Therapeutic application of hydrogen sulfide donors: the potential and challenges

Abstract

Hydrogen sulfide (H₂S), a colorless gas smelling of rotten egg, has long been considered a toxic gas and environment hazard. However, evidences show that H₂S plays a great role in many physiological and pathological activities, and it exhibits different effects when applied at various doses. In this review, we summarize the chemistry and biomedical applications of H₂S-releasing compounds, including inorganic salts, phosphorodithioate derivatives, derivatives of Allium sativum extracts, derivatives of thioaminoacids, and derivatives of anti-inflammatory drugs.

Keywords

hydrogen sulfide / cardiovascular / cancer / hypertension

Introduction

Given continued developments in the life science, the gaseous signal molecule hydrogen sulfide (H₂S) has attracted increasing attention. H₂S, a colorless gas with a rotten egg smell, has long been considered a toxic gas and environment hazard. However, evidences show that H₂S is produced in mammalian cells and plays a great role in many physiological and pathological activities. The aim of this work is to identify the potential and challenges of H₂S donors in therapeutic use.

H₂S production and metabolism

H₂S is a colorless gas with characteristic toxicity and flammability. Whereas low concentrations of H₂S smell of rotten eggs, high concentrations of the molecule result in insipidness because of paralysis of the olfactory nerve. H₂S is soluble in water up to 0.71 g/100 g H₂O at 0 °C and acts as a weak acid. H₂S solution is unstable, and the molecule is spontaneously oxidized by oxygen in water with a half-life of 3 h.

H₂S production

In the natural world, H₂S is produced from the breakdown of organic matter by bacteria in the absence of oxygen via a process called anaerobic digestion in swamps and sewers [ 1]. In mammalian cells, endogenous H₂S is produced from desulfhydration of cysteine catalyzed by two pyridoxal-5-phosphate-dependent enzymes in mammalian tissue, cystathionine b-synthase (CBS, EC 4.2.1.22) and cystathionine g-lyase (CSE, EC 4.4.1.1) [ 2, 3]. CBS and CSE only exist in the cytosol and generate H₂S from cysteine through b-elimination reactions. Endogenous H₂S in the brain and nervous system is produced by CBS, which is also expressed in the liver and kidney. Inhibition of endogenous H₂S generation has been observed in the presence of the CBS inhibitors aminooxy-acetic acid (AOAA) and hydroxylamine (HA) in brain homogenates [ 4]. CSE is expressed in the cardiovascular system and smooth muscle and highly expressed in the liver. Studies show that H₂S production in the cardiovascular system is reduced by approximately 50% in the CSE knockout mice [ 5]. Cysteine is also catalyzed by the tandem enzymes cysteine aminotransferase (CAT, EC 2.6.1.3) and 3-mercaptopyruvate sulfurtransferase (3-MST, EC 2.8.1.2) to form H₂S, which is the main pathway of endogenous H₂S production in mitochondria. 3-MST activity can be detected in mitochondria, and it is expressed in the kidney, liver, heart, and brain. In mitochondria, H₂S can be produced by 3-MST from 3-mercaptopyruvate, which is produced by CAT from cysteine (Fig. 1) [ 3].

Several pharmacological inhibitors, including propargylglycine (PAG), b-cyanoalanine (BCA), AOAA and HA (Fig. 2), can inhibit H₂S biosynthesis [ 1, 6]. PAG is an irreversibly selective inhibitor of CSE with a concentration range of inhibition of 1–10 mmol/L [ 7]; this compound does not inhibit the activity of recombinant human CBS. Co-crystallization of CSE and PAG indicates that PAG could obstruct the accessibility of the active site of the enzyme by occupying the space of the side chain [ 8]. Unlike PAG, BCA is a reversible inhibitor of CSE, and it inhibits the activity of CBS at concentrations as low as 1 mmol/L [ 7]. AOAA is an inhibitor of aminotransferases and it inhibits activities of both CBS and CSE. HA is a product of cellular metabolism and a selective inhibitor of CSE at low concentrations. HA also inhibits CBS at high concentrations [ 9].

