Stimulation Of Soluble Guanylyl Cyclase Research Articles

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities

Herein, we describe the rational design, synthesis and in vitro functional characterization of new heme-dependent, direct soluble guanylyl cyclase (sGC) agonists. These new compounds bear a 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton, modified to enable efficient sGC binding and stimulation. To gain insights into structure–activity relationships, the N6-alkylation of the skeleton was explored, while a pyrimidine ring, substituted with various C5′-polar groups, was installed at position C3. Among the newly synthesized 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones, derivatives 14b, 15b and 16a display characteristic features of sGC “stimulators” in A7r5 vascular smooth muscle cells in vitro. They strongly synergize with the NO donor, sodium nitroprusside (SNP) in inducing cGMP generation in a manner that requires the presence of a reduced heme moiety associated with sGC, and elevate the cGMP-responsive phosphorylation of the protein VASP at Ser239. In line with their sGC stimulating capacity, docking calculations of derivatives 16a, 15(a–c) on a cryo-EM structure of human sGC (hsGC) in an ΝΟ-activated state indicated the implication of 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton in efficient bonding interactions with the recently identified region that binds known sGC stimulators, while the presence of either a N6-H or N6-methyl group pointed to enhanced binding affinity. Moreover, the in vitro functional effects of our newly identified sGC stimulators were compatible with a beneficial role in vascular homeostasis. Specifically, derivative 14b reduced A7r5 cell proliferation, while 16a dampened the expression of adhesion molecules ICAM-1 and P/E-Selectin in Human Umbilical Vein Endothelial Cells (HUVECs), as well as the subsequent adhesion of U937 leukocytes to the HUVECs, triggered by tumor necrosis factor alpha (TNF-α) or interleukin-1 beta (IL-1β). The fact that these compounds elevate cGMP only in the presence of NO may indicate a novel way of interaction with the enzyme and may make them less prone than other direct sGC agonists to induce characteristic hypotension in vivo.

Stimulation of soluble guanylyl cyclase in erythrocytes induces export of cGMP and cardioprotection in type 2 diabetes

Abstract Background Red blood cells (RBCs) are known to export cardioprotective nitric oxide (NO) bioactivity. Although, RBCs contain a functional soluble guanylyl cyclase (sGC), the signalling behind the export of NO bioactivity remains unclear. Impairment of NO signalling in RBCs has been suggested to contribute to augmented cardiac ischemia-reperfusion injury in type 2 diabetes (T2D). We hypothesized that increased NO signaling by stimulation of sGC in RBCs protects from myocardial ischemia-reperfusion injury by release of a cardioprotective signal. Purpose To investigate whether stimulation of sGC, the intracellular NO receptor, in RBCs from T2D patients induces export of a cardioprotective factor and to determine its nature. Methods RBCs were collected from patients with T2D and from gender- and age-matched healthy subjects and incubated with different pharmacological compounds affecting the NO-signalling pathway. The RBCs or their supernatant were then administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure and infarct size were measured. ELISA and immunofluorescence were used to determine levels of cyclic guanosine monophosphate (cGMP) and substrate of protein kinase G activation. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85-23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results RBCs from T2D patients impaired post-ischemic cardiac function and increased infarct size compared to RBCs from healthy subjects, and these effects were prevented by pre-incubation of the RBCs with the sGC stimulator CYR715. Administration of the supernatant from RBCs incubated with the sGC stimulator resulted in similar cardioprotection, indicating release of a cardioprotective factor from the RBCs (Figure 1A and B). The cardioprotection was abolished by inhibition of transport of cGMP with MK571, an inhibitor of cyclic nucleotide transport. Extracellular cGMP levels in the supernatant from RBCs were increased following sGC stimulation, and administration of exogenous cGMP induced cardioprotection (Figure 1C and D). Expression of protein kinase G-dependent phosphorylated vasodilator-specific phosphoprotein in cardiomyocytes was increased following administration of supernatant from RBCs incubated with the sGC stimulator (Figure 1E). Conclusions This study shows that stimulation of sGC in RBCs leads to release of a cardioprotective mediator, identified as cGMP, that activates cardiac protein kinase G and protects from ischemia-reperfusion injury. It further identifies RBC sGC as a novel therapeutic target to prevent cardiovascular injury in T2D.Figure 1

Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

Stimulation of Erythrocyte Soluble Guanylyl Cyclase Induces cGMP Export and Cardioprotection in Type 2 Diabetes

Reduced nitric oxide (NO) bioactivity in red blood cells (RBCs) is critical for augmented myocardial ischemia-reperfusion injury in type 2 diabetes. This study identified the nature of "NO bioactivity" by stimulating the intracellular NO receptor soluble guanylyl cyclase (sGC) in RBCs. sGC stimulation in RBCs from patients with type 2 diabetes increased export of cyclic guanosine monophosphate from RBCs and activated cardiac protein kinase G, thereby attenuating ischemia-reperfusion injury. These results provide novel insight into RBC signaling by identifying cyclic guanosine monophosphate from RBC as a mediator of protection against cardiac ischemia-reperfusion injury induced by sGC stimulation in RBCs.

