Structural insights into the mechanism of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1ß1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the ß1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.

NO rapidly mobilizes cellular heme to trigger assembly of its own receptor.

Nitric oxide (NO) signaling in biology relies on its activating cyclic guanosine monophosphate (cGMP) production by the NO receptor soluble guanylyl cyclase (sGC). sGC must obtain heme and form a heterodimer to become functional, but paradoxically often exists as an immature heme-free form in cells and tissues. Based on our previous finding that NO can drive sGC maturation, we investigated its basis by utilizing a fluorescent sGC construct whose heme level can be monitored in living cells. We found that NO generated at physiologic levels quickly triggered cells to mobilize heme to immature sGC. This occurred when NO was generated within cells or by neighboring cells, began within seconds of NO exposure, and led cells to construct sGC heterodimers and thus increase their active sGC level by several-fold. The NO-triggered heme deployment involved cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-heme complexes and required the chaperone hsp90, and the newly formed sGC heterodimers remained functional long after NO generation had ceased. We conclude that NO at physiologic levels triggers assembly of its own receptor by causing a rapid deployment of cellular heme. Redirecting cellular heme in response to NO is a way for cells and tissues to modulate their cGMP signaling and to more generally tune their hemeprotein activities wherever NO biosynthesis takes place.

Cardiovascular Effects of Stimulators of Soluble Guanylate Cyclase Administration: A Meta-analysis of Randomized Controlled Trials.

PURPOSE OF REVIEW: Heart failure (HF) is one of the main causes of cardiovascular mortality in the western world. Despite great advances in treatment, recurrence and mortality rates remain high. Soluble guanylate cyclase is an enzyme which, by producing cGMP, is responsible for the effects of vasodilation, reduction of cardiac pre- and after-load and, therefore, the improvement of myocardial performance. Thus, a new therapeutic strategy is represented by the stimulators of soluble guanylate cyclase (sGCs). The aim of this meta-analysis was to analyze the effects deriving from the administration of sGCs, in subjects affected by HF. A systematic literature search of Medline, SCOPUS, and Google Scholar was conducted up to December 2022 to identify RCTs assessing the cardiovascular effects, as NT-pro-BNP values and ejection fraction (EF), and all-cause mortality, of the sGCs. Quantitative data synthesis was performed using a random-effects model, with weighted mean difference (WMD) and 95% confidence interval (CI) as summary statistics. RECENT FINDINGS: The results obtained documented a statistically significant improvement in NT-proBNP values (SMD: - 0.258; 95% CI: - 0.398, - 0.118; p < 0.001) and EF (WMD: 0.948; 95% CI: 0.485, 1.411; p < 0.001) in subjects treated with sGCs; however, no significant change was found in the all-cause mortality rate (RR 0.96; 95% CI 0.868 to 1.072; I2, p = 0). The sGCs represent a valid therapeutic option in subjects suffering from HF, leading to an improvement in cardiac performance.

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-1ß). 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.

Loss of Gucy1a3 causes poor post-stroke recovery by reducing angiogenesis via the HIF-1α/VEGFA signaling pathway in mice.

OBJECTIVES: Ischemic stroke is a common and debilitating disease that can cause permanent neurological damage. Gucy1a3, which encodes the α1 subunit of soluble guanylyl cyclase, has been reported to be associated with functional recovery after ischemic stroke. However, the mechanism is still not well understood. In the present study, we investigated the effects of Gucy1a3 on (i) post-stroke recovery; (ii) vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF-1α) expression; and (iii) angiogenesis after ischemic stroke. MATERIALS AND METHODS: Wild-type and Gucy1a3 knockout C57BL/6J male mice were respectively used to establish the models of permanent middle cerebral artery occlusion (pMCAO). Neurological deficit scores were evaluated at 24 h and 96 h after pMCAO. Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. For determining microvessel density, immunohistochemical analysis was performed with CD31. The expression of VEGFA and HIF-1α was detected by western blotting. RESULTS: Our results suggest that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. In addition, the Gucy1a3 knockout brains exhibited significantly lower microvessel densities and VEGFA and HIF-1α expression levels than the wild-type brains at 96 h post-pMCAO. CONCLUSIONS: Our study indicates that GUCY1A3 might be involved in angiogenesis after ischemic stroke. Further investigation of GUCY1A3 will provide a new therapeutic target for stroke.