H₂S metabolism

A dynamic balance can be observed between H₂S and HS^–, and the pKa is around 6.9. Over 1/3 of the endogenous H₂S exists as undissociated gas; the remaining 2/3 exists as HS^– [ 10]. The metabolic pathway of sulfide differs in different organs. In the liver, sulfide is oxidized into sulfate. Sulfide is slowly oxidized into thiosulfate in the lung because of deletion of sulfite oxidase. In the kidney, sulfide is initially oxidized into thiosulfate, and then thiosulfate is reduced into sulfite by thiosulfate reductase and thiosulfate sulfurtransferase. Sulfite is oxidized into sulfate in the presence of glutathione (Fig. 1) [ 11].

Biological effects of H₂S

Evidences show that H₂S exhibits different effects when applied at different doses. At low concentrations (micromole), H₂S shows cytoprotective effects, including anti-apoptosis, anti-necrosis, and cell proliferation effects. At high concentrations (millimole levels), however, H₂S is characterized as presenting cytotoxicity effects, such as stimulating apoptosis and cell death [ 1, 7, 12].

Decreases in the expression and activity of CSE in the thoracic aorta and plasma H₂S levels have been observed in hypertension rats. Treatment with the H₂S donor, for example, attenuated increases in blood pressure and vascular structural remodeling during hypertension development [ 13]. H₂S applied at 500 mmol/L caused a mean dilation from the pre-constricted tension in arterial rings isolated from resections of patients with bronchial carcinoma. It also caused reductions in pulmonary artery pressure and bronchial airway pressure in human isolated perfused lung [ 14]. Stimulation with a H₂S donor increased mRNA levels and the release of vascular endothelial growth factor, thereby resulting in vasodilatation in soluble fms-like tyrosine kinase 1 (sFlt-1)-induced hypertension [ 15]. The hypotensive effects of H₂S occur through the relaxation of resistance blood vessels by the opening of K_ATP channels and the guanosine 3′,5′-cyclic phosphate (cGMP) pathway [ 16]. Reports on H₂S-induced vasodilatation suggest that this action occurs at least in part through the NO pathway. H₂S could react with S-nitrosothiol as a reductant to release NO, thus providing the effect of vasorelaxation [ 17].

Growing evidences indicate that H₂S plays a great role in heart failure. Concentrations of myocardial and circulating H₂S have been observed to significantly decrease in a heart failure mouse model. Deletion of CSE resulted in greater cardiac dilatation and dysfunction than that observed in wild-type mice in a pressure overload-induced heart failure model [ 18]. Coronary heart disease (CHD) accounts for about 2/3 of all heart failure cases [ 19]. Balneotherapy with H₂S has been shown to improve excise tolerance to CHD, reduce the clinical manifestations of the disease, and decrease daily nitrate needs in CHD patients [ 20]. A negative correlation between blood H₂S levels and arterial pathological damage scores has been reported in atherosclerotic rats. CSE-KO mice fed with high-fat diets developed early fatty steak lesions in the aortic root, and treatment with H₂S remarkably inhibited the progression of these lesions to atherosclerosis [ 21].

Treatment with H₂S donors increases numbers of interstitial cells of Cajal, an event that plays a great role in gastrointestinal motility regulation. This protective effect, which occurs through Akt phosphorylation, may be utilized to treat gastrointestinal motility diseases, such as pseudo-obstruction and diabetic enteropathy [ 22]. H₂S also exerts physiological and pathological effects in the oral cavity. Studies have indicated, for example, that H₂S was the principal cause of physiological halitosis. H₂S inhibits epithelial cell proliferation and delays its repair in deep periodontal pockets by inhibiting Rb protein phosphorylation, which could induce periodontitis [ 12].

H₂S donors

Considering the extensive and effective physiological effects of H₂S, several independent-research groups have reported the protective effects of H₂S donors in different disease models. H₂S donors are divided into the following categories according to their chemical structures and sources: inorganic salts, phosphorodithioate derivatives, derivatives of Allium sativum extracts, derivatives of thioaminoacids, and derivatives of anti-inflammatory drugs (Table 1).

