Annals of Pharmacology and Pharmaceutics

Short Communication
Published: 12 Sep, 2020

Inhibition of the Binding of Spike Protein of SARS-CoV-2Coronavirus to Human Angiotensin-Converting Enzyme 2by Chlorine Dioxide
Norio Ogata* and Takanori Miura
Department of R&D, Taiko Pharmaceutical Co. Ltd, Japan

Abstract
Aim: The new Coronavirus (SARS-CoV-2) responsible for COVID-19 disease is still a significant
threat and no effective and convenient method for decontamination of this virus exists as yet. We
aimed to evaluate the effect of a novel method for the decontamination of the virus using aqueous
Chlorine Dioxide (ClO2) solution. SARS-CoV-2 virus uses its “spike protein” to adhere to humancells containing Angiotensin-Converting Enzyme 2 (ACE2), which acts as a receptor protein.
Materials and Methods: In an in vitro experiment, the spike protein was treated with 0.5 mmol/Lchlorine dioxide solution at room temperature for 5 min, and the binding of the spike protein toACE2 was quantitated by a chemiluminescence reaction.
Findings: The binding of the spike protein to ACE2 decreased to 1.9% of the control (no chlorinedioxide) (n=4, p<0.001).
Conclusion: The result strongly indicates the usefulness of chlorine dioxide solution as adecontamination method of SARS-CoV-2 virus.
Keywords: Chlorine dioxide; SARS-CoV-2 virus; COVID-19; Angiotensin-converting enzyme 2Introduction
OPEN ACCESS*Correspondence:
Norio Ogata, Department of R&D, Taiko
Pharmaceutical Co. Ltd, 1-2-1 Hikaridai,
Seikacho, Kyoto 619-0237, Japan, Tel:
81-774-98-2716;
E-mail: nogata7@yahoo.co.jp
Received Date: 22 Jul 2020
Accepted Date: 09 Sep 2020
Published Date: 12 Sep 2020
Citation:Ogata N, Miura T. Inhibition of theBinding of Spike Protein of SARSCoV-2 Coronavirus to Human
Angiotensin-Converting Enzyme 2by Chlorine Dioxide. Ann PharmacolPharm. 2020; 5(5): 1195.
Copyright © 2020 Norio Ogata. This is
an open access article distributed under
the Creative Commons Attribution
License, which permits unrestricted
use, distribution, and reproduction inany medium, provided the original work
is properly cited.

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The worldwide emergence and rapid spread of the new Coronavirus (SARS-CoV-2) responsiblefor COVID-19 disease is a serious threat to human wellbeing. Powerful therapeutic approaches andmethods for decontamination are needed as soon as possible to suppress the ongoing pandemic[1]. The SARS-CoV-2 virus has a positive-sense single-stranded RNA genome surrounded by amembrane with Spike Proteins (S-proteins) consisting of S1 and S2 subunits encoded by the viralgenome [2]. In the case of human infection, the spike protein binds to a receptor, AngiotensinConverting Enzyme 2 (ACE2) on host cells [3]. At this stage of virus-receptor binding, tyrosine
residue 453 (Y453) located in the receptor-binding domain of the spike protein forms a hydrogenbond with Histidine residue 34 (H34) located in an α1 helix of ACE2 (Figure 1) [3].
Chlorine Dioxide (CD) has been used as a gas or aqueous solution to inactivate many different
microbes [4-6]. It inactivates microbes by oxidizing their proteins; the prime targets are tryptophan
and tyrosine residues [7,8]. Tyrosine residues are oxidized to dihydroxyphenylalanine or trihydroxyphenylalanine [7]. As the Y453 residue is exposed on the outside of the spike protein of the virus andis required for the hydrogen-bonding necessary for virus-receptor binding [3], we hypothesized thatCD will inactivate the virus and therefore prevent the spread of viral infection.

