Hydrogen Sulfide-Based Therapeutics: Exploiting a Unique but Ubiquitous Gasotransmitter

REVIEWS

Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous gasotransmitter

John L. Wallace1,2 and Rui Wang3

Abstract | Hydrogen sulfide (H2S) has become recognized as an important signalling molecule throughout the body, contributing to many physiological and pathological

processes. In recent years, improved methods for measuring H2S levels and the availability of

a wider range of H2S donors and more selective inhibitors of H2S synthesis have helped to more accurately identify the many biological effects of this highly reactive gaseous mediator.

Animal studies of several H2S-releasing drugs have demonstrated considerable promise for the safe treatment of a wide range of disorders. Several such drugs are now in clinical trials.

β‑synthase Cystathionine γ‑lyase Over the past 15 years, hydrogen sulfide (H2S) has become (CBS) — catalyse the production of H2S, (CSE). An enzyme that converts recognized as a crucial signalling molecule with a wide ammonia and pyruvate from l‑cysteine and homocyst- l‑cysteine into hydrogen range of physiological functions1,2. It can profoundly affect eine, respectively. A PLP-binding domain is common to sulfide, pyruvate and most organ systems in animals and humans, but it also CSE and CBS, and is crucial for their catalytic activities. ammonia. It requires the cofactor pyridoxal phosphate contributes to many functions in plants and prokaryotes. Unlike CSE, CBS contains a 70‑amino-acid haem domain (vitamin B6) for this activity. Indeed, H2S had important roles in the development of in its amino terminus, which equips CBS to perform life on Earth during the 500 million years before photo- three functions. First, it offers an interaction site for two Cystathionine β‑synthase synthesis3. The realization of the biological importance of other gasotransmitters: nitric oxide (NO) and carbon (CBS). An enzyme that H S in numerous cells, tissues and organs is now shedding monoxide. Second, it can act as a redox sensor to regu- catalyses the conversion 2 of homocysteine to light on the pathogenesis of various human diseases, and late its own production of H2S (notably, without a haem 4 cystathionine (the first step in paving the way for innovative therapeutic interventions. domain, CBS is no longer reactive to oxidative stress) . the trans-sulfuration pathway) In this article, we review the pathways for the synthe- Third, it performs as an oxygen sensor for the regulation and the condensation of sis and metabolism of H S (FIG. 1), its major mechanisms of CBS degradation: an increase in the partial pressure of homocysteine and cysteine 2 to form cystathionine and of action, and some of the key disease processes in which oxygen leads to the oxygenation of the haem group such hydrogen sulfide. H2S appears to participate, as well as the tissues affected. that the conformational change of CBS can be recognized 5 Methods for measuring H2S levels in vivo, which are crucial by Lon protease, triggering its degradation of CBS . BOX 1 cysteine aminotransferase for the study of H2S biology, are discussed in . Also Like CSE and CBS, (CAT) uses discussed are the concerns associated with the reliability PLP as a cofactor and converts cysteine to 3‑mercapto- 1Department of Physiology and accuracy of such measurements, which have triggered pyruvate. Using zinc as a cofactor, 3‑mercaptopyruvate & Pharmacology, University debate on the physiologically relevant endogenous levels of sulfurtransferase (MST) transfers the sulfur in the sulfane of Calgary, Calgary, Alberta T2N 4N1, Canada. H2S and the use of H2S donors in high micromolar concen- group of 3‑mercaptopyruvate to other sulfur acceptors. 2 Department of trations to mimic the physiological actions of H2S. Finally, In essence, MST acts as a sulfur carrier, rather than an Pharmacology & Toxicology, we provide examples of attempts to exploit the actions of H2S producer, as the sequential reactions that are cata- University of Toronto, Toronto, H S in the design of novel therapeutic agents for the treat- lysed by CAT and MST generate sulfane sulfur. This Ontario M5S 2J7, Canada. 2 3Department of Biology, ment of arthritis, inflammatory bowel disease, myocardial bound sulfur has to be released or reduced to liberate H2S Lakehead University, dysfunction and chemoprevention of cancer. (REF. 6). Another sulfur-carrying enzyme in our bodies is Thunder Bay, Ontario rhodanese (also known as thiosulfate sulfurtransferase)7. P7B5E1, Canada. H2S synthesis and metabolism However, the biological and physiological importance of Correspondence to J.L.W. H S synthesis. H S is produced in mammalian cells this enzyme in endogenous H S metabolism has not been e‑mail: 2 2 2 [email protected] mostly through the reverse trans-sulfuration pathway fully determined. In contrast to MST, which is localized doi:10.1038/nrd4433 (FIG. 1). Two pyridoxal 5ʹ-phosphate (PLP)–dependent both in the cytosol and mitochondria, rhodanese is a true Published online 7 April 2015 enzymes — cystathionine γ‑lyase (CSE) and cystathionine mitochondrial protein.

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H2S metabolism and excretion. Unlike NO, H2S is translocation to the mitochondrion have not yet been relatively stable in body fluids. In the circulation identified. It may also be possible that pathophysiological

and in the cytoplasm, free H2S can be scavenged by stimuli such as hypoxia directly increase intracellular methaemoglobin8 or by metallo- or disulfide-containing calcium levels and result in the subsequent translocation macromolecules that function as sulfane-sulfur and of CSE. Interestingly, this mitochondrial translocation of bound-sulfate pools. Oxidation and methylation are CSE is not observed in hepatocytes5.

another two mechanisms of H2S metabolism. H2S is CBS has also been conventionally regarded as being oxidized sequentially in the mitochondrion to thio- compartmentalized to the cytosol. Recent studies in liver sulfate and then to sulfite, with the end-product under cells5 and colon-cancer-derived epithelial cells26 show the physiological conditions being sulfate9. The mitochon- existence of CBS proteins in the mitochondrion under drial enzyme sulfide-quinone reductase has a crucial resting conditions. MST and CAT are located both in the (REF. 10) 6,30 role in the oxidation of H2S . The cytosol is cytosol and in mitochondria .

the major intracellular site of H2S methylation. Thiol Based on the tissue-specific expression patterns of

S‑methyltransferase catalyses the methylation of H2S to various H2S-generating enzymes, one may estimate the

yield methanethiol and dimethylsulfide. major enzymatic sources of H2S in various organs and

H2S is excreted in urine and flatus as free sulfate, cells. H2S production in the cardiovascular system is pre- thiosulfate or free sulfide, and is also exhaled in breath11. dominantly regulated by CSE, whereas CBS is the enzyme

mostly responsible for H2S production in the nervous Tissue-specific distribution and intracellular compart- system. In other systems and organs, multiple enzymes

mentalization of H2S-generating enzymes. Among the may control the production of H2S, and their individual mammalian tissues that express CSE are the cardiovas- contributions may vary depending on the developmen- cular system12,13, liver, kidney, uterus, placenta, pancreatic tal stage and any disturbance of homeostasis. However, islets13,14, lung15, and gastrointestinal tract16–19. Previously, the above estimation clearly oversimplifies the complex

although CSE mRNA had been detected in the brain, the nature of endogenous H2S metabolism. For example, lack of CSE expression in the nervous system was taken even within the central nervous system, different types for granted20. However, a recent study revealed abundant of neurons or glial cells may rely on CSE or MST rather

expression of CSE proteins in the mouse and human than CBS to produce H2S. Furthermore, the endogenous 21 striatum, cortex, and cerebellum . production of H2S depends on substrate availability and The brain is the primary organ in which CBS expres- other properties of the intracellular milieu that may dif- sion is dominant. CBS protein has been identified in the ferentially affect enzyme activities, in addition to the hippocampus, cerebellum, cerebral cortex, and brain- expression and distribution of these enzymes. stem22,23. CBS expression and activities in other tissues,

such as the liver, kidney, pancreas, gastrointestinal tract, Cellular and molecular effects of H2S 5,6,12,17,19,24–26 and lungs, have been confirmed . The expres- H2S participates in the regulation of homeostasis of sion of CBS protein in the cardiovascular system and the numerous systems in our body, including but not lim- functional relevance of such expression have not been ited to the cardiovascular, neuronal, gastrointestinal, convincingly established. A recent study detected a CBS respiratory, renal, liver and reproductive systems. The 27 protein band in cardiac tissues , although such expres- lipid-soluble nature of H2S enables this gasotransmitter sion had not been detected in many previous studies. to easily reach its molecular targets — on the plasma The answer to the controversy may not be simple, but membrane, inside the cytosol or in intracellular orga- certainly the specificity of the antibody used, the real nelles. This ubiquitous membrane permeability under- identify of the protein band detected in western-blot lies the wide scope of the physiological or biological

analysis, and the use of appropriate positive and nega- effects of H2S, but it is its unique chemical reactivity tive controls should be considered. These considerations with certain types of macromolecules in different types 28 and cautions also apply to the detection of CSE protein of cells that makes H2S a selective, specific and powerful and MST protein in western-blot analyses. signalling molecule. MST is expressed in the central nervous system, — mostly in glial cells29, hippocampal pyramidal neurons, Interactions with ion channels. Numerous cellular effects

