Abstract
Significance:
Hydrogen sulfide (H2S) is regarded as the third gasotransmitter along with nitric oxide and carbon monoxide. Extensive studies have demonstrated a variety of biological roles for H2S in neurophysiology, cardiovascular disease, endocrine regulation, and other physiological and pathological processes.
Recent Advances:
Novel H2S donors have proved useful in understanding the biological functions of H2S, with morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) being one of the most common pharmacological tools used. One advantage of GYY4137 over sulfide salts is its ability to release H2S in a slow and sustained manner akin to endogenous H2S production, rather than the delivery of H2S as a single concentrated burst.
Critical Issues:
Here, we summarize recent progress made in the characterization of the biological activities and pharmacological effects of GYY4137 in a range of in vitro and in vivo systems. Recent developments in the structural modification of GYY4137 to generate new compounds and their biological effects are also discussed.
Future Directions:
Slow-releasing H2S donor, GYY4137, and other phosphorothioate-based H2S donors are potent tools to study the biological functions of H2S. Despite recent progress, more work needs to be performed on these new compounds to unravel the mechanisms behind H2S release and pace of its discharge, as well as to define the effects of by-products of donors after H2S liberation. This will not only lead to better in-depth understanding of the biological effects of H2S but will also shed light on the future development of a new class of therapeutic agents with potential to treat a wide range of human diseases.
Introduction
Hydrogen sulfide (H2S) is a small gaseous molecule that together with nitric oxide (NO) and carbon monoxide belong to the gasotransmitter family of molecules (81, 107). Previously considered a hazardous gas with a pungent rotten egg smell, H2S is now recognized as an important signaling molecule capable of modulating a plethora of physiological and pathological processes in a variety of human tissues (37, 46, 56, 69, 102, 108, 112). Furthermore, there is now an emerging body of evidence linking H2S deficiency to the pathogenesis of a wide range of diseases, highlighting many potential applications of H2S donors in therapy (91, 95, 101, 111, 125, 126).
H2S is produced endogenously from homocysteine, cysteine, or 3-mercaptopyruvate. Three enzymes, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), are responsible for the catalysis of this reaction (1, 34, 98). While CBS is predominantly responsible for the production of H2S in the brain and nervous systems, CSE is mainly expressed in peripheral tissues such as the liver, kidney, and smooth muscles (37, 47). Expression of 3-MST has mainly been reported in the brain and vascular endothelium.
In the past decade, considerable efforts have been invested to study how H2S levels affect various physiological processes. Much of this work has been performed using genetic and pharmacological inhibitors of CBS and/or CSE activity in animal models. With the discovery of slow-releasing H2S donor molecules, a combination of inhibition and rescue studies will further aid in the elucidation of the complex cellular intricacies modulated by H2S. Of the many H2S donors reported, morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) is without doubt one of the most utilized slow-releasing H2S compounds in biological studies thus far. Here, we summarize the research carried out to date in the elucidation of the physiological effects of GYY4137. Recent progress in the development of GYY4137-modified compounds and its biological activities will also be discussed.
Development and Characterization of GYY4137
Early studies investigating the physiological and pathological roles of H2S in vivo and in vitro were performed mainly using sulfide salts (e.g., sodium hydrosulfide [NaHS] or sodium sulfide [Na2S]), which release H2S spontaneously as a single concentrated burst once solubilized. These reagents trigger an instant release of a large amount of H2S, the effect is short-lived (typically within only a few seconds) and is, therefore, likely to be a poor representation of in vivo physiological H2S synthesis, which usually occurs in small sustained quantities and at much slower rates. To mimic physiological H2S release, many different H2S donor compounds, which are capable of releasing H2S in low and controlled amounts, have been developed. Of these, GYY4137 is the most widely studied H2S donor compound [reviewed in Refs. (95, 111)].
GYY4137, a water soluble, slow-releasing H2S donor, was first reported to exhibit vasorelaxant activity both in vitro and in vivo (58). Compared with sulfide salts such as NaHS or Na2S, GYY4137 possesses several advantages. First, GYY4137 is soluble in water (>1 mg/mL at pH 7.4), allowing for the preparation of highly concentrated stock solutions without the need for organic solvents (e.g., ethanol, dimethyl sulfoxide [DMSO], dimethylformamide). However, researchers should be cautioned that H2S production by GYY4137 begins upon contact with water and repeated freeze-thaw cycles of stock solutions should be avoided. In addition, despite the water solubility of GYY4137, commercial suppliers of GYY4137 recommend that GYY4137 be stored in organic solvents such as DMSO. On this note, it should be mentioned that Whiteman et al. observed accelerated GYY4137 decomposition rates in DMSO, and hence, a freshly prepared solution of GYY4137 for each experiment immediately before use is strongly advised (111). Nonetheless, the ease of use and high water solubility has, no doubt, contributed to its widespread use and application.
