Decoding signaling mechanisms: unraveling the targets of guanylate cyclase agonists in cardiovascular and digestive diseases.

While sGC stimulators target reduced and heme-containing forms of sGC, sGC activators target oxidized or heme-free sGC. Since the status of these enzymes occurs mainly in the condition of diseases accompanied by oxidative stress, this binding pattern is quite attractive for the clinical use of activator drugs ( Evgenov et al., 2006 ). Cinaciguat, a type of amino dicarboxylic acid, is the first characteristic drug of this new sGC activator class ( Stasch et al., 2002 ). Although other sGC activators have been identified, no sGC activators are available to patients. A 1997 ultrahigh-throughput screening (uHTS) identified cinaciguat as an sGC activator ( Sandner et al., 2021b ). As a direct, NO-independent activator of sGC, cGMP levels directly increase in the presence of heightened oxidative stress and impaired endothelial function, which could yield notable efficacy. However, it increases the risk of low blood pressure ( Mitrovic et al., 2011 ). In a randomized, double-blind, multicenter, multinational phase IIb study ( NCT00559650 ), cinaciguat significantly reduced pulmonary capillary wedge pressure (PCWP) and mean right atrial pressure in patients with decompensated chronic congestive heart failure. There was also a decrease in both pulmonary and systemic vascular resistance, as well as a reduction in mean arterial pressure, and the cardiac index increased ( Mitrovic et al., 2011 ). Cinaciguat was associated with 71% of adverse events, and placebo was associated with 45%. There were no adverse events associated with 30-day mortality. When the dose was increased to 200 g/h, hypotensive events increased, and the trial was terminated ( Erdmann et al., 2013 ). Three phase IIb trials, including COMPOSE 1 ( NCT01065077 ), COMPOSE 2 ( NCT01067859 ), and the COMPOSE Early Trial ( NCT01064037 ), were subsequently conducted to investigate the safety and efficacy of varying doses of cinaciguat 200 μg/h versus placebo in treating patients with acute HF initiated at different time points. However, because hypotensive events occurred and no significant benefit was observed, the clinical development of cinaciguat was discontinued ( Breitenstein et al., 2017 ). The clinical trials led to the discontinuation of cinaciguat. Most of the research since then has been conducted on animals ( Benaldo et al., 2022 ; Dai and Stuehr, 2023 ). Ataciguat, formerly known as HMR 1766, is an anthranilic acid derivative that is a novel sGC activator ( Schafer et al., 2010 ). Researchers found that ataciguat normalized vasodilation and the vascular response to exogenous NO in rats with congestive heart failure and reduced platelet activation ( Schafer et al., 2010 ). In a rat model of inflammatory chronic renal impairment, ataciguat exhibited beneficial BP-independent effects on kidney structure and urinary albumin excretion ( Benz et al., 2007 ). Ataciguat is currently being studied in clinical trials for numerous indications, including changes in tolerated blood pressure and orthostatic tolerance (i.e., ability to stand without passing out) in patients with mild to moderate calcified aortic stenosis ( NCT02049203 ), effectiveness in relieving patients with neuropathic pain ( NCT00799656 ), improving claudication in patients with PAD ( NCT00443287 ), and slowing the progression of valve calcification in patients with moderate calcific aortic valve stenosis ( NCT02481258 ). However, the results of these clinical trials have not yet been made publicly available. MGV354 is a novel sGC activator that effectively lowers IOP in glaucoma models in preclinical studies ( Prasanna et al., 2018 ). Unfortunately, in a clinical trial ( NCT02743780 ), MGV354 did not cause a statistically significant reduction in IOP compared to placebo ( Stacy et al., 2018 ). Mosliciguat (BAY 1237592) is an sGC activator designed for topical application in the lung for the treatment of PAH. Inhalation of mosliciguat specifically activates apo-sGC, leading to a selective effect in the lung ( Becker-Pelster et al., 2022 ). Mosliciguat was shown to activate heme-free NO-GC and improve cardiopulmonary circulation in minipigs and rats ( Becker-Pelster et al., 2022 ). Based on these results, mosliciguat is currently in phase Ib clinical development ( NCT03754660 ) as an inhaled therapy for PAH. As a once-daily oral sGC activator, runcaciguat demonstrated good PK distribution when administered by Bayer ( Hahn et