cerebellar Purkinje cells, and mitral cells in the olfactory of H2S are mediated by its interactions with membrane ion 30 bulb . Both MST and CAT are detected in certain types channels. The ATP-sensitive potassium (KATP) channel was

of vascular endothelium. The expression of MST has the first-identified molecular target of endogenous H2S, 31 13 also been detected in vascular smooth muscle cells , and it mediates H2S-induced vasorelaxation . The activa- 29 29,32 cardiomyocytes , kidney cells and liver cells . tion of KATP channels by H2S has been reported in cardio Cysteine aminotransferase 13,34 35 36 37,38 (CAT). An enzyme that Under physiological conditions, CSE proteins are vascular , endocrine , respiratory , nervous and 33 39,40 catalyses the conversion of mainly localized in the cytosol . Increases in intra gastrointestinal systems . It seems that multiple subunits l‑cysteine and α‑ketoglutarate cellular free calcium levels in vascular smooth muscle of the KATP channel complex are modified by H2S. Using to 3‑mercaptopyruvate and cells, due either to the entry of extracellular calcium or a whole-cell patch-clamp technique and a mutagenesis glutamate. Another enzyme, to the release of intracellular calcium from the endoplas- approach, Jiang et al.41 demonstrated that H S specifically 3‑mercaptosulfurtransferase, 2 can then catabolize the mic reticulum, trigger the translocation of CSE from the acts on the sulfonylurea receptor 1 (SUR1; also known 33 generation of hydrogen sulfide cytosol to the mitochondrion . It should be noted that as ABCC8) subunit of the KATP channel complex to acti- from 3‑mercaptopyruvate. the endogenous substance or substances that elicit CSE vate these channels. Specifically, the amino-acid targets

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Cytosol CSE

Cys CAT MST • Protein modiﬁcation H2S • Inhibition of PDEs • Ion channel interactions

CBS HCY CSE

CSE Stress (for example, 2+ Mitochondria ↓pO2 or ↑[Ca ])

CSE

Cys CAT MST • Biogenesis H2S • ATP production • Antioxidant eﬀects

HCY CBS

Figure 1 | Cytosolic and mitochondrial production and functions of H2S. Depending on the cell type and specific 6 stress conditions, endogenous hydrogen sulphide (H2S) production can occur in the cytosol and/or the mitochondria . Cystathionine ‑lyase (CSE)-mediated H S production usually occurs in the cytosol; however, under stress conditions γ 2 Nature Reviews | Drug Discovery CSE can be translocated from the cytosol into the mitochondrion, where it catalyses H2S production from cysteine (Cys). Cysteine aminotransferase (CAT) and 3‑mercaptopyruvate sulfurtransferase (MST) are located both in the cytosol

and mitochondria, and regulate H2S production. Cystathionine β-synthase (CBS) is located in the cytosol, but can also be found in the mitochondrion of certain cell types under resting conditions. Hypoxia decreases the degradation of

CBS in the mitochondrion, leading to an accumulation of CBS and thus increasing production of H2S in this organelle.

HCY, homocysteine; PDE, phosphodiesterase; pO2, partial pressure of oxygen.

46 of H2S are the Cys6 and Cys26 residues of the extracell NaHS (at concentrations of 10 μM−1 mM) . Indirect ular N terminus of the SUR1 subunit. The SUR2B sub evidence also showed that native T‑type VDCCs in rat

unit of KATP channels in colonic circular smooth muscle cardiomyoblasts (H9c2 cells) were inhibited by H2S, as cells is S‑sulfhydrated by sodium hydrosulfide (NaHS); Ni2+ (which inhibits these channels) abolished NaHS-

this action underlies the increased KATP channel current induced decreases in resting intracellular calcium con- 47 that is induced by NaHS and blocked by the sulfonyl centration . However, the effects of H2S on VDCCs urea drug glybenclamide42. Similarly, the pore-forming appear to be tissue-specific; in neurons, the activities

subunit of the KATP channel (such as Kir6.1) in vascular of Cav3.2 T‑type VDCCs and Cav1.2 L‑type VDCCs are 48,49 smooth muscle cells can be S‑sulfhydrated, leading to enhanced by endogenous and/or exogenous H2S .

increased KATP channel current amplitude and membrane An important advance in vascular physiology in 34,43 hyperpolarization . recent years is the identification of H2S as an endothe- Voltage-dependent calcium channels (VDCCs) are lium-derived hyperpolarizing factor (EDHF)48,49.

also important targets of H2S in various cell types. In iso- H2S activates small- and intermediate-conductance 44 45 lated mouse pancreatic β-cells and rat cardiomyocytes , calcium-activated potassium channels (SKCa channels

H2S inhibits L‑type VDCC currents. The amplitude of and IKCa channels, respectively), and H2S-induced the current through heterologously expressed recombi- endothelium-dependent smooth muscle hyperpolariza-

nant voltage-gated calcium channel 3.2 (Cav3.2)‑subunit tion and vasorelaxation of mouse mesenteric arteries are T‑type VDCCs in HEK293 cells was also inhibited by abolished by the co‑application of charybdotoxin and

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Box 1 | Determination of endogenous H2S levels

Determining endogenous hydrogen sulfide (H2S) levels in the circulation and in tissues and cells is important for

characterizing H2S as a gasotransmitter. It is also needed for evaluating whether exogenous H2S donors mimic the 176 physiological and biological effects of endogenous H2S. As has been systematically reviewed recently , a wide range —

from 0.1 μM to more than 300 μM — of circulating H2S levels has been reported under physiological conditions. This remarkable variation is largely attributable to the wide array of measurement methods that have been used, including the colorimetric methylene blue method, ion-selective or polarographic electrodes, gas chromatography with flame photometry and the monobromobimane assay. Often, the total reactive sulfur pool — which does not

distinguish free H2S from acid-labile sulfide or bound sulfur (also known as sulfane-sulfur) — was measured. The extent of

H2S oxidation and/or H2S scavenging in biological samples may also skew estimations of the true concentrations of H2S.

For example, it was reported that plasma levels of H2S could not be reliably measured, as red blood cells avidly scavenge 177 H2S and remove it from the circulation . These measurement pitfalls have been addressed through the use of more advanced techniques. By measuring the 178 H2S in headspace gas from plasma samples, Shen et al. showed that the human plasma sulfide level is around 3 μM. Using the monobromobimane method coupled with reverse-phase high-performance liquid chromatography, the same authors recorded free hydrogen sulfide levels of 0.2–0.8 μM and acid-labile sulfur levels of 1.8–3.8 μM in plasma from mice 178 and from humans . With a combined modified gas chromatography and mass spectrometry technique, H2S levels of 176 0.5–2.5 μM were measured in pig and mouse blood . Thus, as a conservative estimate, plasma H2S levels in healthy humans or animals are in the higher nanomolar to lower micromolar range178–180. However, there is still no consensus

on the physiological levels of H2S in blood, or on the most acceptable and reliable techniques for measuring H2S in biological samples.

With respect to determining the physiological importance of H2S, does it matter if the circulatory or tissue levels

of endogenous H2S are in the nanomolar or micromolar range? It seems not. At nanomolar or even picomolar concentrations, numerous endogenous substances exert profound physiological effects in different systems.

Indeed, in many cases, it is the relative change in endogenous H2S levels that is more important in determining the physiological and pathophysiological importance of this gasotransmitter. This notion is exemplified by reports of the development of hypertension94, atherosclerosis101, diabetes119, renal ischaemic damage180, colitis155, acute liver failure153,181 and Huntington disease21 owing to reduced gene expression or activity of cystathionine γ‑lyase (CSE).

As far as the issue of free H2S versus total reactive sulfur is concerned, what is important is the indication and interpretation of the measured levels, not the methods themselves. Ideally, we should precisely and reliably detect

changes in each and all components of the total sulfur pool. Given that free H2S, acid-labile sulfide, and sulfane-sulfur are interchangeable under different conditions, an understanding of the mechanisms underlying the changes in this total reactive sulfur pool, and the physiological effects of such changes, carry biological and therapeutic importance.

However, should variations in H2S levels be used as biomarkers for the purpose of diagnosis and prognosis of certain

diseases, it becomes imperative to accurately measure the absolute concentrations of H2S in the circulation and in

tissues. Furthermore, the development of H2S-based therapies will be facilitated by accurately monitoring H2S levels in the circulation and/or in the targeted organs and systems in order to determine the pharmacokinetics and

pharmacodynamics of H2S donors as well as their efficacy and toxicity profiles.

50 apamin . The co‑application of these two compounds The interaction of H2S with ion channels is not

specifically blocks IKCa channels and SKCa channels, restricted to KATP and KCa channels. Delayed-rectifier and, as such, should block the vasorelaxant effect of K+ channels in mouse gastric-muscle cells can be inhib- (REF. 56) 4‑aminopyridine an EDHF. The molecular basis for the role of H2S as an ited by H2S , whereas (4‑AP)- EDHF is the post-translational modification of the tar- sensitive K+ channels in rat coronary artery smooth 57 gets of H2S. H2S can sulfhydrate IKCa channels in primary muscle cells can be activated by H2S . H2S has also been human aortic endothelial cells51. Furthermore, in vascular reported to activate chloride channels, voltage-gated

tissues the expression of SKCa2.3 channels but not IKCa3.1 sodium Nav1.5 channels and the transient receptor

channels was increased by H2S and decreased by a CSE potential cation channels TRPV1 and TRPA1 in different inhibitor or by deletion of the gene encoding CSE50. tissues58.

