Annals of Pharmacology and Pharmaceutics

Research Article
Published: 06 Dec, 2021

Effect of Chlorine Dioxide Gas on the Binding of SARSCoV-2 Coronavirus Spike Protein to a Human Receptor
Norio Ogata* and Takanori Miura
Department of R&D, Taiko Pharmaceutical Co., Ltd, Japan

Abstract
Aim: COVID-19, caused by a new coronavirus SARS-CoV-2, remains a serious threat to humanhealth despite the development of several vaccines. Thus, a safe and effective disinfection systemis urgently needed to prevent the further spread of COVID-19 cases in crowded and/or poorly
ventilated areas. Chlorine dioxide (CD) gas is known to disinfect indoor air in hospital andhealthcare settings. The aim of this work was to perform in vitro experiments to investigate
whether CD gas could be used to block the capacity of SARS-CoV-2 to bind its receptor protein.
Materials and Methods: An assay kit was used to measure binding of the spike protein of SARSCoV-2 virus to the human receptor protein, angiotensin-converting enzyme 2. We first measured
concentration of CD that can dissolve in water. We next performed assays using spike protein that
had been exposed to various concentrations of CD gas.
Results: CD gas dissolves in water in a concentration- and time-dependent manner to reach nearsaturation after 30 min. Almost complete inactivation of the spike protein-receptor protein
binding was found at about 3 parts per million (ppmv) (volume/volume ratio) of gas with a halfInhibition concentration (IC50) of 1.2 ppmv after 30 min at 25°C.
Conclusion: CD gas can dissolve in water in a concentration- and time-dependent manner, and it
can be used as an effective disinfectant against SARS-CoV-2. The mechanism of disinfection involves
inhibition of binding of the virus spike protein to human receptor protein in an aqueous phase.
Keywords: Chlorine dioxide; COVID-19; SARS-CoV-2; Virus; Disinfection; IC50

OPEN ACCESS*Correspondence:
Norio Ogata, Department of R&D,
Taiko Pharmaceutical Co., Ltd., 1-2-1
Hikaridai, Seikacho, 619-0237, Kyoto,
Japan, Tel: 81 774 98 2716; Fax: 81
774 98 2737;
E-mail: nogata7@yahoo.co.jp
Received Date: 04 Nov 2021
Accepted Date: 25 Nov 2021
Published Date: 06 Dec 2021
Citation:
Ogata N, Miura T. Effect of Chlorine
Dioxide Gas on the Binding of SARSCoV-2 Coronavirus Spike Protein toa Human Receptor. Ann Pharmacol
Pharm. 2021; 6(1): 1202.
Copyright © 2021 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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Introduction
The COVID-19 pandemic began in Wuhan, China in late 2019, and then spread rapidly acrossthe entire world [1-3]. The disease is caused by a SARS-CoV-2 virus, which is an enveloped virus
with a positive single-stranded RNA genome [3]. The virus has a “spike protein” comprising 1,273
amino acids on its surface that can bind to a human receptor protein, angiotensin-converting
enzyme 2 (ACE2), to facilitate entry into the host cell [3]. Although several effective vaccines havebeen developed against SARS-CoV-2, mostly modified mRNA vaccines [4,5], the COVID-19
pandemic has not yet ended [1]. Oral anti-virus medicines are under development in many
countries but, to date, none are commercially available [6].
Chlorine dioxide (CD) is a water-soluble free radical gas with strong oxidation activity [7,8].
The gas has been shown to protect against infection from influenza virus in an experiment using
mice and an aerosol suspension of viral particles [9]. CD also inactivates other viruses [10,11], atconcentrations low enough to have no discernable adverse effect on human [12,13]. The
corresponding mechanism of action of CD is related to the oxidation of tryptophan residues inviral proteins [14,15]. We recently demonstrated that CD dissolved in water can inhibit the bindingof SARS-CoV-2 to human ACE2 [16,17]. Moreover, the inhibition of binding of SARS-CoV-2 tohuman ACE2 was shown to be due to inactivation of the viral spike protein of the original Wuhan
strain as well as the subsequent alpha (B.1.1.7) and beta (B.1.351) variants [16,17]. Given that CD
gas can prevent viral infection in animal experiments [9], it seems likely that the gas will dissolve inorinasal aerosols expelled from human. We reasoned the resulting dissolved CD may inactivate viralparticles in the aerosol. The aim of this study was to investigate whether CD gas dissolved in water
can inactivate the capacity of the viral spike protein to bind human ACE2 in the aqueous phase.
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Norio Ogata, et al.,Annals of Pharmacology and Pharmaceutics

Material and Methods
Chemicals and software
A SARS-CoV-2 spike protein-human receptor protein binding
assay kit (BPS Bioscience, San Diego, CA, USA) was used that employs
the Wuhan strain spike protein. In this kit, purified and histidine
tag-labeled human angiotensin-converting enzyme 2 (ACE2) wasused as a receptor protein. Data points were fitted by regression
analysis to a linear, an exponential increase or an exponential decay
curve using GraphPad Prism software (San Diego, CA, USA). Where
appropriate the half life was obtained from the exponential curves.
Solubility measurements of CD in water

Figure 1: Illustration showing the experimental setup for chlorine dioxide
generation, measurement of its concentration and evaluation of the effect ofchlorine dioxide on the binding of SARS-CoV-2 spike protein to angiotensinconverting enzyme 2. Samples were placed in a plastic box (dimensions:
174 mm × 246 mm × 173 mm). A chlorine dioxide-generating canister, anelectric fan, a battery box and a chlorine dioxide detector tube are shown.
Three layers of spike protein-coated or non-coated 96-well microtiter plates
are also shown.

