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Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine β-synthase (CBS), a major H

Hydrogen sulfide (H

The mechanisms underlying temporomandibular disorders following orofacial pain remain unclear. Hydrogen sulfide (H2S), a newly identified gasotransmitter, has been reported to modulate inflammation. Cystathionine γ-lyase (CSE) is responsible for the systemical production of H2S, which exerts both pro- and antinociceptive effects through inflammation. In the current study, we investigated whether the endogenous H2S production pathway contributes to arousal and maintenance of orofacial inflammatory pain, through the investigation of the effects of a CSE inhibitor, propargyglycine (PAG), in a rat CFA (Complete Freund Adjuvant)-induced temporomandibular inflammation model to mimic persistent pain in the orofacial region. For this, rats received either CFA or saline in the temporomandibular joints (TMJs), and after 3 or 14 days, they received a single injection of PAG or saline and were evaluated for nociception with the von Frey and formalin test. Also, pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were analyzed in TMJs and trigeminal ganglion (TG). In this last one, glial cells reactivity was also verified. Endogenous H2S production rate were measured in both, TMJ and TG. Our results indicated decreased allodynia and hyperalgesic responses in rats submitted to CFA after injection of PAG. Moreover, PAG inhibited leucocyte migration to temporomandibular synovial fluid after 3 and 14 days of inflammation. PAG was able to reduce levels of CBS, CSE, TNF-α, and IL-1β in the TMJ and TG, after 13 days of CFA injection. The observed increased activation of glial cells in the trigeminal ganglia on the 14th day of inflammation can be prevented by the highest dose of PAG. Finally, CBS and CSE expression, and endogenous H2S production rate in the TMJ and TG was found higher in rats with persistent temporomandibular inflammation compared to rats injected with saline and PAG was able to prevent this elevation. Our results elucidated the molecular mechanisms by which H2S exerts its pro-inflammatory and pro-nociceptive role in the orofacial region by alterations in both local tissue and TG.

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extra cellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

Hydrogen sulfide (H2S) is recognized as a third gaseous signaling molecule behind nitric oxide (NO) and carbon monoxide (CO). In pancreatic beta-cells, H2S inhibits glucose-induced insulin release. There are multiple underlying mechanisms for this inhibitory process. Apart from these inhibitory effects, H2S also protects pancreatic islets from apoptotic cell death induced by high glucose. Moreover, expression of the H2S-producing enzyme, cystathionine γ-lyase (CSE), is induced by glucose stimulation. These observations suggest that H2S is produced in an inducible manner, as are the other two gaseous signaling molecules, NO and CO. We recently reported that a lack of CSE induces apoptotic beta-cell death and promotes the development of high-fat diet (HFD)-induced diabetes. These findings tempt us to suggest that H2S produced by CSE is part of a homeostatic mechanism used by pancreatic beta-cells to inhibit insulin release and reduce cellular stress evoked by glucose, possibly via the anti-oxidant properties of H2S.

Hydrogen sulfide (H2S) is considered the third gasotransmitter following nitric oxide (NO) and carbon monoxide (CO) in the mammalian body including the brain, heart, blood vessels, liver, kidney, pancreas, lung, gastrointestinal tract and reproductive organs. H2S is formed endogenously from L-cysteine by multiple enzymes, such as cystathionine-γ-lyase, cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase in combination with cysteine aminotransferase, and participates in a variety of biological events through a number of target molecules. Exogenous and/or endogenous H2S enhances the activity of T-type Ca(2+) channels in NG108-15 cells and isolated dorsal root ganglion neurons that abundantly express Cav3.2, and in Cav3.2-transfected HEK293 cells. Cav3.2 mediates not only the H2S-induced enhancement of pain signals in nociceptor neurons, but also neuronal differentiation characterized by neuritogenesis and functional upregulation of high voltage-activated Ca(2+) channels in NG108-15 cells. In this review, we focus on the functional modulation by H2S of primarily Cav3.2 T-type Ca(2+) channels and the molecular mechanisms underlying the H2S-induced neuronal differentiation.

Previously we reported that matrix metalloproteinase-9 (MMP-9) plays an important role in extracellular matrix (ECM) remodeling in diabetic kidney. Induction of NMDA-R and dysregulation of connexins (Cxs) were also observed. We concluded that this was due to decreased H2S production by downregulation of CBS and CSE enzymes. However, the potential role of H2S to mitigate ECM dysregulation and renal dysfunction was not clearly understood. The present study was undertaken to determine whether H2S supplementation reduces MMP-9-induced ECM remodeling and dysfunction in diabetic kidney. Wild type (C57BL/6J), diabetic (Akita, C57BL/6J-Ins2(Akita)), MMP-9 knockout (MMP-9(-/-), M9KO) and double KO of Akita/MMP-9(-/-) (DKO) mice were treated without or with 0.005 g/l of NaHS (as a source of H2S) in drinking water for 30 days. Decreased tissue production and plasma content of H2S in Akita mice were ameliorated with H2S supplementation. Dysregulated expression of MMP-9, CBS, CSE, NMDA-R1 and Cxs-40, -43 was also normalized in Akita mice treated with H2S. In addition, increased renovascular resistive index (RI), ECM deposition, plasma creatinine, and diminished renal vascular density and cortical blood flow in Akita mice were normalized with H2S treatment. We conclude that diminished H2S production in renal tissue and plasma levels in diabetes mediates adverse renal remodeling, and H2S therapy improves renal function through MMP-9- and NMDA-R1-mediated pathway.

Hydrogen sulfide is well known to regulate multiple physiological aspects of vascular function including vasodilation, inflammation, and growth responses that may become dysfunctional during ischemia. However, specific sulfide metabolism responses during chronic vascular ischemia remain poorly understood. Recent work from our laboratory reveals that permanent vascular ischemia due to ligation and surgical removal results in a rapid increase in cystathionine gamma lyase (CSE) expression along with increased hydrogen sulfide bioavailability within ischemic tissues that prefaces restoration of ischemic tissue blood flow. Importantly, as tissue perfusion is restored, expression of CSE is reduced back to basal levels along with hydrogen sulfide metabolites. Consistent with these observations, genetic deletion of CSE significantly blunts ischemic tissue vascular remodeling responses necessary for tissue reperfusion. Whereas, transgenic vascular endothelial cell CSE expression significantly augments ischemic vascular remodeling responses leading to tissue reperfusion. These responses invoke increased nitric oxide formation and vascular endothelial cell growth factor production that are critical requirements for chronic ischemic vascular remodeling. Together, these data reveal that progressive induction of vascular sulfide metabolism during chronic ischemia is an important physiological adaptive response.

Introduction CBS, CSE and 3-MST are the enzymatic source of endogenous hydrogen sulfide (H 2 S), a gaseous signaling molecule implicated in a wide range of physiological processes. We investigated the status of CBS, CSE, and 3-MST as well as the extent of oxidative stress in skeletal muscle tissue obtained from patients suffering from critical limb ischemia (CLI) ± diabetes mellitus (Db). Methods/Results We found that both mRNA and protein levels of CSE, CBS, and 3-MST were significantly decreased in skeletal muscle of CLI (>2 fold, p p Nrf2 ), a factor responsible for the expression of antioxidant response element-regulated genes (2 fold, p P Conclusion The present study suggests that the induction of oxidative stress in skeletal muscle of both CLI and CLI + Db patients may directly down-regulate the expression of the endogenous H 2 S-generating enzymes: CSE, CBS, and 3-MST. Increased oxidative stress and reductions in H 2 S bioavailability may contribute to the pathogenesis of CLI.

Purpose One of the reasons of increasing the risk of cardiovascular diseases during aging is a dysfunction of endothelium. At the same time we can see more and more publications about vascular protection of hydrogen sulfide (H2S). Cystathionine gamma-lyase (CSE) is the main enzyme for H2S synthesis in blood vessels. Methods The segments of aorta from adult and old rats were used. Activation of isolated samples was carried out by phenylephrine (Phe). Acetylcholine (Ach)-mediated vasorelaxation in the aortic vessel were investigated. The activity of CSE was inhibited with propargylglycine (PPG) (incubation 30 min). H2S was determined by N,N-dimethyl-p-phenylenediamine method, NO de novo synthesis – by NOs activity and nitrite determination. Results The experiments on isolated segments of aorta have shown that the effect of Ach endothelium-dependent vasodilator results in less profound relaxation of aortic smooth muscles (SM) in old animals (12.4%) compared to adult ones (66.3%). PPG addition resulted in growing aortic SM relaxation amplitude from 12.4% to 49.5% (p NO 2 - pools. Also increased the activity of cNOS and decreased the activity of iNOS (p Conclusions Therefore, we can state that use of PPG has led to growing production of two powerful vasodilators – H2S and NO and to the restoration of endothelium-dependent aortic SM vasorelaxation in old animals.

Previous studies reported both beneficial and detrimental roles of hydrogen sulfide (H2S) in various models of critical illness. The goal of our study was to compare the outcome of septic shock in wild-type mice and in mice deficient in cystathionine-gamma-lyase (CSE). Mice were subjected to sepsis by cecal ligation and puncture and survival was monitored for 48 h. In a separate study, animals were sacrificed at 24 h post-CLP, vascular function was evaluated ex vivo and indices of organ function were measured. At 30 h, there was 20% mortality in CSE−/− mice, while no mortality in the wild-type controls. By 36 h, the mortality increased to 50% in CSE−/− mice, while it remained low (10%) in the wild-type controls. However, by 46 h, the two survival curves converged, and showed a similar, 60% mortality in both groups. Analysis of the plasma markers of organ injury revealed slight trends for a CLP-induced increase in the liver injury markers ALT and AST and the pancreatic injury marker amylase in wild-type mice. More pronounced increases were noted in the same parameters in the CSE−/− mice. Blood urea nitrogen levels were elevated 3-fold over baseline in wild-type mice subjected to CLP and 4-fold over baseline in CSE−/− mice. There was an impairment of endothelium-dependent relaxation responses in the thoracic aorta after CLP, which was more pronounced in the CSE−/− mice. These data indicate that CSE, and its product, H2S, exerts protective effects against the development of sepsis, especially in the early stages of the disease.

The endogenous gas, hydrogen sulfide (H2S), plays an important role in regulating cardiovascular function and inflammation. It also contributes to the pathogenesis of insulin resistance and glucose metabolism [1] , [2] , [3] . Recently, it has been reported that large amounts of H2S are formed from L-cysteine by cystathionine gamma lyase (CSE) in fat tissues and that H2S reduces glucose uptake in rat adipose tissue [3] . However, the biological roles of H2S in adipocytes and its contribution to adipogenesis are not yet clear. Here, we investigate whether H2S-synthesizing enzymes; cystathionine beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), are endogenously expressed in adipocytes and play a role in adipogenesis. In the present experiments, we stimulated the mouse fibroblast-like cell line, 3T3L1, to differentiate into adipocytes for up to one week as a model of adipogenesis in vitro. Over 7 days differentiation, whole cell lysate at day 0, 1, 3, 5 and 7 were collected for Western blotting of CBS, CSE and 3-MST. Endogenous CBS and 3-MST in adipocytes peaked at day 5 of differentiation whereas CSE protein expression was up-regulated in a time-dependent manner and maximally expressed at day 7 (n = 3, P [4] , [5] , [6] , but that these enzymes are also present in large amounts in adipose tissue and that their up-regulation during adipogenesis promotes adipocyte maturation and prevent lipid degradation. Thus, it is possible that modulation of the H2S system may be useful for obesity treatment.

Background Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin3-gene. The polyQ expansion leads to neuronal dysfunction and cell death. Besides proteotoxic stress, mitochondrial dysfunction, oxidative stress, inflammation, apoptosis and a disturbed calcium homeostasis are thought to contribute to the multifaceted pathogenesis of SCA3. Hydrogen sulfide (H2S) is endogenously produced by cystathionine γ-lyase (CSE), cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase and has crucial physiological functions in the central nervous system. It has anti-oxidative, anti-inflammatory and anti-apoptotic properties and regulates calcium homeostasis, making it an attractive candidate to intervene in the damaging processes of SCA3. We therefore investigated the protective role of overexpression of endogenous CSE using a Drosophila melanogaster model of SCA3. Methods In human, CSE mRNA and protein expression was investigated by qRT-PCR and immunohistochemistry of the pons of SCA3 (n = 7) and control (n = 7) patients. Drosophila Melanogaster culture and crosses were performed at 25 °C following standard protocols. CSE mRNA overexpression in CSE transgenic flies was confirmed by qRT-PCR. For eye degeneration experiments, fly stocks expressing the mutated human ataxin3 gene with 78 repeats in the presence or absence of overexpression of CSE in eye tissue were used. Isogenic wild type flies were used as controls. To inhibit CSE, the crosses were performed on food containing propargylglycine (1 mM). Twenty-four hours after eclosion, each male eye was scored for degeneration. Per cross, at least 150 eyes were examined. Results In SCA3 patients CSE mRNA levels were downregulated by 36% compared to control patients (p Conclusion CSE mRNA expression in SCA3 patients is decreased. Using a Drosophila melanogaster model for SCA3, we show that manipulation of CSE expression influences the degree of eye degeneration. Possibly, a higher production of endogenous H2S underlies the protective role of CSE overexpression. In the absence of effective therapeutic interventions in the devastating course of polyglutamine disorders, manipulation of CSE might be a promising target for future interventions.

Introduction H 2 S plays key roles in various physiological processes. So, compounds which can regulate activities of H 2 S producing enzymes are expected to be useful as chemical tools for life sciences and medicine. These compounds also would be able to contribute to the progress of H 2 S research. Here we show the discovery of small molecule inhibitors for 3-mercaptopyruvate sulfurtransferase (3MST) and cystathionine gamma lyase (CSE) by high-throughput screening (HTS). Method For screening, we used a novel fluorescence probe for H 2 S (HSip-1), which has been developed by our group (J. Am. Chem. Soc. 133 (2011) 18003). By using this probe, we established an assay system. In the assay system, the increase of the fluorescence intensity is suppressed, when the enzyme activity is inhibited by a compound and the production of H 2 S decreases. We then performed 3MST and CSE inhibitor screening of a chemical library containing about 160,000 compounds. Results We selected compounds which could suppress the fluorescence increase of HSip-1. After the repeatability test, we excluded HS − reactive compound as false positive compounds, and then found hit compounds. These compounds were selective against targeted H 2 S producing enzymes. Conclusion As a result of screening, we could discover both selective 3MST and CSE inhibitors. We will examine the utility of these compounds by applying them to living cells and we expect these compounds would be used as research tools.

Hydrogen sulfide (H2S) regulates smooth muscle tone, cell metabolism and growth, apoptosis and migration. It has, thus, been implicated in a plethora of physiological and pathophysiological processes including angiogenesis, cardioprotection, atherosclerosis, inflammation and cancer. H2S is produced by cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST). Aminoethoxyvinylglycine (AVG), β-cyano- l -alanine (BCA) and propargylglycine (PAG) selectively inhibit CSE over CBS and 3-MST. All of them, exhibit limited potency and block other PLP-dependent enzymes, too. It should be emphasized that no selective CBS or 3-MST inhibitors are currently available. During this presentation data obtained from a screen of chemical libraries against H2S-synthesizing enzymes will be presented. The available donors can be divided based on their chemical nature and rate of release to inorganic vs organic compounds and fast vs slow releasers. The most widely used inorganic compounds to generate H2S are NaSH and Na2S. These compounds dissociate rapidly in solution leading to immediate H2S formation. The organic donors available can be divided according to their source of origin into naturally occurring and synthetic. Contrary to inorganic salts, the organic donors release H2S more slowly; the rate of H2S liberation may differ substantially depending on the absence or presence of biological material. Mitochondrial-targeted donors and hybrid molecules between known drugs and H2S-releasing moieties are special subclasses of H2S donors. During the second part of the presentation data on signalling, pharmacological profiles and biological activities in the context of cardiovascular disease of different classes of H2S-generating compounds will be analysed.