Inorganic salts

Inorganic salts, including sodium hydrosulfide (NaHS), sodium sulfide (Na₂S) and calcium sulfide (CaS), are considered exogenous donors of H₂S. These inorganic salts form HS⁻ and H₂S through hydrolytic actions in physiological buffer and increase H₂S concentrations rapidly. However, the duration of the H₂S effective dose afforded by these salts is short because volatilization of H₂S in solution occurs rapidly. This phenomenon somehow limits the use of the salts in clinical settings. Toombs et al. demonstrated a rat model that could detect exhaled H₂S after intravenous administration of IK-1001 (Na₂S in sterile solution). The group also extended these observations to humans, finding a significant increase in blood H₂S levels and exhaled H₂S concentrations with a short half time (<5 min) after a single intravenous administration [ 23].

Phosphorodithioate derivatives

Morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) was first synthesized by Ling Li et al. (Fig. 3A). GYY4137 is a water-soluble and slowly released H₂S donor and it released H₂S relay on hydrolysis; in fact, 1 mmol/L GYY4137 only releases 40–50 mmol/L H₂S within 25 minutes at physical temperatures and pH relay on hydrolysis. Administration of 133 mmol/kg GYY4137 intravenously or intraperitoneally increases plasma H₂S level from 30 mmol/L to 80 mmol/L within 30 minutes and these levels remain elevated for over 3 hours in anesthetized rats. In vitro, an increase in moderate H₂S levels (about 20 mmol/L) is observed up to 7 days after exposure to 400 mmol/L GYY4137 in human breast adenocarcinoma MCF-7 cells. By contrast, increases in moderate H₂S levels occur for less than 2 hours after exposure of MCF-7 cells to NaHS [ 24].

Evidence shows that GYY4137 exhibits anti-hypertensive activity by relaxing rat aortic rings and dilating the perfused rat renal vasculature through opening vascular smooth muscle K_ATP channels [ 25]. The compound neither exerts significant cytotoxic effects on cultured rat vascular smooth muscle cells nor affects cardiac function. Paradoxically, GYY4137 could increase the blood pressure after lipopolysaccharide (LPS)-induced hypotension. It also decreases TNF-a, IL-1b, IL-6, nitrite/nitrate, and C-reactive proteins and increases plasma concentrations of the anti-inflammatory cytokine IL-10 after LPS treatment [ 26].

GYY4137 significantly induces death in cancer cells, such as human cervical carcinoma HeLa cells, colorectal carcinoma HCT-116 cells, hepatocellular carcinoma Hep G2 cells, human promyelocytic leukemia HL-60 cells, human acute myelocytic leukemia MV4-11 cells, and osteosarcoma U2OS cells, by decreasing glycolysis and lactate overproduction, but exerts no significant cytotoxicity or cell death in non-cancer cells, such as human diploid lung fibroblast IMR90 cells and WI-38 cells [ 24, 27]. However, a structural analog of GYY4137 (ZYJ1122), which was unable to release H₂S because it lacked sulfur, did not show similar anti-cancer effects. Treatment with GYY4137 (400 mmol/L) for 5 days caused increases in cleaved-PARP and cleaved-caspase 9, indicating initiation of apoptosis. Treatment with GYY4137 (100–300 mg/(kg·d)) for 14 days also caused decreases in tumor growth in mice xenograft studies with HL-60 and MV4-11 cells. Thus, GYY4137 administration can be considered a novel and selective anti-cancer strategy.

