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Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide.

ABSTRACT: 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.

SUBMITTER: Hanaoka K

PROVIDER: S-EPMC5228037 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide.

Hanaoka Kenjiro K Sasakura Kiyoshi K Suwanai Yusuke Y Toma-Fukai Sachiko S Shimamoto Kazuhito K Takano Yoko Y Shibuya Norihiro N Terai Takuya T Komatsu Toru T Ueno Tasuku T Ogasawara Yuki Y Tsuchiya Yukihiro Y Watanabe Yasuo Y Kimura Hideo H Wang Chao C Uchiyama Masanobu M Kojima Hirotatsu H Okabe Takayoshi T Urano Yasuteru Y Shimizu Toshiyuki T Nagano Tetsuo T

Scientific reports 20170112

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 sc ...[more]

PMID: 28079151

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Project description:Background and purposeDuring angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis.Experimental approachTo assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds.Key results3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome.Conclusion and implications3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis.Linked articlesThis article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Project description:Endogenous hydrogen sulfide (H2S), which is primarily generated by 3-mercaptopyruvate sulfurtransferase (3-MST) in Escherichia coli (E. coli) under aerobic conditions, renders bacteria highly resistant to oxidative stress. However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H2S in E. coli under anaerobic conditions. A significant decrease in H2S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (?mstA). Furthermore, the H2S-producing activity of recombinant IscS using L-cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H2S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be -329 ± 1.6 mV. Moreover, in E. coli cells H2S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (?iscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 ?M Na2S (an H2S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 ?M Na2S to culture medium stimulates ATP synthesis in the mutant E. coli (?iscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H2S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions.

Dataset Information

Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide.

Publications

Discovery and Mechanistic Characterization of Selective Inhibitors of H<sub>2</sub>S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide.

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