The inhibitory effects of H2S on the α‑subunit of As the physiological level of endogenous H2S in the

big-conductance calcium-activated potassium (BKCa) circulation or in various types of cells is unclear, many channels in heterologously transfected HEK293 cells of the aforementioned studies, such as those describing 52 have been reported . Likewise, H2S inhibited native the effects of H2S on BKCa channels, cannot be used as

BKCa channels in type 1 glomus cells from the isolated evidence for a physiological role of H2S in the regula- mouse carotid body53. By sharp contrast, ~300 μM NaHS tion of different ion channels. Indeed, establishing such

(an H2S donor) increased whole-cell BKCa currents and a physiological role of H2S would require a comparison

enhanced single-channel BKCa activity in rat pituitary of the characteristics of ion channels in the presence and 4‑aminopyridine 54 tumour cells . This stimulatory effect of NaHS (10 μM) absence of endogenous H2S. This forethought should (4‑AP). One of three isomeric on BK channels was also observed in endothelial cells55. be taken into account in all other studies that use H S amines of pyridine, which is Ca 2 widely used as a research tool These differential effects may be attributable to specific donors to deduce the physiological actions of H2S. to characterize the subtypes subtypes of BKCa channels in different types of cells and Moreover, ion channels are diversified in their amino-acid of potassium channels. to the concentrations of H2S donors. composition, voltage dependency, gating mechanisms,

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ion selectivity and sensitivity to different endogenous The abundance of S‑sulfhydrated proteins in vivo has signalling molecules. This diversity forms the basis of also been questioned. Whereas a previous study using the

the variable responses of ion channels to H2S exposure. biotin-switch assay reported that 10–25% of liver proteins are S‑sulfhydrated43, another study using a tag-switch Interactions with classical second messengers. Although method revealed a much lower abundance of protein 67 H2S can directly act on its target proteins without the S‑sulfhydration . The pH of the cellular environment, engagement of second messengers, this gasotransmitter and the proximity of the target cysteine amino acids to also affects the levels of several known second messen- the active centrum of the protein would affect the extent gers. Non-selective inhibition of phosphodiesterases of protein S‑sulfhydration. Therefore, special attention

(PDEs) by H2S leads to decreased degradation of cyclic should be paid to the sample preparation, including GMP and cyclic AMP, thereby increasing net cGMP the oxidative state and level of protonation of cysteine and cAMP levels59–61. For example, with regard to thiol groups, in order to more accurately determine the

cAMP levels, it has been demonstrated that H2S inhibits extent of protein S‑sulfhydration. the mitochondrial-matrix-localized PDE2A in isolated S‑sulfhydration can affect numerous proteins68. For rat liver mitochondria — leading to increased cAMP example, Kelch-like ECH-associated protein 1 (KEAP1) levels — as well as inhibiting the recombinant PDE2A is a negative regulator of the activity of nuclear factor 51 enzyme in vitro . Conversely, H2S can inhibit adenylyl erythroid 2‑related factor 2 (NRF2). H2S-induced 62 cyclase in the rat kidney . Thus, the net effect of H2S on S‑sulfhydration of KEAP1 at Cys151 facilitates the dis- cAMP levels could be determined by assessing the bal- sociation of KEAP1 from NRF2, thereby resulting in an ance between the inhibition of PDEs and the inhibition enhancement of NRF2‑mediated antioxidant responses14. of adenylyl cyclase. The post-translational modification of nuclear Intracellular free calcium is another second messen- factor‑κB (NF‑κB) serves as an example of the differ- ger for cellular signal transduction. Via its effects on ence in functional effects between S‑nitrosylation and plasma membrane ion channels and on intracellular S‑sulfhydration. NF‑κB is a transcription factor that

calcium pools, H2S can increase intracellular calcium regulates the expression of many inflammation- and levels in vascular endothelial cells by stimulating cal- apoptosis-responsive genes. S‑nitrosylation of the Cys38 cium influx63 or by releasing calcium from an ATP- and residue of the p65 subunit of NF‑κB inhibits the bind- 4‑chloro‑3‑ethylphenol (4‑CEP)-sensitive intracellular ing of NF‑κB to the cytokine-responsive sites in the pool64. The consequent elevation of intracellular calcium promoter of the gene that encodes inducible NO syn- levels may activate many calcium-dependent signalling thase, resulting in decreased expression of this enzyme69. pathways and enzymes such that endothelial proliferation S‑sulfhydration of the same cysteine residue, however,

and function can be regulated. H2S decreases intracell enhances the binding of NF‑κB to ribosomal protein S3, ular calcium levels in cardiomyocytes by inhibiting both which increases the transcriptional activity of p65 in L‑type and T‑type VDCCs, but it increases intracellular the nucleus. Consequently, cell apoptosis is greatly calcium release in the same cell preparation47. The func- inhibited68.

tional consequence of this biphasic effect of H2S is not For a signal transduction process to be regulated, calcium sparks clear. H2S increases in smooth muscle both ‘turn‑on’ and ‘turn-off’ mechanisms are required. cells of intact piglet cerebral arterioles65 and mesenteric For example, the removal of NO from S‑nitrosylated arteries55. Increased calcium sparks activate transient proteins occurs through the de‑nitrosylation process.

KCa channels and smooth muscle hyperpolarization; S‑nitrosoglutathione reductase (GSNOR), a de‑nitrosyla- eventually, global intracellular free calcium levels drop tion enzyme, reduces the product of protein S‑nitrosylation, and vasodilation ensues65. GSNO, to glutathione hydroxysulfenamide70. Thioredoxin, another de‑nitrosylation enzyme71, has two redox-active Protein S‑sulfhydration. At the amino-acid level, NO cysteine residues (in the sequence Cys-Gly-Pro-Cys) in its

and H2S both modify sulfhydryl groups of certain pro- active site. Thioredoxin breaks the disulfide bonds of its teins, but often generate opposite effects66. NO covalently target protein and then binds with the same protein; this modifies free sulfhydryls (–SH) of the cysteine residues activity accounts for its antioxidant function. By de‑nitro of these targeted proteins to form S‑nitrosothiols. Such sylating its substrates, thioredoxin can reverse the func- S‑nitrosylated proteins usually have decreased functions. tion of S‑nitrosylated proteins. However, our knowledge Low-molecular-weight thiols or cysteine residues of pro- of protein de‑sulfhydration is very limited. Mitochondrial teins can also be modified via S‑sulfhydration, in which persulfide dioxygenase (ETHE1) has been shown to cata- Calcium sparks the –SH from a sulfhydryl donor is transferred to cysteine lyse the conversion of glutathione persulfide (GSSH) to Intracellular Ca2+ release sulfhydryls, forming a covalent persulfide (–SSH) in the sulfite in vitro. This process has important implications events that play an important 51 72 role in excitation–contraction target protein . for mitochondrial sulfur catabolism and redox balance . coupling. The mechanisms for H2S-induced protein S‑sulfhydra However, the physiological substrate for ETHE1 is not

tion are under debate. As the sulfur in H2S and in –SH known, although the persulfide bound in the active-site Tag-switch method groups is at its lowest oxidative state, H2S may not be peptide of sulfide-quinone oxidoreductase might be one A technique used to measure able to directly interact with the –SH group of cysteine candidate. Moreover, little is known about the de‑sulf protein S‑sulfhydration, whereby S‑sulfhydrated to form a persulfide, and the oxidation of cysteine and/or hydration of S‑sulfhydrated proteins in the cytosol or residues are labelled to the production of polysulfides may be prerequisite for non-mitochondrial organelles. It is conceivable, however, form thioether conjugates. protein S‑sulfhydration. that the S‑sulfhydrated proteins may be de‑sulfhydrated

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in the presence of excess reductants and, as such, the Oxygen sensing and mitochondrial bioenergetics. level of oxygen will be a factor in the de‑sulfhydration Changes in the oxygenation status of cells affect endog-

process. enous H2S levels in different ways. Under hypoxic condi-

tions, the oxidation of H2S in mitochondria is decreased,

Role in redox balance. Redox is the integration of reduc- leading to a net elevation of H2S levels. In vascular tion and oxidation processes, in which electrons are trans- smooth muscle cells, hypoxia or calcium overloading ferred between species. Redox balance reflects a dynamic also leads to the translocation of CSE from the cytosol state in which oxidant and antioxidant levels are balanced. to mitochondria, where CSE uses approximately three-