Another advantage of GYY4137 is that it releases H2S in a slow and sustained manner, which more closely resembles endogenous H2S production. GYY4137 is a derivative of Lawesson's reagent and can be synthesized by reacting Lawesson's reagent with morpholine in methylene chloride at room temperature (Fig. 1). Similar to Lawesson's reagent, GYY4137 releases H2S upon hydrolysis. GYY4137 releases low quantities of H2S over a sustained period (hours to days) in aqueous solution (pH 7.4, 37°C) (58). It is a slow process with ≈4% to 5% of H2S generated from a starting concentration of 1 mM within 25 min.
A recent study by Alexander et al. examined the hydrolysis kinetics of GYY4137 with 31P nuclear magnetic resonance (NMR) spectroscopy and demonstrated a two-step hydrolytic decomposition process (Fig. 2) (2). The first step involves sulfur–oxygen exchange with water to give an arylphosphonamidothiate 1 and the second slower step results in arylphosphonate 2 after complete hydrolysis. It is of note that complete decomposition of GYY4137 in aqueous solution was too slow to be monitored by NMR spectroscopy. Organic solvents (e.g., acetone or chloroform) result in faster hydrolysis and complete hydrolysis requires 71 days. Furthermore, H2S release from GYY4137 is pH- and temperature-dependent, with more release at acidic pH and less release at low temperatures. Under physiological conditions, H2S production from GYY4137 is sustained at low levels (less than 10%) for 7 days (51). When GYY4137 (133 μmol/kg) was administered intravenously or intraperitoneally to anesthetized male Sprague-Dawley rats, plasma H2S concentration was increased after 30 min and remained elevated over the next 180 min (58).
Bearing in mind that hydrolysis of phosphorodithioate is affected by pH change and hydrolysis under highly acidic conditions is much faster than at neutral pH, it is now realized that the traditional methylene blue method with involvement of strong acidic conditions is not suitable for the evaluation of H2S-release levels of phosphorodithioate-based donors including GYY4137. Recently, a number of fluorescent probes (15, 21, 63, 67, 118) have been developed for the detection of H2S, and these probes are used at neutral pH and thus useful for evaluating H2S release by its donors.
When performing experiments using H2S donors, the effect of “decomposed” or “time-expired” donor controls should also be considered to ensure that any observed effects are truly due to the “released” H2S and not a consequence of the unhydrolyzed parent compound or any by-product molecules (after H2S release). This is particularly important for slow-releasing H2S donors such as GYY4137 where concentrations of at least 100 μM are typically used to reach the effective dose of H2S in various in vivo and in vitro experimental conditions. In this context, control experiments with a time-expired GYY4137 or structural analogue such as the sulfur-lacking GYY4137 backbone ZYJ1122 (Fig. 3, top left panel) can be used to circumvent these issues. It is worth mentioning that time-expired GYY4137 will contain its hydrolysis products as well as the morpholinium counterion. To avoid potential complications due to the presence of the morpholinium, a recent study has reported a strategy to prepare a pharmaceutically more acceptable sodium salt equivalent of GYY4137 (2).
Biological Implications of GYY4137
GYY4137 effects on cardiovascular system
Modulation of vasodilation and antihypertensive function
The discovery that H2S dilated blood vessels in synergy with NO (34) first ignited interest in the field of H2S biology. Li et al. (58) were the first to demonstrate the effect of GYY4137 on vasodilation in rat aortic rings and antihypertensive effect in intact rats. This effect was mediated by the opening of vascular smooth muscle KATP channels. Since then, similar KATP-mediated vasorelaxing activities of GYY4137 on smooth muscle cells have been described in various tissues, including human and rat pregnant myometrium (92), isolated bovine ciliary artery (17, 96), as well as in the bladder neck and intravesical ureter of pigs (24, 25). In addition to direct stimulation of the KATP channel, GYY4137 also relaxes precontracted airway smooth muscle cells by inhibiting Ca2+ (calcium ion) release through its effects on the inositol-1,4,5-trisphosphate receptors (14).
Age-dependent hypertension and loss of endothelium-dependent vasorelaxation were first shown in CSE knockout mice (120), and it was later reported that the administration of CSE inhibitors to pregnant mice induced abnormal placentation and maternal hypertension (106). The latter report further showed that such placental defects and hypertensive phenotype could be rescued by GYY4137 treatment, and this rescue was mediated by the inhibition of circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels, in turn restoring the fetal growth that was compromised by CSE inhibition.
Cardioprotection
Cardioprotective effects of GYY4137, such as the attenuation of myocardial ischemia–reperfusion injury and infarction, anti-atherosclerosis, and antithrombosis, have been described in various in vitro and in vivo animal models. GYY4137 has also been reported to exert cardioprotective mechanism against ischemia–reperfusion injury by enhancing endothelial nitric oxide synthase (eNOS) phosphorylation and PI3K/Akt signaling (45). Similarly, GYY4137 activates the PHLPP-1/Akt/Nrf2 pathway to protect against diabetic myocardial ischemia–reperfusion injury (89). Furthermore, GYY4137 confers protection against myocardial ischemia–reperfusion injuries through the attenuation of oxidative stress and apoptosis (77) as well as via enhanced early postischemic endogenous natriuretic peptide activation (60).