Role of the Coiled-Coil Domain in Allosteric Activity Regulation in Soluble Guanylate Cyclase.

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in higher eukaryotes, including humans. NO-dependent signaling via sGC is associated with important physiological effects in the vascular, pulmonary, and neurological systems, and sGC itself is an established drug target for the treatment of pulmonary hypertension due to its central role in vasodilation. Despite isolation in the late 1970s, high-resolution structural information on full-length sGC remained elusive until recent cryo-electron microscopy structures were determined of the protein in both the basal unactivated state and the NO-activated state. These structures revealed large-scale conformational changes upon activation that appear to be centered on rearrangements within the coiled-coil (CC) domains in the enzyme. Here, a structure-guided approach was used to engineer constitutively unactivated and constitutively activated sGC variants through mutagenesis of the CC domains. These results demonstrate that the activation-induced conformational change in the CC domains is necessary and sufficient for determining the level of sGC activity.

BAY58-2667 Activates Different Soluble Guanylyl Cyclase Species by Distinct Mechanisms that Indicate Its Principal Target in Cells is the Heme-Free Soluble Guanylyl Cyclase-Heat Shock Protein 90 Complex.

Nitric oxide (NO)-unresponsive forms of soluble guanylyl cyclase (sGC) exist naturally and in disease can disable NO-sGC-cGMP signaling. Agonists like BAY58-2667 (BAY58) target these sGC forms, but their mechanisms of action in living cells are unclear. We studied rat lung fibroblast-6 cells and human airway smooth muscle cells that naturally express sGC and HEK293 cells that we transfected to express sGC and variants. Cells were cultured to build up different forms of sGC, and we used fluorescence and FRET-based measures to monitor BAY58-driven cGMP production and any protein partner exchange or heme loss events that may occur for each sGC species. We found that: (i) BAY58 activated cGMP production by the apo-sGCß-Hsp90 species after a 5-8 minute delay that was associated with apo-sGCß exchanging its Hsp90 partner with an sGCα subunit. (ii) In cells containing an artificially constructed heme-free sGC heterodimer, BAY58 initiated an immediate and three times faster cGMP production. However, this behavior was not observed in cells expressing native sGC under any condition. (iii) BAY58 activated cGMP production by ferric heme sGC only after a 30-minute delay, coincident with it initiating a delayed, slow ferric heme loss from sGCß We conclude that the kinetics favor BAY58 activation of the apo-sGCß-Hsp90 species over the ferric heme sGC species in living cells. The protein partner exchange events driven by BAY58 account for the initial delay in cGMP production and also limit the speed of subsequent cGMP production in the cells. Our findings clarify how agonists like BAY58 may activate sGC in health and disease. SIGNIFICANCE STATEMENT: A class of agonists can activate cyclic guanosine monophosphate (cGMP) synthesis by forms of soluble guanylyl cyclase (sGC) that do not respond to NO and accumulate in disease, but the mechanisms of action are unclear. This study clarifies what forms of sGC exist in living cells, which of these can be activated by the agonists, and the mechanisms and kinetics by which each form is activated. This information may help to hasten deployment of these agonists for pharmaceutical intervention and clinical therapy.

Atrophy signaling pathways in respiratory and limb muscles of guinea pigs exposed to chronic cigarette smoke: role of soluble guanylate cyclase stimulation.