Materials and Methods
We used a binding assay kit (SARS-CoV-2 Spike: ACE2 Inhibitor Screening Assay Kit) fromBPS Bioscience (San Diego, California, USA) and followed its manual. Spike protein was first mixed
with various concentrations of CD (obtained from Taiko Pharmaceutical Co., Ltd., Osaka, Japan)
at room temperature for 5 min. The reaction with CD was terminated by adding two-fold molar
excess of sodium thiosulfate. Next, 3.5 µg/ml of ACE2 protein was mixed with the CD-treatedspike protein and left at room temperature for 30 min. The amount of ACE2 bound to the treatedspike protein was next quantitated by adding horseradish peroxidase-labeled anti-ACE2 antibody.
Chemiluminescence of the substrate of the horseradish peroxidase was then added to the mixture tomeasure the enzyme activity of the horseradish peroxidase. Chemiluminescence was immediatelymeasured with a luminometer (model SH-9000, Corona Electric Co., Ltd., Hitachinaka, Ibaraki,12020 | Volume 5 | Issue 5 | Article 1195

Norio Ogata, et al.,Annals of Pharmacology and Pharmaceutics

Conclusion

Table 1: Effect of chlorine dioxide aqueous solution on the binging of the viral
spike protein to human Angiotensin-Converting Enzyme 2.
p
Concentration of chlorine dioxide
Chemiluminescencevaluea(mmol/L)(count/s, × 103)
06097 ± 708

0.17122 ± 967

0.14

0.25

3107 ± 709<0.0010.5

116 ± 17<0.001The spike protein of SARS-CoV-2 virus was treated with CDsolution. It inactivated the biding ability of the viral protein to humanACE2 protein, suggesting that the virus cannot bind to humancells. The result strongly indicates the usefulness of this solution todecontaminate SARS-CoV-2 virus.

Acknowledgement

Each value represents an average ± standard deviation of four experiments. a vs.
0 mM chlorine dioxide

The authors are employees of Taiko Pharmaceutical Co., Ltd. This
work was supported by the company.

References
1. Wang C, Horby PW, Hayden FG, Gao GF. A novel coronavirus outbreakof global health concern. Lancet. 2020;395(10223):470-3.
2. Guo Y-R, Cao Q-D, Hong Z-S, Tan Y-Y, Chen S-D, Jin H-J, et al. Theorigin, transmission and clinical therapies on Coronavirus Disease 2019
(COVID-19) outbreak-an update on the status. Mil Med Res. 2020;7(1):11.
3. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for therecognition of SARS-CoV-2 by full-length human ACE2. Science.
2020;367(6485):1444-8.
4. Ogata N. Chlorine dioxide gas for the prevention of infectious diseases.
Eur J Pharm Med Res. 2017;4(8):678-85.
5. Ogata N, Shibata T. Protective effect of low-concentration chlorine dioxide
gas against influenza A virus infection. J Gen Virol. 2008;89(Pt 1):60-7.

Figure 1: Binding of SARS-CoV-2 coronavirus spike protein to human
Angiotensin-Converting Enzyme 2 (ACE2). The figure shows an enlarged
view of the binding domain of the viral spike protein (brown) to an α1 helix ofthe ACE2 (blue) demonstrating hydrogen-bonding of tyrosine 453 (Y453) of
the spike protein and histidine 34 (H34) (Protein Data Bank, EMD-30046).

6. Ogata N, Sakasegawa M, Miura T, Shibata T, Takigawa Y, Taura K, etal. Inactivation of airborne bacteria and viruses using extremely low
concentrations of chlorine dioxide gas. Pharmacology. 2016;97(5-6):301-6.
7. Ogata N. Denaturation of protein by chlorine dioxide: oxidative
modification of tryptophan and tyrosine residues. Biochemistry.
2007;46(16):4898-911.

Japan). The difference in luminescence intensity was statisticallyevaluated by Student t test and considered significant if p<0.05.

8. Ogata N. Inactivation of influenza virus haemagglutinin by chlorine
dioxide: Oxidation of the conserved tryptophan 153 residue in thereceptor-binding site. J Gen Virol. 2012;93(Pt 12):2558-63.

Results and Discussion
As shown in Table 1, CD at 0.25 mmol/L or 0.5 mmol/L
was effective in inactivating the binding of the spike protein toACE2 (p<0.001). However, CD was not effective at 0.1 mmol/Lconcentration. The result strongly suggests that CD in sufficientconcentration is effective in inactivating the spike protein of SARSCoV-2 coronavirus and thus preventing its binding to human cells.
CD aqueous solution will be a useful disinfection measure to preventthe spread of the virus.

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