mediated aggregation better than ADPinduced aggregation, but has no eect on the thrombin pathway. BAY 41-8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations. 7 These results suggest that BAY 41-8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique pro®le may oer a novel approach for treating cardiovascular diseases. IntroductionGuanylyl cyclases (GTP pyrophosphate-lyase [cyclizing]; EC 4.6.1.2) catalyse the biosynthesis of cyclic GMP from GTP. While the membrane bound forms are monomers which are stimulated by the natriuretic peptides, the soluble guanylyl cyclases exist as heterodimers consisting of an a-and a bsubunit and contain a heme as a prosthetic group (Wedel & Garbers, 1997). By formation of cyclic GMP as a second messenger, sGC plays an important role in smooth muscle cell relaxation (Lincoln, 1989), inhibition of platelet aggregation, retinal signal transduction (Moncada & Higgs, 1995) and synaptic transmission (Zhuo & Hawkins, 1995). The sGC is the intracellular receptor for the ubiquitous biological messenger NO (Moncada et al., 1991;Furchgott, 1999;Murad, 1999;Ignarro, 1999) and is also activated by the benzylindazole derivative YC-1 (Ko et al., 1994; MuÈ lsch et al., 1997;Friebe et al., 1996;Hoenicka et al., 1999). In several studies, YC-1 was shown to inhibit platelet aggregation by elevation of cyclic GMP causing VASP phosphorylation (Wu et al., 1995;1997;Ko et al., 1994;Friebe et al., 1998;Becker et al., 2000) and to relax precontracted aortic rings (MuÈ lsch et al., 1997). Interestingly, in addition to the direct activation of the puri®ed sGC by YC-1, an overadditive eect was observed by the c...

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