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is characterized by a significant reduction in muscle strength and endurance. Preclinical studies show that stimulation of the soluble guanylate cyclase (sGC)-cGMP pathway attenuates muscle mass loss and prevents cigarette smoke-induced oxidative stress, indicating that pharmacological activation of the guanylyl cyclase pathway in COPD may provide a beneficial therapeutic strategy that reaches beyond the lung. In this study, conducted in an animal model of COPD, we first set out to assess the effect of cigarette smoke (CS) on biomarkers of muscle fatigue, such as protein degradation and its transcriptional regulation, in two types of muscles with different energy demands, i.e., the diaphragm and the gastrocnemius muscle of the limbs. Second, we evaluated the administration of an sGC stimulator on these markers to study the potential efficacy of such treatment in the recovery of skeletal muscle function. Exposure to CS led to weight loss, which was associated in the gastrocnemius with increased levels of proteolytic markers of muscle atrophy (MURF-1, Atrogin-1, proteasome C8 subunit 20 s, and total protein ubiquitination), whereas the size of fast-twitch muscle fibers decreased significantly. Long-term treatment with the sGC stimulator BAY 41-2272 resulted in a significant reduction in gastrocnemius levels of the aforementioned proteolytic markers, concomitant with a weight recovery and increased cGMP levels. Remarkably, levels of some of the analyzed biomarkers differed between respiratory and limb muscles. In conclusion, targeting sGC might exert beneficial effects on muscle alterations in patients with COPD.

Vericiguat in Patients with Heart Failure and Reduced Ejection Fraction.

BACKGROUND: The effect of vericiguat, a novel oral soluble guanylate cyclase stimulator, in patients with heart failure and reduced ejection fraction who had recently been hospitalized or had received intravenous diuretic therapy is unclear. METHODS: In this phase 3, randomized, double-blind, placebo-controlled trial, we assigned 5050 patients with chronic heart failure (New York Heart Association class II, III, or IV) and an ejection fraction of less than 45% to receive vericiguat (target dose, 10 mg once daily) or placebo, in addition to guideline-based medical therapy. The primary outcome was a composite of death from cardiovascular causes or first hospitalization for heart failure. RESULTS: Over a median of 10.8 months, a primary-outcome event occurred in 897 of 2526 patients (35.5%) in the vericiguat group and in 972 of 2524 patients (38.5%) in the placebo group (hazard ratio, 0.90; 95% confidence interval [CI], 0.82 to 0.98; P = 0.02). A total of 691 patients (27.4%) in the vericiguat group and 747 patients (29.6%) in the placebo group were hospitalized for heart failure (hazard ratio, 0.90; 95% CI, 0.81 to 1.00). Death from cardiovascular causes occurred in 414 patients (16.4%) in the vericiguat group and in 441 patients (17.5%) in the placebo group (hazard ratio, 0.93; 95% CI, 0.81 to 1.06). The composite of death from any cause or hospitalization for heart failure occurred in 957 patients (37.9%) in the vericiguat group and in 1032 patients (40.9%) in the placebo group (hazard ratio, 0.90; 95% CI, 0.83 to 0.98; P = 0.02). Symptomatic hypotension occurred in 9.1% of the patients in the vericiguat group and in 7.9% of the patients in the placebo group (P = 0.12), and syncope occurred in 4.0% of the patients in the vericiguat group and in 3.5% of the patients in the placebo group (P = 0.30). CONCLUSIONS: Among patients with high-risk heart failure, the incidence of death from cardiovascular causes or hospitalization for heart failure was lower among those who received vericiguat than among those who received placebo. (Funded by Merck Sharp & Dohme [a subsidiary of Merck] and Bayer; VICTORIA ClinicalTrials.gov number, NCT02861534.).

Nonclinical Cardiovascular Assessment of the Soluble Guanylate Cyclase Stimulator Vericiguat.

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action-related dose-dependent changes in systolic arterial blood pressure (up to -20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (-6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1. SIGNIFICANCE STATEMENT: There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.

The Novel, Clinical-Stage Soluble Guanylate Cyclase Activator BI 685509 Protects from Disease Progression in Models of Renal Injury and Disease.