al., 2021 ). Preclinical studies have demonstrated that runcaciguat may be effective in preventing CKD caused by hypertension, diabetes, and obesity ( Benardeau et al., 2021 ). The oral sGC activator runcaciguat is currently in a phase II clinical program for patients with proteinuric CKD ( NCT04507061 ). BI 685509 is an orally bioavailable, potent sGC activator that exhibits significant renal protection properties and antifibrotic activity in preclinical models of kidney disease and injury ( Reinhart et al., 2023 ). In addition, in a preclinical rat model of thioacetamide-induced nodularity, hepatic fibrosis, portal hypertension and portosystemic shunts, BI 685509 reduced Sirius red morphometry (SRM) by 38%, alpha-smooth muscle actin (αSMA)-positive area by 55%, portal pressure by 26% and portal shunt by 10%; hence, it could be used as a potential treatment for cirrhosis-associated portal hypertension ( Jones et al., 2023 ). BI 685509 effectively inhibited the induction effect of activated platelet-rich plasma on the SSC-related chemokine CXCL4, more strongly than riociguat did ( Nabozny et al., 2023 ). These findings suggest that BI 685509 is a new drug to treat SSc that is superior to riociguat. In a phase Ib study ( NCT03165227 ), BI 685509 was generally well tolerated in patients with diabetic kidney disease (DKD) ( Cherney et al., 2023 ). BI 685509 is currently in phase II trials for CKD ( NCT04736628 ) and DKD ( NCT04750577 ). Studies are currently underway on the indications of PAH and CTEPH ( NCT03754660 ) and SSc ( NCT05559580 ). The sGC activators BI 703704 and GSK2181236A have demonstrated sustained protection against preclinical models of CKD ( Costell et al., 2012 ; Stasch et al., 2015 ; Hu et al., 2022 ). The results of these preclinical studies need to be confirmed in humans before these agents can be considered alternatives to current recommended treatments.

GC is widely distributed throughout the human body, mostly as sGC and GC-C, which catalyze GTP and thus increase the intracellular content of the second messenger cGMP, leading to the modulation of various intracellular physiological regulatory processes. The development of drugs on the basis of functional modulation of the GC pathway has been a fast-moving area of research over the past few years. A series of clinical studies have validated the therapeutic potential of sGC stimulators in patients with HF and PAH. Due to promising clinical trial results, riociguat and vericiguat have been approved by the US FDA for the treatment of chronic heart failure and pulmonary hypertension. In addition, the role of sGC stimulants in improving cGMP signaling might enable them to play an active role in a wide range of clinical indications, such as metabolic diseases, fibrotic diseases, urinary diseases, and neurodegenerative diseases. However, clinical studies have shown that the benefit for dcSSc patients and patients with diabetic nephropathy is debatable, and more clinical studies are needed to support its use or nonuse in them. In contrast to sGC stimulants, the treatment potency of sGC activators is not fully clarified, although they can bind to sGC when the body is in a disease condition involving oxidative stress. The development of this class of drugs is still at the clinical study stage. In intestinal epithelial cells, GC-C is the dominant supplier of cGMP. The GC-C molecule is important for maintaining fluid and ion homeostasis in the intestinal tract, and linaclotide and plecanatide are the main drugs targeting it. Preclinical and clinical evidence shows that modulation of GC-C may improve symptoms and be tolerated by patients with IBS-C and CIC. Recent studies have shown that GC-C is also relevant to intestinal inflammation, dysbiosis and cancer, the specific mechanisms of which remain to be explored. In conclusion, cGMP has a broad spectrum of physiological effects, which gives it considerable development prospects. We systematically reviewed the transduction procedures of the NO-sGC-cGMP signaling pathway and the potential use of sGC stimulators and GC-C stimulators. Novel compounds are being developed based on the structures of sGC and GC-C, some of which are being studied in clinical trials. These clinical results may open up new therapeutic approaches for cardiovascular, renal and other diseases. Various questions remain regarding the mechanisms of their effects and the therapeutic potential of these treatments for diseases besides PAH and HF, and much more research is needed in the future.

