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

Kenjiro Hanaoka,Kazuhito Shimamoto,Chao Wang,Hirotatsu Kojima,Toshiyuki Shimizu,Yoko Takano,Sachiko Toma-Fukai,Yusuke Suwanai,Takuya Terai,Takayoshi Okabe,Toshimitsu Komatsu ,Hideo Kimura,Yukihiro Tsuchiya,Norihiro Shibuya,Masanobu Uchiyama,Takahiro Ueno ,Kiyoshi Sasakura,Yuki Ogasawara,Yasuo Watanabe,Yasuteru Urano,Tetsuo Nagano

doi:10.1038/srep40227

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.

Highlights

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 recently shown to produce sulfane sulfur (H2Sn)
Based on crystal structure determinations of 3MST complexes with two of the inhibitors, as well as theoretical calculations, we suggest that a unique long-range electrostatic interaction between the inhibitor and the persulfurated cysteine residue located in the enzyme active site plays a key role in inhibitor binding
We adopted an E. coli expression system in order to obtain a sufficient amount of GST-fused m3MST (GST-3MST) for the high-throughput screening (HTS)

Summary

Introduction

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 recently shown to produce sulfane sulfur (H2Sn). It has been proposed that sulfane sulfurs[5], i.e., sulfur atoms with oxidation state 0 or −1 , existing in the form of polysulfides (H2Sn), glutathione persulfide (GSSH), glutathione trisulfide (GSSSG), cysteine persulfide (CysSSH) and so on, mediate some of the reported biological activities previously thought to be regulated by H2S6–11. To study the physiological roles and redox biology of these reactive sulfur species, we require inhibitors able to regulate the activities of the above three enzymes. Based on crystal structure determinations of 3MST complexes with two of the inhibitors, as well as theoretical calculations, we suggest that a unique long-range electrostatic interaction between the inhibitor and the persulfurated cysteine residue located in the enzyme active site plays a key role in inhibitor binding. We discuss the implications of our findings for the reaction mechanism of the enzyme

Methods

Results

Conclusion