Activation of soluble guanylate cyclase (sGC) to restore cyclic guanosine monophosphate (cGMP) and improve functionality of nitric oxide (NO) pathways impaired by oxidative stress is a potential treatment of diabetic and chronic kidney disease. We report the pharmacology of BI 685509, a novel, orally active small molecule sGC activator with disease-modifying potential. BI 685509 and human sGC α1/ß1 heterodimer containing a reduced heme group produced concentration-dependent increases in cGMP that were elevated modestly by NO, whereas heme-free sGC and BI 685509 greatly enhanced cGMP with no effect of NO. BI 685509 increased cGMP in human and rat platelet-rich plasma treated with the heme-oxidant ODQ; respective EC50 values were 467 nM and 304 nM. In conscious telemetry-instrumented rats, BI 685509 did not affect mean arterial pressure (MAP) or heart rate (HR) at 3 and 10 mg/kg (p.o.), whereas 30 mg/kg decreased MAP and increased HR. Ten days of BI 685509 at supratherapeutic doses (60 or 100 mg/kg p.o., daily) attenuated MAP and HR responses to a single 100 mg/kg challenge. In the ZSF1 rat model, BI 685509 (1, 3, 10, and 30 mg/kg per day, daily) coadministered with enalapril (3 mg/kg per day) dose-dependently reduced proteinuria and incidence of glomerular sclerosis; MAP was modestly reduced at the higher doses versus enalapril. In the 7-day rat unilateral ureteral obstruction model, BI 685509 dose-dependently reduced tubulointerstitial fibrosis (P < 0.05 at 30 mg/kg). In conclusion, BI 685509 is a potent, orally bioavailable sGC activator with clear renal protection and antifibrotic activity in preclinical models of kidney injury and disease. SIGNIFICANCE STATEMENT: BI 685509 is a novel small soluble guanylate cyclase (sGC) molecule activator that exhibits an in vitro profile consistent with that of an sGC activator. BI 685509 reduced proteinuria and glomerulosclerosis in the ZSF1 rat, a model of diabetic kidney disease (DKD), and reduced tubulointerstitial fibrosis in a rat 7-day unilateral ureteral obstruction model. Thus, BI 685509 is a promising new therapeutic agent and is currently in phase II clinical trials for chronic kidney disease and DKD.

Soluble guanylate cyclase stimulation fosters angiogenesis and blunts myofibroblast-like features of systemic sclerosis endothelial cells.

OBJECTIVES: In SSc, angiogenesis impairment advances in parallel with the development of fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-mesenchymal transition (EndoMT). Soluble guanylate cyclase (sGC) stimulation has shown antifibrotic effects in SSc skin fibroblasts and mouse models. Here, we investigated the effects of pharmacological sGC stimulation on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-MVECs). METHODS: To determine whether sGC stimulation affected cell viability/proliferation, SSc-MVECs and healthy dermal MVECs (H-MVECs) were challenged with the sGC stimulator (sGCS) MK-2947 and assayed by annexin V/propidium iodide flow cytometry and the water-soluble tetrazolium salt (WST-1) assay. To study angiogenesis and EndoMT, MK-2947-treated SSc-MVECs were subjected to wound healing and capillary morphogenesis assays and analysed for the expression of endothelial/myofibroblast markers and contractile ability. RESULTS: MK-2947 treatment did not affect H-MVEC viability/proliferation, while it significantly increased SSc-MVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-MVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in the expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 mesenchymal markers was also found in MK-2947-treated SSc-MVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-MVECs to contract collagen gels and reduced phosphorylated-extracellular signal-regulated kinases 1 and 2 protein levels. CONCLUSION: These findings demonstrate for the first time that pharmacological sGC stimulation effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-MVECs, thus providing new evidence for repurposing sGCSs for SSc.

Mechanisms and Models in Heart Failure: A Translational Approach.