Increased oxidative stress and/or decreased antioxidant fold higher concentrations of l‑cysteine to produce H2S capability under various pathological situations disrupt (REF. 33). In addition, whereas under normoxic conditions the redox balance and cause damage to molecules and CBS in hepatocytes is degraded inside mitochondria by 5 cells. The protective, antioxidant actions of H2S have been Lon protease , upon hypoxic stress Lon protease can- well established, particularly with respect to the vascula- not recognize the deoxygenated haem group in CBS, ture. In cultured vascular smooth muscle cells, oxidative and therefore CBS is not degraded. The accumulation stress that was induced by high levels of homocysteine of CBS protein in the mitochondrion leads to increased (REF. 5) or methylglyoxal was markedly reduced by NaHS at mitochondrial production of H2S . concentrations of 30–90 μM (REFS 73,74). Moreover, the It appears that different tissues handle changes in the accumulation of lipid peroxidation products (including partial pressure of oxygen differently by altering their

lipid hydroperoxide, the main contributor to the cytotoxic H2S production and oxidation, as well as by changing

effect both of oxidized low-density lipoprotein (LDL) and their responses to H2S. For example, H2S dilates systemic of lipid derived from atheroma) in human umbilical vein blood vessels82 and/or promotes angiogenesis to increase endothelial cells (HUVECs) was inhibited or prevented by blood supply to hypoxic tissues83. By contrast, in hypoxic

NaHS at 1–20 μM; endothelial cytotoxicity mediated rat lungs, H2S constricts vascular smooth muscle in order by hydrogen peroxide and oxidized LDL was similarly to achieve regional ventilation–perfusion matching, in a inhibited or prevented by NaHS at these concentrations75. mechanism known as hypoxic pulmonary vasoconstric- 84,85 Oxidative stress-mediated death of amyloid-β‑treated tion . In this way, H2S helps the diversion of blood from microglial cells was also inhibited by NaHS at concentra- oxygen-deprived areas to oxygen-supplied areas86. At the (REF. 76) tions as low as 25 μM . Although the physiological same time, H2S dilates airway smooth muscle to increase 36 relevance of the antioxidant effect of H2S at these concen- lung ventilation (in humans) and to reduce airway resist- 15 trations is unclear, the therapeutic value of H2S donors in ance (in mice) . These compensatory changes induced by

this regard is certainly apparent. H2S in response to hypoxia help improve the efficiency

In addition to its reducing nature, H2S influences of pulmonary gas exchange. Moreover, H2S mediates the the oxidant–antioxidant balance. Expression of the response of carotid-body chemoreceptors to hypoxia by 53 antioxidant enzyme thioredoxin 1 in vascular endothe- modulating BKCa channels . As far as bioenergy produc- (REF. 77) lial cells is upregulated by H2S , whereas the tion is concerned, under hypoxic conditions the upregu-

expression of NAD(P)H oxidase, a key source of extra- lated mitochondrial production of H2S — which can act as mitochondrial oxidant species, is downregulated in an electron donor in the mitochondrial respiratory chain (REF. 78) 79 osteoblastic cells that have been exposed to H2S . — contributes to the generation of bioenergy . Thus, in fac- The production of glutathione (GSH), a strong scavenger ing a hypoxic challenge, mitochondrial production of ATP 33,86,87 of free radicals, is also enhanced by H2S. It was shown can be maintained by hypoxia-increased H2S levels . that incubation of cultured mouse embryonic fibroblasts It should be mentioned that the notion of an ‘oxygen 88 with NaHS induces nuclear translocation of NRF2 by sensor’ role of H2S does not go without challenge . A S‑sulfhydrating KEAP1 at Cys151. As such, there was decrease in the partial pressure of oxygen in pulmonary

an increase in the binding of NRF2 to the antioxidant- circulation does not always lead to an increase in H2S

response element (ARE), and an increase in ARE- levels, and H2S does not always cause the constriction of mediated transcription of the genes involved in GSH pulmonary arteries. For instance, in hypoxic pulmonary

synthesis and maintenance. The consequent increase in hypertension, hypoxia and a reduction of H2S levels in 89 GSH levels contributes to the protective effect of H2S the pulmonary circulation occur in parallel . In another against cellular senescence induced by oxidative stress14. example, in contrast to the assumed vasoconstrictive effect

The mitochondrion is the primary source of oxy- of H2S on pulmonary arteries, one recent study showed that,

gen-derived free radicals and the organelle that is most under normoxic conditions, H2S actually dilated pre-con- affected by oxidative stress. Oxidative stress suppresses stricted human lobar pulmonary artery rings and reduced mitochondrial electron transport and bioenergetics, but pulmonary artery pressure90. It is not yet clear whether the (REF. 79) these effects can be reversed by exogenous H2S . It vasoactive effects of H2S on pulmonary circulation, and

was recently reported that nanomolar concentrations of the interaction of H2S with hypoxia, are species-specific.

a novel mitochondrion-targeted H2S donor, AP39, inhibited oxidative damage in microvascular endothelial cells H2S in disease processes 80 in vitro . GYY4137, a non-mitochondrion-targeted, slow- H2S levels contribute to homeostasis of the organism,

releasing H2S donor, produced protective effects similar to and abnormally increased or decreased endogenous

those produced by AP39, but only at a 1,000‑fold higher H2S production is associated with various diseases. concentration than that of AP39 (REF. 81). Among the most-studied diseases related to abnormal

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H2S metabolism are those involving the cardiovascular, induced hypertension and promoted abnormal laby- endocrine, gastrointestinal and nervous systems. For the rinthine vascularization in the placenta98. Women with

pathological roles of H2S in other mammalian systems, pre‑eclampsia exhibit decreased plasma levels of H2S readers are referred to other recent review articles2,5,91. and reduced expression of CSE in the placenta compared with gestational, age-matched controls98, and the

Hypertension and vascular remodelling. As an EDHF, H2S therapeutic value of H2S donors (NaHS or GYY4137) in induces the relaxation of many systemic blood vessels. pre‑eclampsia has been shown in mice98 and rats99. When it is produced in smooth muscle cells or other non-

endothelial cells, H2S also dilates blood vessels by directly Angiogenesis and atherosclerosis. H2S increases vascular relaxing smooth muscle cells12,92. These endothelium- endothelial cell proliferation and migration, micro

dependent and -independent vasorelaxant effects of H2S vessel formation and the healing of wounds and ulcers

are linked to the role of H2S in blood pressure regulation. both in vivo and in vitro. These pro-angiogenic effects 93 Since the early study by Zhao et al. in rats that showed of H2S are proposed to be mediated through the phos- that pharmacological inhibition of CSE elevated blood phorylation of AKT, extracellular signal-regulated kinase

pressure, the role of H2S in regulating blood pressure (ERK) and p38, as well as through the activation of KATP has become increasingly clear. Mice that are genetically channels18,83,92,100. The interaction of vascular endothe-

deficient in CSE (CSE‑knockout mice) exhibit an age- lial growth factor (VEGF) and H2S constitutes another dependent development of hypertension that is largely important pro-angiogenesis mechanism. Rats treated with

due to diminished endothelial production of H2S and NaHS (50 μmol per kg, twice daily) showed increased free endothelium-dependent relaxation of peripheral resist- plasma levels of VEGF and upregulated renal expression ance arteries82. Similarly to CSE‑knockout mice, sponta- of Veg fa mRNA. In vitro incubation of podocytes with neously hypertensive rats (SHRs) exhibit age-dependent NaHS resulted in VEGF release and upregulation of Veg fa development of hypertension that correlates with dimin- mRNA levels99. In turn, VEGF stimulates the release of 83 ished CSE expression and H2S production in aortic tis- H2S from vascular endothelial cells . Thus, H2S and sues82,94. The treatment of CSE‑knockout mice with NaHS VEGF individually and synergistically stimulate angio-

at 3.9 μmol per kg, 19.5 μmol per kg, and 39 μmol per kg genesis. Similarly, the interaction between H2S and NO (intravenous administration) or of SHRs with NaHS at can affect angiogenesis60,66,100. NaHS was shown to pro- 56 μmol per kg (intraperitoneal (i.p.) administration) sup- mote the phosphorylation of endothelial NO synthase in pressed the development of hypertension, decreased vas- cultured HUVECs, leading to increased production of cular damage and prevented vascular remodelling82,94,95. NO, which further contributed to endothelial cell prolifer 100 Altered H2S metabolism has been linked to pulmonary ation and tube formation . hypertension. After high pulmonary blood flow-induced Atherosclerosis is a chronic circulatory disease that is pulmonary hypertension was established in rats after an characterized by the build-up of fatty or high-cholesterol abdominal aorta–inferior cava vein shunt operation, the plaques on the inner surface of large- to medium-sized

plasma level of H2S and CSE mRNA levels in lung tissues blood vessels. The pathogenesis of atherosclerosis were significantly lower than in normotensive control is not fully understood, but recent studies have indi- 96 rats . In another rat model of hypoxia-induced pulmonary cated that altered H2S metabolism is involved in both hypertension, the animals were exposed to normobaric the initiation and progression of this vascular disorder. hypoxia (10% oxygen) in a transparent plastic hypoxia Atherosclerosis in apolipoprotein E (APOE)-knockout

chamber for 3 weeks. In these hypertensive rats, H2S pro- mice is accompanied by decreased H2S levels in the duction and CSE expression in lung tissues decreased sig- blood, and treatment of these mice with NaHS was nificantly compared with that in control rats89,97. In rats shown to attenuate the thickening and stiffening of arte- with hypoxic pulmonary hypertension, daily treatment rial vessels101. Mani et al.102 showed that CSE‑knockout with NaHS at 14 μmol per kg (i.p. administration) provided mice that were fed an atherogenic paigen-type diet for anti-hypertensive protection in these animals and reduced 12 weeks developed early fatty-streak lesions in the the extent of remodelling of pulmonary arteries89,97. aortic root, increased oxidative stress and expression In the two-kidney-one-clip model of renovascular hyper- of adhesion molecules, and enhanced aortic intimal tension, upregulated renin expression and increased proliferation. By contrast, wide-type mice fed the same activity of plasma renin and angiotensin II are markers atherogenic diet, or CSE‑knockout mice fed a nor- of the severity of the disorder. As reflected by these mal diet, did not develop any atherosclerotic damage.