Tylophora indica (Burm. f.) Merr alleviates tracheal smooth muscle hyperresponsiveness in ovalbumin-induced allergic-asthma model in guinea-pigs: Evidences from ex vivo, in silico and in vivo studies.

Tylophora indica (Burm. f.) Merr is a climbing perennial plant reported in Indian traditional system of medicine for its use in allergy and asthma. However, only few scientific studies have been performed in the past to validate its antiasthmatic potential. The present study deals with investigation of airway smooth muscle relaxant and antiasthmatic potential of extract and subsequent fractions prepared from T.indica. The most active fraction of T.indica leaves selected through bio-guided activity was subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for chemical profiling. The binding affinity of identified compounds in fraction towards M3 and H1 receptors was determined by molecular docking study. F-2 (chloroform fraction prepared from methanolic extract of T.indica leaves) was examined for its smooth muscle relaxant properties using isolated trachea of guinea-pig. Further, F-2 was evaluated through in vivo studies employing ovalbumin-induced asthma model in guinea-pigs. F-2 was found most effective in bioassay-guided fractionation. Characterization by LC-MS analysis revealed presence of five major bioactive compounds in F-2 that showed good docking interactions with M3 and H1 receptors. The ex vivo study demonstrated that F-2 could significantly relax tracheal rings via targeting multiple signalling pathways videlicet, namely, noncompetitive antagonism of the histamine and muscarinic receptors, β2-adrenergic stimulation and activation of soluble guanylyl cyclase. In in vivo studies, F-2 ameliorated airway hyperresponsiveness and decreased broncho alveolar lavage fluid (BALF) levels of inflammatory cytokines and immunoglobulin E (IgE). These results confirm the traditional use of T.indica as an antiasthmatic agent which are evidenced through ex vivo, in silico and in vivo studies.

First-in-human trial to assess safety, tolerability, pharmacokinetics, and pharmacodynamics of zagociguat (CY6463), a CNS-penetrant soluble guanylyl cyclase stimulator.

Soluble guanylate cyclase (sGC) and its product, cyclic guanosine monophosphate, play a role in learning and memory formation. Zagociguat (CY6463) is a novel stimulator of sGC being developed for the treatment of neurodegenerative disease. Single zagociguat doses of 0.3, 1, 3, 10, 20, 30, and 50 mg were administered once to healthy participants in a single-ascending-dose phase; then zagociguat 2, 5, 10, and 15 mg was administered q.d. for 14 days in a multiple-ascending-dose phase; and, finally, zagociguat 10 mg was administered once in both fed and fasted state in a food-interaction phase. Safety of zagociguat was evaluated by monitoring treatment-emergent adverse events, suicide risk, vital signs, electrocardiography, and laboratory tests. Pharmacokinetics of zagociguat were assessed through blood, urine, and cerebrospinal fluid sampling. Pharmacodynamic effects of zagociguat were evaluated with central nervous system (CNS) tests and pharmaco-electroencephalography. Zagociguat was well-tolerated across all doses evaluated. Zagociguat exposures increased in a dose-proportional manner. Median time to maximum concentration ranged from 0.8 to 5 h and mean terminal half-life from 52.8 to 67.1 h. CNS penetration of the compound was confirmed by cerebrospinal fluid sampling. Zagociguat induced up to 6.1 mmHg reduction in mean systolic and up to 7.5 mmHg reduction in mean diastolic blood pressure. No consistent pharmacodynamic (PD) effects on neurocognitive function were observed. Zagociguat was well-tolerated, CNS-penetrant, and demonstrated PD activity consistent with other sGC stimulators. The results of this study support further development of zagociguat.

Soluble Guanylyl Cyclase Activators-Promising Therapeutic Option in the Pharmacotherapy of Heart Failure and Pulmonary Hypertension.