Despite multiple attempts to develop a unifying hypothesis that explains the pathophysiology of heart failure with a reduced ejection fraction (HFrEF), no single conceptual model has withstood the test of time. In the present review, we discuss how the results of recent successful phase III clinical development programs in HFrEF are built upon existing conceptual models for drug development. We will also discuss where recent successes in clinical trials do not fit existing models to identify areas where further refinement of current paradigms may be needed. To provide the necessary structure for this review, we will begin with a brief overview of the pathophysiology of HFrEF, followed by an overview of the current conceptual models for HFrEF, and end with an analysis of the scientific rationale and clinical development programs for 4 new therapeutic classes of drugs that have improved clinical outcomes in HFrEF. The 4 new therapeutic classes discussed are ARNIs, SGLT2 (sodium-glucose cotransporter 2) inhibitors, soluble guanylate cyclase stimulators, and myosin activators. With the exception of SGLT2 inhibitors, each of these therapeutic advances was informed by the insights provided by existing conceptual models of heart failure. Although the quest to determine the mechanism of action of SGLT2 inhibitors is ongoing, this therapeutic class of drugs may represent the most important advance in cardiovascular therapeutics of recent decades and may lead to rethinking or expanding our current conceptual models for HFrEF.

Hydrogen sulfide regulates the redox state of soluble guanylate cyclase in CSE-/- mice corpus cavernosum microcirculation.

The corpus cavernosum (CC) is a highly vascularized tissue and represents an excellent example of microcirculation. Indeed, erectile dysfunction is considered an early index of cardiovascular disease. Hydrogen sulfide (H2S) at the vascular level is endogenously produced from L-cysteine mainly by the action of cystathionine-γ-lyase (CSE) and plays a role in CC vascular homeostasis. Here we have evaluated the involvement of the endogenous H2S in the regulation of the soluble guanylate cyclase (sCG) redox state. The lack of CSE-derived endogenous H2S, in CSE-/- mice, disrupted the eNOS/NO/sGC/PDE pathway. Indeed, the absence of CSE-derived endogenous H2S caused a significant reduction of the relaxant response to riociguat, an sGC redox-dependent stimulator. Conversely, the response to cinaciguat, an sGC redox-independent activator, was not modified. The relevance of the role played at the redox level of the endogenous H2S was confirmed by the findings that in CC harvested from CSE-/- mice there was a significant reduction of GCß1 expression coupled with a decrease in CYP5R3, a reductase involved in the regulation of the redox state of sGC. These molecular changes driven by the lack of endogenous H2S translate into a significant reduction in cGMP levels. The replenishment of the lack of H2S with an H2S donor rescued the relaxant response to riociguat in CC of CSE-/- mice. In conclusion, the endogenous CSE-derived H2S plays a physiological key role in the regulation of the redox state of sGC in CC microcirculation.

Randomized placebo-controlled crossover study to assess tolerability and pharmacodynamics of zagociguat, a soluble guanylyl cyclase stimulator, in healthy elderly.

AIMS: Dysfunction of nitric oxide-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate signalling is implicated in the pathophysiology of cognitive impairment. Zagociguat is a central nervous system (CNS) penetrant sGC stimulator designed to amplify nitric oxide-cyclic guanosine monophosphate signalling in the CNS. This article describes a phase 1b study evaluating the safety and pharmacodynamic effects of zagociguat. METHODS: In this randomized crossover study, 24 healthy participants aged ≥65 years were planned to receive 15 mg zagociguat or placebo once daily for 2 15-day periods separated by a 27-day washout. Adverse events, vital signs, electrocardiograms and laboratory tests were conducted to assess safety. Pharmacokinetics of zagociguat were evaluated in blood and cerebrospinal fluid (CSF). Pharmacodynamic assessments included evaluation of cerebral blood flow, CNS tests, pharmaco-electroencephalography, passive leg movement and biomarkers in blood, CSF and brain. RESULTS: Twenty-four participants were enrolled; 12 participants completed both treatment periods, while the other 12 participants completed only 1 treatment period. Zagociguat was well-tolerated and penetrated the blood-brain barrier, with a CSF/free plasma concentration ratio of 0.45 (standard deviation 0.092) measured 5 h after the last dose of zagociguat on Day 15. Zagociguat induced modest decreases in blood pressure. No consistent effects of zagociguat on other pharmacodynamic parameters were detected. CONCLUSION: Zagociguat was well-tolerated and induced modest blood pressure reductions consistent with other sGC stimulators. No clear pharmacodynamic effects of zagociguat were detected. Studies in participants with proven reduced cerebral blood flow or CNS function may be an avenue for further evaluation of the compound.