In spontaneously hypertensive rats, Meng et al. reported that GYY4137 inhibits myocardial hypertrophy by downregulating Krüppel-like factor 5 (KLF5) transcription activity by enhancing S-sulfhydration on specificity protein 1 in cardiomyocytes (78). Using the same hypertensive rat model, intraperitoneal administration of GYY4137 daily for 4 weeks exhibited protective effects against cardiac fibrosis by attenuating oxidative stress and suppressing the TGF-β1/Smad2 signaling pathway, and alpha-smooth muscle actin expression (79).
A potential protective role of GYY4137 for diabetic cardiomyopathy has also been described (109). In this context, hyperglycemia increased the production of reactive oxygen species (ROS) and induced a chronic proinflammatory environment, leading to cardiac damage. GYY4137 protected cardiac myoblast cells (H9c2) against cytotoxicity brought about by high glucose levels via the activation of the AMPK/mTOR signaling pathway. In diabetic mice, GYY4137 was further found to protect against cardiac fibrosis both by reducing ROS-ERK1/2-MAPkinase-activation (127) and by modulation of the nuclear translocation and activation of transcription factor, Forkhead box protein O1 (122).
In addition to its antihypertensive and cardioprotective effects, many in vivo animal studies have also demonstrated the antithrombotic and anti-atherosclerotic activity of GYY4137. GYY4137 regulates thrombogenesis in mice by inhibiting the thrombin receptor agonist peptide-induced adhesion molecule expression and platelet activation (29). Furthermore, GYY4137 decreases thrombus stability and reduces platelet–leukocyte aggregation, promoting endogenous thrombolysis (28). In ApoE-knockout mice fed a high-fat diet, the administration of GYY4137 for 30 days reduced aortic atherosclerotic plaque formation and partially restored aortic endothelium-dependent relaxation (70). In this study, the authors showed that the expression of aortic ICAM-1, TNF-α, IL-6, and LOX-1 was reduced, and eNOS phosphorylation, PI3K expression, and Akt phosphorylation were elevated in GYY4137-treated mice.
Recent molecular evidence suggests that the effect of GYY4137 on the cardiovascular system is also mediated at least in part by the process of S-sulfhydration. For instance, Xie et al. demonstrated that GYY4137 curtailed diabetes-accelerated atherosclerosis in low-density lipoprotein receptor (LDLr)-knockout mice, but not in LDLr and Nrf2 double-knockout mice, suggesting that the anti-atherosclerotic activity of GYY4137 is mediated by Nrf2 activation (116). They further show that S-sulfhydration of Keap1 at Cys151 upon GYY4137 treatment is likely to be essential in the activation of the Nrf2 pathway. In a more recent study, either GYY4137 or NaHS treatment enhanced the stability of histone deacetylase Sirtuin-1 (SIRT1) by direct S-sulfhydration, thereby reducing atherosclerotic plaque formation, macrophage infiltration, aortic inflammation, and plasma lipid levels in ApoE-knockout atherosclerotic mice (19). Interestingly, other mechanisms by which GYY4137 mediates an anti-atherosclerotic action have also recently been suggested by Potor et al. They found that GYY4137 mitigated hemoglobin–lipid interactions and subsequent endothelial responses, which are required for atherosclerotic plaque formation (86).
GYY4137 and inflammatory response
Effects of H2S on the cardiovascular system are closely linked to its importance in the regulation of inflammatory and immunoregulatory processes within the cardiovascular system. Various studies on the role of H2S in the regulation of the inflammatory response have emerged, although with conflicting conclusions. There is now some consensus that pro- or anti-inflammatory characteristics of H2S are very much dependent on the dose, release rate, and the time of administration of the gasotransmitter. Despite the dispute between the “friend or foe” role of H2S in inflammation, the anti-inflammatory role GYY4137 has been widely studied and reported in a variety of in vitro and in vivo models of inflammation.
The first suggestion of a different mode of action between slow (GYY4137) and fast (NaHS) H2S donors in inflammation was presented by Whiteman et al., who showed that pretreatment of lipopolysaccharide (LPS)-treated murine macrophage with GYY4137 exhibited a concentration-related inhibition of NF-κB activation (110). These authors also demonstrated that GYY4137-induced NF-κB inhibition reduced secretion of proinflammatory mediators such as IL-1β, IL-6, TNF-α, NO, and prostaglandin E2 (PGE2). In contrast, NaHS exhibits a biphasic effect on proinflammatory mediators. At low concentrations, NaHS reduced proinflammatory mediator secretion while increased proinflammatory mediator synthesis was detected at high NaHS concentrations.