The amount of dissolved CD at various concentrations of CDgas was determined at 25°C spectrophotometrically from absorption
measurements at 358 nm using a molar extinction coefficient of1570 L∙mol-1∙cm-1.
Binding assay
The binding assay kit was used according to the protocol outlinedby the manufacturer (BPS Bioscience) with some modifications.
Specifically, 50 µl of purified water, rather than Immuno Buffer 1
from the kit was aliquoted into each well (depth, 11.25 mm; innerdiameter, 6.75 mm) of a flat bottomed 96-well microtiter plate shortly
after coating with spike protein. CD gas was generated in a plastic box(dimensions: 246 mm × 175 mm × 174 mm) using a commercial tabletop CD-generating canister (Cleverin, 150 g; Taiko Pharmaceutical,
Osaka, Japan) (Figure 1).
The rate of release of CD gas was adjusted by covering the topof the canister with aluminum foil and then making a few pinholes
in the foil. The 96-well plate containing aliquots of water was placed
into the plastic box containing CD gas for 30 min at 25°C unlessotherwise indicated (Figure 1). After 30 min, 20 µl of 10 mmol/L
Na2S2O3 was added to each well to terminate the reaction of CD withthe spike protein. Specifically, CD reacts with Na2S2O3 to generate
ClO2–[14]. The extent of spike protein-ACE2 binding was measured
by chemiluminescence generated by horseradish peroxidase using aluminometer (model SH-9000; Corona Electric, Hitachinaka, Japan).
CD gas concentration in the plastic box was measured using a CD
detector (23 M tube; GASTEC, Ayase, Japan). The concentration of
CD dissolved in the 50 µl aliquot of water could also be measured
using a 96-well microtiter plate without spike protein coating.

Figure 2: Concentration of chlorine dioxide in water as a function of chlorine
dioxide gas concentration (A) when the microtiter plates were exposed for 30
min. Time course of the increase of chlorine dioxide concentration in water(B) when the microtiter plates were exposed to 6.2 ppmv (filled circles) or 1.5
ppmv (open circles) chlorine dioxide gas. Each data point represents a single
experiment. All experiments were performed at 25°C.

Results
CD gas dissolved in the water in a concentration- and timedependent manner (Figure 2). As shown in Figure 2A, the amount ofCD gas dissolved in the purified water increased almost linearly upon
the increase of CD partial pressure.

Figure 3: Effect of chlorine dioxide gas of various concentrations on
the binding of the SARS-CoV-2 spike protein to angiotensin-converting
enzyme 2. The spike protein-coated microtiter plate was treated withvarious concentrations of chlorine dioxide gas at 25°C for 30 min, and thebinding ability of the spike protein to the receptor protein was subsequently
measured by chemiluminescence. Each data point represents an average ±standard deviation of four independent experiments. Data points were fitted
to an exponential decay curve, whose 50% decay point (IC50) was found tobe 1.2 ppmv.

Thus, the amount of dissolved CD in the water was dependent
on the partial pressure of CD gas, and therefore obeyed Henry’s law[18,19]. In the equilibrium between the gaseous and liquid phases of
the gas, the amount of CD gas in the liquid phase obeys the equation
C=kP, where C is the concentration of CD in the liquid phase, P ispartial pressure of CD gas in the gaseous phase and k is the equilibrium
constant. From Figure 2A, the slope k measured between the CD gas
concentration from 2.0 to 6.1 ppmv after 30 min at 25°C was equivalent
to approximately 2.8 × 10-6 mol•L-1•ppmv-1 or 2.8 × 10-5 mol•L-1•Pa-1.
As shown in Figure 2B, the concentration of CD in water reached anear plateau after 30 min under the experimental conditions used inthis study. The data points in Figure 2B were fitted to exponential
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increase curves, with a half increase point of 4.6 min (filled circles)
and 8.3 min (open circles). Subsequent experiments were performed
30 min after exposure to the gas. Different concentrations of CD gas
were used in the experimental setup to expose a 96-well microtiterplate containing 50 µl water in each well. The binding ability of the22021 | Volume 6 | Issue 1 | Article 1202

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

spike protein to ACE2 was subsequently measured. Binding wascompletely inhibited at 3 ppmv of CD gas (Figure 3).

4. Trivedi N, Verma A, Kumar D. Possible treatment and strategies for
COVID-19: Review and assessment. Eur Rev Med Pharmacol Sci.
2020;24(23):12593-608.

