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Herein, we describe the identification, chemical optimization, and preclinical characterization of novel soluble guanylate cyclase (sGC) stimulators. Given the very broad therapeutic opportunities for sGC stimulators, new tailored molecules for distinct indications with specific pharmacokinetics, tissue distribution, and physicochemical properties will be required in the future. Here, we report the ultrahigh-throughput (uHTS)-based discovery of a new class of sGC stimulators from an imidazo[1,2- a ]pyridine lead series. Through the extensive and staggered optimization of the initial screening hit, liabilities such as potency, metabolic stability, permeation, and solubility could be substantially improved in parallel. These efforts resulted ultimately in the discovery of the new sGC stimulators 22 and 28 . It turned out that BAY 1165747 (BAY-747, 28 ) could be an ideal treatment alternative for patients with hypertension, especially those not responding to standard anti-hypertensive therapy (resistant hypertension). BAY-747 ( 28 ) demonstrated sustained hemodynamic effects up to 24 h in phase 1 studies.

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.

Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators.

Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing β1-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities.

Direct stimulation of soluble guanylate cyclase (sGC) is emerging as a potential new approach for the treatment of renal disorders. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), deficiency of which is implicated in the pathogenesis of chronic kidney disease (CKD). Therefore, new classes of drugs - sGC stimulators and activators - are being investigated in preclinical models under conditions where nitric oxide is deficient. In preclinical models with different etiologies of CKD, the sGC stimulators BAY 41-2272, BAY 41-8543, BAY 60-4552, riociguat and vericiguat and the sGC activators cinaciguat, ataciguat, BI 703704 and GSK2181236A have shown consistently renoprotective effects. Clinical trials are required to confirm these findings in humans, and to ascertain whether these agents could provide a future alternative to guideline-recommended treatments., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Human variation in susceptibility to hypoxia-induced pulmonary hypertension is well recognized. High-altitude residents who do not develop pulmonary hypertension may host protective gene mutations., Methods and Results: Exome sequencing was conducted on 24 unrelated Kyrgyz highlanders living 2400 to 3800 m above sea level, 12 (10 men; mean age, 54 years) with an elevated mean pulmonary artery pressure (mean±SD, 38.7±2.7 mm Hg) and 12 (11 men; mean age, 52 years) with a normal mean pulmonary artery pressure (19.2±0.6 mm Hg) to identify candidate genes that may influence the pulmonary vascular response to hypoxia. A total of 140 789 exomic variants were identified and 26 116 (18.5%) were classified as novel or rare. Thirty-three novel or rare potential pathogenic variants (frameshift, essential splice-site, and nonsynonymous) were found exclusively in either ≥3 subjects with high-altitude pulmonary hypertension or ≥3 highlanders with a normal mean pulmonary artery pressure. A novel missense mutation in GUCY1A3 in 3 subjects with a normal mean pulmonary artery pressure encodes an α1-A680T soluble guanylate cyclase (sGC) variant. Expression of the α1-A680T sGC variant in reporter cells resulted in higher cyclic guanosine monophosphate production compared with the wild-type enzyme and the purified α1-A680T sGC exhibited enhanced sensitivity to nitric oxide in vitro., Conclusions: The α1-A680T sGC variant may contribute to protection against high-altitude pulmonary hypertension and supports sGC as a pharmacological target for reducing pulmonary artery pressure in humans at altitude., (© 2014 American Heart Association, Inc.)