GC-C is a transmembrane protein receptor that has received increasing attention for its importance in digestive diseases. It plays a key role in regulating water and electrolyte balance, maintaining gastrointestinal function, relieving abdominal pain, controlling inflammation, regulating intestinal ecology, inhibiting tumor growth and regulating cell proliferation and is considered a potential therapeutic target for digestive system diseases ( Waldman and Camilleri, 2018 ). It was discovered in 1970 as a receptor for heat-stable endotoxins of exogenous diarrhea-causing bacteria and can be detected not only in intestinal mucosal cells but also in primary and metastatic colorectal cancer, peripheral blood, lymph nodes and liver tissues ( Smith and Gyles, 1970 ). The signaling pathway of GC-C/cGMP has a significant impact on digestive disorders, and agonists are on the market to treat these associated gastrointestinal conditions. GC-C agonists include natural and synthetic ligands; natural ligands include endogenous ligands such as uroguanylin and guanylin, exogenous ligands such as heat-resistant enterotoxin, and synthetic ligands such as linaclotide, plecanatide and dolcanatide ( Kuhn, 2016 ). The current progress of GC-C agonists is summarized in Table 3 . The current progress on guanylate cyclase-C agonists. IBS-C, irritable bowel syndrome with constipation; AEs, adverse events; SAEs, serious adverse events; TEAEs, treatment-emergent adverse events; OIC, opioid-induced constipation; SBMs, spontaneous bowel movements; CIC, chronic idiopathic constipation; CSBM, complete spontaneous bowel movement; CC, chronic constipation. As shown in Figure 2 , intestinal guanylin and uroguanylin are effective regulators of fluid ion homeostasis. They are secreted by various cells of the intestinal mucosa, including intestinal chromaffin cells, epithelial cells, goblet cells, and Pan’s cells. These peptide hormones act as receptor GC-C ligands that produce intracellular cGMP and activate PKGII. PKGII phosphorylates the cystic fibrosis transmembrane conduction regulator (CFTR) and increases the secretion of chloride ions (CI − ) into the intestinal lumen. cGMP can also increase the content of cyclic adenosine monophosphate (cAMP) by inhibiting the activity of PDE3. cAMP activates PKA and coactivates CFTR along with PKGII. Promoting the discharge of chloride ions and bicarbonate ( Vaandrager, 2002 ), cGMP activates HCO 3 − secretion through an unknown mechanism. In addition, cGMP can inhibit the sodium/hydrogen exchanger NHE3, thereby reducing the absorption of sodium, preventing hypernatremia and unnecessary hypervolemic shock, and maintaining the fluid balance in the intestine. By these mechanisms, cGMP can maintain the hydration state of colon mucus and ion homeostasis ( Brierley, 2012 ; Waldman and Camilleri, 2018 ; Samanta and Chaudhuri, 2021 ). In addition, it can regulate the intestinal immune barrier and increase the levels of IL-2 and IFN-γ and paracellular permeability ( Xing et al., 2021 ). Abdominal pain is a major symptom of inflammatory bowel disease (IBD), and therapies modulating the GC-C/cGMP pathway promote visceral analgesia in patients with these diseases in animal models and clinical trials ( Waldman and Camilleri, 2018 ). The mechanism of its analgesia is mainly through the afferent pathway involved in the regulation of gastrointestinal pain. After receptor–ligand binding in epithelial cells, elevated intracellular cGMP can be transported to the extracellular space through the silencing of the multidrug resistance-related protein 4 cyclic nucleoglycine efflux pump located on the basolateral membrane, reducing the excitation of submucosal afferent neurons and relieving abdominal pain. Another mechanism underlying this action is the prevention of intraluminal factors from affecting pain afferents and immune mechanisms in the lamina propria and other areas to indirectly promote analgesia ( Shailubhai et al., 2015 ; Brierley et al., 2022 ). It can also inhibit the proliferation of intestinal