Recent developments in cGMP research: From mechanisms to medicines and back
2022-03-25

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 Cyclic guanosine monophosphate (cGMP) is a ubiquitous second messenger of wide-ranging physiological, pathophysiological, and pharmacological significance (Hofmann, 2020; Petraina et al., 2021; Sandner et al., 2021). Cellular receptors causing a rise in cGMP include two types of guanylyl cyclase, which convert GTP to cGMP and are activated by important cardiovascular and neuronal ligands. Transmembrane or “particulate” guanylyl cyclases (pGCs) are homodimers stimulated by extracellular peptides such as atrial, brain, or C-type natriuretic peptide (ANP, BNP, or CNP, respectively). Nitric oxide (NO)-sensitive guanylyl cyclases (NO-GCs) are heterodimeric cytosolic enzymes activated by binding of NO to their prosthetic haem group. The discovery of small-molecule modulators of NO-GC was a milestone in the field of cGMP pharmacology. NO-GC stimulators are allosteric regulators that enhance the activity of the reduced, haem-containing NO-GC. These compounds act independently of NO and synergistically with endogenous NO. NO-GC activators specifically bind to, and activate, the oxidized, haem-free form of NO-GC. cGMP targets downstream effectors such as cGMP-dependent protein kinases (cGKs, also known as PKGs) and cyclic nucleotide-gated cation channels. cGMP-hydrolysing phosphodiesterases (PDEs) control the duration, amplitude, and spread of an intracellular cGMP signal. Some PDEs degrade both cGMP and cyclic adenosine monophosphate (cAMP) and some cAMP-PDEs are regulated by cGMP, providing a mechanism for cGMP-cAMP crosstalk. Pharmacological approaches targeting the NO/NO-GC and natriuretic peptide/pGC cascades have advanced to clinical management of various diseases such as erectile dysfunction (Goldstein et al., 1998), pulmonary arterial hypertension (Galie et al., 2005; Ghofrani et al., 2013), and more recently heart failure (HF) (Armstrong et al., 2020; McMurray et al., 2014). Indeed, out of 50 novel drugs approved by the U.S. Food and Drug Administration in 2021, two target the cGMP signalling system, that is, the NO-GC stimulator vericiguat to treat chronic HF with reduced ejection fraction and the pGC stimulator vosoritide to treat achondroplasia, a genetic disorder leading to short stature (visit Novel Drug Approvals for 2021 at https://www.fda.gov/drugs/). Both drugs are classified as first-in-class, having mechanisms of action different from those of existing medicines. Thus, the translational potential of preclinical findings with regard to new indications for cGMP-modulating drugs should be enormous. Although our knowledge about NO- and natriuretic peptide-dependent cGMP signal transduction and its diverse cellular effects has substantially increased in the last few decades, critical gaps remain. These gaps mostly concern the mechanisms and consequences of subcellular cGMP compartmentation as well as the identity and regulation of effectors, that mediate cGMP's effects in vivo (Cuello & Nikolaev, 2020). In addition to its short-term effects on, for example, smooth muscle contractility, cGMP also plays an important role in the homeostatic control of cell growth and survival. However, the positive and potentially also negative effects of acutely or chronically altered cGMP concentrations on tissue remodelling, degeneration and regeneration are not completely understood. Current research aims at identifying the relevant cell types and molecular mechanisms targeted by cGMP pathway modulation. To guide the future development of cGMP-based drugs, it appears important to consider the dynamics of endogenous cGMP profiles under the respective pathophysiological condition. This British Journal of Pharmacology themed issue on cGMP signalling in cell growth and survival assembles five review articles followed by eight original research papers from leading scientists that improve our understanding of the effects of endogenous and pharmacologically modulated cGMP signals on tissue repair and maintenance. We hope that the presented insights will lead to new strategies for the management of several age-related disorders including diseases of the heart, lungs, kidneys, nervous system, and hearing organ. The review section of the themed issue begins with a contribution by Friebe and Englert focusing on cGMP in the lung (Friebe & Englert, 2022). This organ expresses particularly high levels of NO-GC, but neither the cell types expressing NO-GC in the lung (e.g., pericytes, smooth muscle cells, and epithelial cells) nor the NO-GC-mediated