sGC Activity Research Articles

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

Soluble Guanylate Cyclase Stimulators and Activators Attenuate Placental Production of sFlt‐1

Preeclampsia (PE) is a pregnancy specific disorder associated with maternal hypertension due to widespread endothelial dysfunction. Placental ischemia leads to increases in the circulating anti‐angiogenic factor, sFlt‐1 and a PE‐like phenotype. PE and chronic sFlt‐1 excess is also associated with depletion of nitric oxide (NO), which, during normal pregnancy, binds to soluble guanylate cyclase (sGC), and synthesizes cGMP, to facilitate vasodilation.. In fact, studies in reduced uterine perfusion pressure (RUPP) model of placental ischemia demonstrate that restoring the angiogenic imbalance, administration of placental growth factor or VEGF abolishes hypertension. A novel drug, sGC stimulators and activators, which increase sGC activity independently of NO, are currently in clinical trials for the treatment of hypertension, however, whether this drug could be beneficial during PE is not known. We tested the hypothesis that sGC stimulators (Riociguat, Sigma‐Aldrich) and activators (Cinaciguat, Sigma‐Aldrich) attenuate sFlt‐1 production by the placenta. Placentas were isolated from normal pregnant rats on gestational day 19. Villous bundles were excised and plated in 24‐well plate coated with Matrix Matrigel Basement Membrane. Increasing concentrations of each the sGC stimulator and activator (0.1 μM, 1 μM, 10 μM, 30 μM) were added to the media for treatments. The explants were then exposed to a hypoxic environment (1% oxygen) to determine whether sFlt‐1 production, directly by the placenta, is attenuated by sGC stimulators and activators in a dose‐dependent manner. Interestingly, each of the doses administered significantly reduced sFlt‐1 production in response to hypoxia compared to the untreated group. However, the 30 μM sGC activator treatment produced the most profound effect (Untreated, n=6, 4699±942 pg/mL; 30 μM sGC stimulator‐treated, n=6, 3647±1385 pg/mL, 30 μM sGC activator‐treated, n=6, 3247±1402 pg/mL; P<0.01). The results of this study demonstrate that increasing sGC activity attenuates sFlt‐1 production from the placenta and could improve the angiogenic imbalance seen in this condition. In conclusion, these findings suggest there is a therapeutic potential for treating preeclampsia with sGC activatorsSupport or Funding InformationFunding source: NIH HL51971This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Nitric Oxide‐Dependent Mechanisms in Early Sunitinib‐Induced Hypertension

Sunitinib is an antiangiogenic receptor tyrosine kinase inhibitor used in cancer treatment. A major adverse effect of sunitinib is a rapidly developing hypertension associated with reduced renal nitric oxide (NO) availability and increased renal fractional sodium reabsorption (FRNa). We tested if NO‐dependent regulation of renal hemodynamics and FRNa are compromised and if nitrate supplementation has antihypertensive effects in early sunitinib‐induced hypertension.Male Wistar rats were treated with sunitinib at 15 mg/(kg*d). Four days after initiation of sunitinib treatment, renal hemodynamics and renal sodium excretion were investigated. Radiotelemetric experiments were performed to study the effects of nitrate supplementation and soluble guanylate cyclase (sGC) activation on arterial pressure. Nitric oxide synthase (NOS) as well as sGC mRNA and protein abundances were studied in renal tissue.Within four days, sunitinib elicited a 20–25 mmHg arterial pressure rise. NOS inhibition elicited similar reductions in total and renal medullary blood flow in sunitinib‐ and vehicle‐treated rats. NO‐dependent and independent components of endothelium‐dependent renal vasodilation were not compromised in early sunitinib‐induced hypertension. Low dose intrarenal NOS inhibition did not differentially affect FRNa in sunitinib‐ and vehicle‐treated rats. In sunitinib‐treated rats, renal nitrite/nitrate excretion was 60 per cent less than in controls (p < 0.05). However, nitrate supplementation did not reduce arterial pressure and renal sodium balance in sunitinib‐treated rats. While sunitinib treatment had no effects on renal NOS mRNA abundances, it significantly reduced renal cortical sGC α‐ and β‐subunit mRNA abundances as well as renal cortical guanylate cyclase protein expression. Pharmacological sGC activation caused a rapid 15–20 mmHg arterial pressure fall in sunitinib‐treated rats (p < 0.05).We conclude that the NO‐dependent regulation of renal hemodynamics is largely unaltered in early sunitinib‐induced hypertension despite evidence for reduced NO formation. Reduced renal sGC expression and activity may contribute to the early arterial pressure rise in sunitinib‐induced hypertension and cause its nitrate resistance.Support or Funding InformationSupported by the German Research Foundation, Grant‐No. KO 4936/2‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GENIe: A bright new genetically‐encoded fluorescent biosensor for monitoring cGMP