Endogenous nitric oxide (NO)-dependent vascular relaxation plays a leading role in the homeostasis of the cardiovascular, pulmonary, and vascular systems and organs, such as the kidneys, brain, and liver. The mechanism of the intracellular action of NO in blood vessels involves the stimulation of the activity of the soluble cytosolic form of guanylyl cyclase (soluble guanylyl cyclase, sGC), increasing the level of cyclic 3'-5'-guanosine monophosphate (cGMP) in smooth muscle and subsequent vasodilation. In recent years, a new group of drugs, soluble guanylyl cyclase stimulators, has found its way into clinical practice. Based on the CHEST-1 and PATENT-1 trials, riociguat was introduced into clinical practice for treating chronic thromboembolic pulmonary hypertension (CTEPH). In January 2021, the FDA approved the use of another drug, vericiguat, for the treatment of heart failure.

Responses in Blood Pressure and Kidney Function to Soluble Guanylyl Cyclase Stimulation or Activation in Normal and Diabetic Rats

Introduction: Agonists of soluble guanylate cyclase (sGC) are being developed as treatment for cardiovascular disease. Most effects of nitric oxide (NO) on glomerular and tubular function are mediated through sGC, but whether sGC agonists mimic these effects is unknown. Methods: Renal clearance and micropuncture studies were performed in Wistar-Froemter rats (WF), with or without streptozotocin diabetes (STZ-WF), and in Goto-Kakizaki rats (GK) with mild type 2 diabetes to test for acute effects of the sGC “stimulator” BAY 41-2272, which synergizes with endogenous NO, and the “activator” runcaciguat, which generates cGMP independent of NO. Results: Both sGC agonists reduced arterial blood pressure (MAP). For MAP reductions <10%, the drugs increased GFR in WF and STZ-WF but not in GK. Larger MAP reductions outweighed this effect and GFR declined, with better preserved GFR in STZ-WF. Changes in GFR could not be accounted for by changes in RBF, suggesting parallel changes in ultrafiltration pressure and/or ultrafiltration coefficient. The doses chosen for micropuncture in WF and GK reduced MAP by 2–10%, and the net effect on single nephron GFR and ultrafiltration pressure was neutral. The effects of the drugs on tubular reabsorption were dominated by declining MAP, and no natriuretic effect was observed at any dose. Discussion/Conclusion: sGC agonists impact kidney function directly and because they reduce MAP. The direct tendency to increase GFR is most apparent for MAP reductions <10%. The direct effect is otherwise subtle and overridden when MAP declines more. Effects of sGC agonists on tubular reabsorption are dominated by the effects on MAP.

Congenital Heart Disease: The State-of-the-Art on Its Pharmacological Therapeutics.

Congenital heart disease is one of the most common causes of death derived from malformations. Historically, its treatment has depended on timely diagnosis and early pharmacological and surgical interventions. Survival rates for patients with this disease have increased, primarily due to advancements in therapeutic choices, but mortality remains high. Since this disease is a time-sensitive pathology, pharmacological interventions are needed to improve clinical outcomes. Therefore, we analyzed the applications, dosage, and side effects of drugs currently used for treating congenital heart disease. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and potassium-sparing diuretics have shown a mortality benefit in most patients. Other therapies, such as endothelin receptor antagonists, phosphodiesterase-5 inhibitors, prostaglandins, and soluble guanylyl cyclase stimulators, have benefited patients with pulmonary artery hypertension. Likewise, the adjunctive symptomatic treatment of these patients has further improved the outcomes, since antiarrhythmics, digoxin, and non-steroidal anti-inflammatory drugs have shown their benefits in these cases. Conclusively, these drugs also carry the risk of troublesome adverse effects, such as electrolyte imbalances and hemodynamic compromise. However, their benefits for survival, symptom improvement, and stabilization outweigh the possible complications from their use. Thus, cases must be assessed individually to accurately identify interventions that would be most beneficial for patients.