measures, systemic hypertension was either prevented The anti-atherosclerotic effects of H2S are manifested or attenuated by daily administration of NaHS (at doses through: the inhibition of neointimal hyperplasia and of 30–100 μmol per kg) to rats in this model62. smooth muscle proliferation; a decrease in levels of oxi-

The role of H2S in gestational hypertension has also dized LDLs, vascular calcification and vascular inflam- been investigated. Hypertension is one of the major mation; and a suppression of the adhesion of monocytes characteristics of pre-eclampsia — a disorder that affects to endothelial cells102,103. Two-kidney-one-clip model women during pregnancy, but that has no clear aetiology. An enhanced understanding of the anti-atherosclerotic A model of hypertension Endogenous H S is required for healthy placental vas- role of H S has helped advance H S-based therapies induced by chronically 2 2 2 constricting one renal artery culature, whereas decreased CSE or H2S activity may targeting atherosclerosis. In APOE-knockout mice, treat- while the other renal artery contribute to the pathogenesis of pre‑eclampsia. In preg- ment with NaHS (56 μmol per kg per day, i.p. admin- remains fully perfused. nant mice, the inhibition of CSE with propargylglycine istration) or GYY4137 (133 μmol per kg per day, i.p.

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administration) reduced the atherosclerotic plaque Diabetes. Diabetes mellitus is caused by decreased load and partially restored acetylcholine-induced endo bioavailability of insulin from the pancreas and/or dimin- 101,103 thelium-dependent relaxation of the aorta . H2S ished insulin sensitivity of the peripheral tissues. Altered

donors also suppressed the expression of intercellular metabolism of H2S in the pancreas and in peripheral adhesion molecule 1 (ICAM1), tumour necrosis factor tissues is involved in both the pathogenesis of diabetes (TNF) and interleukin‑6 (IL‑6) in the aorta; the expres- and its complications (FIG. 2). Overexpression of CSE and

sion of CX3C‑chemokine receptor 1 (CX3CR1) and the consequential overproduction of H2S in pancreatic CX3C‑chemokine ligand 1 (CX3CL1) in macrophages β-cells, as detected in genetic diabetic rat models (Zucker and lesion plaques; and the generation of superoxides diabetic rats), constitute pathogenic factors for diabetes114. in the aorta of these animals101,103,104. Moreover, in these Loss of functional β-cell mass is directly responsible for mice, early treatment with NaHS at a dose of 1 mg per kg the pathogenesis of type 1 diabetes and progression of 115 per day (i.p. administration) generated better therapeutic type 2 diabetes . Although a β-cell-killing effect of H2S benefit against atherosclerotic damage than did NaHS after has not been directly demonstrated in vivo, cultured atherosclerosis had fully developed104. insulin-secreting INS‑1E cells undergo increased apop

tosis in response to H2S — a finding that is certainly 116 Lipid metabolism disorders and liver diseases. The indicative of the potential impact of H2S on β-cell mass .

liver has essential roles in lipid metabolism, and hepatic Also relevant to pancreatic insulin release is the H2S-

production of H2S plays a key part in managing lipid induced stimulation of KATP channels in β-cells, and their metabolism. Indeed, genetic deficiencies in either CBS subsequent membrane hyperpolarization35,114. In addition, hyperhomocysteinaemia or CSE result in . CBS‑knockout H2S has been shown to inhibit L‑type VDCCs in mouse 44 mice that are fed normal chow exhibit abnormal lipid pancreatic β-cells . H2S-induced membrane hyper- metabolism, with increased serum and hepatic levels polarization and inhibition of VDCCs synergistically of triglycerides and non-essential fatty acids, as well decrease calcium entry and insulin release from β-cells44. 105,106 as spontaneous hepatic fibrosis and steatosis . Following the consideration of the negative effects of H2S Nevertheless, liver morphology and function seem on pancreatic insulin production and insulin release, a 107 to be normal in chow-fed CSE‑knockout mice . The causative role of pancreatic H2S in diabetes was proposed impact of CSE deficiency on lipid metabolism becomes in 2004 (REFS 114,117). Zucker diabetic fatty rats have a detectable only after these mice are fed a high-fat athero type 2 diabetic phenotype. Pancreatic β-cell expression

genic diet: with such an atherogenic feeding regime, of the gene encoding CSE and β-cell production of H2S they exhibit increased plasma levels of total and LDL were both found to be significantly higher in Zucker cholesterol, decreased high-density lipoprotein (HDL) diabetic fatty rats than in non-diabetic Zucker lean or cholesterol and early development of atherosclerosis102. non-diabetic Zucker fatty rats114,117. Patients with type 1

Strikingly, in humans, plasma H2S levels are positively or type 2 diabetes (but without nephropathy) had signifi- correlated with HDL cholesterol, and negatively correlated cantly enhanced activities of CSE and CBS compared with with the ratio of LDL to HDL cholesterol108. healthy controls118,119. Further study of Zucker diabetic Downregulation of CSE expression and decreased fatty rats has shown that inhibition of CSE activity signifi-

endogenous H2S levels in the liver are common conse- cantly decreased the production of H2S, increased plasma quences observed in bile duct-ligated rats with cirrhosis insulin levels, and lowered hyperglycaemia114. Diabetes and associated portal hypertension109 or in rats with induced by streptozotocin was much slower to develop in carbon tetrachloride-induced liver cirrhosis110. Several CSE‑knockout mice than in wild-type mice120. Moreover,

studies have shown the beneficial effects of H2S supple- streptozotocin induced a higher amount of pancreatic H2S mentation therapy in these models of liver diseases109–111. production and killed more β-cells in wild-type mice than 120 However, there are circumstances of elevated H2S produc- in CSE‑knockout mice . Therefore, at least some of the

tion in which treatment with H2S donors can be detri- diabetes-inducing effects of streptozotocin are mediated

mental. For example, when mice with septic shock were by CSE and/or H2S, and an increased level of pancreatic

treated with NaHS at a dose of 14 μmol per kg (i.p. admin- H2S is a causative factor for diabetes development. istration) this increased liver damage112, whereas phar- What happens in the pancreas in terms of the metabo Hyperhomocysteinaemia macological inhibition of CSE with dl‑propargylglycine lism and functions of H2S is quite different to what A condition characterized by (50 mg per kg, i.p. administration) protected these animals happens in other parts of the body. Once diabetes is abnormally high levels of homocysteine in the blood. from liver damage. In rat endotoxaemia, portal infusion of established, various diabetic complications can develop, The major causes of this sodium sulfide (Na2S) through a splenic cannula at a rate including cardiomyopathy, nephropathy and vascular condition are deficiencies of of 2 μmol per kg per minute for 10 minutes caused hepatic disorders associated with endothelial dysfunction. vitamins B6, B9 and B12, vasoconstriction and increased portal pressure in vivo113. These complications are closely related to systemic H S and mutations in the gene 2 encoding the enzyme Conversely, treatment of the rats with dl‑propargylglycine deficiency, as hyperglycaemic cells oxidize and consume (REFS 121,122) 5‑methyltetrahydrofolate. (50 mg per kg, i.p. administration) significantly attenuated more H2S . Overexpression of CSE or

the vasoconstrictive effect of endothelin 1 in endotoxin- treatment with H2S prevented dysfunction of cultured Streptozotocin treated animals. The proposed underlying mechanism for microvascular endothelial cells that was induced by high A chemical commonly used to the detrimental effects of H S in these situations is that the levels of glucose; improved hyperglycaemia-impaired induce diabetes in laboratory 2 animals, as it is toxic to the perturbed hepatic sinusoidal perfusion during sepsis is endothelium-dependent aortic vascular relaxations 123 insulin-producing β-cells in further decreased by H2S supplementation, thus leading in vitro and ex vivo ; and accelerated wound healing the pancreas. to exacerbated tissue hypoxia113. by restoring the functions of endothelial progenitor

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a Diabetes onset b Diabetic complications

↑H2S ↑H2S in ↓H2S in in liver pancreas peripheral tissues

Gluconeogenesis Glycogenolysis Insulin Insulin Endothelial Nephropathy production release dysfunction