GENIe is a Genetically Encoded Nucleotide Indicator for monitoring cGMP, a second messenger in cell signaling that mediates many important physiological processes. Phosphodiesterase (PDE) inhibitors and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP.We have developed a new, very bright mNeonGreen fluorescent biosensor to monitor cGMP levels in any living cell type. The sensor produces robust changes in fluorescence intensity that are readily detected on fluorescence imaging systems or plate readers. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP in HEK293 cells expressing either the cGMP biosensor (GENIe) or the cAMP biosensor (cADDis). These sensors produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.We tested GENIe in HEK293T cells on a BioTek Synergy MX automated fluorescence plate reader. cGMP production was initiated by adding sodium nitroprusside (SNP), a nitric oxide donor, in both the presence and absence of the PDE5 selective inhibitor, Sildenafil. Fluorescence intensity changed dramatically in the presence of Sildenafil, as degradation of cGMP is inhibited by the drug. To optimize the response in HEK293T cells, we over‐expressed sGC, treated with SNP, and observed a significant improvement in the amplitude of the signal accompanied by faster kinetics and an increased sensitivity to the nitric oxide donor SNP. We expect that endogenous levels of sGC will vary in different cell types and these parameters should be varied accordingly.To examine PDE inhibitor specificity, we monitored responses from both cADDis and GENIe in the presence of different PDE inhibitors. IBMX, a non‐specific inhibitor, produced similar responses from both sensors. Sildenafil and Rolipram indicated cGMP vs cAMP specificity. Sildenafil exhibited selective inhibition of the cGMP‐specific PDE5, while Rolipram exhibited selective inhibition of the cAMP specific PDE4.Support or Funding InformationFunding from NSF SBIR Phase IIB 1430878This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of soluble guanylyl cyclase with inhibition of multidrug resistance protein inhibitor-4 (MRP4) as a new antiplatelet therapy

The intracellular levels of cyclic GMP are controlled by its rate of formation through nitric oxide-mediated stimulation of soluble guanylate cyclase (sGC) and its degradation by phosphodiesterases. Multidrug resistance protein 4 (MRP4) expressed in human platelets pumps cyclic nucleotides out of cells. In search for new antiplatelet strategies, we tested the hypothesis that sGC activation concomitant with MRP4 inhibition confers higher antiplatelet efficacy compared with monotherapy alone. This study was undertaken to investigate the pharmacological association of the sGC activator BAY 60-2770 with the MRP4 inhibitor MK571 on human washed platelets. Collagen- and thrombin-induced platelet aggregation and ATP-release reaction assays were performed. BAY 60-2770 (0.001–10 µM) produced significant inhibitions of agonist-induced platelet aggregation accompanied by reduced ATP-release. Pre-incubation with 10 µM MK571 alone had no significant effect on platelet aggregation and ATP release, but it produced a left displacement by about of 10–100-fold in the concentration-response curves to BAY 60-2770. Pre-incubation with MK571increased and decreased, respectively, the intracellular and extracellular levels of cGMP to BAY 60-2770, whereas the cAMP levels remained unchanged. The increased VASP-serine 239 phosphorylation in BAY 60-2770-treated platelets was enhanced by MK571. In Fluo-4-loaded platelets, BAY 60-2770 reduced the intracellular Ca2+ levels, an effect significantly potentiated by MK571. Flow cytometry assays showed that BAY 60-2770 reduces the αIIbβ3 integrin activation, which was further reduced by MK571 association. Blocking the MRP4-mediated efflux of cGMP may be a potential mechanism to enhance the antiplatelet efficacy of sGC activators.

In Endothelial Cells, the Activation or Stimulation of Soluble Guanylyl Cyclase Induces the Nitric Oxide Production by a Mechanism Dependent of Nitric Oxide Synthase Activation.

