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BAY 41-8543 is a novel, highly specific and so far the most potent NO-independent stimulator of sGC. Here we report the effects of BAY 41-8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation. BAY 41-8543 stimulates the recombinant sGC concentration-dependently from 0.0001 microM to 100 microM up to 92-fold. In combination, BAY 41-8543 and NO have synergistic effects 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. 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. In the rat heart Langendorff preparation, BAY 41-8543 potently reduces coronary perfusion pressure from 10(-9) to 10(-6) g ml(-1) without any effect on left ventricular pressure and heart rate. BAY 41-8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate-tolerant rats. BAY 41-8543 is a potent inhibitor of collagen-mediated aggregation in washed human platelets (IC(50)=0.09 microM). In plasma, BAY 41-8543 inhibits collagen-mediated aggregation better than ADP-induced aggregation, but has no effect 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. 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 profile may offer a novel approach for treating cardiovascular diseases.

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.

Background: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase., Results: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL., Conclusions: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the alpha and beta1 subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min(-1) mg(-1) in the presence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg2+, whereas the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 microM. The combination of DEA/NO (10 microM) and YC-1 (100 microM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg2+, resulting in a specific activity of 121 micromol min(-1) mg(-1). The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 microM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators.