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Hydrogen sulfide (H 2 S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H 2 S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H 2 S donor (SPD-2) that utilizes xanthone photochemistry to control H 2 S release. H 2 S generation from SPD-2 was completely dependent on UVA-irradiation (325–385 nm), as confirmed by methylene blue assay and by the use of a H 2 S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H 2 S delivery in live cells, and should be suitable for various biological applications.

Hydrogen sulfide (H2S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection. Cytotoxicity of H2S in neurodegenerative diseases may be mediated by N-methyl-D-aspartate receptor (NMDAR) activation. To exploit cytoprotective effects of H2S while minimizing its toxicity, we synthesized a series of H2S-releasing NMDAR antagonists and examined their effects against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death, a cellular model of Parkinson's disease. We observed that cytoprotective effects of H2S-releasing NMDAR antagonists correlated with their ability to increase intracellular sulfane sulfur, but not H2S, levels. These studies suggest that H2S-donor compounds that increase intracellular sulfane sulfur levels are potentially useful neuroprotective agents against neurodegenerative diseases.

Very recent studies indicate that sulfur atoms with oxidation state 0 or −1, called sulfane sulfurs, are the actual mediators of some physiological processes previously considered to be regulated by hydrogen sulfide (H2S). 3-Mercaptopyruvate sulfurtransferase (3MST), one of three H2S-producing enzymes, was also recently shown to produce sulfane sulfur (H2Sn). Here, we report the discovery of several potent 3MST inhibitors by means of high-throughput screening (HTS) of a large chemical library (174,118 compounds) with our H2S-selective fluorescent probe, HSip-1. Most of the identified inhibitors had similar aromatic ring-carbonyl-S-pyrimidone structures. Among them, compound 3 showed very high selectivity for 3MST over other H2S/sulfane sulfur-producing enzymes and rhodanese. The X-ray crystal structures of 3MST complexes with two of the inhibitors revealed that their target is a persulfurated cysteine residue located in the active site of 3MST. Precise theoretical calculations indicated the presence of a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety of the inhibitor. Our results also provide the experimental support for the idea that the 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism.

We report the design, synthesis and application of a directly photocontrollable hydrogen sulfide (H2S) donor, which releases H2S proportionally to the intensity and duration of photoirradiation. Photocontrolled H2S release from this donor was also demonstrated in bovine serum. This H2S donor should be suitable for use in various biological systems.

Hydrogen sulfide (H2S) exerts a host of biological effects ranging from cytotoxicity to cytoprotection. Cytotoxicity of H2S in neurodegenerative diseases may be mediated by N-methyl-D-aspartate receptor (NMDAR) activation. To exploit cytoprotective effects of H2S while minimizing its toxicity, we synthesized a series of H2S-releasing NMDAR antagonists and examined their effects against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death, a cellular model of Parkinson’s disease. We observed that cytoprotective effect of H2S-releasing NMDAR antagonists correlated with their ability to increase intracellular sulfane sulfur, but not H2S, levels. These studies suggest that H2S-donor compounds that increase intracellular sulfane sulfur are potentially useful neuroprotective agents against neurodegenerative diseases.

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

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.

Recently, we have reported a novel fluorescence probe for H 2 S, HSip-1, based on azamacrocyclic Cu 2+ complex chemistry (J. Am. Chem. Soc., 133 (2011) 18003) (Figure A). HSip-1 can selectively detect H 2 S in aqueous solution with high selectivity over biothiols, inorganic sulfur compounds, ROS and RNS, and has excellent photophysical properties for biological applications (Φ fl = 0.019 and 0.78 in the absence or the presence of H 2 S, respectively), deriving from its fluorescein scaffold. We also confirmed that HSip-1 could be used both for detection of pseudoenzymatic H 2 S release in a cuvette and of H 2 S production by 3-mercaptopyruvate sulfurtransferase (3MST) or lysate of 3MST-expressing cells, and 3-mercaptopyruvate, i.e., H 2 S-producing enzyme and its substrate. So, we anticipated that HSip-1 would be useful for not only fluorescence imaging, but also high-throughput screening of CBS (cystathionine β -synthase), CSE (cystathionine γ -lyase) and 3MST inhibitors, which should be required for detailed investigation of a wide range of biological functions of H 2 S. To investigate whether the probe can be used to monitor recombinant enzymatic H 2 S generation, we examined its efficacy with recombinant human CSE, which was purchased from Cayman and 3MST produced by Escherichia coli BL21 strain using a plasmid (3MST/pGEX) expressing 3MST with GST tag. H 2 S production by CSE (20 μg/mL) was readily detected in the presence of millimolar concentrations of the thiol substrate (cysteine; 2.5 mM) and pyridoxal phosphate (50 μM) in 30 mM HEPES buffer (pH 7.4). The fluorescence increment was suppressed by pretreatment with PAG (propargylglycine; 1 mM), which is an inhibitor of CSE (Figure B). Moreover, H 2 S production by 3MST (3.8 μg/mL) was also detected in the presence of thiol substrates (50 μM 3MP and 40 μM DTT) in 30 mM HEPES buffer (pH 7.4) (Figure C). We further confirmed that these systems could be applied to 384-well plate assay. By utilizing HSip-1 as an indicator for H 2 S, we then performed 3MST inhibitor screening of a chemical library containing about 150,000 compounds obtained from Open Innovation Center for Drug Discovery, The University of Tokyo. In this conference, we will show the detailed protocols and results in our poster.