In endothelial cells, investigate if the soluble guanylate cyclase (sGC) activation or stimulation is able to potentiate the relaxation in vessels. Aortic and coronary rings with and without endothelium were placed in a myograph and cumulative concentration-effect curves for DETA-NO or ataciguat were performed. Nitric oxide (NO) were measured by fluorescence or by selective electrode in human umbilical endothelial cells (HUVECs) in response to some treatments, including ataciguat, 8-Br-cGMP and A23187. The presence of the endothelium potentiated the relaxation induced by DETA-NO in aortic and coronary rings. In addition, in aortic rings the endothelium potentiated the relaxation induced by ataciguat. In the presence of nitric oxide synthase (NOS) inhibitor, the endothelium effect was abolished to DETA-NO or ataciguat, in both vessels. Ataciguat, 8-Br-cGMP and A23187 were able to induce NO production in HUVECs cells. In the presence of NOS inhibitor, the NO production induced by ataciguat and 8-Br-cGMP was abolished. Our results suggest that in aortic and coronary rings the endothelium potentiates the relaxation induced by activation or stimulation of sGC through a mechanism dependent of NOS activation.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3′,5′-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H2O2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H2O2-, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H2O2, but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H2O2, were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na+-K+ ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na+-K+ ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H2O2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators.

Prevention of PKG-1α Oxidation Suppresses Antihypertrophic/Antifibrotic Effects From PDE5 Inhibition but not sGC Stimulation.

Stimulation of sGC (soluble guanylate cyclase) or inhibition of PDE5 (phosphodiesterase type 5) activates PKG (protein kinase G)-1α to counteract cardiac hypertrophy and failure. PKG1α acts within localized intracellular domains; however, its oxidation at cysteine 42, linking homomonomers, alters this localization, impairing suppression of pathological cardiac stress. Because PDE5 and sGC reside in separate microdomains, we speculated that PKG1α oxidation might also differentially influence the effects from their pharmacological modulation. Knock-in mice expressing a redox-dead PKG1α (PKG1αC42S) or littermate controls (PKG1αWT) were subjected to transaortic constriction to induce pressure overload and treated with a PDE5 inhibitor (sildenafil), sGC activator (BAY602770 [BAY]), or vehicle. In PKG1αWT controls, sildenafil and BAY similarly enhanced PKG activity and reduced pathological hypertrophy/fibrosis and cardiac dysfunction after transaortic constriction. However, sildenafil failed to protect the heart in PKG1αC42S, unlike BAY, which activated PKG and thereby facilitated protective effects. This corresponded with minimal PDE5 activation in PKG1αC42S exposed to transaortic constriction versus higher activity in controls and little colocalization of PDE5 with PKG1αC42S (versus colocalization with PKG1αWT) in stressed myocytes. In the stressed heart and myocytes, PKG1α C42-disulfide formation contributes to PDE5 activation. This augments the pathological role of PDE5 and so in turn enhances the therapeutic impact from its inhibition. PKG1α oxidation does not change the benefits from sGC activation. This finding favors the use of sGC activators regardless of PKG1α oxidation and may help guide precision therapy leveraging the cyclic GMP/PKG pathway to treat heart disease.

Multiple Avenues of Modulating the Nitric Oxide Pathway in Heart Failure Clinical Trials.

This review discusses the integral role of the nitric oxide (NO) pathway in the pathophysiology of heart failure (HF). We emphasize potential therapeutic targets in the NO pathway and review contemporary clinical trials evaluating these novel therapeutic options. Nitrates, neprilysin inhibitors, and phosphodiesterase (PDE) inhibitors have all proven to be efficacious in HF patients with systolic dysfunction, with the former two classes of medications producing a net mortality benefit. However, neither PDE inhibitors nor nitrates have demonstrated significant clinical benefit in patients with HF with preserved ejection fraction (HFpEF), and neprilysin inhibitors have yet to be evaluated in this population. Soluble guanylate cyclase (sGC) stimulators have shown significant promise in all HF patients, leading to improvements in both quality of life scores and exercise capacity. Conversely, sGC activators have limited clinical utility in HF, owing largely to safety concerns of hypotension. Inorganic nitrates and nitrites, meanwhile, may be emerging as potential therapies for the HFpEF population. The advent of novel therapies targeting the NO pathway is beginning to create a paradigm shift in the treatment of the HF patient. These therapies offer a promising outlook for the future, with hopes of reducing HF-associated morbidity and mortality.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 μM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy.