mechanisms that affect lung physiology and pathophysiology are clear. The article discusses how pharmacological NO-GC activation in the lung may benefit patients suffering from obstructive airway diseases, acute respiratory distress syndromes, pulmonary hypertension, and pulmonary fibrosis. Interestingly, despite this obviously huge therapeutic potential, only a few drugs targeting cGMP in the lung have been successful so far, perhaps because NO-GC exists during disease progression in different isoforms and redox states, which are differentially targeted by NO-GC stimulators and activators. Lukowski et al. address the cytoprotective functions of the cGMP pathway in the heart, a topic that has attracted a lot of interest in recent years (Lukowski et al., 2022). They provide an up-to-date overview of the underlying molecular and cellular mechanisms, highlighting that cGMP-mediated protection of cardiac muscle during ischaemia and reperfusion (I/R) injury in vivo requires activation of distinct K+ channels present in the inner compartment of the cardiomyocyte's mitochondria (Frankenreiter et al., 2017). Interestingly, cGMP signals are usually restricted to the cytosol and/or sub-sarcolemmal regions, which are distinct from the mitochondrial compartment of the cardiomyocyte. The article deals with the question of how signalling between cGMP-generating guanylyl cyclases and cGMP effectors in mitochondria can be established during cardiac I/R. The themed issue continues with two comprehensive review articles on cGMP-regulated neuronal and neurosensory functions. In the first contribution, Schmidt et al. discuss a CNP-cGMP-cGKI pathway that is critical for the correct branching of axons during embryonic development of the spinal cord, enabling neurons to connect with different targets in their vicinity (Schmidt et al., 2022). While genetic loss-of-function studies have clearly demonstrated the requirement of CNP, the CNP receptor GC-B, and cGKI for axon bifurcation, downstream mechanisms and targets phosphorylated by cGKI in these neurons remain poorly understood. The second part of the review summarizes how distinct cell populations in the spinal cord and dorsal root ganglia contribute to cGMP-controlled processing of pain and itch during adulthood. It appears that in addition to the CNP-cGMP-cGKI pathway alternative cGMP signalling cascades also modulate nociception in the adult somatosensory system. The contribution by Marchetta et al. describes in detail what is known about the functions of the NO-GC- and pGC-stimulated pathways in the auditory system (Marchetta et al., 2022). They present evidence that basal hearing is largely unaffected by the cGMP cascade. Interestingly, however, ageing- and noise injury-induced dysfunctions, as well as the integrity of sound processing pathways in peripheral and central parts of the auditory system, are differentially affected by NO-GC- and pGC-induced cGMP formation. Overall, Marchetta et al. conclude that local delivery of NO-GC or pGC modulators to the inner ear may counteract hearing loss. However, such strategies need to consider that the manipulation of discrete cGMP pools may yield different and even opposite functional effects in different parts of the auditory system. A question that must be answered in future is if otoprotective functions of cGMP stem from neurons and/or the vasculature in close vicinity to the cochlea. The last review article discusses genetically encoded biosensors that can be used to monitor cGMP dynamics in living cells and organisms (Feil et al., 2022). Single-cell imaging of cGMP has revealed a pronounced heterogeneity of cGMP signalling both within a cell (also referred to as “compartmentation” or “microdomains”) as well as between different cells of a tissue or organ (also referred to as “cell-to-cell variability”). cGMP imaging has been instrumental for the recent discoveries of intercellular cGMP transfer and mechanosensitive cGMP generation, and it can be used to characterize the mode of action of cGMP-elevating drugs. The article summarizes the wide range of cGMP biosensors and their applications in cardiomyocytes, vascular smooth muscle cells, and platelets, and discusses innovative future directions for optical mapping of cGMP in vitro and in vivo. The second part of this themed issue presents eight research papers that describe pharmacological cGMP modulation to preserve cellular functions in different physiological systems such as heart and kidney, new mechanisms of cGMP signalling, and novel optogenetic tools for manipulating cGMP locally in living cells by light. Previous work has concluded that