Attenuated cardiac post-ischemic injury and endothelial dysfunction by stimulation of soluble guanylyl cyclase in red blood cells from patients with type 2 diabetes

Abstract Background Reduced bioavailability of nitric oxide (NO) contributes to ischemic heart disease in type 2 diabetes (T2D). Red blood cells (RBCs) are known to produce NO bioactivity and to contain a functional soluble guanylyl cyclase (sGC) that is activated by NO. Recent studies revealed that RBCs from patients with T2D exacerbates ischemia-reperfusion (I/R) injury and induces endothelial dysfunction via a mechanism that is dependent on reduced export of NO bioactivity from RBCs. It remains unknown whether stimulation of sGC in RBCs from patients with T2D protects against myocardial I/R injury and endothelial dysfunction. Purpose To test the hypothesis that stimulation of sGC in RBCs from T2D patients protects against myocardial I/R injury and improves endothelial function. Methods RBCs collected from T2D patients and healthy subjects were incubated with vehicle or the sGC stimulator CYR715 before being administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure (LVDP) and infarct size were determined. In addition, isolated rat aortic rings were incubated with RBCs subsequent determination of endothelium-dependent relaxation (EDR). Results Administration of RBCs from T2D patients impaired post-ischemic recovery of LVDP and induced endothelial dysfunction in comparison with RBCs from healthy subjects (P&amp;lt;0.001). Pre-incubation of RBCs from patients with T2D with CYR715 prior to administration to the isolated heart enhanced the recovery of LVDP (Fig.1A), reduced infarct size (Fig.1B), and attenuated endothelial dysfunction (Fig.1C). CYR715 did not induce cardioprotection in the absence of RBCs. The sGC inhibitor ODQ did not significantly affect cardiac recovery per se but totally abolished the protective effect of CYR715 (Fig.1A). Conclusions Stimulation of sGC in RBCs from patients with T2D protects against cardiac I/R injury and prevents development of endothelial dysfunction. Stimulation of the NO-sGC pathway in RBCs appears to be an attractive therapeutic strategy to prevent cardiovascular injury in T2D. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation, the Swedish Research Council

SGC stimulation lowers elevated blood pressure in a new canine model of resistant hypertension

Therapy-resistant hypertension is a serious medical problem, causing end-organ damage, stroke, and heart failure if untreated. Since the standard of care fails in resistant hypertension patients, there is still a substantial unmet medical need for effective therapies. Active stimulation of soluble guanylyl cyclase via novel soluble guanylyl cyclase stimulators might provide an effective treatment option. To test this hypothesis, we established a new experimental dog model and investigated the effects of the soluble guanylyl cyclase-stimulator BAY 41-2272. In beagle dogs, a resistant hypertension phenotype was established by combining unilateral renal wrapping with the occlusion of the renal artery in the contralateral kidney. The most frequently used antihypertensive drugs were administered orally, either alone or in combination, and their acute effect on telemetric measured blood pressure was assessed and compared with that of BAY 41-2272. The chosen disease stimulus led to a moderate and stable increase in blood pressure. Even high doses of standard-of-care antihypertensives only slightly decreased blood pressure. In contrast, the administration of the soluble guanylyl cyclase stimulator BAY 41-2272 as standalone therapy led to a dose-dependent reduction in blood pressure (−14.1 ± 1.8 mmHg). Moreover, BAY 41-2272 could also further decrease blood pressure in addition to a triple combination of standard-of-care antihypertensives (−28.6 ± 13.2 mmHg). BAY 41-2272 was highly efficient as a standalone treatment in resistant hypertension but was also effective in addition to standard-of-care treatment. These data strongly suggest that soluble guanylyl cyclase stimulators might provide an effective pharmacologic therapy for patients with resistant hypertension.

Olinciguat, a stimulator of soluble guanylyl cyclase, attenuates inflammation, vaso-occlusion and nephropathy in mouse models of sickle cell disease.

Background and PurposeReduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso‐occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP‐amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα‐induced inflammation in wild‐type C57BL/6 mice and in ‘humanised’ mouse models of SCD.Experimental ApproachEffects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα‐induced and systemic inflammation associated with SCD.Key ResultsAcute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte‐endothelial cell interactions in TNFα‐challenged mice. Co‐treatment with hydroxyurea, an FDA‐approved SCD therapeutic agent, further augmented the anti‐inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα‐induced vaso‐occlusive crisis, a single dose of olinciguat attenuated leukocyte‐endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle‐treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat‐ than in vehicle‐treated mice. In addition, kidney mass, water consumption, 24‐h urine excretion, plasma levels of cystatin C and urinary excretion of N‐acetyl‐β‐d‐glucosaminidase and neutrophil gelatinase‐associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle‐treated mice.Conclusion and ImplicationsOur results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso‐occlusion and kidney injury in mouse models of SCD and systemic inflammation.