epithelial cells and maintain genomic stability, thus inhibiting the occurrence of intestinal tumors. Following the activation of GC-C, the level of cGMP increases to activate cyclic phospho-dependent PKGII, which can inhibit the protein kinase B (Akt)-related signaling pathway and thus upregulate the expression of the tumor suppressor p53. p38 MAPK is also activated, which increases the phosphorylation of the transcription factor Sp1, leading to the accumulation of p21 in cells, which promotes cell aging and reduces the risk of cancer ( Basu et al., 2014 ). Activation of GC-C induces apoptosis by promoting the degradation of β-catenin and opposing the pro-proliferative Wnt/β-catenin/Tcf-4 signaling pathway ( Thompson et al., 2000 ). Because Akt enhances β-catenin activity either directly or indirectly, the suppression of Akt signaling may also be associated with the reduction of β-catenin/T-cell factor (TCF) transcriptional activity mediated by GC-C ( Fang et al., 2007 ). As the GMP concentration increases, cyclic nucleotide-gated ion channels (CNGs) are activated, Ca 2+ inflow is promoted, and cytostatic activity is mediated ( Kazerounian et al., 2005 ). GC-C signaling can also block metastasis by inhibiting matrix metalloproteinase-9 (MMP-9), which is produced by colorectal cancer cells ( Lubbe et al., 2009 ). PKGII can also promote DNA repair in intestinal epithelial cells, thereby protecting intestinal epithelial tight junction proteins, maintaining intestinal wall integrity and reducing the risk of tumor development ( Rappaport and Waldman, 2020 ; Loretah et al., 2021 ). Schematic diagram of the GC-C/cGMP signaling pathway. GC-C, guanylate cyclase C; cGMP, cyclic guanosine monophosphate; PDEs, phosphodiesterases; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; PKG, protein kinase; CNGs, cyclic nucleotide-gated ion channels; CFTR, cystic fibrosis transmembrane regulator; NHE3, Na + /H + exchanger 3; MRP4, multidrug resistance-associated protein 4; Akt, protein kinase B; p38 MAPK, p38 mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; IL-2, interleukin 2; IFN-γ, Interferon γ. Linaclotide, a 14-amino acid peptide, was approved by the FDA in 2012 as an oral drug for the treatment of chronic constipation (CC) and constipation-type irritable bowel syndrome (IBS-C). It acts on the GC-C receptor on the luminal membrane, resulting in increased intracellular phosphorylation of cGMP and pVASpser239. The mechanisms underlying its effects include increased bicarbonate secretion during CFTR expression or functional loss, increased chloride and bicarbonate secretion into the intestine, inhibited sodium ion absorption, and thus increased water secretion into the lumen and improved defecation ( Love et al., 2014 ; Ahsan et al., 2017 ; Sarthi et al., 2023 ). The pharmacological activity of linaclotide is limited to the gastrointestinal tract, and its oral bioavailability is low, so systemic side effects are unlikely. After oral linaclotide is taken, approximately 3%–5% of the active peptide is excreted in the stool. Linaclotide and its metabolites are excreted through the kidneys (33%–45%) and biliary tract (48%–59%). Its dosage should be adjusted with particular care in patients with moderate liver impairment or mild to severe liver impairment (data are not available for patients with severe liver impairment). It is metabolized by multiple cytochrome P450 (CYP) enzymes and has no effect on major CYP subtypes, giving it a low risk of clinically relevant drug interactions ( Frey et al., 2018 ). A therapeutic dose of linaclotide should be taken at least 30 min before meals, as effectiveness and tolerability can be affected by high-fat foods ( Bassotti et al., 2018 ). Linaclotide was evaluated at doses between 62.5 g and 600 g for efficacy and safety in patients ( n = 4,107) with CC in a meta-analysis. More patients completed spontaneous defecation (CSBM) at different doses, which was more significant in the very low-dose group, and there were no adverse reactions in the high-dose group ( Yang and