The Role of sGC Stimulators and Activators in Heart Failure With Reduced Ejection Fraction

Over the past decade, soluble guanylate cyclase (sGC) activators and stimulators have been developed and studied to improve outcomes in patients with heart failure with reduced ejection fraction (HFrEF). The sGC enzyme plays an important role in the nitric oxide (NO)-sGC-cyclic guanosine monophosphate (cGMP) pathway, that has been largely untargeted by current guideline directed medical therapy (GDMT) for HFrEF. Disruption of the NO-sCG-cGMP pathway can be widely observed in patients with HFrEF leading to endothelial dysfunction. The disruption is caused by an oxidized state resulting in low bioavailability of NO and cGMP. The increase in reactive oxygen species can also result in an oxidized, and subsequently heme free, sGC enzyme that NO is unable to activate, furthering the endothelial dysfunction. The novel sGC stimulators enhance the sensitivity of sGC to NO, and independently stimulate sGC, while the sGC activators target the oxidized and heme free sGC to stimulate cGMP production. This review will discuss the pathophysiologic basis for sGC stimulator and activator use in HFrEF, review the pre-clinical and clinical data, and propose a place in the HFrEF armamentarium for this novel pharmacotherapeutic class.

Current Advances of Nitric Oxide in Cancer and Anticancer Therapeutics

Nitric oxide (NO) is a short-lived, ubiquitous signaling molecule that affects numerous critical functions in the body. There are markedly conflicting findings in the literature regarding the bimodal effects of NO in carcinogenesis and tumor progression, which has important consequences for treatment. Several preclinical and clinical studies have suggested that both pro- and antitumorigenic effects of NO depend on multiple aspects, including, but not limited to, tissue of generation, the level of production, the oxidative/reductive (redox) environment in which this radical is generated, the presence or absence of NO transduction elements, and the tumor microenvironment. Generally, there are four major categories of NO-based anticancer therapies: NO donors, phosphodiesterase inhibitors (PDE-i), soluble guanylyl cyclase (sGC) activators, and immunomodulators. Of these, NO donors are well studied, well characterized, and also the most promising. In this study, we review the current knowledge in this area, with an emphasis placed on the role of NO as an anticancer therapy and dysregulated molecular interactions during the evolution of cancer, highlighting the strategies that may aid in the targeting of cancer.

Homogeneous single-label cGMP detection platform for the functional study of nitric oxide-sensitive (soluble) guanylyl cyclases and cGMP-specific phosphodiesterases

Cardiovascular diseases are the number one death worldwide. Nitric oxide (NO)—NO-sensitive (soluble) guanylyl cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway regulates diverse set of important physiological functions, including maintenance of cardiovascular homeostasis. Resting and activated sGC enzyme converts guanosine triphosphate to an important second messenger cGMP. In addition to traditional NO generators, a number of sGC activators and stimulators are currently in clinical trials aiming to support or increase sGC activity in various pathological conditions. cGMP-specific phosphodiesterases (PDEs), which degrade cGMP to guanosine monophosphate, play key role in controlling the cGMP level and the strength or length of the cGMP-dependent cellular signaling. Thus, PDE inhibitors also have clear clinical applications. Here, we introduce a homogeneous quenching resonance energy transfer (QRET) for cGMP to monitor both sGC and PDE activities using high throughput screening adoptable method. We demonstrate that using cGMP-specific antibody, sGC or PDE activity and the effect of small molecules modulating their function can be studied with sub-picomole cGMP sensitivity. The results further indicate that the method is suitable for monitoring enzyme reactions also in complex biological cellular homogenates and mixture.

Abstract P093: Cytochrome B5 Reductase 3 And Xanthine Oxidase Coordinately Regulate Soluble Guanylyl Cyclase Physiological Redox State

In cardiovascular disease, oxidative stress can drive soluble guanylyl cyclase (sGC) heme oxidation resulting in the loss of the sGC heme (apo-sGC), the impairment of nitric oxide (NO) binding and cGMP production, and vasoconstriction. Consequently, a new class of therapeutic compounds sGC activators have been developed which target oxidized and apo-sGC to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. While sGC activators have had varied clinical success, surprisingly few studies have defined the impact of NO-independent sGC activation on vascular physiology in healthy conditions. We found mesenteric and pulmonary arteries are two log orders more sensitive to NO-independent sGC activator BAY 58-2667 induced vasodilation than aorta; no difference in NO-dependent sGC vasodilation between vessels was observed. These data indicate the presence of an activatable physiological pool of oxidized and/or apo-sGC in pulmonary and mesenteric arteries. We recently published that smooth muscle cell cytochrome b5 reductase 3 (CYB5R3) acts to reduce oxidized heme sGC back to its NO-sensitive reduced heme state during vascular disease. We found transgenic CYB5R3 overexpression (CYB5R3 OE) mice were more resistant to BAY 58-2667 mesenteric artery vasodilation and blood pressure lowering compared to wild-type controls (n=5-9) under physiologic conditions. Also, healthy CYB5R3 OE pulmonary arteries had a near complete loss of BAY 58-2667 vasodilation suggesting both mesenteric and pulmonary arteries contain a pool of oxidized sGC. We next asked if physiological H 2 O 2 production accounts for changes in BAY 58-2667 responsiveness. We found using mitochondrial-specific catalase overexpression mice, that BAY 58-2667 vasodilation did not differ from controls in any vascular bed (n=4-6). We next tested whether xanthine oxidase (XO), which can produce H 2 O 2 at the endothelial cell surface of vessels, can impact physiological BAY 58-2667 vasodilation. We found that Febuxostat, a XO inhibitor, led to a significant decrease in mesenteric artery BAY 58-2667 induced vasodilation from ~70% to ~30% (n=6). Combined, these data provide evidence for CYB5R3 and XO as regulators of physiological sGC resistance artery vasodilation.