Background: Cinaciguat (BAY 58-2667), an NO- and heme-independent sGC activator, was shown to be more effective when the heme-group of sGC is oxidized in vascular tissue. In apo-sGC mice (sGCβ1 (His105Phe) knockin) both sGC isoforms (sGCα1 β1 and sGCα2 β1 ) are heme-deficient and can no longer be activated by NO; these mice, showing decreased gastrointestinal nitrergic relaxation and decreased gastric emptying, can be considered as a model to study the consequence of heme-oxidation in sGC. Our aim was to compare the influence of cinaciguat, on in vitro muscle tone of gastrointestinal tissues, and on gastric emptying in WT and apo-sGC mice., Methods: Gastrointestinal smooth muscle strips were mounted in organ baths for isometric force recording and cGMP levels were determined by enzyme immunoassay. Protein levels of sGC subunits were assessed by immunoblotting. Gastric emptying was determined by phenol red recovery., Key Results: Although protein levels of the sGC subunits were lower in gastrointestinal tissues of apo-sGC mice, cinaciguat induced concentration-dependent relaxations and increased cGMP levels in apo-sGC fundus and colon to a similar or greater extent than in WT mice. The sGC inhibitor ODQ increased cinaciguat-induced relaxations and cGMP levels in WT fundus and colon. In apo-sGC antrum, pylorus and jejunum, cinaciguat was not able to induce relaxations. Cinaciguat did not improve delayed gastric emptying in apo-sGC mice., Conclusions & Inferences: Cinaciguat relaxes the fundus and colon efficiently when sGC is in the heme-free condition; the non-effect of cinaciguat in pylorus explains its inability to improve the delayed gastric emptying in apo-sGC mice., (© 2014 John Wiley & Sons Ltd.)

We studied the influence of soluble guanylate (sGC) on renal blood flow (RBF), glomerular filtration rate (GFR), and RBF autoregulation and its role in mediating the hemodynamic effects of endogenous nitric oxide (NO). Arterial pressure (AP), heart rate (HR), RBF, GFR, urine flow (UV), and the efficiency and mechanisms of RBF autoregulation were studied in anesthetized rats during intravenous infusion of sGC activator cinaciguat before and (except GFR) also after inhibition of NO synthase (NOS) by Nω-nitro-L-arginine methyl ester. Cinaciguat (0.1, 0.3, 1, 3, 10 μg·kg(-1)·min(-1), n=7) reduced AP and increased HR, but did not significantly alter RBF. In clearance experiments (FITC-sinistrin, n=7) GFR was not significantly altered by cinaciguat (0.1 and 1 μg·kg(-1)·min(-1)), but RBF slightly rose (+12%) and filtration fraction (FF) fell (-23%). RBF autoregulatory efficiency (67 vs. 104%) and myogenic response (33 vs. 44 units) were slightly depressed (n=9). NOS inhibition (n=7) increased AP (+38 mmHg), reduced RBF (-53%), and greatly augmented the myogenic response in RBF autoregulation (97 vs. 35 units), attenuating the other regulatory mechanisms. These changes were reversed by 77, 78, and 90% by 1 μg·kg(-1)·min(-1) cinaciguat. In vehicle controls (n=3), in which cinaciguat-induced hypotension was mimicked by aortic compression, the NOS inhibition-induced changes were not affected. We conclude that sGC activation leaves RBF and GFR well maintained despite hypotension and only slightly impairs autoregulation. The ability to largely normalize AP, RBF, RBF autoregulation, and renovascular myogenic response after NOS inhibition indicates that these hemodynamic effects of NO are predominantly mediated via sGC., (Copyright © 2014 the American Physiological Society.)

Rationale: Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available., Objectives: To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions., Methods: Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done., Measurements and Main Results: The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils., Conclusions: The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion during maturation of sGC to its NO-responsive active form. Here, we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response (i) required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGC-β1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60-2770; and (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90-dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGC-α1 in response to NO is unprecedented and represent new steps by which cells can modulate the heme content and activity of sGC for signaling cascades., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step toward this goal is the identification of high-affinity ligands that stabilize an sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGCα1β1 by 38 purine- and pyrimidine-nucleotides with 2,4,6,-trinitrophenyl and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with that for the structurally related membranous adenylyl cyclases (mACs) 1, 2, 5 and the purified mAC catalytic subunits VC1:IIC2. TNP-GTP [2',3'-O-(2,4,6-trinitrophenyl)-GTP] was the most potent sGCα1β1 inhibitor (Ki, 10.7 nM), followed by 2'-MANT-3'-dATP [2'-O-(N-methylanthraniloyl)-3'-deoxy-ATP] (Ki, 16.7 nM). Docking studies on an sGCαcat/sGCβcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of (M)ANT- and TNP-nucleotides with sGCα1β1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1β1 require a 3'-endo ribosyl conformation (versus 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGCα1β1 versus mACs 1, 2, 5 correlated poorly. Collectively, we identified highly potent sGCα1β1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.