More studies indicating the anti-inflammatory role of GYY4137 followed with Li et al. confirming the anti-inflammatory effects of GYY4137 in a complete Freund's adjuvant (CFA) mouse model of acute joint inflammation (55). They also showed that in human synoviocytes and articular chondrocytes, GYY4137 (0.1–0.5 mM) reduced LPS-induced production of nitrite, PGE2, TNF-α, and IL-6 and decreased LPS-induced NF-κB activation and levels of inducible NO synthase and cyclooxygenase-2 (55). Similarly, in IL-1β-stimulated chondrocytes isolated from osteoarthritis tissue, both NaHS and GYY4137 resulted in reduction in release of NO, PGE2, IL-6, and MMP13, and the observed anti-inflammatory and anti-catabolic properties were attributed in part to the reduction of NF-κB activation (8, 9). More recently, Faller et al. demonstrated that GYY4137 pretreatment ameliorated LPS-induced acute lung injury inflammation by restricting neutrophil transmigration, inflammation, and oxidative burst (20). Another study found that GYY4137 can alleviate infrarenal aortic cross-clamping-induced acute lung injury via inhibition of inflammation and angiopoietin 2 release (104).
The anti-inflammatory role of GYY4137 has also been well documented in a variety of in vivo animal studies. In mice, the effective dose of GYY4137 has been documented to be around 50–100 mg/kg when administrated intraperitoneally and confers protection against acute endotoxemia-associated lung injury, intestinal barrier injury, and histamine-mediated acute pruritus and cutaneous inflammation (16, 93, 123). GYY4137 also inhibited oral mucosal wound-induced macrophage activation via the NF-κB pathway in mouse models (129). Furthermore, GYY4137 was anti-inflammatory in a rat model of endotoxic shock (57). Interestingly, in CFA-treated acute joint inflammation mouse, 1 h pretreatment of GYY4137 increased knee joint swelling while an anti-inflammatory effect was apparent when GYY4137 was injected 6 h after CFA. Therefore, it is worth noting that in addition to the dose and rate of release of H2S by its donors, the time of administration should also be considered when utilizing H2S donors.
Recent studies have begun to decipher the role of H2S in the regulation of the inflammatory response during bacterial and viral infections. In cellular models of acute infection with mycoplasma, GYY4137 exerted an anti-inflammatory effect simultaneously through the suppression of the NF-κB-mediated proinflammatory response and stimulation of Nrf2-mediated antioxidative effects (6, 7). In the context of viral-induced lung inflammation, treatment of respiratory syncytial virus (RSV)-infected airway epithelial cells with GYY4137 significantly reduced viral replication and mitigated the production of proinflammatory mediators both in vitro and in vivo (38, 54). Of note, GYY4137 treatment did not affect RSV genome replication or viral mRNA and protein synthesis but inhibited syncytium formation and virus assembly/release. In contrast, in a recent report using a model of influenza infection, GYY4137 was found to exert antiviral activity by reducing viral protein and mRNA expression and was postulated to inhibit an early step of influenza viral replication (4, 5). Although, clinically, RSV and the influenza viral infections often present with similar symptoms, they have very different incubation and propagation rates (3). The observation that RSV possesses much slower replication rates compared with the influenza virus may be speculated to account for the differences observed between the responses of these two viruses to GYY4137, but this needs to be proven in a rigorous scientific study.
Mechanistically, inflammation is a complex multistep process, which is time-dependent, and involves the recruitment of various cell types and cellular factors. Despite the well-documented fact that GYY4137 modulation of the inflammatory response is through the suppression of NF-κB activation, other possible mechanism of action should not be ruled out. Apart from its effect on the NF-κB pathway, a recent study involving monosodium urate crystal-activated NLRP3 inflammasomes suggests that GYY4137 also inhibits inflammasome activation as well as cytokine production and secretion both in vivo and in vitro (13). More work needs to be performed to better understand the involvement of other signaling pathways such as activation of Nrf2 and the inhibition of inflammasome activation by GYY4137.
GYY4137 and kidney health and disease
H2S is synthesized in the kidneys, and dysregulation of H2S production has been implicated in chronic kidney disease and renal pathologies (71). In a recent study, Luo et al. showed that inhibition of endogenous H2S production increased urine output and reduced urine osmolality in mice and that GYY4137 improved urine concentration via the upregulation of aquaporin-2 expression in the collecting duct principal cells, likely through the cAMP-PKA pathway (73). Before this study, the protective effects of GYY4137 were demonstrated in prolonged unilateral ureteral obstruction (61, 62). It was suggested that GYY4137 mitigated fibrosis by attenuating TGF-β1-mediated epithelial–mesenchymal transition. In another study, using NaHS as the H2S donor, it was suggested that H2S-mediated inhibition of Smad and mitogen-activated protein kinase-dependent pathway had protective effects in renal cells (99).