Derivatives of Allium sativum extracts

Several researchers have committed to extract sulfur compounds from natural products to obtain better H₂S donors. In traditional Chinese medicine, Allium sativum (garlic) is used to treat a number of diseases including cardiovascular disease and cancer. A possible mechanism of the protective effect of garlic involves organic polysulfides. S-allycysteine (SAC) is a water-soluble organosulfur compound extracted from garlic that can inhibit triglyceride and cholesterol generation in hepatocytes (Fig. 3B). Treatment with SAC (240 mg/kg) caused a decrease in tumor growth via inhibition of cancer cell proliferation and PI3K/Akt, NF-kB and cyclooxygenase-2 (COX-2) expression in nude mice implanted with non-small cell lung carcinoma A549 cells [ 28]. A double blind parallel-randomized placebo-controlled trial was designed to assess the antihypertensive effects of garlic extract. The systolic blood pressure (SBP) of patients with uncontrolled hypertension (SBP≥140 mmHg at baseline) decreased after receiving 4 capsules of garlic extract (960 mg containing 2.4 mg of SAC) daily for 12 weeks. SBP did not show significant changes in patients with SBP<140 mmHg at baseline [ 29]. Pre-treatment with SAC (50 mg/(kg·d)) for 7 days remarkably decreased mortality and infarct size, and PAG, the CSE inhibitor, eliminated the protective effects of SAC in an acute myocardial infarction rat model [ 30].

Researchers have synthetized a series of derivatives to improve the therapeutic effects of organosulfur compounds, such as S-propargyl-cysteine (SPRC). SPRC is a cysteine analog (Fig. 3C) that can increase the expression and activities of CSE and CBS to enhance H₂S levels in vivo and in vitro. In an acute myocardial infarction rat model, treatment with SPRC caused increases in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and glutathione (GSH) levels, as well as decreases in the lipid peroxidation product malondialdehyde (MDA) in ventricular tissue [ 31]. Treatment with SPRC also improved cell proliferation, adhesion, and tube formation in primary human umbilical vein endothelial cells (HUVECs) through phosphorylation of signal transducer and activator of transcription 3 (STAT3). Co-crystallization assay showed a possible direct interaction between SPRC and STAT3 [ 32]. In LPS induced-rat embryonic ventricular myocardial H9c2 cells, SPRC decreased NF-kB p65 phosphorylation and Ikba degradation as well as suppressed ERK1/2 phosphorylation and intracellular reactive oxygen species (ROS) production. Thus, SPRC exerts anti-inflammatory effects through generation of H₂S by impairing Ikba/NF-kB signaling and activating the PI3K/Akt signaling pathway [ 33]. In the nervous system, SPRC exerts protective effects on LPS-induced spatial learning and memory impairment through TNF-a and TNFR1 inhibition and amyloid-b (Ab) generation as well as increases in NF-kB phosphorylation and of Ikba degradation [ 34]. In vivo, high doses of SPRC (100 mg/kg) reduced the weight and volume of tumors in nude mice implanted with human gastric cancer SGC-7901 cells. SPRC also induced apoptosis in cancer tissue with increasing p53 and Bax levels [ 35]. SPRC combined with leonurine (designated as LEO, Fig. 3D), a plant alkaloid present in Chinese motherwort (Leonurus artemisia and L. heterophyllus), exerts protective effects against hypoxia-induced neonatal rat ventricular myocytes damage at a lower dose than SPRC (100-fold less) [ 36].

When garlic is crushed, the alliin it contains produces allicin (diallyl thiosulfinate) via the enzyme alliinase. Allicin is unstable in aqueous solution and rapidly decomposes to diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and ajoene (Fig. 4A) [ 37]. Unlike SPRC, DADS and DATS increase H₂S levels but not through endogenous H₂S produced-enzymes. DADS and DATS produce H₂S when they react with GSH, and NADPH is necessary to support the activity of GSH reductase, which, in turn, is necessary to maintain normal concentrations of GSH (Fig. 4B) [ 16]. Preclinical studies have indicated that DAS, DADS, and DATS could inhibit liver, prostate, lung, and skin cancer development [ 38]. Treatment with DAS (10 mg/kg, 3 times/week, for 28 weeks) stimulated apoptosis and decreased tumor growth by increasing the expression of the proteins p53, p21/waf1, and bax and decreasing the expression of survivin and bcl-2 in dimethylbenzanthracene-induced skin cancer in Swiss albino mice [ 39]. DATS administration (10 and 50 mg/kg, once for 4 days, for 4 weeks) resulted in decreases in tumor weight and volume through induction of apoptosis in mice colorectal cancer CT26 cells on a murine allograft animal model [ 40]. Treatment of neuroblastoma SH-SY5Y cells with DADS resulted in activation of mitochondrial pathway apoptosis through induction of cell cycle arrest at the G₂/M phase and increases in ROS production [ 41].