The 50% inhibitory concentration (IC50) was 1.2 ppmv for 30
min at 25°C. Noteworthy, at a gas concentration of 3 ppmv the CDconcentration in water was 4.1 µmol/L after 30 min (Figure 2A),
which is consistent with the results shown in Figure 3.

5. Wibawa T. COVID-19 vaccine research and development: Ethical issues.
Trop Med Int Health. 2021;26(1):14-9.
6. Meng L, Hua F, Bian Z. Coronavirus Disease 2019 (COVID-19):
Emerging and future challenges for dental and oral medicine. J Dent Res.
2020;99(5):481-7.

Discussion

7. Ogata N. Chlorine dioxide gas for the prevention of infectious diseases.
Eur J Pharma Med Res. 2017;4(8):678-85.

Here, we confirmed that gaseous CD dissolves in water with anestimated Henry's equilibrium constant of 2.8 × 10-5 mol•L-1•Pa-1.
This value is close to 1.3 × 10-5 mol•L-1•Pa-1 as reported by Ishi [19].
CD gas is known to dissolve in water with a maximal achievable
concentration of 8 g/L [20]. Using a CD concentration of 6.2 ppmv
the maximum concentration of CD in water was 7.2 µmol/L (Figure2). Indeed, Ishi reported [19] that at a CD gas concentration of
6.2 ppmv dissolved CD in water was 8.1 µmol/L, which is in good
agreement with the present results.

8. Shimakura H, Ogata N, Kawakita Y, Ohara K, Takeda S. Determination of
the structure of liquids containing free radical molecules: Inter-molecular
correlations in liquid chlorine dioxide. Mol Phys. 2013;111(8):1015-22.
9. 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.
10. Morino H, Fukuda T, Miura T, Shibata T. Effect of low-concentration
chlorine dioxide gas against bacteria and viruses on a glass surface in wet
environments. Lett Appl Microbiol. 2011;53(6):628-34.

We recently reported that a CD aqueous solution added to a
96-well microtiter plate containing spike protein from the Wuhan
strain of SARS-CoV-2 virus or the alpha (B.1.1.7) and beta (B.1.351)
variants showed inhibition in their binding to ACE2 protein [16,17].
IC50 concentrations of this inhibition for the alpha (B.1.1.7) and beta
(B.1.351) variants were, 7.6 and 5.8 µmol/L, respectively [16,17].
These values closely correspond to the IC50 values determined in thepresent study. These findings strongly indicate that CD gas, once
dissolved in water, can inactivate binding of the spike protein to its
receptor as found in the previous aqueous phase-only experiments[16,17]. Aerosols expelled from virus-infected patients may containother components, such as proteins and salts. However, we speculatethat these components do not interfere with CD from modifying
spike protein to block receptor protein binding as reported in thisstudy because their concentrations in the expelled aerosols are very
low [21].

11. Morino H, Fukuda T, Miura T, Lee C, Shibata T, Sanekata T. Inactivationof feline calicivirus, a norovirus surrogate, by chlorine dioxide gas.
Biocontrol Sci. 2009;14(4):147-53.
12. 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.
13. Akamatsu A, Lee C, Morino H, Miura T, Ogata N, Shibata T. Six-month
low level chlorine dioxide gas inhalation toxicity study with two-week
recovery period in rats. J Occup Med Toxicol. 2012;7:2.
14. Ogata N. Denaturation of protein by chlorine dioxide: Oxidative
modification of tryptophan and tyrosine residues. Biochemistry.
2007;46(16):4898-911.
15. Ogata N. Inactivation of influenza virus haemagglutinin by chlorine
dioxide: oxidation of the conserved tryptophan 153 residue in the receptorbinding site. J Gen Virol. 2012;93(Pt 12):2558-63.

Conclusion

16. Ogata N, Miura T. Inhibition of the binding of spike protein of SARSCoV-2 coronavirus to human angiotensin-converting enzyme 2 bychlorine dioxide. Ann Pharmacol Pharm. 2020;5(5):1195.

CD gas can inhibit the binding of the spike protein of SARSCoV-2 virus to the human receptor protein ACE2 after it is dissolvedin water in a concentration-dependent manner. The IC50 of CD was
1.2 ppmv after exposure to the gas at 25°C for 30 min. These findings
strongly justify the use of CD gas to prevent the spread of SARSCoV-2 virus in enclosed spaces.

17. Ogata N, Miura T. Inhibition of the binding of variants of SARS-CoV-2
coronavirus spike protein to a human receptor by chlorine dioxide. Ann
Pharmacol Pharm. 2021;6(1):1199.
18. Dukes AD III. Measuring the Henry's law constant for carbon dioxide andwater with UV-visible absorption spectroscopy. Anal Sci. 2020;36(8):9715.

Acknowledgement

19. Ishi G. Solubility of chlorine dioxide. Chem Eng Japan. 1958;22(3):153-4.

This work was supported by Taiko Pharmaceutical Co., Ltd.

20. Chlorine dioxide. Center for disease control and prevention. 1999; ICSC
0127-Chlorine dioxide (ilo.org).

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