Aims: Guanylyl cyclase-cyclic guanosine monophosphate signalling plays an important role in endogenous cardioprotective signalling. The aim was to assess the potential of direct pharmacological activation and stimulation of soluble guanylyl cyclase, targeting different redox states of the enzyme, to limit myocardial necrosis during early reperfusion., Methods and Results: Rat isolated hearts were subjected to reversible left coronary artery occlusion (ischaemia-reperfusion) and infarct size was assessed by the tetrazolium staining technique. Administration during early reperfusion of BAY 41-2272, an NO-independent, haem-dependent stimulator of soluble guanylyl cyclase targeting the reduced state, or BAY 60-2770, an NO-independent, haem-independent activator targeting the oxidized state, significantly limited infarct size. Inhibition of NO synthesis did not abrogate this protection, but exogenous perfusion of NO with BAY 41-2272 produced a synergistic effect. The haem site oxidiser, ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidized forms of sGC together did not afford additive protection., Conclusions: Targeting either reduced or oxidized forms of sGC during early reperfusion affords cardioprotection, providing support for the concept that direct sGC manipulation at reperfusion has therapeutic potential for the management of acute myocardial infarction.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1β1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: Stimulation of the nitric oxide (NO)--soluble guanylate (sGC)--protein kinase G (PKG) pathway confers protection against acute ischaemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI) heart failure are less defined. The aim of this study was to not only determine whether the sGC stimulator riociguat reduces infarct size but also whether it protects against the development of post-MI heart failure., Methods and Results: Mice were subjected to 30 min ischaemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l) were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI) and (18)F-FDG-positron emission tomography (PET) were performed to determine infarct size. In the riociguat-treated mice, the resulting infarct size was smaller (8.5 ± 2.5% of total LV mass vs. 21.8% ± 1.7%. in controls, p = 0.005) and LV systolic function analysed by MRI was better preserved (60.1% ± 3.4% of preischaemic vs. 44.2% ± 3.1% in controls, p = 0.005). After 28 days, LV systolic function by echocardiography treated group was still better preserved (63.5% ± 3.2% vs. 48.2% ± 2.2% in control, p = 0.004)., Conclusion: Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size and better long-term preservation of LV systolic function. These findings suggest that sGC stimulation during reperfusion therapy may be a powerful therapeutic treatment strategy for preventing post-MI heart failure.

Aims: Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation., Results: Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770., Innovation and Conclusion: These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.

The vasodilatory properties of nitric oxide (NO) have been utilized in pharmacotherapy for more than 130 years. Still today, NO-donor drugs are important in the management of cardiovascular diseases. However, inhaled NO or drugs releasing NO and organic nitrates are associated with noteworthy therapeutic shortcomings, including resistance to NO in some disease states, the development of tolerance during long-term treatment, and nonspecific effects, such as post-translational modification of proteins. The beneficial actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC), a heme-containing enzyme which produces the intracellular signaling molecule cyclic guanosine monophosphate (cGMP). Recently, two classes of compounds have been discovered that amplify the function of sGC in a NO-independent manner, the so-called sGC stimulators and sGC activators. The most advanced drug, the sGC stimulator riociguat, has successfully undergone Phase III clinical trials for different forms of pulmonary hypertension., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC's salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-Nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.

The number of annual hospitalizations for heart failure (HF) and the mortality rates among patients hospitalized for HF remains unacceptably high. The search continues for safe and effective agents that improve outcomes when added to standard therapy. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway serves an important physiologic role in both vascular and non-vascular tissues, including regulation of myocardial and renal function, and is disrupted in the setting of HF, leading to decreased protection against myocardial injury, ventricular remodeling, and the cardio-renal syndrome. The impaired NO-sGC-cGMP pathway signaling in HF is secondary to reduced NO bioavailability and an alteration in the redox state of sGC, making it unresponsive to NO. Accordingly, increasing directly the activity of sGC is an attractive pharmacologic strategy. With the development of two novel classes of drugs, sGC stimulators and sGC activators, the hypothesis that restoration of NO-sGC-cGMP signaling is beneficial in HF patients can now be tested. Characterization of these agents in pre-clinical and clinical studies has begun with investigations suggesting both hemodynamic effects and organ-protective properties independent of hemodynamic changes. The latter could prove valuable in long-term low-dose therapy in HF patients. This review will explain the role of the NO-sGC-cGMP pathway in HF pathophysiology and outcomes, data obtained with sGC stimulators and sGC activators in pre-clinical and clinical studies, and a plan for the further clinical development to study these agents as HF therapy.