Glucose utilization

Cardiomyopathy Neuropathy ↑Glucose levels ↓Insulin levels

Figure 2 | The pathogenic roles of H2S at different stages of diabetes development. Elevated endogenous hydrogen sulfide (H S) levels in pancreatic -cells and hepatocytes have crucial roles in the onset of diabetes. At late stages of 2 β Nature Reviews | Drug Discovery the disease, endogenous H2S levels are lower in the affected organs and tissues, such as in vascular endothelial cells or

cardiomyocytes, contributing to the development of diabetic complications in these organs. Correspondingly, H2S-based therapy for diabetes should be staged in two phases. At the onset phase of diabetes (part a), selective inhibition of the

endogenous production of H2S in pancreatic β-cells and the liver would both increase insulin availability and decrease

glucose production. By contrast, at later stages (part b), selective delivery of H2S donors to the peripheral organs would be beneficial for preventing and treating diabetic complications.

cells and the activation of angiopoietin 1 signalling124. (HD), an autosomal-dominant disease associated with Notably, in rats with streptozotocin-induced diabetes, a mutation in the gene encoding huntingtin21. In this NaHS treatment at 100 μmol per kg per day (i.p. admin- study, CSE deficiency was found in brain tissues (the istration) also improved cardiac function and reversed striatum and cerebral cortex) but not in the cerebel- the diabetes-related cardiac morphological changes by lum of patients with HD, keeping in line with the rela- protecting cardiomyocytes against oxidative damage tive susceptibility of these brain regions to HD damage. and preserving mitochondrial functions125. Furthermore, in murine models of HD (Q175 mice and R6/2 HD mice), CSE expression was downregulated

Neurodegenerative diseases. Brain levels of H2S in indi- in the striatum, cortex, hippocampus, hypothalamus viduals with Alzheimer disease are lower than in age- and brainstem, but not in the cerebellum. Interestingly, matched healthy people, although the expression levels CSE‑knockout mice display impaired rotarod perfor- of CBS between these groups are not different126. As mance and an abnormal hindlimb clasping and clenching Alzheimer disease is associated with reduced produc- phenotype that is reminiscent of murine models of HD.

tion of H2S, there may be an associated decrease in neu- These HD‑related phenotypic changes were reversed by ronal cytoprotection, such that the deleterious effects of exogenously supplied cysteine21. damage and neuroinflammation induced by amyloid‑β In traumatic spinal cord injury, neuronal damage is and oxidative stress are increased127–129. Whether the low initially caused by the trauma itself, but considerable

levels of H2S in the brain observed in Alzheimer disease additional damage is caused by the ensuing inflamma- are a cause or a consequence of the disorder is not clear. tory reaction. In a mouse model of spinal cord injury, In a rat model of ischaemic vascular dementia, plasma Campolo et al.133 demonstrated the potential of using

H2S levels were lower and inversely correlated with the H2S to decrease the inflammatory component of the decrease in the number of viable neurons in the hippo injury, and to accelerate the recovery of lost motor campus. Intraperitoneal injection with NaHS (14 μmol function. Post-trauma treatment with a nonsteroidal per kg) markedly protected against neuronal injury and anti-inflammatory drug (NSAID), naproxen, enhanced improved the performance of learning and memory, recovery of lost motor function in the mice, and tested by the Morris water maze, in these animals130. decreased several indices of spinal cord inflammation.

Parkinson disease is another neurodegenerative dis- However, in mice that were treated with an H2S-releasing

ease in which the metabolism of H2S may be involved. derivative of naproxen (ATB‑346), there was a marked

In a mouse model of Parkinson disease, H2S levels in the acceleration in the recovery of lost motor function and substantia nigra and striatum were significantly lower further enhancement of anti-inflammatory effects. 131 131 than in control mice . H2S that was given via injection 132 or inhalation impeded or prevented Parkinson-disease- Gastrointestinal disorders. H2S, including that produced like abnormalities, including movement dysfunction and by enteric bacteria (BOX 2), has been implicated as a microglial activation. mediator of several physiological functions in the diges- Unexpectedly — given the dominant expression of tive tract. There is emerging evidence of the utility of

CBS in the brain — a recent study revealed the impor- H2S donors in treating several gastrointestinal disorders, tance of CSE for the manifestation of Huntington disease particularly those associated with inflammation (FIG. 3).

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | MAY 2015 | 337

For example, abdominal pain is a common but poorly mediator of gastric mucosal defence; that is, the ability treated condition, and chronic abdominal pain is often of the gastric mucosa to resist injury induced by endog- described as irritable bowel syndrome. enous and exogenous substances139,140. Pharmacological

The role of H2S in modulating visceral pain is contro- inhibition of H2S synthesis increases the susceptibility of

versial: conflicting data suggest that it is both pro- and the stomach to injury, whereas H2S donors (such as NaHS anti-nociceptive39,91,134–137. However, these differences may or diallyl disulfide) can protect the stomach from dam- be related to the models used and the types and doses of age139–141. The underlying mechanism of the cytoprotec- 91 H2S donors that were used . Several studies have sug- tive action of H2S probably involves its ability to inhibit 142 gested potent anti-nociceptive effects of H2S donors in leukocyte adherence to the vascular endothelium rodent models of visceral pain39,91,134,136. For example, — a key event in the pathogenesis of NSAID-induced 143 colonic distention-induced pain in rats was substantially gastric mucosal damage . H2S can also trigger gastric

attentuated by several H2S donors, the actions of which and duodenal secretion of bicarbonate, which neutral- were mediated, at least in part, through the activation of izes excess mucosa-damaging acid144,145. Moreover, 39 KATP channels . Similarly, gastric distention-induced pain H2S increases gastric mucosal blood flow, enhancing 139 in rats was markedly reduced by H2S donors and exacer- mucosal resistance to injury . H2S-releasing derivatives 91 bated by an inhibitor of CSE activity . An H2S-releasing of NSAIDs have been shown to produce markedly less salt of trimebutine (an opioid anti-spasmotic drug) was gastrointestinal damage than their corresponding parent shown to be safe and well tolerated in a Phase I clini- NSAIDs146–151. True gastric ulcers, which penetrate into cal trial (ClinicalTrials.gov identifier: NCT01738425)138, the submucosal layer of the stomach wall, take many days and is now being tested as an abdominal analgesic in or weeks to heal, and clinically the healing of such ulcers Phase II clinical trials (ClinicalTrials.gov identifiers: can be accelerated to some extent by drugs that suppress NCT01926444 and NCT02276768). gastric acid secretion. This healing is partially dependent

Acute damage to the lining of the stomach can be upon H2S, the synthesis of which is increased at the induced by certain drugs (such as NSAIDs), stress and margins of the ulcer, where there is increased expression 152 gastric ischaemia–reperfusion. H2S is an important of CSE and CBS . Oral administration of l‑cysteine

or H2S donors (in this case, Lawesson’s reagent or 4‑hydroxythiobenzamide) to rats with gastric ulcers 152 Box 2 | H2S and bacterial–epithelial signalling resulted in a significant acceleration of ulcer healing . Whereas NSAIDs are known to retard the healing of gas- The greatest source of hydrogen sulfide (H2S) synthesis ‘within’ the human body may actually be an external source: the microbiota. In addition to contributing to the pool of tric ulcers in humans and animals, H2S-releasing NSAIDs 182,183 have been shown to accelerate ulcer healing in mice148. H2S, polysulfides and bound sulfane-sulfur , bacteria-derived H2S has the capacity to influence many physiological and pathophysiological processes, particularly in Treatment with the H2S donor NaHS has also been shown

the gastrointestinal tract. Bacteria-derived H2S may contribute to several aspects of to significantly reduce the severity of reflux oesophagitis mucosal defence and repair, and certainly to the bioenergetics of gastrointestinal in rats, and inhibition of CSE in these animals led to the epithelial cells184. Conversely, H S donors appear to be able to influence the 153 2 exacerbation of tissue injury . 155,185 microbiota — an effect that might be exploited in drug design to promote health. NSAIDs also induce notable ulceration and bleeding in Sulfate-reducing bacteria (SRBs) that produce H S reside in the gastrointestinal 2 the small intestine, and there are no preventive or curative tract, where there are also sulfate-consuming bacteria. In a study of healthy individuals in the United States, approximately 50% of those studied had their gut colonized by treatments available that have been proven to be effective 149,154 SRBs, with Desulfovibrio piger — a member of the class of δ‑Proteobacteria — being for this potentially lethal condition . Treatment with 186 187 the H S donor diallyl disulfide can substantially reduce the primary H2S producer . Bacteria can also make polysulfides . Just as it is now 2 NSAID-induced enteropathy in rats155, and H S-releasing clear that circulating levels of H2S in mammals were markedly over-estimated in the 2 188 past , the concentrations of H2S produced in the lumen of the gut were grossly NSAIDs induce negligible damage to the small intestine