PDE-5 and PDE-9 inhibition requires cGKI to oppose cardiac hypertrophy and fibrosis. In line with this model, cardiomyocyte-restricted deletion of cGKI provoked a more severe remodelling phenotype in mouse models of cardiac hypertrophy induced by angiotensin II or pressure overload. The beneficial effects attributed to cardiac cGKI are at odds with other studies suggesting that PDE-5 and cGKI in murine cardiomyocytes do not play major roles in pathological cardiac remodelling (Frantz et al., 2013; Lee et al., 2015; Lukowski et al., 2014; Patrucco et al., 2014). In this issue, Schwaerzer et al. (2022) add another piece to the puzzle of regulatory functions mediated by cGMP in the aged and diseased heart. In a cGKI mutant mouse model that expresses a constitutively active cGKI (cGKIR177Q/+ or Prkg1R177Q/+ mice), they find a mild acceleration of cardiac muscle fibrosis and more apoptotic myocytes in elderly (12-month-old) animals. In addition, left ventricular mass and other parameters of the cardiac structure were also affected by the presence of active cGKI in an age-related manner. Moreover, when young cGKIR177Q/+ animals were subjected to experimental models of cardiac hypertrophy, they developed more severe structural changes affecting cardiac chambers and walls, contractile dysfunction, cardiomyocyte hypertrophy, and cell death than control mice. Contrary to what has been known so far, the data of Schwaerzer et al. suggest that prolonged activation of cGKI may be harmful to aged or hemodynamically overloaded hearts. From a clinical perspective, caution is needed when considering long-term (over-)activation of cGKI for HF therapy. While the pathophysiological role of cGKI in myocardial hypertrophy is under debate, the preclinical study by Rüdebusch et al. (2022) substantiates the therapeutic potential of NO-GC stimulators such as riociguat in HF. Riociguat, clinically approved for patients with certain forms of pulmonary hypertension, acts as a NO sensitizer and also stimulates NO-GC in a NO-independent manner. In mice with established HF, riociguat (administered daily by oral gavage for 8 weeks) improved cardiac ejection fraction, decreased pathological myocardial fibrosis and myocyte cross-sectional area, and led to a reduction in heart weight as compared with non-treated mice. At the cellular level, riociguat opposed angiotensin II-induced pro-proliferative and pro-hypertrophic effects in cardiac fibroblasts and myocytes. Taken together, the results of Rüdebusch et al. support the concept that HF is associated with reduced bioavailability of NO and, therefore, pharmacological stimulation of NO-GC may prove beneficial under this condition. cGMP signals are usually terminated by the activity of cGMP-hydrolysing PDEs. In addition to this mechanism, Grange et al. (2022) followed up on the idea that cGMP efflux might also contribute to the kinetics of a cGMP-driven response. To test whether efflux of cGMP involves multidrug resistance-associated proteins (MRPs), the authors applied MK571, an inhibitor of MRP4 and MRP5, to human coronary artery smooth muscle cells, murine aorta, and a mouse model of pressure overload-induced HF. The key findings of this study are as follows: (i) cGMP generated via the ANP-pGC pathway in coronary artery smooth muscle cells is subject to significant MK571-sensitive cGMP efflux; (ii) vasorelaxant responses to ANP are amplified by MK571; (iii) although MK571 alone did not alter blood pressure, hypotensive responses to ANP were significantly enhanced by this compound; and (iv) MK571 reversed multiple parameters of cardiac dysfunction in mice with pressure overload-induced HF. These beneficial effects of MK571 were associated with a reduced plasma cGMP concentration, which indicates a relevant contribution of cGMP efflux via MRPs to pathological changes in the intracellular and extracellular cGMP equilibrium. It will be exciting to see if pharmacological MRP inhibition can be established to enhance or optimize the benefits of cGMP-based therapies. Compared with NO-GC stimulators, the clinical development of NO-GC activators is less advanced. NO-GC activators bind to the haem-free NO-unresponsive NO-GC, which might be increasingly formed under conditions of pathological oxidative stress. Chronic kidney disease (CKD) is associated with decreased NO bioavailability and increased oxidative stress, and NO-GC gain of function has been linked to a higher glomerular filtration rate and lower risk for CKD (Emdin et al., 2018). As such, CKD is an interesting indication, particularly for NO-GC activators. NO-GC stimulators and activators have been