Interestingly, in the diabetic kidney, an alternative regulation mechanism has been proposed. It was hypothesized that in the kidney of diabetic patients, GYY4137 reduced renal fibrosis through the modulation of miR-194-depedent collagen deposition and realignment (42). In another report, it was suggested that GYY4137 protects the diabetic kidney from injury by blocking mitochondrial Ca2+ permeability through attenuation of the N-methyl-
Cisplatin is a commonly used chemotherapeutic drug, and nephrotoxicity is a known side effect of the drug. Recently, Cao et al. reported that renal toxicity caused by cisplatin is likely through the downregulation of CSE, which impairs H2S production (11). H2S donors such as NaHS and GYY4137, but not mitochondria-targeted H2S donor AP39, alleviated cisplatin-induced renal proximal tubule cell death and nephrotoxicity. Such a protective effect of H2S donors was mediated by the suppression of intracellular ROS generation and inhibition of NADPH oxidase activity through p47phox persulfidation. Their findings suggest that H2S may have potential translational applications in negating cisplatin-induced nephrotoxicity.
GYY4137 and cancer
While there is a vast amount of literature showing the effect and therapeutic potential of GYY4137 in diseases of the cardiovascular and inflammatory systems, the role of H2S in cancer cell proliferation and apoptosis is only just starting to unfold. As in inflammation, a paradox seems to exist in the literature in terms of the anticancer effects of H2S. While inhibition of H2S biosynthesis by targeting CBS in colon and ovarian cancers causes anticancer effects (32), many other studies have shown that numerous H2S donors exhibit anticancer properties (48, 51). This phenomenon can perhaps be explained again by the biphasic regulation or bell-shaped dose–response pharmacology of H2S, whereby low endogenous H2S production promotes cellular bioenergetics, while exogenous addition of high amounts of H2S by its donors tends to inhibit cancer cell proliferation (32). In addition, the choice of H2S donors and their H2S release rates (e.g., a fast-release H2S donor NaHS with a very short half-life in cell culture media vs. a prolonged and continuous H2S donor GYY4137) should be taken into consideration when studying the anticancer properties of H2S (95). Here, we focus on the effects of GYY4137 on cancer proliferation and apoptosis.
The antigrowth effects of GYY4137 were observed in a range of different cancer types including cervix, colon, bone, leukemia, liver, and breast cancer-derived cell lines but not in normal human diploid cell lines (51). Incubation with GYY4137 over 3–5 days induced apoptosis, evident by the generation of cleaved poly (ADP-ribose) polymerase and cleaved caspase 9. Compared with GYY4137, fast-release H2S donor (NaHS) was a less potent anticancer agent, and this was mainly due to the fact that NaHS and GYY4137 possess very different H2S-releasing profiles. Nonetheless, in an independent study by Wu et al., NaHS decreased the proliferation of colon cancer cells and induced autophagy via the AMPK pathway in a dose-dependent manner (114). In another study conducted on hepatocellular carcinoma, GYY4137 exerted anticancer activity by inhibiting the activation of STAT3 and the expression of its downstream target genes, leading ultimately to apoptosis in vitro and in vivo (72). Furthermore, a recent report also suggests that a variety of H2S donors, including GYY4137, suppressed the growth of human breast cancer cells in vitro and in vitro, and this inhibitory effect of H2S on breast cancer cells was mediated through the suppression of the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling pathways (18).
Besides the effects of GYY4137 on apoptosis and survival signaling pathways, GYY4137 also altered cellular metabolism. In human liver (HepG2) and breast (MCF7) cancer cell lines, GYY4137 treatment increased glycolysis, resulting in an overproduction of lactate, and simultaneously decreased anion exchanger and sodium/proton exchanger (NHE) activity (50). The combinatorial dysregulation of both components induced by H2S caused uncontrolled intracellular acidification and cancer cell death. In this context, the magnitude of the intracellular pH decrease is a key determinant of cancer cell sensitivity to H2S. Therefore, coupling GYY4137 with either known monocarboxylate transporter 4 inhibitor simvastatin or metformin, to further boost glycolysis, enhanced intracellular hyperacidification-mediated cancer cell death (49). Similarly, an increase in glycolysis and lactate production by low concentrations of GYY4137 has also been observed in HCT116 colon cancer cells (105). Interestingly, the silencing of lactate dehydrogenase A (LDHA) failed to attenuate GYY4137-induced glycolysis but successfully mitigated GYY4137-stimulated mitochondrial respiration and lactate production. This was explained by the observation that GYY4137 can stimulate the LDHA activity via S-sulfhydration on LDHA (105).
