The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione

Mana Murae; Yoshimi Shimizu; Yuichiro Yamamoto; Asuka Kobayashi; Masumi Houri; Tetsuya Inoue; Takuya Irie; Ryutaro Gemba; Yosuke Kondo; Yoshio Nakano; Satoru Miyazaki; Daisuke Yamada; Akiyoshi Saitoh; Isao Ishii; Taishi Onodera; Yoshimasa Takahashi; Takaji Wakita; Masayoshi Fukasawa; Kohji Noguchi

doi:10.1016/j.bbrc.2022.01.106

The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione

Biochem Biophys Res Commun. 2022 Mar 15:597:30-36. doi: 10.1016/j.bbrc.2022.01.106. Epub 2022 Jan 29.

Authors

Mana Murae¹, Yoshimi Shimizu², Yuichiro Yamamoto³, Asuka Kobayashi³, Masumi Houri³, Tetsuya Inoue³, Takuya Irie¹, Ryutaro Gemba⁴, Yosuke Kondo⁵, Yoshio Nakano⁵, Satoru Miyazaki⁵, Daisuke Yamada⁶, Akiyoshi Saitoh⁶, Isao Ishii⁷, Taishi Onodera⁸, Yoshimasa Takahashi⁸, Takaji Wakita⁹, Masayoshi Fukasawa¹⁰, Kohji Noguchi¹¹

Affiliations

¹ Laboratory of Molecular Target Therapy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan; Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
² Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan; Department of Pharmaceutical Sciences, Teikyo Heisei University, 4-21-2 Nakano, Nakano-ku, 164-8530, Japan.
³ Laboratory of Molecular Target Therapy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan.
⁴ Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
⁵ Department of Medical and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan.
⁶ Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan.
⁷ Department of Health Chemistry, Showa Pharmaceutical University, Tokyo, 194-8543, Japan.
⁸ Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
⁹ National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
¹⁰ Laboratory of Molecular Target Therapy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan; Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. Electronic address: fuka@nih.go.jp.
¹¹ Laboratory of Molecular Target Therapy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki 2641, Noda, Chiba, 278-8510, Japan; Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. Electronic address: noguchi-kj@rs.tus.ac.jp.

Abstract

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

Keywords: Cysteine; SARS-CoV-2; Spike.