tested in several preclinical models of kidney diseases and resulted in reduced proteinuria and/or albuminuria and decreased renal glomerulosclerosis and fibrosis. In this issue, Harloff et al. (2022) explore the first-generation NO-GC activator cinaciguat in a mouse model of diabetic nephropathy, the leading cause of end-stage renal disease worldwide. Cinaciguat inhibited the progression of renal damage by attenuating glomerular and fibrotic changes, particularly in diabetic mice lacking endothelial NO synthase. In primary mesangial cells exposed to diabetic conditions, cinaciguat increased the level of NO-GC, presumably by binding to its haem pocket and thereby leading to the stabilization of the protein. Furthermore, cinaciguat suppressed the expression of thrombospondin 1, a key activator of transforming growth factor β and profibrotic processes in the diseased kidney. The clinical value of cinaciguat has recently been challenged in patients with acute HF (ClinicalTrials.gov Identifier: NCT01064037). Thus, cinaciguat appears to be useful to elucidate molecular mechanisms in preclinical models, but it may be not suitable to treat patients. New NO-GC activators are currently in development which show improved pharmacokinetics and a lower potential to cause hypotension (Sandner et al., 2021). Runcaciguat is a novel potent and selective second-generation NO-GC activator, which has great potential to prevent the decline of kidney function (Benardeau et al., 2021). In this issue, Stehle et al. (2022) studied the kidney-specific mechanisms behind the renoprotective effects of runcaciguat. They measured cGMP levels in real time in kidney slices of transgenic cGMP sensor mice and show that the runcagiguat-like compound BAY-453 indeed increases the glomerular cGMP concentration in the presence of oxidative stress. Moreover, runcaciguat induced dilation of glomerular arterioles and improved renal blood flow under disease-relevant oxidative stress conditions. Thus, runcaciguat's mode of action appears to be the dilation of renal blood vessels, presumably by activation of NO-GC in vascular smooth muscle cells. This may increase renal blood flow, thereby, slowing down the progression of kidney disease. NO-GC activators could represent a new therapeutic approach for the treatment of CKD patients. Runcaciguat is currently being tested for this indication in a phase 2 clinical trial (NCT04507061). A study by Galley et al. (2022) advances our understanding of renal hypertension and the regulation of cGMP signalling within the renal vascular wall. They analysed the potential crosstalk of the renin-angiotensin-aldosterone system and the cGMP signalling pathway using the murine two-kidney-one-clip model. In this model, reduced renal blood flow triggers activation of the renin-angiotensin-aldosterone system leading to renovascular hypertension. The authors show that in response to elevated levels of angiotensin II, NO-GC expression is increased in the renal smooth muscle through an angiotensin II type 1 receptor- and FoxO transcription factor-dependent mechanism. The increased vascular cGMP signalling improves renal blood flow and maintains electrolyte balance, thereby, providing a previously unrecognized protective response to counteract angiotensin II-dependent increases in systemic blood pressure. These findings also offer a rationale for the use of NO-GC modulators for the treatment of renal hypertension. In addition to kidney diseases, reduced bioavailability of NO may contribute to sickle cell disease (SCD), a condition that is characterized by chronic inflammation driven by ongoing haemolysis and hypoxia. Activation of the vascular endothelium and upregulation of endothelial adhesion receptors on the surface of the vascular wall enables interactions between sickled red blood cells, activated leukocytes, and endothelial cells. These events lead to occlusion of the venous microcirculation and eventual organ damage, with the kidney being one of the most affected organs in SCD patients. In a recent regular issue of the British Journal of Pharmacology, Tchernychev et al. (2021) tested the hypothesis that treatment with the NO-GC stimulator olinciguat attenuates endothelial cell activation, inflammation, and renal injury associated with SCD. Using various mouse models, they observed that olinciguat reduced the adhesion of leukocytes to vascular endothelium activated by tumour necrosis factor α-induced inflammation. Co-treatment with hydroxyurea, an approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In addition, olinciguat improved blood flow, mitigated kidney damage, and prolonged