The possibility that GYY4137 may regulate calcium homeostasis and that this is relevant for its anticancer activity has also been suggested. In a recent study, treatment of colorectal (DLD1) and ovarian (A2780) cancer cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner, leading to apoptosis induction (103). Mechanistic studies revealed that intracellular acidification is likely due to the uncoupling of the sodium/calcium exchanger 1 (NCX1)/NHE1 complex through internalization of NHE1. Furthermore, NCX1 is a ubiquitously expressed membrane protein essential in calcium homeostasis. In addition to the disintegration NCX1/NHE1 complex, GYY4137 has also been shown to upregulate NCX1 expression in HeLa cells (76). Upregulation of NCX1 is accompanied by elevated cAMP levels, leading to the upregulation of β1 and β3 adrenergic receptors and stimulation of apoptosis. Likewise, GYY4137 elevates inositol 1,4,5-trisphosphate receptor R1 and R2 expression, resulting in the depletion of calcium storage in the endoplasmic reticulum (ER), subsequent activation of ER stress and apoptosis induction (52). GYY4137-induced ER stress and induction of apoptosis was further exacerbated under hypoxic conditions in ovarian cancer cells (53). Taken together, these findings suggest that GYY4137 may induce ER stress through the regulation of calcium transport and homeostasis.
Although the current literature suggests a potential application for GYY4137 as an anticancer agent, this is still very much an emerging field and much remains to be carried out. The precise molecular mechanism of GYY4137 action remains obscure. Furthermore, most studies involving the anticancer effects of GYY4137 have centered on the use of in vitro models. Thus, the effective delivery and assessment of antitumor effects of GYY4137 in vivo need to be studied in greater detail to better define its translational potential, if any.
GYY4137 in aging and aging-related aliments
GYY4137 in the prevention of cellular aging?.
Aging is intrinsically linked to many H2S-related pathophysiological diseases. In fact, H2S levels in humans have been found to decline after the age of 50 years and low levels of plasma H2S are associated with several age-associated diseases, such as diabetes (39), atherosclerosis (75), and hypertension (106, 119). Thus, it is rational to postulate that H2S may play a fundamental role in the aging process.
Miller and Roth first implicated H2S in the aging process in an elegant study using the nematode Caenorhabditis elegans where they showed that C. elegans exposed to H2S (50 ppm) appeared to be thermotolerant and long-lived (80). The finding of the age-dependent decline of human plasma H2S levels supports this hypothesis (16). Several studies now support a role for H2S in aging. These reports implicate H2S in the induction of genes associated with longevity and protection against cellular senescence, oxidative stress, and mitochondria dysfunction (85). It is shown that several members of the SIRT family of proteins (SIRT1, SIRT3, and SIRT6) are involved in the regulation of longevity and healthy aging, and H2S may regulate the expression and activity of SIRTs. SIRT1 in the human umbilical vein endothelial cells (100, 128), SIRT3 in human gingival epithelium (10), hydrogen peroxide (H2O2)-exposed EA.hy926 endothelial cells (115), and in diabetic mice with lung ischemia–reperfusion injury (40), as well as SIRT6 expression in brain endothelial cells (36) are all reportedly regulated by H2S. Atherosclerosis is a common age-related aliment, and a recent study by Du et al. also demonstrated that H2S is a novel SIRT1 activator, which mediates anti-atherogenesis (19). These authors showed that endogenous H2S directly sulfhydrated SIRT1 to promote its deacetylation activity and increase its stability, thereby reducing atherosclerotic plaque formation. In summary, despite the variation in cell types and H2S treatment regimens, it seems likely that the modulation of the sirtuin activity by H2S is associated with its protective role against senescence, apoptosis, and oxidative stress.
The potential anti-aging role of slow-releasing H2S donor GYY4137 is still a work in progress with only a handful of reports examining its effects on the aging process. To date, there are only two in vivo studies by Qabazard et al. (87, 88) linking GYY4137 to the aging process. In their studies with the C. elegans model, GYY4137 promoted induction of several genes involved in anti-aging, stress response, and antioxidation all leading to a reduction in the incidence and severity of cellular aging. They also presented evidence that GYY4137 prolonged lifespan in short-lived mev-1 mutant and protect wild-type C. elegans against paraquat poisoning (87, 88). The field of H2S and GYY4137 biology in the aging process or anti-aging is still in its infancy with most of the work performed using the nematode model. However, the results thus far are intriguing and have positioned H2S and its donors as potential key players in aging and associated diseases.
GYY4137 in the prevention of osteoporosis
Characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased risk of fractures, osteoporosis or porous bone is a common disease associated with aging. While initial studies have suggested that H2S maintained mesenchymal stem cell function and bone homeostasis via the regulation of Ca2+ channel activity (68), only after the study conducted in MC3T3-E1 osteoblast-like cell line, where GYY4137 stimulated osteoblastic cell proliferation and bone differentiation via an ERK1/2-dependent antioxidant mechanism (74), did the therapeutic potential of GYY4137 in the treatment and prevention of osteoporosis came to light.