Background Myocardial infarction (MI) is a leading cause of death globally. MicroRNAs (miRs) have been identified as a novel class of MI injury regulators. Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates cardiovascular function. The purpose of this study was to explore the role of the miR-30 family in protecting against MI injury by regulating H2S production. Methods and Results Two days after coronary artery ligations in rats, the expression of miR-30 family was significantly upregulated in the non-infarct and border zone regions. However, the CSE expression was just the opposite. The overexpression of each miR-30 family member decreased mRNA and protein levels of CSE and reduced H2S production. In contrast, silencing the whole miR-30 family resulted in elevated CSE expression and increased H2S production. Forced expression of the miR-30 family aggravated hypoxia-induced myocardial cell injury. This was reversed by locked nucleic acid (LNA)-miR-30 family inhibitor transfection. Systemic delivery of a LNA-miR-30 family inhibitor correspondingly repressed the miR-30 family in mouse cardiac tissue and increased CSE expression and H2S production. The therapeutic targeting of the miR-30 family in mice reduced infarct size, decreased apoptotic cell number in the peri-infarct region, and improved cardiac function in response to MI, whereas miR-30b overexpression by lentivirus in vivo exacerbated MI injury. Conclusion The inhibition of miR-30 family expression in the heart can protect against mouse cardiac ischemic injury by increasing CSE expression and H2S production, indicating the therapeutic potential of miR-30 for cardiac diseases associated with MI injury.

Primary cilium is associated with numbers of kidney diseases including polycystic kidney diseases and acute kidney injury. Recently, we reported that the length of primary cilium largely depends on oxidative stress and intracellular signaling molecules such as ERK in the kidney tubular epithelial cells. Hydrogen sulfide (H2S), a novel biological gas, plays a signaling molecule in kidney cells. Here, we investigated the role of H2S in primary ciliogenesis and its underlying mechanisms in the Madin Darby Canine Kidney (MDCK) cell. Primary cilia were gradually lengthened overtime after cells were confluent. Expressions of H2S-producing enzymes, cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE), increased overtime with increases of H2S levels in the cells and medium. Treatment of NaHS, a donor of H2S, lengthened primary cilia, whereas propargylglycine (PAG, an inhibitor of CSE) and hydroxylamine (HA, an inhibitor of CBS) shortened the primary cilia. NaHS increased levels of activated ERK. Treatment of U0126, an inhibitor of ERK signal, shortened primary cilia. U0126 prevented elongation of primary cilia induced by NaHS. In addition NaHS treatment increased expressions of ADP-ribosylation factor-like 13B (Arl13b) and Sec10 which regulate cilia formation and elongation. In conclusion, H2S elongates the length of primary cilia through ERK activation and increase of Sec10 and Arl13b expression, and these results providing H2S as a novel target for the regulation of primary cilia length in kidney tubule cells.

Introduction Hydrogen sulfide (H2S) is the endogenous signaling gasotransmitter, offering protective effects on oxidative stress including ischemia–reperfusion and hypertrophy/heart failure (HF). CSE is believed to be the major H2S-generating enzyme in the heart. Our previous study investigated that CSE KO mice are more susceptible to oxidative stress. NF-kappaB is a marker of cellular oxidative stress and its activation has been found to increase in ischemia–reperfusion and hypertrophy/HF. The purpose of the present study was to investigate the role of NF-kappaB in response to CSE. Methods/results We found that both mRNA and protein levels of NF-kappaB-p65 were significantly increased in the heart tissues of CSE KO mice compared with control as they were judged by RT-qPCR and immunoblot, respectively. Interestingly, phosphorylation of p65 was increased (1.4 fold, p Conclusion Since we observed that the activation of NF-kappaB occurred in the heart tissues of CSE KO mice, our findings suggest that CSE may protect the heart from oxidative injury, such as that which occurs during ischemia–reperfusion, hypertrophy/HF.