Stimulation of soluble guanylate cyclase exerts antiinflammatory actions in the liver through a VASP/NF-κB/NLRP3 inflammasome circuit.

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine triphosphate into cyclic guanosine-3',5'-monophosphate, a key second messenger in cell signaling and tissue homeostasis. It was recently demonstrated that sGC stimulation is associated with a marked antiinflammatory effect in the liver of mice with experimental nonalcoholic steatohepatitis (NASH). Here, we investigated the mechanisms underlying the antiinflammatory effect of the sGC stimulator praliciguat (PRL) in the liver. Therapeutic administration of PRL exerted antiinflammatory and antifibrotic actions in mice with choline-deficient l-amino acid-defined high-fat diet-induced NASH. The PRL antiinflammatory effect was associated with lower F4/80- and CX3CR1-positive macrophage infiltration into the liver in parallel with lower Ly6CHigh- and higher Ly6CLow-expressing monocytes in peripheral circulation. The PRL antiinflammatory effect was also associated with suppression of hepatic levels of interleukin (IL)-1ß, NLPR3 (NACHT, LRR, and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), and active cleaved-caspase-1, which are components of the NLRP3 inflammasome. In Kupffer cells challenged with the classical inflammasome model of lipopolysaccharide plus adenosine triphosphate, PRL inhibited the priming (expression of Il1b and Nlrp3) and blocked the release of mature IL-1ß. Mechanistically, PRL induced the protein kinase G (PKG)-mediated phosphorylation of the VASP (vasodilator-stimulated phosphoprotein) Ser239 residue which, in turn, reduced nuclear factor-κB (NF-κB) activity and Il1b and Nlrp3 gene transcription. PRL also reduced active cleaved-caspase-1 levels independent of pannexin-1 activity. These data indicate that sGC stimulation with PRL exerts antiinflammatory actions in the liver through mechanisms related to a PKG/VASP/NF-κB/NLRP3 inflammasome circuit.

Soluble Guanylate Cyclase Activators and Stimulators in Patients with Heart Failure.

PURPOSE OF REVIEW: This review sought to summarize the current emerging soluble guanylate cyclase activators and stimulators in patients with heart failure, both heart failure with reduced and preserved ejection fraction, to provide a reference for soluble guanylate cyclase activators and stimulators discovery. RECENT FINDINGS: Heart failure is a common disease with considerable morbidity, hospitalization, and mortality Soluble guanylate cyclase is a key enzyme in the nitric oxide signaling pathway and has attracted rapidly growing interest as a therapeutic target in heart failure. Currently, several soluble guanylate cyclase agonists are in clinical development. Cinaciguat and praliciguat have shown no clear clinical benefit in patients with heart failure in clinical trials. Riociguat increased the 6-min walk distance, cardiac index, and stroke volume index as well as decreased N-terminal pro-B-type natriuretic peptide. Although these populations cover nearly every range of ejection fractions, these were not clinical trials directly in patients with heart failure but were designed in patients with pulmonary hypertension. Vericiguat is recommended in the latest American guideline for patients with heart failure with reduced ejection fraction; however, it gets mixed results in patients with heart failure with preserved ejection fraction. To date, only vericiguat reduces the composite of death from cardiovascular causes or first hospitalization for heart failure in patients with heart failure with reduced ejection fraction and riociguat might improve clinical symptoms and quality of life in patients with heart failure, both heart failure with reduced and preserved ejection fraction. We need more exploration about soluble guanylate cyclase activators and stimulators in patients with heart failure.

Anti-Fibrotic and Anti-Inflammatory Role of NO-Sensitive Guanylyl Cyclase in Murine Lung.