Novel Therapies in Heart Failure with Reduced Ejection Fraction: from Soluble Guanylyl Cyclase Stimulators to Cardiac Myosin Activators

In this review, our goal was to provide a brief background on the evolution of therapeutic targets for heart failure with reduced ejection fraction (HFrEF), summarize the results of recent clinical trials with diverse pharmacological targets, discuss potential limitations of these findings, and provide future perspectives. Despite advances in pharmacological and device therapy, HFrEF still carries a considerably high morbidity and mortality. For the past four decades, the neurohormonal model has been pivotal in the development of efficacious pharmacotherapies for HFrEF. However, recent clinical trials with sodium-glucose cotransporter 2 inhibitors (SGLT2i), soluble guanylate cyclase stimulators, and cardiac myosin activators have yielded positive results and created a new spectrum of pharmacologic targets to further improve outcomes in HFrEF. Specifically for SGLT2i, the data point to a class disease-modifying effect with mortality benefit in HFrEF, in addition to a substantial reduction in healthcare resources utilization. We are currently witnessing a paradigm shift away from neurohormonal inhibition as the sole line of disease-modifying pharmacotherapies in HFrEF, as encouraging data emerge about alternative pathophysiologic pathways. Further research is needed to identify the optimal target subpopulations for these therapies and to improve outcomes in patients with HFrEF decompensation.

Stimulation of soluble guanylyl cyclase (sGC) by riociguat attenuates heart failure and pathological cardiac remodelling

Heart failure is associated with an impaired NO-soluble guanylyl cyclase (sGC)-cGMP pathway and its augmentation is thought to be beneficial for its therapy. We hypothesized that stimulation of sGC by the sGC stimulator riociguat prevents pathological cardiac remodelling and heart failure in response to chronic pressure overload. Transverse aortic constriction or sham surgery was performed in C57BL/6N mice. After 3weeks of transverse aortic constriction when heart failure was established, animals receive either riociguat or its vehicle for 5 additional weeks. Cardiac function was evaluated weekly by echocardiography. Eight weeks after surgery, histological analyses were performed to evaluate remodelling and the transcriptome of the left ventricles (LVs) was analysed by RNA sequencing. Cell culture experiments were used for mechanistically studies. Transverse aortic constriction resulted in a continuous decrease of LV ejection fraction and an increase in LV mass until week 3. Five weeks of riociguat treatment resulted in an improved LV ejection fraction and a decrease in the ratio of left ventricular mass to total body weight (LVM/BW), myocardial fibrosis and myocyte cross-sectional area. RNA sequencing revealed that riociguat reduced the expression of myocardial stress and remodelling genes (e.g. Nppa, Nppb, Myh7 and collagen) and attenuated the activation of biological pathways associated with cardiac hypertrophy and heart failure. Riociguat reversed pathological stress response in cultivated myocytes and fibroblasts. Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Solution structures of the Shewanella woodyi H-NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP-051.

Heme-nitric oxide/oxygen binding (H-NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H-NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H-NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H-NOX and coiled-coil domains. Here, we report the full NMR structure for CO-ligated Sw H-NOX in the presence and absence of stimulator compound IWP-051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi-empirical quantum potential energy approach. Although IWP-051 binding is weak, a single binding conformation was found at the interface of the two H-NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix-connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure-based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H-NOX proteins. The largest dynamical loop lies at the interface between Sw H-NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.

BAY 41-2272 Attenuates CTGF Expression via sGC/cGMP-Independent Pathway in TGFβ1-Activated Hepatic Stellate Cells.

Activation of hepatic stellate cells (HSCs) is a critical pathogenic feature of liver fibrosis and cirrhosis. BAY 41-2272 is a canonical non-nitric oxide (NO)-based soluble guanylyl cyclase (sGC) stimulator that triggers cyclic guanosine monophosphate (cGMP) signaling for attenuation of fibrotic disorders; however, the impact of BAY 41-2272 on HSC activation remains ill-defined. Transforming growth factor (TGF)β and its downstream connective tissue growth factor (CTGF or cellular communication network factor 2, CCN2) are critical fibrogenic cytokines for accelerating HSC activation. Here, we identified that BAY 41-2272 significantly inhibited the TGFβ1-induced mRNA and protein expression of CTGF in mouse primary HSCs. Indeed, BAY 41-2272 increased the sGC activity and cGMP levels that were potentiated by two NO donors and inhibited by a specific sGC inhibitor, ODQ. Surprisingly, the inhibitory effects of BAY 41-2272 on CTGF expression were independent of the sGC/cGMP pathway in TGFβ1-activated primary HSCs. BAY 41-2272 selectively restricted the TGFβ1-induced phosphorylation of Akt but not canonical Smad2/3 in primary HSCs. Together, we illustrate a unique framework of BAY 41-2272 for inhibiting TGFβ1-induced CTGF upregulation and HSC activation via a noncanonical Akt-dependent but sGC/cGMP-independent pathway.