Lei, 2021 ). Japanese researchers ( NCT02809105 ) found that linaclotide at a dose of 0.5 mg/day was effective and safe in patients with CC, with mild and occasional adverse reactions being the most common ( Fukudo et al., 2019 ). A prospective study from China evaluated patients taking linaclotide ( n = 97) on bowel movements, abdominal symptoms, IBS Symptom Severity Scale (IBS-SSS), and IBS Quality of Life Questionnaire (IBS-QOL) and found a significant increase in weekly bowel movements and a significant improvement in patients’ quality of life. Diarrhea occurred in 11 cases (11.3%). IBS-C symptoms and severity were improved with linaclotide, and the drug was safe and effective ( Liu et al., 2022 ). Another phase III clinical trial involving 659 Chinese IBS-C patients also showed that linaclotide (290 µg/day) was effective and well tolerated in Chinese IBS-C patients with rapid onset of action ( NCT01880424 ) ( Peng et al., 2022 ). Another study found that linaclotide reduced reaction time in patients with IBS-C ( Brenner et al., 2023 ). In a trial in the elderly population, the most common side effect was diarrhea, but the incidence of diarrhea and constipation in elderly patients, even at reduced doses, did not differ significantly from that in nonelderly patients, and multivariate analysis showed that age, sex, and dose were not associated with diarrhea caused by linaclotide treatment. Thus, linaclotide was effective and safe in elderly patients ( Chang et al., 2021 ; Ishigo et al., 2021 ). In special pediatric populations, AEs are relatively common, although studies have found that nearly half of children with FC or IBS-C benefit from linaclotide treatment, so further research is needed ( Baaleman et al., 2021 ). In a cohort study of patients with SSc, 31 patients were treated with linaclotide. Twenty-eight of the 31 patients responded to treatment, while only three (9.7%) reported ineffective or intolerable side effects. Diarrhea, cramping, and bloating were the most commonly reported side effects (11/31, 35%). Linaclotide is a well-tolerated, effective drug that can be used to treat refractory symptoms of a low GI score on SSc ( Dein et al., 2021 ). A multicenter phase II clinical study evaluated the efficacy and safety of linaclotide in the treatment of opioid-induced constipation (OIC) in patients with chronic noncancer pain syndrome ( NCT02270983 ). Compared with placebo, linaclotide significantly improved stool consistency, diarrhea, bloating, and treatment satisfaction scores ( p < 0.05). Linaclotide significantly improved OIC symptoms and was well tolerated in patients with chronic noncancerous pain ( Brenner et al., 2020 ). Linaclotide treatment was found to reduce vaginal hyperalgesia and mechanical hyperalgesia associated with endometriosis through viscerovaginal crosstalk ( Ge et al., 2019 ). In patients with colon cancer, a US phase I clinical trial ( NCT01950403 ) showed that administration of linaclotide (870 μg/d) for 7 days after oral preparation of the intestine with polyethylene glycol increased the level of cGMP and reduced the proportion of Ki-67-positive colon epithelial cells (a higher proportion of Ki-67-positive cells suggested a faster rate of cell proliferation). These results suggest that linaclotide inhibits colonic epithelial cell proliferation in human colons ( Weinberg et al., 2017 ). An oral GC-C agonist, plecanatide, consists of a 16-amino acid synthetic peptide equivalent to human uroguanylin and is used to treat gastrointestinal (GI) disorders. For the treatment of chronic idiopathic constipation (CIC) in adults, plecanatide received its first global approval in 2017 ( Al-Salama and Syed, 2017 ; Rao, 2018 ). Plecanatide metabolism occurs in the gastrointestinal tract. After oral administration of 3 mg of plecanatide, blood levels of plecanatide and its active metabolites were lower than detectable levels. Standard pharmacokinetic parameters cannot be calculated, and the amount of plecanatide or its metabolites in the tissue is negligible due to the