Novel guanylate cyclase stimulators are disclosed. Design, synthesis, SAR, and pharmacological profile of the compounds are discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of the selective Rho-associated protein kinase (ROCK) inhibitor azaindole-1 on erectile function under physiologic and pathophysiologic conditions in the rat., Methods: The effect of intracavernosal (i.c.) injections of azaindole-1 on change in intracavernous pressure (ICP), ICP/mean arterial pressure (MAP), area under the curve (AUC), and response duration were investigated in the anesthetized rat under control conditions and when nonadrenergic noncholinergic neurotransmission and cholinergic function or soluble guanylyl cyclase (sGC) were inhibited or after cavernosal nerve crush injury., Results: The i.c. injections of azaindole-1 produced dose-related increases in ICP/MAP and AUC that were long-lasting at the highest doses studied compared with the prototypical ROCK inhibitor fasudil. Erectile responses were not altered by 7-nitroindazole and atropine in doses that reduced the response to cavernosal nerve stimulation by 86%, indicating that they were independent of NO release by cavernosal nerves or activation of muscarinic receptors in the corpora cavernosa. Erectile responses to azaindole-1 were not altered by the sGC inhibitor ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside, indicating that they were independent of an action on sGC. The erectile response to i.c. injections of azaindole-1 or Y-27632, which was reported to be NO/cyclic guanosine monophosphate-dependent, was not attenuated after cavernosal nerve crush injury., Conclusion: The present studies indicate that azaindole-1 has long-lasting erectile activity that is independent of NO release, muscarinic receptor, or sGC activation or the integrity of the cavernosal nerves., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Soluble guanylate cyclase (sGC) is a key enzyme in the nitric oxide (NO) signalling pathway. On binding of NO to its prosthetic haem group, sGC catalyses the synthesis of the second messenger cyclic guanosine monophosphate (cGMP), which promotes vasodilation and inhibits smooth muscle proliferation, leukocyte recruitment, platelet aggregation and vascular remodelling through a number of downstream mechanisms. The central role of the NO-sGC-cGMP pathway in regulating pulmonary vascular tone is demonstrated by the dysregulation of NO production, sGC activity and cGMP degradation in pulmonary hypertension (PH). The sGC stimulators are novel pharmacological agents that directly stimulate sGC, both independently of NO and in synergy with NO. Optimisation of the first sGC stimulator, YC-1, led to the development of the more potent and more specific sGC stimulators, BAY 41-2272, BAY 41-8543 and riociguat (BAY 63-2521). Other sGC stimulators include CFM-1571, BAY 60-4552, vericiguat (BAY 1021189), the acrylamide analogue A-350619 and the aminopyrimidine analogues. BAY 41-2272, BAY 41-8543 and riociguat induced marked dose-dependent reductions in mean pulmonary arterial pressure and vascular resistance with a concomitant increase in cardiac output, and they also reversed vascular remodelling and right heart hypertrophy in several experimental models of PH. Riociguat is the first sGC stimulator that has entered clinical development. Clinical trials have shown that it significantly improves pulmonary vascular haemodynamics and increases exercise ability in patients with pulmonary arterial hypertension (PAH), chronic thromboembolic PH and PH associated with interstitial lung disease. Furthermore, riociguat reduces mean pulmonary arterial pressure in patients with PH associated with chronic obstructive pulmonary disease and improves cardiac index and pulmonary vascular resistance in patients with PH associated with left ventricular systolic dysfunction. These promising results suggest that sGC stimulators may constitute a valuable new therapy for PH. Other trials of riociguat are in progress, including a study of the haemodynamic effects and safety of riociguat in patients with PH associated with left ventricular diastolic dysfunction, and long-term extensions of the phase 3 trials investigating the efficacy and safety of riociguat in patients with PAH and chronic thromboembolic PH. Finally, sGC stimulators may also have potential therapeutic applications in other diseases, including heart failure, lung fibrosis, scleroderma and sickle cell disease.