Pulmonary fibrosis is a chronic and progressive disease with limited therapeutic options. Nitric oxide (NO) is suggested to reduce the progression of pulmonary fibrosis via NO-sensitive guanylyl cyclase (NO-GC). The exact effects of NO-GC during pulmonary fibrosis are still elusive. Here, we used a NO-GC knockout mouse (GCKO) and examined fibrosis and inflammation after bleomycin treatment. Compared to wildtype (WT), GCKO mice showed an increased fibrotic reaction, as myofibroblast occurrence (p = 0.0007), collagen content (p = 0.0006), and mortality (p = 0.0009) were significantly increased. After fibrosis induction, lymphocyte accumulations were observed in the lungs of GCKO but not in WT littermates. In addition, the total number of immune cells, specifically lymphocytes (p = <0.0001) and neutrophils (p = 0.0047), were significantly higher in the bronchoalveolar lavage fluid (BALF) of GCKO animals compared to WT, indicating an increased inflammatory response in the absence of NO-GC. The pronounced fibrotic response in GCKO mice was paralleled by significantly increased levels of transforming growth factor ß (TGFß) in BALF (p = 0.0207), which correlated with the total number of immune cells. Taken together, our data show the effect of NO-GC deletion in the pathology of lung fibrosis and the effect on immune cells in BALF. In summary, our results show that NO-GC has anti-inflammatory and anti-fibrotic properties in the murine lung, very likely by attenuating TGFß-mediated effects.

A Systematic Review of the Effect of Vericiguat on Patients with Heart Failure.

Despite recent advances in heart failure (HF) therapy, the risk of cardiovascular (CV) mortality, morbidity, and HF hospitalization (HFH) are major challenges in HF treatment. We aimed to review the potential of vericiguat as a treatment option for HF. A systematic literature review was performed using the PubMed database and ClinicalTrials.gov. Four randomized controlled trials were identified, which study the safety and efficacy of vericiguat in HF patients. Vericiguat activates soluble guanylate cyclase (sGC) by binding to the beta-subunit, bypassing the requirement for NO-induced activation. The nitric oxide (NO)-sGC-cyclic guanosine monophosphate (cGMP) pathway plays an essential role in cardiovascular (CV) regulation and the protection of healthy cardiac function but is impaired in HF. Vericiguat reduced the risk of CV death and HFH in HF patients with reduced ejection fraction (HFrEF) but showed no therapeutic effect on HF with preserved ejection fraction (HFpEF). The trials demonstrated a favorable safety profile with most common adverse events such as hypotension, syncope, and anemia. Therefore, vericiguat is recommended for patients with HFrEF and a minimum systolic blood pressure of 100 mmHg. Treatment with vericiguat is considered when the individual patient experiences decompensation despite being on guideline-recommended medication, e.g., angiotensin-converting inhibitor/AT1 receptor antagonist, beta-adrenoceptor antagonist, spironolactone, and sodium-glucose transporter 2 inhibitors. Furthermore, larger studies are required to investigate any potential effect of vericiguat in HFpEF patients. Despite the limitations, vericiguat can be recommended for patients with HFrEF, where standard-of-care is insufficient, and the disease worsens.

Inhaled Nitric Oxide Promotes Angiogenesis in the Rodent Developing Brain.

Inhaled nitric oxide (iNO) is a therapy used in neonates with pulmonary hypertension. Some evidence of its neuroprotective properties has been reported in both mature and immature brains subjected to injury. NO is a key mediator of the VEGF pathway, and angiogenesis may be involved in the reduced vulnerability to injury of white matter and the cortex conferred by iNO. Here, we report the effect of iNO on angiogenesis in the developing brain and its potential effectors. We found that iNO promotes angiogenesis in the developing white matter and cortex during a critical window in P14 rat pups. This shift in the developmental program of brain angiogenesis was not related to a regulation of NO synthases by exogenous NO exposure, nor the VEGF pathway or other angiogenic factors. The effects of iNO on brain angiogenesis were found to be mimicked by circulating nitrate/nitrite, suggesting that these carriers may play a role in transporting NO to the brain. Finally, our data show that the soluble guanylate cyclase/cGMP signaling pathway is likely to be involved in the pro-angiogenetic effect of iNO through thrombospondin-1, a glycoprotein of the extracellular matrix, inhibiting soluble guanylate cyclase through CD42 and CD36. In conclusion, this study provides new insights into the biological basis of the effect of iNO in the developing brain.