Soluble Guanylate Cyclase Stimulators and Activators.

When Furchgott, Murad, and Ignarro were honored with the Nobel prize for the identification of nitric oxide (NO) in 1998, the therapeutic implications of this discovery could not be fully anticipated. This was due to the fact that available therapeutics like NO donors did not allow a constant and long-lasting cyclic guanylyl monophosphate (cGMP) stimulation and had a narrow therapeutic window. Now, 20years later, the stimulator of soluble guanylate cyclase (sGC), riociguat, is on the market and is the only drug approved for the treatment of two forms of pulmonary hypertension (PAH/CTEPH), and a variety of other sGC stimulators and sGC activators are in preclinical and clinical development for additional indications. The discovery of sGC stimulators and sGC activators is a milestone in the field of NO/sGC/cGMP pharmacology. The sGC stimulators and sGC activators bind directly to reduced, heme-containing and oxidized, heme-free sGC, respectively, which results in an increase in cGMP production. The action of sGC stimulators at the heme-containing enzyme is independent of NO but is enhanced in the presence of NO whereas the sGC activators interact with the heme-free form of sGC. These highly innovative pharmacological principles of sGC stimulation and activation seem to have a very broad therapeutic potential. Therefore, in both academia and industry, intensive research and development efforts have been undertaken to fully exploit the therapeutic benefit of these new compound classes. Here we summarize the discovery of sGC stimulators and sGC activators and the current developments in both compound classes, including the mode of action, the chemical structures, and the genesis of the terminology and nomenclature. In addition, preclinical studies exploring multiple aspects of their in vitro, ex vivo, and in vivo pharmacology are reviewed, providing an overview of multiple potential applications. Finally, the clinical developments, investigating the treatment potential of these compounds in various diseases like heart failure, diabetic kidney disease, fibrotic diseases, and hypertension, are reported. In summary, sGC stimulators and sGC activators have a unique mode of action with a broad treatment potential in cardiovascular diseases and beyond.

Nitric Oxide: From Good to Bad.

This essay summarizes a lecture presented on October 19th, 2017, during the 58th Annual Meeting of the Japanese College of Angiology in Nagoya, Japan. The lecture summarizes several instances where the absence of relaxations of isolated blood vessels in response to endothelium-dependent vasodilator agonists, which cause activation of endothelial nitric oxide synthase (eNOS) and consequent production of endothelium-derived nitric oxide (NO) and stimulation of soluble guanylyl cyclase (sGC) in underlying vascular smooth muscle, or hypoxia are curtailed or reversed to endothelium-dependent contractions. Chosen examples include selective dysfunction of eNOS activation in regenerated endothelial cells, unresponsiveness of vascular smooth muscle cells to NO during subarachnoid hemorrhage, and biased activation of sGC in vascular smooth muscle cells during acute exposure to hypoxia. The main message of this essay is that absence, blunting, or reversal of endothelium-dependent relaxations in response to vasodilator agonists cannot necessarily be interpreted as a sign of endothelial dysfunction. (This is a review article based on the invited lecture of the 58th Annual Meeting of Japanese College of Angiology.)

Biased activation of soluble guanylyl cyclase by quinones causes contractions of isolated arteries: Role of NADPH: quinone oxidoreductase-1

Background Previous experiments in isolated arteries indicate that quinones, including thymoquinone (a para-quinone) and β-lapachone [an ortho-quinone and high-affinity substrate of NADPH:quinone oxidoreductase-1 (NQO-1)], causes augmentations of contraction. The augmentation depends on the activation of soluble guanylyl cyclase (sGC) to generate cyclic inosine monophosphate (cIMP) rather than the canonical cyclic guanosine monophosphate (cGMP). The present study aims to identify cellular target of the quinones in mediating the biased activation of sGC.