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H(2)S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

Background: The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63-2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO-sGC-cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH., Methods and Results: Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55 ± 0.02, p<0.05), increased cardiac output (60.8 ± .8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03 ± 0.3 mmHg min(-1) ml(-1) 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05)., Conclusion: Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α(1)- and β(1)-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H(2)O(2)). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively (P < 0.01 vs. controls). Hyperoxia or H(2)O(2) enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.

Objective: The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy., Methods/results: Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients., Conclusion: We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.

Background: A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension., Methods and Results: Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1., Conclusions: Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.

Objectives: The nitric oxide-soluble guanylate cyclase (sGC)-cGMP signal transduction pathway is impaired in different cardiovascular diseases, including pulmonary hypertension, heart failure and arterial hypertension. Riociguat is a novel stimulator of soluble guanylate cyclase (sGC). However, little is known about the effects of sGC stimulators in experimental models of hypertension. We thus investigated the cardio-renal protective effects of riociguat in low-renin and high-renin rat models of hypertension., Methods: The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate tolerant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model)., Results: In both animal models, riociguat treatment improved survival and normalized blood pressure. Moreover, in the L-NAME study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target organ damage as indicated by lower plasma creatinine and urea, less glomerulosclerosis and less renal interstitial fibrosis. In the 5/6 nephrectomy study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight, lower myocyte diameter and lower arterial media/lumen ratio, and reduced renal target organ damage as indicated by improved creatinine clearance and less renal interstitial fibrosis., Conclusion: We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the alphabeta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both alpha and beta subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.

Aims: BAY 58-2667 (BAY-58) directly activates soluble guanylyl cyclase without tolerance in a nitric oxide (NO)-independent manner, and its haemodynamic effect is similar to that of nitroglycerin. We tested whether BAY-58 could make both rabbit and rat hearts resistant to infarction when given at the end of an ischaemic insult., Methods and Results: All hearts were exposed to 30 min regional ischaemia followed by 120-(isolated hearts) or 180-(in situ hearts) min reperfusion. BAY-58 (1-50 nM) infused for 60 min starting 5 min before reperfusion significantly reduced infarction from 33.0 +/- 3.2% in control isolated rabbit hearts to 9.5-12.7% (P < 0.05). In a more clinically relevant in situ rabbit model, infarct size was similarly reduced with a loading dose of 53.6 microg/kg followed by a 60 min infusion of 1.25 microg/kg/min (41.1 +/- 3.1% infarction in control hearts to 16.0 +/- 4.4% in treated hearts, P < 0.05). BAY-58 similarly decreased infarction in the isolated rat heart, and protection was abolished by co-treatment with a protein kinase G (PKG) antagonist, or a mitochondrial K(ATP) channel antagonist. Conversely, N(omega)-nitro-L-arginine-methyl-ester-hydrochloride, a NO-synthase inhibitor, failed to block BAY-58's ability to decrease infarction, consistent with the latter's putative NO-independent activation of PKG. Finally, BAY-58 increased myocardial cGMP content in reperfused hearts while cAMP was unchanged., Conclusion: When applied at reperfusion, BAY-58 is an effective cardioprotective agent with a mechanism similar to that of ischaemic pre-conditioning and, hence, should be a candidate for treatment of acute myocardial infarction in man.

Cardiac fibrosis is a hallmark of cardiovascular remodeling associated with hypertension. The purpose of this study was to explore the effect and mechanism of soluble guanylate cyclase (sGC) stimulator BAY 41-2272, leading to intracellular cyclic guanosine monophosphate (cGMP) elevation, on the remodeling process induced by pressure overload. Seven-week-old male Wistar rats with hypertension induced by suprarenal aortic constriction (AC) were treated orally with 2 mg kg(-1) day(-1) of BAY 41-2272 for 14 days. BAY 41-2272 had no effects on blood pressure, but decreased AC-induced collagen accumulation in the left ventricle (LV), inhibiting the number of myofibroblasts and gene expressions of transforming growth factor-beta1 and type 1 collagen. In addition, the antifibrotic action of BAY 41-2272 was accompanied by reducing AC-induced angiotensin-converting enzyme (ACE) mRNA and its enzymatic activity, and angiotensin II concentration in LV. In cultured cardiac fibroblasts, BAY 41-2272 inhibited ACE synthesis and myofibroblast transformation, accompanied by elevating the intracellular cGMP concentration. These results suggest that sGC stimulator BAY 41-2272 might be effective to reduce fibrosis in hypertensive heart disease by attenuating angiotensin II generation through myofibroblast transformation.