survival time compared with vehicle-treated SCD mice. These preclinical findings suggest that olinciguat could be useful to attenuate inflammation and organ damage in patients with SCD. However, recent phase 2 trials evaluating olinciguat in SCD patients (NCT03285178) and praliciguat, another NO-GC stimulator, in diabetic nephropathy (NCT03217591) did not meet their primary endpoints. Thus, further studies are needed to better understand the effect of NO-GC stimulators on inflammation and organ protection in SCD and beyond. An ongoing clinical phase 2 trial with riociguat in patients with SCD (NCT02633397) is expected to be completed in summer 2022. The molecular mechanisms that drive NO-GC maturation, inactivation and recovery are not completely understood. As outlined above, in many disease states, NO-GC may become insensitive towards NO activation. Clarifying what changes occur to NO-GC during its inactivation in living cells may direct strategies to preserve or recover NO-dependent cGMP signalling in disease. In this issue, Dai and Stuehr (2022) studied NO-GC inactivation triggered by the oxidant ODQ or by the NO donor NOC12 in HEK293 cells expressing a fluorescent NO-GC reporter construct allowing tracking of its haem content. They found that either inactivation regime led to dissociation of the NO-GC α/β-heterodimer and binding of the β subunit to heat shock protein 90. Interestingly, oxidation of ferrous haem to ferric was already sufficient to cause dissociation of the NO-GC heterodimer in cells. A connection between the NO-GC haem redox state and its heterodimer status has not been considered before in the literature. Most surprisingly, no haem was lost during or following the inactivation process, contradicting current dogma that posits haem loss as a common feature of NO-GC inactivation in tissues and cells. This study also helps to clarify how NO-GC activators may function in living systems. While it cannot be excluded that NO-GC activators are able to displace haem from inactive NO-GC in stressed cells, the authors also suggest a revised model that these compounds act by binding to the empty haem pocket of immature NO-GC that is complexed with heat shock protein 90 and is present in both healthy and stressed cells. As in other research fields, breakthroughs in cGMP research have often been initiated by new technological developments. In addition to real-time cGMP imaging in living cells and tissues (Feil et al., 2022) other powerful tools have been developed such as light-regulated cGMP generators and degraders. These optogenetic approaches enable the modulation of biological processes in a spatiotemporally highly defined manner within living cells and animals. This themed issue concludes with an original research article by Henß et al. (2022), who present a comprehensive optogenetic toolbox for cGMP manipulation. It allows fine-tuning of cGMP levels in living cells by light as well as two-component optogenetics by the combination of a guanylyl with K+ channels. Although the were in these novel optogenetic tools will be in higher organisms to gain exciting insights cGMP-mediated processes such as cell growth and survival. the clinical of riociguat and the recent of the therapeutic potential for NO-GC stimulators in lung diseases and chronic HF is clearly the clinical effects of these and many other cGMP-elevating drugs are to acute of smooth muscle cells. As outlined in this themed issue, recent preclinical evidence indicates that these drugs offer an even potential for the treatment of a wide range of diseases associated with altered cell growth and such as conditions, fibrotic diseases, and However, should in that cGMP may not only have beneficial In the need to improve our understanding of the potential effects of cGMP-based their long-term As in this themed issue, of the cGMP pathway might et al., 2022). studies that it might also vascular damage et al., 2021; et al., 2013; Schwaerzer et al., and et al., et al., et al., et al., the development of et al., et al., et al., and (Emdin et al., and of lung et al., 2021). these findings that our understanding of the positive and negative effects of cGMP-elevating drugs is from for further comprehensive of this signalling pathway at the and the increasingly heterogeneity of cGMP signalling both between and within cells of a tissue or organ (Feil et al., the development of cell and/or drugs, although might further improve the and of cGMP-based in the protein targets and in this article are to in the to and are in the to et al., et al., 2021; et al., 2021). 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