Derivatives of thioaminoacids

Thioaminoacids are a type of amino acid derivate commonly used as materials for peptide synthesis. At mild alkaline pH, thioaminoacids are reversibly transformed into a-amino acid N-carboxyanhydrides and release H₂S. Thus, thioaminoacids are considered as ideal H₂S donors. Thioglycine and thiovaline (Fig. 4C and 4D) are derivatives of thioaminoacids and can release H₂S by slowly mimicking the production of endogenous H₂S. Thioglycine could increase plasma levels of H₂S in rat at a dose of 5 mg/kg and caused dilatation in mouse aortic rings through cGMP stimulation [ 42].

Derivatives of anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs for anti-inflammation in the clinical setting. The gastrointestinal side effects of NSAIDs are considered as the major problem related to the use of these drugs, and the relevant mechanisms remain incompletely understood. Generalized inhibition of prostaglandin (PG) production is a major factor contributing to the side effects of NSAIDs. However, COX-2 selective inhibitors, which can reduce this generalized inhibition, continue to exert gastrointestinal side effects. Evidence shows that NSAIDs, including COX-2 selective inhibitors, increase the adherence of leukocytes to the vascular endothelium in the gastrointestinal tract, which has been proven to contribute to the pathogenesis of NSAID-induced gastrointestinal damage. Precisely because H₂S can inhibit leukocyte adherence, researchers have been able to design a series of H₂S-releasing NSAIDs to limit gastrointestinal side effects (Fig. 5A and 5B). ATB-337, for example, is a derivate of diclofenac that can release H₂S. Data show that ATB-337 does not increase leukocyte adherence or gastrointestinal side effects, similar to observations made during diclofenac administration [ 43]. ATB-343, a derivate of indomethacin, exerts protective effects similar to those of ATB-337. Besides reducing gastrointestinal side effects, H₂S-releasing NSAIDs have also been shown to exert better anti-inflammatory effects than the original NSAIDs. ATB-337, for example, can significantly decrease paw edema after injection of carrageenan in rats at a dose of 10 mmol/L. By comparison, diclofenac must be administered at a dose 3 times that of ATB-337 to produce the same reduction of paw swelling [ 43].

Numerous preclinical and clinical studies indicate that NSAIDs can be used as anti-cancer drugs. These molecules can restore normal apoptosis and inhibit angiogenesis in various cancer cell lines. However, the adverse side effects of NSAIDs, including gastrointestinal and renal effects, limit their clinical applications in cancer patients [ 44]. Therefore, the use of H₂S-releasing NSAIDs has been proposed as an approach to reduce the side effects of NSAIDs in the cancer therapy. Chattopadhyay et al. tested the effect of four H₂S-releasing NSAIDs (i.e., HS-aspirin, HS-sulindan, HS-ibuprofen, and HS-naproxen) in different human cancer cell lines (Fig. 5C–5F) and showed that HS-NSAIDs induced cell apoptosis through G₀/G₁ cell cycle arrest and inhibited the proliferation of cancer cell lines, with the potencies 30–3000-fold higher than those of the original NSAIDs. In this study, HS-aspirin was the most potent anti-cancer agent among the drugs studied, and its cell growth inhibition IC₅₀ was (1.6±0.7)−(4.2±1.1) mmol/L. The potency of HS-ibuprofen, HS-sulindan, and HS-naproxen were 2–20-fold lower than that of HS-aspirin [ 45]. The anti-cancer effect of HS-NSAIDs is COX-independent because the drugs showed the same effects on COX-1- and COX-2-positive human colon cancer HT-29 cells as well as on COX-null human colorectal carcinoma HCT-15 cells [ 46].