The protective effects of nitric oxide (NO), a physiological activator of soluble guanylyl cyclase (sGC), have been reported in ischemia-reperfusion (I/R) syndrome of the lung. Therefore, we studied the effects of BAY 41-2272, a novel sGC stimulator, on I/R injury of the lung in an isolated intact organ model. Lung injury was assessed by measuring weight gain and microvascular permeability (capillary filtration coefficient, K(fc)). Release of reactive oxygen species (ROS) into the perfusate was measured during early reperfusion by electron spin resonance (ESR) spectroscopy. Rabbit lungs were treated with BAY 41-2272, N(G)-monomethyl-L-arginine (L-NMMA), or NO to evaluate the effects on I/R-induced lung injury. In untreated lungs, a dramatic rise in K(fc) values and weight gain during reperfusion were observed, and these results were associated with increased ROS production. Both, BAY 41-2272 and L-NMMA significantly attenuated vascular leakage and suppressed ROS release. Additional experiments showed that BAY 41-2272 diminished PMA-induced ROS production by NADPH oxidase. A pharmacological inhibition of the enzyme with consequent reduction in ROS levels decreased I/R injury. NO had only marginal effect on I/R injury. Thus BAY 41-2272 protects against I/R-induced lung injury by interfering with the activation of NADPH oxidases.

The nitric oxide (NO) signalling pathway is altered in cardiovascular diseases, including systemic and pulmonary hypertension, stroke, and atherosclerosis. The vasodilatory properties of NO have been exploited for over a century in cardiovascular disease, but NO donor drugs and inhaled NO are associated with significant shortcomings, including resistance to NO in some disease states, the development of tolerance during long-term treatment, and non-specific effects such as post-translational modification of proteins. The development of pharmacological agents capable of directly stimulating the NO receptor, soluble guanylate cyclase (sGC), is therefore highly desirable. The benzylindazole compound YC-1 was the first sGC stimulator to be identified; this compound formed a lead structure for the development of optimized sGC stimulators with improved potency and specificity for sGC, including CFM-1571, BAY 41-2272, BAY 41-8543, and BAY 63-2521. In contrast to the NO- and haem-independent sGC activators such as BAY 58-2667, these compounds stimulate sGC activity independent of NO and also act in synergy with NO to produce anti-aggregatory, anti-proliferative, and vasodilatory effects. Recently, aryl-acrylamide compounds were identified independent of YC-1 as sGC stimulators; although structurally dissimilar to YC-1, they have a similar mode of action and promote smooth muscle relaxation. Pharmacological stimulators of sGC may be beneficial in the treatment of a range of diseases, including systemic and pulmonary hypertension, heart failure, atherosclerosis, erectile dysfunction, and renal fibrosis. An sGC stimulator, BAY 63-2521, is currently in clinical development as an oral therapy for patients with pulmonary hypertension. It has demonstrated efficacy in a proof-of-concept study, reducing pulmonary vascular resistance and increasing cardiac output from baseline. A full, phase 2 trial of BAY 63-2521 in pulmonary hypertension is underway.

Oxidative stress, a risk factor for several cardiovascular disorders, interferes with the NO/sGC/cGMP signalling pathway through scavenging of NO and formation of the strong intermediate oxidant, peroxynitrite. Under these conditions, endothelial and vascular dysfunction develops, culminating in different cardio-renal and pulmonary-vascular diseases. Substituting NO with organic nitrates that release NO (NO donors) has been an important principle in cardiovascular therapy for more than a century. However, the development of nitrate tolerance limits their continuous clinical application and, under oxidative stress and increased formation of peroxynitrite foils the desired therapeutic effect. To overcome these obstacles of nitrate therapy, direct NO- and haem-independent sGC activators have been developed, such as BAY 58-2667 (cinaciguat) and HMR1766 (ataciguat), showing unique biochemical and pharmacological properties. Both compounds are capable of selectively activating the oxidized/haem-free enzyme via binding to the enzyme's haem pocket, causing pronounced vasodilatation. The potential importance of these new drugs resides in the fact that they selectively target a modified state of sGC that is prevalent under disease conditions as shown in several animal models and human disease. Activators of sGC may be beneficial in the treatment of a range of diseases including systemic and pulmonary hypertension (PH), heart failure, atherosclerosis, peripheral arterial occlusive disease (PAOD), thrombosis and renal fibrosis. The sGC activator HMR1766 is currently in clinical development as an oral therapy for patients with PAOD. The sGC activator BAY 58-2667 has demonstrated efficacy in a proof-of-concept study in patients with acute decompensated heart failure (ADHF), reducing pre- and afterload and increasing cardiac output from baseline. A phase IIb clinical study for the indication of ADHF is currently underway.