sGC Stimulators and Activators

Nitric oxide (NO)-soluble guanylate cyclase(sGC)-cGMP signalling is impaired in HF syndromes, which could predispose to vascular oxidative stress. Nitrates directly stimulate cGMP, but are limited by tolerance. Therapeutic targets that aim at increasing cGMP concentrations have therefore been explored. Recently, two classes of drugs have been discovered, the sGC activators and the sGC stimulators, which target two different redox states of sGC: the NO-sensitive reduced (ferrous) sGC and NO-insensitive oxidized (ferric) sGC, respectively. Cinaciguat is an activator and riociguat and vericiguat are sGC stimulators. Vericiguat is the most advanced agent in its clinical trial programme with two completed phase IIb studies, SOCRATES -REDUCED in HFrEF and SOCRATES-PRESERVED in HFpEF, with mixed results on NT-proBNP. The ongoing VICTORIA trial in HFrEF will study 4,872 participants with a mortality/morbidity end-point and VITALITY HFpEF trial will study 735 participants, with a quality of life end-point.

The Potential of sGC Modulators for the Treatment of Age-Related Fibrosis: A Mini-Review

Fibrotic diseases cause high rates of morbidity and mortality, and their incidence increases with age. Despite intense research and development efforts, effective and well-tolerated antifibrotic treatments are scarce. Transforming growth factor-β signaling, which is widely considered the most important profibrotic factor, causes a pro-oxidant shift in redox homeostasis and a concomitant decrease in nitric oxide (NO) signaling. The NO/cyclic guanosine monophosphate (cGMP) signaling cascade plays a pivotal role in the regulation of cell and organ function in whole-body hemostasis. Increases in NO/cGMP can lead to relaxation of smooth muscle cells triggering vasorelaxation. In addition, there is consistent evidence from preclinical in vitro and in vivo models that increased cGMP also exerts antifibrotic effects. However, most of these findings are descriptive and the molecular pathways are still being investigated. Furthermore, in a variety of fibrotic diseases and also during the natural course of aging, NO/cGMP production is low, and current treatment approaches to increase cGMP levels might not be sufficient. The introduction of compounds that specifically target and stimulate soluble guanylate cyclase (sGC), the so called sGC stimulators and sGC activators, might be able to overcome these limitations and could be ideal tools for investigating antifibrotic mechanisms in vitro and in vivo as they may provide effective treatment strategies for fibrotic diseases. These drugs increase cGMP independently from NO via direct modulation of sGC activity, and have synergistic and additive effects to endogenous NO. This review article describes the NO/cGMP signaling pathway and its involvement in fibrotic remodeling. The classes of sGC modulator drugs and their mode of action are described. Finally, the preclinical in vitro and in vivo findings and antifibrotic effects of cGMP elevation via sGC modulation are reviewed. sGC stimulators and activators significantly attenuate tissue fibrosis in a variety of internal organs and in the skin. Moreover, these compounds seem to have multiple intervention sites and may reduce extracellular matrix formation, fibroblast proliferation, and myofibroblast activation. Thus, sGC stimulators and sGC activators may offer an efficacious and tolerable therapy for fibrotic diseases, and clinical trials are currently underway to assess the potential benefit for patients with systemic sclerosis.

Abstract P175: Soluble Guanylate Cyclase Activator Attenuates Tumor Necrosis Factor- α Induced Production of Endothelin-1 from Human Glomerular Endothelial Cells

Preeclampsia (PE) is a disorder associated with maternal hypertension, endothelial dysfunction and reductions in renal hemodynamics. Placental ischemia leads to increases in circulating maternal anti-angiogenic and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) that induce endothelin-1 (ET-1), a potent vasoconstrictor. PE is also associated with depletion of nitric oxide, a facilitator of vasodilation, which binds to soluble guanylate cyclase (sGC), and synthesizes cGMP. In addition to promoting vasodilation, sGC activators and stimulators inhibit smooth muscle proliferation, leukocyte recruitment and platelet aggregation and are therefore, currently in clinical trials for treating cardiopulmonary disease. Although it is known that activating the nitric oxide signalling pathway induces vasodilation, its ability to inhibit TNF-α induced renal glomerular endothelial ET-1 production is unknown. We tested the hypothesis that cinaciguat, a sGC activator, attenuates ET-1 production induced by TNF-α in conditionally immortalized human glomerular endothelial cells. Cells were cultured; starved for 48 h; and treated for 12 h resulting in the following 4 groups having N=6/group: 1) Untreated, 2) 10 ng TNF-α 3) 10 μM cinaciguat + 10 ng TNF-α, and 4) 20 μM cinaciguat + 10 ng TNF-α. TNF-α (10 ng, 67.25±3.2 pg/mL) significantly increased ET-1 production compared to the untreated group (43.6±4.3 pg/mL, P<0.01). Interestingly, both cinaciguat treatment groups attenuated TNF-α induced ET-1 production, with significant reductions at a higher dose (20 μM, 57.38±1.42 pg/mL, P=0.02; 10 μM, 58.6±2.32 pg/mL, P=0.07). The results of this study demonstrate that activating sGC can attenuate ET-1 production. In conclusion, these findings suggest there is a therapeutic potential for treating preeclampsia with sGC activators.