title: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Sasakura
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suwanai
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Toma-Fukai
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Shimamoto
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Takano
authors.affiliation: Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Shibuya
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|PRESTO (Japan) Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ogasawara
authors.affiliation: High Technology Research Center, Pharmacology, Showa Pharmaceutical University, Machidashi 194-8543, Tokyo, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tsuchiya
authors.affiliation: High Technology Research Center, Pharmacology, Showa Pharmaceutical University, Machidashi 194-8543, Tokyo, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Kimura
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Advanced Elements Chemistry Research Team, RIKEN Center for Sustainable Resource Science, and Elements Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Advanced Elements Chemistry Research Team, RIKEN Center for Sustainable Resource Science, and Elements Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Uchiyama
authors.affiliation: Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Kojima
authors.affiliation: Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Okabe
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|CREST (Japan) Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D011485:Protein Binding; D011487:Protein Conformation; D013466:Sulfurtransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D004220:Disulfides; D004791:Enzyme Inhibitors; C116840:cysteine persulfide; D013466:Sulfurtransferases; C021550:3-mercaptopyruvate sulphurtransferase; D003545:Cysteine