Riociguat ameliorates kidney injury and fibrosis in an animal model

Chronic kidney disease (CKD) is one of the greatest health burdens with an increasing global prevalence. Renal fibrosis (RF) is the hallmark of all forms of CKD which shows a strong positive correlation with severity of the disease. However, there are no therapeutic options available for treatment of RF. In the present study, we used an animal model based on unilateral ureteral obstruction (UUO), for renal injury and fibrosis. The UUO animals were treated with soluble guanylyl cyclase (sGC) stimulator, riociguat (RIO) (1, 3 and 10 mg/kg) to investigate its possible renoprotective effects. Kidneys of animals treated with RIO were found to show less abnormalities as compared to UUO control. Further, the levels of proinflammatory cytokines were reduced in RIO treated group. Furthermore, administration of RIO reduced expression of collagen-1, TGF-β, CTGF, α-SMA, vimentin along with transcription factors including Snail and Slug. The results of the present study provided strong evidence to support the antifibrotic activity of RIO.

Beneficial Effects of Soluble Guanylyl Cyclase Stimulation and Activation in Sickle Cell Disease Are Amplified by Hydroxyurea: In Vitro and In Vivo Studies.

The complex pathophysiology of sickle cell anemia (SCA) involves intravascular hemolytic processes and recurrent vaso-occlusion, driven by chronic vascular inflammation, which result in the disease’s severe clinical complications, including recurrent painful vaso-occlusive episodes. Hydroxyurea, the only drug frequently used for SCA therapy, is a cytostatic agent, although it appears to exert nitric oxide/soluble guanylyl cyclase (sGC) modulating activity. As new drugs that can complement or replace the use of hydroxyurea are sought to further reduce vaso-occlusive episode frequency in SCA, we investigated the effects of the sGC agonists BAY 60-2770 (sGC activator) and BAY 41-2272 (sGC stimulator) in the presence or absence of hydroxyurea on SCA vaso-occlusive mechanisms and cell recruitment both ex vivo and in vivo. These agents significantly reduced stimulated human SCA neutrophil adhesive properties ex vivo in association with the inhibition of surface β2-integrin activation. A single administration of BAY 60-2770 or BAY 41-2272 decreased tumor necrosis factor cytokine–induced leukocyte recruitment in a mouse model of SCA vaso-occlusion. Importantly, the in vivo actions of both agonists were significantly potentiated by the coadministration of hydroxyurea. Erythroid cell fetal hemoglobin (HbF) elevation is also a major goal for SCA therapy. BAY 41-2272 but not BAY 60-2770 at the concentrations employed significantly induced γ-globin gene transcription in association with HbF production in cultured erythroleukemic cells. In conclusion, sGC agonist drugs could represent a promising approach as therapy for SCA, for use either as stand-alone treatments or in combination with hydroxyurea.SIGNIFICANCE STATEMENTThis preclinical study demonstrates that stimulators and activators of sGC are potent inhibitors of the adhesion and recruitment of leukocytes from humans and in mice with sickle cell anemia (SCA) and may represent a promising approach for diminishing vaso-occlusive episode frequency in SCA. Hydroxyurea, a drug already frequently used for treating SCA, was found to potentiate the beneficial effects of sGC agonists in in vivo studies, implying that these classes of compounds could be used alone or in combination therapy.

HP-3-5 Possible Future Therapies in Patients With Erectile Dysfunction NOT Responding to Phosphodiesterase Type 5 Inhibitors

Erectile dysfunction is a disorder with a prevalence of up to 52% in men aged 40 years and above. About half of the affected men are not satisfied by the common treatment with phosphodiesterase type 5 inhibitors which inhibit cyclic guanosine monophosphate (cGMP) degradation. This low driven cGMP production could be attributable to comorbidities like diabetes. This study aims to investigate a new pharmacological mechanism, which enhances cGMP production independently of nitric oxide (NO). A total of 68 experiments were conducted on samples of human corpus cavernosum obtained from 12 men with erectile dysfunction who underwent penile implant surgery. The samples underwent ex vivo organ-bath assay and were exposed to one of the following three chemicals: soluble guanylyl cyclase stimulator (sGC-S; BAY 41-2272), soluble guanylyl cyclase activator (sGC-A; BAY 60-2270), or the control dimethylsulfoxide. When relaxation of the tissue occurred, the compound is more likely to generate an erection.