Postmenopausal osteoporosis, due to the decline in estrogen levels, is a common occurrence in women. Recent studies showing a link between H2S levels and postmenopausal bone loss have highlighted the potential applications of H2S level restoration as a strategy for this problem in older women. Utilizing the ovariectomized (OVX) model of postmenopausal bone loss, Grassi et al. showed a decrease in serum H2S levels and bone marrow (BM) levels of CBS and CSE in OVX mice (30). Treatment with GYY4137 normalized serum H2S in these mice resulting in increased bone formation and completely prevented the loss of trabecular bone. Mechanistic studies revealed that GYY4137 increased murine osteoblastgenesis through activation of the Wnt signaling pathway by increasing the production of Wnt ligands Wnt16, Wnt2b, Wnt6, and Wnt10b in the BM (30). Similar beneficial effects of GYY4137 in increasing bone mineral density (BMD) in ovariectomy-treated rats have also been reported by Xu et al. (117). They demonstrated that the administration of GYY4137 increased BMD and attenuated the reduction in blood calcium, blood phosphate, and calcitonin in these ovariectomy-treated rats. In another study, in which osteoporosis was induced by hind limb suspension in rats, the administration of GYY4137 (25 mg/kg/day, i.p.) also attenuated bone loss (121). Mechanistically, GYY4137 preserved bone structure and promoted osteoblastic differentiation by upregulating runt-related transcription factor 2 transcriptional activities in MC3T3-E1 (121).
Taken together, restoration of H2S levels by supplying GYY4137 to the bone may be a potential novel therapeutic approach for postmenopausal osteoporosis. With these studies in mind, Gambari et al. very recently described a silk fibroin (SF) porous scaffold, which can be loaded with GYY4137 (26). This H2S-releasing SF scaffold, when introduced to the bone, supported cell adhesion and viability, and activated genes and pathways required for bone formation, thereby showing great promise as a therapeutic strategy for bone healing and regeneration (26).
Effects of GYY4137 in plants
Apart from the many biological implications for H2S in human health and disease, there is now increasing evidence of a role for H2S in the plant kingdom. Sulfur assimilation pathway and
Garcia-Mata and Lamattina had previously shown that both NaHS and GYY4137 induced stomatal closure possibly through the regulation of ATP-binding cassette transporters in guard cells of epidermal strips of three plant species (Vicia faba, Arabidopsis thaliana, and Impatiens walleriana) (27), and their findings have been supported by data from other groups (35, 41, 69). In contrast, Lisjak et al. reported that the stomata opened in response to treatment with GYY4137 or NaHS through the reduction of NO accumulation in guard cells (64), and similar findings have been confirmed in a crop plant, Capsium annuum (66). The disparity in results may be resolved by a study from Honda et al. (33) who showed that GYY4137 elicited stomatal closure transiently. GYY4137 first induced stomatal closure after 90 min of treatment at all concentrations (1–100 μM). After reaching maximal closure, the stomatal aperture gradually increased in size and the stomata fully reopened after 120 min of treatment with 100 μM GYY4137. Mechanistic studies revealed that NO-mediated H2S-induced stomatal closure by mutual interaction to induce the synthesis of 8-mercapto-cGMP, which triggers stomatal closure.
Cross talk between NO and H2S has also been suggested in an earlier study (59) in which the application of GYY4137 was demonstrated to enhance heat tolerance in maize induced by the NO donor, sodium nitroprusside, indicating H2S as a downstream signaling molecule in NO-induced heat tolerance in maize seedlings. Interestingly, a recent study described that the beneficial or detrimental long-term effects of dosing pea, radish, and lettuce plants with GYY4137 were very much dependent on how GYY4137 was applied to the plants (12). The addition of GYY4137 to lettuce plants via potting mix caused growth reduction and death, whereas GYY4137 application to the leaves of lettuce plants increased the harvest weight significantly.
The regulatory role of H2S in plant physiology is an emerging and exciting new research area, and how we can exploit H2S and its donor compounds in the modulation of plant physiology in the field to improve drought resistance, promote harvest weight, and protect crops from postharvest spoilage warrants further investigation.
Structural Modifications of GYY4137 and Their Effects
GYY4137 is, without a doubt, the most well-investigated synthetic H2S donor, and extensive studies using this compound have confirmed its biological and physiological relevance. To broaden its applications in H2S research and therapeutic potential, modifications of GYY4137 in an attempt to alter its pharmacokinetics, pharmacodynamics, and toxicity have been attempted by several groups in the past few years (22, 23, 43, 44, 83, 84, 111, 113, 124).
Structurally, GYY4137 possesses a phosphorodithioate core, which undergoes hydrolysis to release H2S. Park et al. replaced the C-P bond of the phenyl-phosphorus linkage in GYY4137 with an O-substitution (Fig. 3) and explored H2S production rates from the resulting analogues (83, 84). They reported that the O-aryl substitution donors 5a–5c (Fig. 3, top right panel) released H2S in a slow and sustainable manner in solution and in cells very much like GYY4137 and exhibited protective effects against H2O2-induced oxidative damage in H9c2 cardiomyocytes.