Unlabelled: Liver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellate cell activation. Activation of hepatic stellate cells is reduced by an increase in cyclic guanosine monophosphate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to the reduced activity of the endothelial nitric oxide synthase., Objective: Here we report that the novel activator of soluble guanylate cyclase (sGC), BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid), which increases the activity of sGC in a nitric oxide-independent manner, attenuates liver fibrosis in two rat models., Methods: The compound was studied in the pig serum model and the carbon tetrachloride model. Fibrosis was assessed by estimating the increase in fibrous collagen by micromorphometry of histological sections stained with Sirius Red/Fast Green and by measuring total hepatic collagen., Results: BAY 60-2770, on a recombinant sGC reporter cell line, stimulated the luminescence signals with an EC50 value of 5.4 +/- 1.2 nmol/L. In the presence of [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 pmol/L) the EC50 was shifted to 0.39 +/- 0.11 nmol/L. In both fibrosis models, once daily oral administration of BAY 60-2770 concomittantly with the fibrotic stimulus prevented 60-75% of fibrosis, the lowest effective dose being 0.1 mg/kg in the pig serum model and 0.3 mg/kg in the carbon tetrachloride model. The treatment was well tolerated by all animals. The doses used were devoid of any significant influence on systemic blood pressure., Conclusion: Nitric oxide-independent activation of sGC might be an innovative therapeutic approach for the treatment of liver fibrosis of necro-inflammatory and immunological origin.

The ubiquitously expressed nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in signal transduction. Binding of NO to the N-terminal prosthetic heme moiety of sGC results in approximately 200-fold activation of the enzyme and an increased conversion of GTP into the second messenger cGMP. sGC exists as a heterodimer the dimerization of which is mediated mainly by the central region of the enzyme. In the present work, we constructed deletion mutants within the predicted dimerization region of the sGC alpha(1)- and beta(1)-subunit to precisely map the sequence segments crucial for subunit dimerization. To track mutation-induced alterations of sGC dimerization, we used a bimolecular fluorescence complementation approach that allows visualizing sGC heterodimerization in a noninvasive manner in living cells. Our study suggests that segments spanning amino acids alpha(1)363-372, alpha(1)403-422, alpha(1)440-459, beta(1)212-222, beta(1)304-333, beta(1)344-363, and beta(1)381-400 within the predicted dimerization region are involved in the process of heterodimerization and therefore in the expression of functional sGC.

Soluble guanylate cyclase is a heterodimeric enzyme with a prosthetic heme group that, on binding of its main ligand, NO, generates the second messenger cGMP. Unlike conventional nitrovasodilators, the novel direct NO- and heme-independent soluble guanylate cyclase activator BAY 58-2667 is devoid of non-cGMP actions, lacks tolerance development, and preferentially activates NO-insensitive heme-free or oxidized soluble guanylate cyclase. BAY 58-2667, therefore, represents a novel therapeutic advance in mediating vasodilation. To date, its cardiorenal actions in congestive heart failure (CHF) are undefined. We, therefore, hypothesized that BAY 58-2667 would have beneficial preload- and afterload-reducing actions in experimental severe CHF together with renal vasodilating properties. We assessed the cardiorenal actions of intravenous administration of 2 doses of BAY 58-2667 (0.1 and 0.3 microg/kg per minute, respectively) in a model of tachypacing-induced severe CHF. In CHF, BAY 58-2667 dose-dependently reduced mean arterial, right atrial, pulmonary artery, and pulmonary capillary wedge pressure (from baseline 19+/-1 to 12+/-2 mm Hg). Cardiac output (2.4+/-0.3 to 3.2+/-0.4 L/min) and renal blood flow increased. Glomerular filtration rate and sodium and water excretion were maintained. Consistent with cardiac unloading, atrial and B-type natriuretic peptide decreased. Plasma renin activity (P=0.31) and aldosterone remained unchanged (P=0.19). In summary, BAY 58-2667 in experimental CHF potently unloaded the heart, increased cardiac output and renal blood flow, and preserved glomerular filtration rate and sodium and water excretion without further neurohumoral activation. These beneficial properties make direct soluble guanylate cyclase stimulation with BAY 58-2667 a promising new therapeutic strategy for cardiovascular diseases, such as heart failure.