Similar to H₂S, nitric oxide (NO) is also a gaseous transmitter; evidence suggests that the use of NO-releasing NSAIDs is another strategy to reduce gastrointestinal side effects. Based on the physiological relevance between NO and H₂S, researchers have postulated that a new hybrid of NSAIDs releasing both NO and H₂S may be more potent and effective than either drug alone (Fig. 6). NOSH-aspirin, a NO and H₂S-releasing compound, did not show any cellular toxicity, which is determined by lactate dehydrogenase release; this drug also exerted anti-inflammatory effects similar to those of aspirin in reducing swelling in the paw edema model [ 47]. In HT-29 cells, NOSH-aspirin could inhibit cell growth with an IC₅₀ ranging from 50 nmol/L to 280 nmol/L within 24 h. This drug is>100 000-fold more potent than aspirin in HT-29 cells at the IC₅₀, particularly in terms of cell growth inhibition. Treatment with NOSH-aspirin for 18 days caused reductions in tumor volume and weight in nude mice with a human colon cancer xenograft [ 47].

Others

Zhao et al. [ 48] investigated a series of cysteine-activated H₂S donors, N-(benzoylthio)benzamides, that could release H₂S in a controlled manner (Fig. 7A). These donors were stable in aqueous buffer and did not react with cellular molecules. Moreover, in the presence of excess cysteine, the unstable S–N bonds were easy to break to generate H₂S. Researchers also proved that structural modifications could control the H₂S release rate of these donors; thus, these donors could be useful for further H₂S research.

Besides biologically activated H₂S donors, some compounds based on photolysis have been reported to release H₂S in living cells. Such donors cannot release H₂S until external stimuli, like UV rays, are applied to them. Thus, they can provide H₂S at specific time points and cellular locations [ 49]. For example, Chen et al. [ 50] synthesized a new H₂S donor, propane-2,2-diylbis((1-(4,5-dimethoxy-2-nitro-phenyl)ethyl)sulfane), and loaded it on the surface of upconversion nanoparticles; a simple night-time ozone profile light could react with this system and release H₂S in target tissue (Fig. 7B). This technology presents a new strategy for developing controlled and targeted H₂S donors.

Future challenges

For decades, hundreds of H₂S donors have been developed and used in biological studies. However, H₂S-related therapy continues to face a number of challenges, including discovery of new H₂S donors with good tissue specificity, development of novel indications of H₂S donors, and so on. The physiological and pathological mechanisms of H₂S are not clearly understood. H₂S has long been considered a toxic gas that can cut off respiration chains, cause mitochondrial dysfunction, and generate endogenous free radical and oxidant species [ 51]. Paradoxically, some evidence shows that H₂S can positively regulate cellular energy production through oxidation and phosphorylation. To determine the mechanisms and pathways of H₂S, as well as the safe use of H₂S, new methods with low limits of detection must be developed to allow measurement of H₂S concentrations in different tissue and diseases, as well as the therapeutic effects of H₂S donors in delivering H₂S to targets. Novel drug delivery is another approach that has been proposed to improve the use of H₂S donors. Compared with normally released SPRC, control-released SPRC shows better cardio protective effects and lower toxicity [ 52].

H₂S is considered a gasotransmitter, together with NO and carbon monoxide (CO). Complex interactions are known to occur between gasotransmitters during regulation of cell function [ 2, 53, 54]. Studies show that treatment with NO donors could enhance the generation of endogenous H₂S [ 10]. Low concentrations of H₂S could promote the NO production in the vascular endothelium to enhance the vasorelaxant effects of NO donors [ 55]. H₂S can increase HO-1 expression, which, in turn, decreases CO production [ 56]. Additional studies are necessary to understand the various interactions between these three gasotransmitters.

As mentioned above, the road ahead for researchers is long in terms of evaluating the biological mechanisms of H₂S and developing novel H₂S donors. Although challenges and opportunities coexist, the therapeutic applications of H₂S donors are expected to undergo further development in the near future.

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