Further successful development of a series of GYY4137 derivatives came from Whiteman et al. (111) who showed that, in physiological buffer or in human synoviocytes, GYY4137 derivatives released H2S at a greater rate than GYY4137 (AP67>AP72>AP105>AP106>GYY4137, Fig. 3, bottom right panel). Importantly, these authors demonstrated that the biological effects of H2S in a variety of cells and tissues, including inhibition of LPS-stimulated formation of NO and PGE2 in murine RAW264.7 macrophages and human synovial fibroblasts, induction of cGMP accumulation in rat arterial smooth muscle cells, and relaxation of phenylephrine-precontracted mouse aortic rings, were dependent on the rates at which the compounds generated H2S.
Similarly, Feng et al. developed a series of dithiophosphorus H2S donors and cyclization analogues (22, 23). They reported that FW1256 is more potent in terms of its anticancer activity followed by FW1131 and GYY4137 (Fig. 3, bottom left panel). Antiproliferative potencies of these compounds, as shown by inhibition of breast cancer MCF7 cell proliferation and 3D tumor spheroid formation and subsequent induction of apoptosis, are in line with the intracellular H2S levels which they cause. Further development of the cyclic structure using FW1256 as a lead compound identified an even more potent compound
In addition to the development of GYY4137-related derivatives with a range of H2S production rates, designs for the spatiotemporal control of H2S release from these thiophosphorous agents have also garnered much interest. To this end, Kang et al. (43) developed a series of pH-controlled slow-releasing phosphonamidothioate-based H2S donors (Fig. 4, top left panel). Here, a novel strategy of pH-dependent intramolecular cyclization was employed to promote H2S release from the donors, and structure modifications further fine-tuned H2S-releasing properties. For instance, JK-1 was found to be a donor that releases H2S only at weakly acidic pHs (5 and 6), whereas JK-5 was found not to release H2S at all. They further demonstrated that H2S released from JK-1 and JK-2 donors provided significant cardioprotection in both cellular models of oxidative damage and in vivo murine models of myocardial ischemia–reperfusion injury (43).
Recently, Woods et al. (113) developed a novel red light-activated H2S donor complex where GYY4137 is coordinated to a photoactive [Ru(tpy)(biq)L]n+ scaffold (Fig. 4, bottom panel). This coordination can inhibit GYY4137s spontaneous hydrolysis until it is released by irradiation with red light. They further demonstrated that this compound increases intracellular H2S concentration only upon irradiation with red light and can protect H9c2 cardiomyoblasts from an in vitro model of ischemia–reperfusion injury (113). Since red light can effectively penetrate biological tissue and is nontoxic, the use of red light-activated H2S-releasing agents is valuable tool both for studies into the biological roles of H2S and the therapeutic utility of this gasotransmitter.
Despite extensive studies into the biological roles of H2S and the development of H2S-releasing agents, substantial scrutiny of the pharmacokinetics and cytotoxic properties of H2S donors in vivo remains limited. Zhang et al. (124) evaluated two series of thiophosphamide H2S donors and found that the H2S-releasing rate and amount of these compounds decreased with increasing pH, suggesting that these compounds may release H2S differently in different organs and tissue in vivo. The pharmacokinetic properties of these compounds were shown to vary with their structure. For instance, compound
Despite the fact that elucidation of the mechanisms leading to different H2S release rates between GYY4137 and its various derived donors still requires more detailed chemical studies, the efforts made so far have led to the development of some promising new compounds. These “new generation” compounds may serve as useful tools, in addition to GYY4137 and other sulfide salts, in the unraveling of the complex physiological and pharmacological implications of H2S biology.
Conclusion
Among all the synthetic H2S donors, GYY4137 is probably the most widely used and has proven to be an extremely versatile experimental tool in the field of H2S research. Much progress has been made over the past few years and a variety of biological effects of this donor have been reported. The effect of GYY4137 on vasodilation, cytoprotection, and inflammation has been comprehensively evaluated in numerous in vitro and in vivo models, highlighting its promise as a potential therapeutic agent in cardiovascular diseases, acute and chronic inflammation, and diabetes. Emerging reports on its effects in aging, cancer, and osteogenesis are of great interest and warrant further investigation. Several interesting reports showing a range of biological activities of GYY4137 in plants and crop preservation further broaden the potential application of GYY4137 taking it from the biomedical to the agricultural field. Moving forward, progress using GYY4137 as a prototype to develop a range of analogues with the ability to release H2S at a well-defined rate in the cells, tissues, and organs will be important to fully characterize its physiological and pharmacological effects.
Footnotes
Funding Information
This work was supported by the Ministry of Education Academic Research Fund Tier 1 and Tier 2 (R183-000-405-114 and R183-000-415-112) to L.-W.D.