overestimated for decades, contributing to the notion that H2S was a primary driver of — even when such NSAIDs are co‑administered with colonic inflammation and cancer189–191. At present, the balance of evidence drugs such as aspirin and proton-pump inhibitors, which suggests that H S is an important metabolic fuel for the epithelial cells that line the 2 can markedly exacerbate NSAID-induced intestinal gastrointestinal tract and that seem to be particularly well adapted to this purpose, damage and bleeding149. perhaps because of the hypoxic environment in which they reside86,184. The epithelium H S appears to have a particularly important role can therefore function not only as a physical barrier against potentially harmful agents 2 that might pass from the lumen of the gut into the body, but also as a metabolic barrier, as an anti-inflammatory, pro-healing molecule in the oxidizing bacteria-derived H S and, in doing so, generating ATP151. Bacterial H S can colon. In experimental colitis in rats, the local synthesis 2 2 18 also stimulate the proliferation of gastrointestinal epithelial cells, and may contribute of H2S is dramatically upregulated at the sites of ulcera- 192 156 to the repair of any damage to the epithelial layer . tion . In this model, H2S promotes the resolution of 18,156 Conversely, the microbiota can be the target of actions of H2S. Studies in rodents have colonic inflammation and the healing of ulcers . demonstrated that intestinal microbiota in the colon form linear biofilms that appear to Inhibition of H2S synthesis in healthy rats led to gastro promote harmonious coexistence of the bacteria with the gastrointestinal mucosa185. intestinal mucosal inflammation, reduced expression When the mucosa is inflamed, the production of mucus is decreased and microbiota of cyclooxygenase 2 and reduced synthesis of prosta- biofilms become fragmented. Delivery of H S into the colon promoted the resolution of 2 glandin in the mucosa18,91. Administration of the H S inflammation, increased production of mucus, and restored normal biofilm structure, 2 donors Lawesson’s reagent, NaHS or diallyl trisulfide to and this biofilm restoration was accompanied by reduced growth of planktonic 185 rats or mice with colitis resulted in faster resolution of bacteria . In another study, administration of an H2S donor to rats was shown to cause profound shifts in the microbiota, correcting the detrimental dysbiosis that had been inflammation and colonic tissue injury healing, as well triggered by the chronic administration of a nonsteroidal anti-inflammatory drug155. as a downregulation of pro-inflammatory cytokine and chemokine expression relative to control animals18,157.

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Rolling NRF2–KEAP1 Adhesion H S pathway 2 Resolution of inﬂammation

H2S

NRF2 KEAP1 • Inhibits leukocyte– M1 M2 endothelial cell adhesion • Reduces oedema formation Dissociation NRF2 ↓NF-κB activation Pro-inﬂammatory Repair from KEAP1 cytokines

Transcriptionally Gene H2S NF-κB NRF2 MAF H2S active complex transcription mRNA ARE

PDE

Antioxidant e e e e e e e Vasodilation H2S Neutrophil apoptosis e e e e e e e e e ATP generation ATP Analgesic

Figure 3 | Anti-inflammatory and cytoprotective targets of H2S. Hydrogen sulfide (H2S) is thought to act through several pathways, some of which are illustrated above, to reduce inflammation and protect tissues from injury (such as Nature Reviews | Drug Discovery ulceration in the gastrointestinal tract). H2S can suppress leukocyte adherence to the vascular endothelium, leukocyte extravasation and consequent formation of oedema. It can substitute for oxygen in driving mitochondrial respiration,

thereby attenuating oxidative-stress-related tissue injury. The ability of H2S to inhibit the activity of phosphodiesterases (PDEs) can contribute to its ability to relax vascular smooth muscle, resulting in enhanced blood flow. Resolution of

inflammation can be enhanced by H2S through actions such as the promotion of neutrophil apoptosis, and driving

macrophage differentiation towards the M2 (anti-inflammatory) phenotype. H2S can modulate the activity of a number of transcription factors: it inhibits nuclear factor‑κB (NF‑κB), leading to a reduced production of pro-inflammatory cytokines, and sulfhydrates Kelch-like ECH-associated protein 1 (KEAP1), which then releases active nuclear factor erythroid 2‑related factor 2 (NRF2), resulting in increased expression of antioxidant-response elements (AREs).

Increased production of H2S occurs around sites of damage, such as around ulcers in the gastrointestinal tract, and can accelerate the healing of such damage via the stimulation of angiogenesis. Anti-nociceptive effects of

H2S have also been demonstrated. Adapted from REF. 193, American Physiological Society.

Impaired colonic H2S synthesis, as observed in rats Conversely, an upregulation of the expression of CBS,

with hyperhomocysteinaemia, was associated with and the accompanying increase in H2S production, have marked exacerbation of colitis, which could be reversed been suggested to play a key part in colonic tumour 158 26 by administration of the H2S donor diallyl disulfide . growth, in part by driving angiogenesis . IL‑10‑deficient mice, which spontaneously develop

colitis, exhibited a similar defect in colonic H2S produc- Human genetic diseases linked to H2S-generating enzymes. tion that could be reversed by administration of IL‑10 Mutations in the human CSE gene can cause heredi- (REF. 158). Thus, there is a substantial body of evidence tary cystathioninuria and hypercystathioninaemia159,160. suggesting that the anti-inflammatory or pro-resolution Similarly, inborn errors in the CBS gene are associated

effects of H2S are mediated to a large extent via downreg- with the human hereditary diseases hyperhomocystein ulation of the expression of a range of pro-inflammatory aemia and homocystinuria161. As well as causing higher cytokines (for example, IL‑1β, TNF, interferon-γ (IFNγ), levels of cystathionine or homocysteine in the blood IL‑12 and IL‑23), although the expression of IL‑10 is and urine, these diseases are associated with systemic either spared or increased147,157,158. inflammation, cardiovascular complications and dam- As discussed further below, several studies have age to other organs. Conversely, overexpression of the demonstrated notable chemopreventive effects of CBS gene is a major cause of human Down syndrome,

H2S-releasing drugs in animal models of colon cancer. which is characterized by low levels of homocysteine in

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | MAY 2015 | 339

the blood162. In addition, intronic polymorphisms and NSAID linked to both a NO‑releasing moiety and an

nonsense mutations in MST have been suggested to be H2S-releasing moiety, is the treatment and chemopreven- responsible for a rare inheritable disorder known as tion of cancer. As well as being effective in in vitro and ani- mercaptolactate-cysteine disulfiduria163. mal models of various cancers, these compounds exhibit significantly reduced adverse gastrointestinal effects168,169. (TABLE 1) H2S-based therapeutics The lead drug of this group is NBS‑1120 . An appreciation of the physiological and pathological GIcare Pharma is attempting to exploit the reported 39,134,136 importance of H2S has been followed very quickly by ability of H2S to reduce visceral pain . Its lead attempts to develop novel therapeutics that aimed to drug, GIC‑1001, is a salt — the counter-ions being

deliver H2S, or to suppress its endogenous production thiobenzamide and trimebutine. Trimebutine is an (TABLE 1). Furthermore, the impetus for the founding opioid anti-spasmodic that has been used to treat a

of Ikaria Therapeutics was the finding that H2S given range of gastrointestinal conditions, including irritable via inhalation could induce a state of ‘suspended ani- bowel syndrome, for over 40 years. Shown to be safe mation’ in mice164 by markedly reducing the meta- in a Phase I clinical trial (ClinicalTrials.gov identifier:

bolic rate. This company attempted to develop Na2S NCT01738425), this drug is now in Phase II clinical trials for critical-care applications, such as for the reduction as a pre-colonoscopy analgesic (ClinicalTrials.gov identi- of myocardial injury following infarction165. However, fier: NCT01926444 and NCT02276768). The rationale in 2011, two clinical trials (ClinicalTrials.gov identi- for this is that sedation during endoscopy is a routine fiers: NCT01007461 and NCT00858936) were halted, practice, but can also be expensive and inconvenient. and Ikaria Therapeutics does not appear to be pursuing The use of GIC‑1001 as pre-colonoscopy analgesic is

H2S-based therapeutics at present. proposed to reduce or even remove the need for sedation, Over the past decade, several other companies that possibly allowing the patient to leave hospital sooner, and focus on exploiting the potent anti-inflammatory and translating to considerable savings for both the patient (TABLE 1) cytoprotective actions of H2S have been founded . and the health-care provider. Trimebutine has been used Antibe Therapeutics, CTG Pharma and Sulfhydris have as a treatment for irritable bowel syndrome170, raising the

all developed H2S-releasing derivatives of a number of possibility that the use of GIC‑1001 may be extended to drugs, with a major focus on NSAIDs148–150,157. The main that indication in the future.