Many prokaryotic species generate hydrogen sulfide (H2S) and nitric oxide (NO) enzymatically, from cysteine and arginine, respectively, in their natural environments. Both gases are small freely diffusible signaling molecules that are known to be involved in numerous physiological and pathological processes in mammals. However the biochemistry and physiological role of these gases in bacteria remains largely unknown. We have shown that inactivation of H2S producing enzymes (cystathionine beta-synthase, cystathionine gamma lyase, or 3-mercaptopyruvate sulfurtransferase) and NO-synthase in several Gram (+) and Gram (−) bacteria render them highly sensitive to different classes of antibiotics (Gusarov et al., Science 325 (2009) 1380–1384; Shatalin et al. Science 334 (2011) 986–990). We also presented evidence that Bacillus anthracis-derived NO is critical at the early stage of infection (Shatalin et al. PNAS 105 (2008) 1009–1013). Here we show that: (1) cbs/cse and nos mutations change Bacilli global gene transcription profile; (2) apore formation process in cbs/cse and nos mutants ofB. anthracis is affected; (3) virulence of cbs/cse and nos mutants of B. anthracis is diminished. These results demonstrate that bacterial H2S and NO are an important virulence factors, and that enzymes generated these gases may serve as an attractive target for antimicrobial therapy.

Retina is one of the tissues that exposed to highest oxidative stress due to its high metabolic rate, abundant photosensitizing activity and visible light exposure. With these high chances of oxidative damage, antioxidative capacity of retinal tissues is essential for maintaining the proper functions of retina. Cytoprotective roles of hydrogen sulfide (H2S) have been documented and were largely attributed to its antioxidative activity. However, whether are these three H2S-producing enzymes (cystathionine-gamma-lyase (CSE), cystathionine-beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) plays antioxidative roles in retinal tissue is unclear. In this study, we examined the role of CSE, CBS and MPST enzymes in human retinal pigmented epithelial cell line ARPE19. ARPE19 expressed comparatively similar protein levels of the three enzymes. Surprisingly, gene silencing of CSE sensitized the cells to hydrogen peroxide (H2O2) challenge to a much greater extent as compared to gene silencing in CBS or MPST. We also observed that loss of CSE in ARPE19 resulted in decreased level of glutathione, higher oxidative stress and accelerated senescence. Taken together, our data suggest that CSE is essential for retinal functions by maintaining its redox balance.

Hydrogen sulfide (H2S) has emerged as a gaseous signaling molecule with important roles in neuromodulation, blood pressure regulation, angiogenesis, cardioprotection, cellular energetics and apoptosis. H2S is produced endogenously by three enzymes in the sulfur metabolic network, namely cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Although several compounds that inhibit CSE are available, no selective inhibitors for CBS or 3MST currently exist. In the present study we screened the LOPAC library for inhibitors of H2S-producing enzymes. The methylene blue assay was used to measure H2S production from recombinant enzymes. HCT116 and HT29 cells were used for MTT-based proliferation assays. Screening of the LOPAC library yielded several hit compounds that blocked H2S-synthesizing enzymes to varying extents. Among those, doxycyclin and demeclocycline were found to inhibit 3MST by approximately 50% at 100 uM, with doxycyclin being a more potent 3MST inhibitor than demeclocycline in the in vitro activity assay. Tetracyclin, meclocyclin, oxycyclin, methacyclin, tigecyclin and minocyclin did not affect 3MST activity when used up to 300 uM. From the above-mentioned tetracyclines, tigecyclin at 100 uM caused only a slight inhibition of both CBS and CSE; tigecyclin additionally inhibited CSE. In a cell-based assay, demeclocyclin, but not doxycyclin, inhibited the growth of HCT116 and HT29, two colon cancer cell lines in which CBS is highly expressed and its product, H2S supports cell proliferation. We conclude that based on its selectivity towards 3MST, demeclocyclin could serve as a lead compound that after undergoing optimization could yield future selective 3MST inhibitors.