It is unknown whether long-term pharmacological stimulation of soluble guanylate cyclase (sGC), elevating intracellular cGMP levels, has a beneficial effect on hypertension. The purpose of this study is to investigate the effects of BAY41-2272, an orally available sGC stimulator, on cardiovascular remodeling in hypertensive rats. Eight-week-old male Wistar rats with hypertension induced by angiotensin II infused subcutaneously at 250 ng/kg per minute were treated orally with a low ([L] 2 mg/kg per day) or high ([H] 10 mg/kg per day) dose of BAY41-2272 for 14 days. BAY41-2272-H partially suppressed the rise in blood pressure and reduced the heart weight (4.20+/-0.34 versus 3.68+/-0.20 mg/g; P<0.01), whereas BAY41-2272-L had no effect. However, both doses decreased the angiotensin II-induced left ventricular accumulation of collagen in the perivascular area (L, -20%, P<0.05; H, -30%, P<0.01) and myocardial interstitium (L, -21%, P<0.05; H, -38%, P<0.01), reducing the number of activated fibroblasts surrounding coronary arteries (L, -74%; H, -79%; P<0.05). BAY41-2272 downregulated the angiotensin II-induced left ventricular gene expression of type 1 collagen (L, -41%, P<0.05; H, -49%, P<0.01) and transforming growth factor-beta1 (L, -49%, P<0.05; H, -65%, P<0.01). cGMP levels were elevated by BAY41-2272 not only in the left ventricle, but also in cultured cardiac fibroblasts, resulting in reduced thymidine incorporation into the cells. Thus, stimulation of sGC by BAY41-2272 attenuates fibrosis of the left ventricle in rats with angiotensin II-induced hypertension partly in a pressure-independent manner, suggesting an important role for sGC generating cGMP in inhibiting cardiovascular remodeling.

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.

1. Chronic renal disease is associated with oxidative stress, reduced nitric oxide (NO) availability and soluble guanylate cyclase (sGC) dysfunction. Recently, we discovered BAY 58-2667, a compound activating heme-deficient or oxidized sGC in a NO-independent manner. 2. We assessed potential of BAY 58-2667 in preventing cardiac and renal target organ damage in rats with 5/6 nephrectomy. 3. Male Wistar rats were allocated to three groups: 5/6 nephrectomy, 5/6 nephrectomy treated with BAY 58-2667 and sham operation. Study period was 18 weeks: blood pressure and creatinine clearance were assessed repeatedly. At study end blood samples were taken and hearts and kidneys harvested for histological studies. 4. BAY 58-2667 markedly lowered blood pressure in animals with 5/6 nephrectomy (untreated versus treated animals: 189+/-14 versus 146+/-11 mmHg, P<0.001). Left ventricular weight, cardiac myocyte diameter as well as cardiac arterial wall thickness significantly decreased in comparison to untreated animals with 5/6 nephrectomy. Natriuretic peptide plasma levels were also improved by BAY 58-2667. Kidney function and morphology as assessed by creatinine clearance, glomerulosclerosis, interstitial and perivascular fibrosis of intrarenal arteries were likewise significantly improved by BAY 58-2667. 5. This is the first study showing that BAY 58-2667 effectively lowers blood pressure, reduces left ventricular hypertrophy and slows renal disease progression in rats with 5/6 nephrectomy by targeting mainly oxidized sGC. Therefore, BAY 58-2667 represents a novel pharmacological principle with potential clinical value in treatment of chronic renal disease.

Background: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling., Methods and Results: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3(+/+)) or NOS3(-/-) mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3(-/-) mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes., Conclusions: Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.

Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.