targets for these drugs are pain and inflammation, with Several H2S-based therapeutics target disorders that

the primary benefit of H2S release being the reduction are characterized by oxidative stress and associated tis- in the gastrointestinal ulceration that is normally caused sue injury. For example, largely on the basis of research by NSAIDs. (However, CTG Pharma and Sulfhydris no by Elrod and colleagues171, SulfaGENIX (in New longer appear to be active.) Orleans, Louisiana, USA) is developing zerovalent sul- The lead drug of Antibe Therapeutics (ATB‑346; a fur (SG‑1002) as a medicinal food, with the initial aim of naproxen derivative; TABLE 1) is being developed for the targeting heart failure. Preclinical studies in relevant ani- treatment of osteoarthritis, and has completed Phase I mal models of heart failure have confirmed that SG‑1002 clinical trials in healthy volunteers (see the Antibe is effective in decreasing infarct size, improving cardiac Therapeutics press release for further information). function, increasing angiogenesis, decreasing inflamma- In animal studies of disorders such as adjuvant-induced tion and downregulating oxidative stress after infarction. arthritis, the drug produced anti-inflammatory effects A Phase I trial of SG‑1002 (ClinicalTrials.gov identi- that were comparable to those achieved with equimolar fier: NCT02278276) evaluated doses of 200 mg, 400 mg doses of the parent drug148,149. However, unlike the par- and 800 mg per day in healthy volunteers and reported

ent drug, ATB‑346 produced negligible damage in the dose-dependent increases in plasma H2S levels, and only gastrointestinal tract, even at very high doses and in ani- minor adverse effects. A second study has been designed mals with impaired mucosal defence148,149,166. Naproxen (ClinicalTrials.gov identifier: NCT01989208) to evalu-

was selected as the base NSAID, because it is the only ate the ability of SG‑1002 to elevate plasma H2S levels

member of the NSAID class that does not significantly (in effect, reversing the defects in circulating H2S) and increase the risk of serious cardiovascular events such as to reduce markers of oxidative stress and heart failure. myocardial infarction and stroke167. In a Phase I clinical Reducing oxidative stress is also the foundation trial, ATB‑346 administration to healthy subjects in of a series of novel compounds developed by Wood, doses escalating from 25 mg to 2,000 mg did not pro- Whiteman and colleagues at the University of Exeter,

duce any notable adverse events, or any irregular cardio UK. Their compounds comprise an H2S-releasing group vascular, renal, haematological or hepatic effects. Antibe linked to a mitochondrion-targeting group. Mitochondria Therapeutics has additional drugs in preclinical devel- have a critical role in determining whether a cell sur- 81 opment for treating acute pain (such as that associated vives or dies and, as discussed, H2S can confer benefits with gout or sports injuries) and for veterinary pain and to mitochondria by acting as an electron donor and inflammation, as well as a novel anti-thrombotic drug that downregulating the antioxidant-response pathway. The exhibits greatly increased gastrointestinal safety (TABLE 1). University of Exeter has patented (WO2013045951 A1)

H2S-releasing NSAID derivatives are also the principal such H2S-releasing compounds for the treatment of focus of drug development efforts by a group at the humans, animals or plants, and the authors propose that City University of New York, USA. However, the main the compounds would be useful for the treatment of dis- therapeutic application for these drugs, which have an orders such as hypertension and haemorrhagic shock,

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Table 1 | H2S-based therapeutics in development Institution Structure Clinical Lead Comment Stage of (location) indications drug development

Antibe S Osteoarthritis ATB‑346 Naproxen Phase I Therapeutics derivative H3CO (Toronto, O NH2 Ontario, Acute pain ATB‑352 Ketoprofen Preclinical derivative Canada) O Veterinary ATB‑338 Diclofenac Preclinical ATB-346 (pain) derivative Thrombosis ATB‑350 Aspirin Preclinical derivative

City University S Cancer NBS‑1120 Aspirin Preclinical of New York derivative (New York, USA) S O S

ONO2 O

GIcare Pharma O Colonic pain GIC‑1001 Trimebutine Phase II for (Montreal, salt; licensed analgesia H CO Quebec, 3 O from Antibe during Canada) +NH Therapeutics colonoscopy* H3CO H3C CH3 O– H3CO O

NH2

S National Hypertension, GYY4137 Slow-releasing Unknown S H University of + inflammation, H S donor – N H 2 Singapore O P S cancer O (Singapore) N

O Sova No structure available Pain, metabolic Unknown Inhibitor of CSE Unknown Pharmaceuticals disorders activity (La Jolla, California, USA

SulfaGENIX S Oxidative SG‑1002 Polyvalent sulfur Phase II for S S ‡ (New Orleans, S S S stress heart failure Louisiana, USA) S S

University of S Inflammation, AP39 Mitochondrion- Preclinical Exeter (Exeter, S oxidative stress targeted H S S 2 UK) O release P+ O

Br–

‡ CSE, cystathionine γ‑lyase; H2S, hydrogen sulfide. *ClinicalTrials.gov identifiers: NCT01926444 and NCT02276768. ClinicalTrials.gov identifier: NCT01989208.

as well as conditions characterized by inflammation the H2S donor GYY4137, which is now widely used as a 80 and oedema . AP39 is the most advanced of these com- research tool to study the effects of H2S. Compared with (TABLE 1) pounds . It has been shown to elevate H2S levels conventional H2S donors, GYY4137 releases H2S more within endothelial mitochondria, protect cells against slowly. It is not clear whether this compound is in devel-

oxidant-induced damage and prevent damage to mito- opment or whether it is just a prototype for an H2S-based chondrial DNA in vitro80. therapeutic. GYY4137 has been shown to exert anti- Another university-based drug development pro- hypertensive actions in SHRs172, and to reduce inflam-

gramme with an H2S focus is that of Moore et al. at the mation through its ability to reduce circulating levels of National University of Singapore. This group developed various pro-inflammatory cytokines and mediators173.

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | MAY 2015 | 341

In this regard, GYY4137 has been shown to be an effective to elicit marked anti-inflammatory effects in rodents142,

anti-inflammatory agent in a murine adjuvant-induced although roles for H2S in mediating its above-mentioned arthritis model174. The current status of the development clinical effects have not yet been established.

of an H2S donor by this group is unclear, but a patent is Future directions held for the use of a slow-releasing H2S donor for the

treatment of cancer (WO2014018569 A1). Given the ubiquitous nature of H2S, it is not surprising Sova Pharmaceuticals is a company that was that it has important roles in a wide range of physiologi- co‑founded by Snyder, and is developing inhibitors of cal and pathophysiological processes. The speed with

CSE for respiratory and metabolic disorders, as well as which many H2S-based therapies have been developed for diseases characterized by pain and inflammation, is a reflection of the excitement that this unique media- such as osteoarthritis and rheumatoid arthritis. The tor has ignited and of the promising data that have been premise for this programme is that overproduction of generated in preclinical and early clinical testing. The

H2S contributes to the pathogenesis of several diseases novel therapeutics that have been developed thus far — in particular, neuropathic and neurodegenerative dis- offer the promise of increased efficacy, reduced toxicity, eases — as well as to inflammation and inflammatory or both, compared with existing therapies. They range pain. The current status of drug development by Sova from very simple (for instance, zerovalent sulfur) to quite

Pharmaceuticals is unclear; however, the company filed sophisticated approaches (such as H2S release targeted to a patent for CSE inhibitors in July 2013. specific organelles). There are also several therapeutics on the market that In the near future, it is likely that organ-specific delivery

were not developed specifically as H2S-releasing drugs, of H2S will be achieved, and with that, the possibility of

but that could contribute to beneficial effects by releasing disease-specific H2S donors. It is also likely that the devel-

H2S. For example, anethole trithione (sold under a num- opment of pH-, oxygen-, and free radical-sensitive donors

ber of brand names, including Sialor (Pendopharm) and will further facilitate the selective delivery of H2S. Agents

Sulfarlem (EurekaSante)) is a drug that has been used for that could selectively activate the different H2S-generating decades for treating dry mouth. Anethole trithione can enzymes (namely, CSE, CBS and MST) are another

generate H2S, and is the same moiety that has been incor- intriguing possibility. Progress in the H2S field has so far

porated into H2S-releasing drugs (for example, NBS‑1120 been hampered by the lack of availability of selective inhib- and AP39 in TABLE 1) for preventing cancers, reducing itors of the various enzymes that contribute to the synthesis oxidative stress and attenuating inflammation. Whether of this gasotransmitter. With enzymes such as CSE being

or not H2S contributes to the desired clinical effects of identified as potential therapeutic targets, there will be anethole trithione is not clear. Like anethole trithione, increased motivation to develop potent and highly selec- oltipraz is a member of the dithiolethione class, and is tive inhibitors that will hopefully be of value in research as used as a schistosomicide; however, it is not clear whether well as for diagnostic and therapeutic applications.

H2S release contributes to its mechanism of action. As drugs that release H2S or modulate its synthesis Zofenopril (Menarini) is an inhibitor of angiotensin- progress through the development path towards wide- converting enzyme (ACE), and Bucci et al.175 demonstr spread clinical use, there will be a growing need to better

ated that a large component of the anti-hypertensive understand the mechanisms of action of H2S, and to

effects of this drug occur independently of ACE inhibi- be able to accurately monitor levels of H2S in vivo. In

tion, and are instead attributable to the H2S released by addition to characterizing the effects of H2S in various this drug. N‑acetylcysteine is a mucolytic drug used in tissues and organs, attention will need to be paid to the

the treatment of cystic and pulmonary fibrosis and also potential impact that modulation of H2S levels has on the as an antidote for acetaminophen-induced liver damage. microbiota (BOX 2), and in turn, the influence of any such

This drug can also generate H2S, and has been shown changes on health and disease.

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