The biological roles of endogenous H 2 S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H 2 S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H 2 S is derived from two pyridoxal-5′-phosphate (PLP)-dependent enzymes responsible for the metabolism of l -cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H 2 S from l -cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H 2 S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H 2 S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H 2 S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H 2 S biosynthesis include propargylglycine (PAG), β -cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H 2 S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of l -aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H 2 S production is up regulated contributing to disease progression), scavenging of H 2 S would be the preferred approach. Our efforts to develop H 2 S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H 2 S-synthesizing enzymes, as well as H 2 S scavengers is an unmet need for the field, so that the role of H 2 S-regulated pathways in the development and treatment of disease can be evaluated.

Cystathionine gamma lyase (CSE) also known as γ-cystathionase, is a key enzyme in the transsulfuration pathway leading to the synthesis of glutathione, the major antioxidant in the cell. CSE converts cystathionine to cysteine, the availability of which is the rate limiting step in glutathione biosynthesis. In addition, CSE utilizes cysteine to generate hydrogen sulfide (H2S), which is one of the three gasotransmitters, the other two being nitric oxide and carbon monoxide. H2S plays an important role in diverse signaling pathways. One of the mechanisms by which H2S signals is via the formation of a persulfide (-SSH) bond at reactive Cys residues, a process designated sulfhydration. Using a modified biotin switch assay, we have shown that sulfhydration of target proteins such as cyclooxygenase 2 (COX2) and nuclear factor κB (NF-κB) modulates their activity thereby regulating prosurvival and inflammatory responses. Using CSE knockout mice we show that H2S functions as an anti-inflammatory and antioxidant molecule in vivo. Depletion of CSE led to a decrease in levels of glutathione and increase in oxidative stress as revealed by protein carbonylation, lipid peroxidation and DNA damage detection assays. Primary hepatocytes and mouse embryo fibroblasts isolated from CSE knockout mice had higher levels of reactive oxygen species and exhibited a higher susceptibility to oxidants such as hydrogen peroxide. Our studies reveal an important role for CSE in the maintenance of redox homeostasis in the cell.

Background Cystathionine gamma-lyase (CSE) is the major H2S-generating enzyme in myocardial cell. H2S has been reported to be cardioprotective during myocardial infarction (MI) injury. MiRNAs are identified as crucial gene regulators in response to myocardial infarction. However, there is little evidence indicating a functional role for miRNA in regulating the generation of H2S. Methods and Results The MI rat model was induced by ligating the left anterior descending coronary artery. Real-time PCR and western bolt results show that the expression of CSE mRNA and protein were increased in the infarct region and reduced in non-infarct and border zone regions 48 h after MI induction. The concentration of H2S in plasma was reduced significantly in MI model. Correspondingly, using real time PCR, we demonstrated that the miR-30 family, which includes 5 closely related miRNAs (miR-30a, miR-30b-5p, miR-30, miR-30d, miR-30e) were significantly down regulated in the infarct region and highly expressed in the non-infarct and border zone regions. To determine the potential roles of miR-30 family members in CSE gene expression, we transfected miRNA mimics into neonatal rat cardiomyocytes to upregulate the expression of miR-30 family numbers. Interestingly, overexpression of each number of the miR-30 family significantly repressed the protein expression of CSE tested by western blot and immunofluorescence, and reduced the H2S production in culture media. The mRNA expression of CSE was also inhibited by each number of the miR-30 family, respectively. In contrast, downregulation of all miR-30 family numbers by LNA modified miR-30 family inhibitor significantly increased the level of CSE mRNA and protein and the H2S concentration in culture media. We further demonstrated that miR-30 family members repressed CSE protein expression by directly targeting at CSE untranslational regions by luciferase activity assay. This result was consistent with computational prediction that CSE is a target of miR-30 family members. Conclusion Take together, these results suggest that miR-30 family participate in regulating the production of endogenous H2S by targeting CSE.