Nitric oxide-cGMP-PKG signaling in the bed nucleus of the stria terminalis modulates the cardiovascular responses to stress in male rats

The bed nucleus of the stria terminalis (BNST) constitutes an important component of neural substrates of physiological and behavioral responses to aversive stimuli, and it has been implicated on cardiovascular responses evoked by stress. Nevertheless, the local neurochemical mechanisms involved in BNST control of cardiovascular responses during aversive threats are still poorly understood. Thus, the aim of the present study was to assess the involvement of activation in the BNST of the neuronal isoform of the enzyme nitric oxide synthase (nNOS), as well as of signaling mechanisms related to nitric oxide effects such as soluble guanylate cyclase (sGC) and protein kinase G (PKG) on cardiovascular responses induced by an acute session of restraint stress in male rats. We observed that bilateral microinjection of either the nonselective NOS inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME), the selective nNOS inhibitor Nω-Propyl-L-arginine (NPLA) or the sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) into the BNST enhanced the tachycardic response and decreased the drop in tail cutaneous temperature evoked by acute restraint stress, but without affecting the increase on blood pressure. Bilateral BNST treatment with the selective PKG inhibitor KT5823 also facilitated the heart rate increase and decreased the drop in cutaneous temperature, in addition to enhancing the blood pressure increase. Taken together, these results provide evidence that NO released from nNOS and activation of sGC and PKG within the BNST play an inhibitory influence on tachycardia to stress, whereas this signaling mechanism mediates the sympathetic-mediated cutaneous vasoconstriction.

BAY 60–2770 activates two isoforms of nitric oxide sensitive guanylyl cyclase: Evidence for stable insertion of activator drugs

Nitric oxide sensitive guanylyl cyclase (NOsGC), a hemoprotein and the major physiological receptor for nitric oxide (NO), is a heterodimer with the α1/β1 and α2/β1 isoforms known to be important for NO-signaling and conversion of GTP to cGMP in humans. Two innovative classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent sGC stimulators, that stimulate NOsGC directly and also increase the affinity towards NO, and the heme-independent sGC activators, that are thought to bind to oxidized and heme-free NOsGC in tissues exposed to oxidative stress. In the current study, we evaluate the effects of the sGC activators BAY 58–2667 (cinaciguat) and BAY 60–2770 on the isoforms α1/β1 and α2/β1 expressed in Sf9 cells. Western blot analysis of cytosolic fractions revealed a decrease in overexpressed NOsGC in the presence of sGC activators, which is dependent on an intact catalytic site of the enzyme. For both isoforms, we show a higher efficacy for BAY 60–2770 compared to cinaciguat after purification of NOsGC by affinity and size exclusion chromatography. Using a new experimental strategy of expression of NOsGC with activator and subsequent purification, we demonstrate a stable insertion of activator drugs into the enzyme during protein biosynthesis independent of the heme redox state. We postulate that the balance between stable insertion of activator during de novo synthesis and replacement of NOsGC ferric heme in tissues exposed to oxidative stress can be influenced by the dosage regimen.

Stimulators and activators of soluble guanylate cyclase for urogenital disorders.

Lower urinary tract symptoms (LUTS), comprising storage (such as urinary incontinence and urinary frequency), voiding, and postmicturition symptoms, are highly prevalent conditions that affect millions of people worldwide. LUTS have a profound effect on quality of life and are a considerable cost to health care systems. In men specifically, BPH commonly leads to LUTS. Clinical studies also show an association of LUTS with erectile dysfunction (ED). Nitric oxide (NO) has long been recognized as an important nonadrenergic, noncholinergic (NANC) transmitter in bladder, urethra, prostate, and corpus cavernosum smooth muscle. Data from clinical and basic research show that oxidation and degradation of soluble guanylate cyclase (sGC; also known as GCS) and reduced cyclic GMP (cGMP) levels are involved in the physiopathology of genitourinary diseases. The NO-sGC-cGMP signalling pathway has a role in disease pathophysiology of the bladder, urethra, prostate, and corpus cavernosum in animal models and humans. Advances in targeting sGC directly to enhance cGMP production independently of endogenous NO have been made using NO-independent stimulators and activators of sGC. These molecules are potential therapeutics in the treatment of LUTS and ED.

Irreversible Activation and Stabilization of Soluble Guanylate Cyclase by the Protoporphyrin IX Mimetic Cinaciguat.

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of ~0.2 μM and maximal cGMP formation at 10 μM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one–oxidized (ferric) sGC was moderate, reaching ~10%–15% of maximal activity. Dilution experiments of cinaciguat/Tween 20–preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide–sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.