small amount of drug absorbed ( Miner, 2020 ). In two large randomized, double-blind, placebo-controlled studies evaluating the efficacy and safety of plecanatide (3 mg, 6 mg) vs placebo in patients with CIC, plecanatide treatment also significantly reduced the severity of other CIC symptoms (tension, consistent stools, bloating). In addition, the satisfaction and quality of life of patients treated with plecanatide improved significantly. The low incidence of adverse reactions after plecanatide treatment shows that it is safe and effective for CIC. In addition, plecanatide combined with acid suppressants is safe and effective in patients with CIC ( DeMicco et al., 2017 ; Miner et al., 2017 ). A meta-analysis evaluating the efficacy and tolerability of GC-C agonists included eight randomized controlled trials with 10,369 patients. Both drugs were effective in treating CIC, and the incidence of diarrhea was higher than that in the placebo group. Linaclotide and plecanatide were similar in efficacy and tolerability to IBS-C and CIC. There was no difference in the incidence of adverse reactions (diarrhea) between linaclotide and plecanatide ( Shah et al., 2018 ). Dolcanatide (SP-333), an oral uroguanylinoid, is replaced by selected D-amino acids to enhance stability and extend persistence, activating GUCY2C in the small and large intestines. A phase I double-blind, placebo-controlled trial ( NCT03300570 ) of 27 mg dolcanatide administered orally daily for 7 days in healthy volunteers did not show activation of GUCY2C in distal rectal epithelial cells, as quantified by the accumulation of its product cGMP. These data suggest that the high stability of dolcanatide and its persistence along the rostral–caudal axis of the small and large intestines are insufficient to regulate GUCY2C throughout the colorectal region to prevent tumorigenesis. These results highlight the importance of developing GUCY2C anticancer agonists that target colorectal release and activity ( Weinberg et al., 2021 ). Current research on dolcanatide focuses on its potential use for the treatment of colon cancer.

Guanylate cyclase (GC) is a protease that catalyzes the conversion of guanylate triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Based on the properties of the distributed form of the enzyme, it can be divided into two categories: membrane-bound guanylate cyclase and soluble guanylate cyclase (sGC). Both are widely distributed in the human body, including the lung, brain, kidney, blood vessels, and other tissues ( Garbers, 1990 ). The agonists of membrane-bound guanylate cyclase are peptides (natriuretic peptides A, B, and C), and the agonists of sGC are gaseous mediators (NO and CO) ( Grzesk et al., 2023 ). sGC is a heterodimer with an α-subunit and β-subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain ( Argyriou et al., 2021 ). As a result of its affinity for nitric oxide (NO), this enzyme has also been called an NO-sensitive guanosyl cyclase. NO binding to sGC heme increases GTP cyclase activity, resulting in the production of cGMP, which regulates multiple signaling pathways in cells ( Stasch et al., 2011 ). The cardiovascular, pulmonary, and neurological systems, as well as organs like the kidney, brain, and liver, are highly dependent on NO-sGC-cGMP regulation. Regarding its role in regulation, fibroblasts, cardiomyocytes, platelets, neurons, and immune cells are also affected by cGMP, and it controls fibrosis, the inflammatory response, and neurotransmission ( Derbyshire and Marletta, 2009 ; Grzesk and Nowaczyk, 2021 ; Nowaczyk et al., 2021 ). Guanylate cyclase C (GC-C) is a member of the membrane-bound GC family. It consists of an extracellular domain (ECD) and an intracellular domain, connected by a single strand across the membrane region. In the cell, it synthesizes cGMP and regulates an array of intracellular physiological functions ( Hasegawa and Shimonishi, 2005 ). Reviewing recent progress in research on sGC agonists and GC-C agonists, along with their mechanisms, is the purpose of this paper.