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. NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | MAY 2015 | 329 © 2015 Macmillan Publishers Limited. All rights reserved REVIEWS 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