Oxoammonium salts exert antiviral effects against coronavirus via denaturation of their spike proteins | Scientific Reports

Oct 17, 2024

Scientific Reports volume 14, Article number: 23934 (2024) Cite this article

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Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV2) infection has forced social changes worldwide. Development of potent antiviral agents is necessary to prevent future pandemics. Titanium oxide, a photocatalyst, is a long-acting antiviral agent; however, its effects are weakened in the dark. Therefore, new antiviral substances that can be used in the dark are needed. Two types of nitroxyl radicals, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) and 2-azaadamantane N-oxyl (AZADO), are commonly used as oxidation catalysts utilizing oxygen in the air as the terminal oxidant. Therefore, in this study, we aimed to evaluate the potential of these radicals as antiviral compounds with sustained activity even in the dark. We evaluated the antiviral effects of oxoammonium salts corresponding to TEMPO and AZADO (TEMPO-Oxo and AZADO-Oxo, respectively), which are the active forms of nitroxyl radicals in oxidation reactions. TEMPO-Oxo and AZADO-Oxo inhibited the binding of SARS-CoV2 spike protein receptor-binding domain (S-RBD) to angiotensin-converting enzyme 2. Notably, AZADO-Oxo exhibited a 10-fold stronger inhibitory effect than TEMPO-Oxo. TEMPO-Oxo and AZADO-Oxo also denatured S-RBD; however, effects of AZADO-Oxo were 10-fold stronger than those of TEMPO-Oxo and did not change in the dark. Some S-RBD peptides treated with AZADO-Oxo were cleaved at the N-terminal side of tyrosine residues. TEMPO-Oxo and AZADO-Oxo exhibited concentration-dependent antiviral effects against feline coronavirus. In conclusion, active forms of the nitroxyl radicals, TEMPO-Oxo and AZADO-Oxo, exerted antiviral effects by denaturing S-RBD, regardless of the presence or absence of light, suggesting their potential as novel antiviral agents.

Since its emergence, coronavirus disease 2019 (COVID-19) have rapidly spread worldwide. The World Health Organization reported more than 700 million confirmed cases of COVID-19, including nearly 7 million deaths, till October, 2023. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is the coronavirus causing COVID-19. Coronaviruses have caused many pandemics, such as SARS caused by SARS-CoV1 in 2002–2003 and Middle East respiratory syndrome (MERS) caused by MERS-CoV in 20121. Effective infection prevention methods need to be developed to prevent such pandemics. Chemical disinfectants play a vital role in preventing infections.

Currently, alcohols, quaternary ammonium salts, and sodium hypochlorite are applied on exterior walls, furniture, medical equipment, and electrical appliances for disinfection. These chemicals inactivate bacteria and viruses by destroying their lipid membranes or denaturing their proteins. However, alcohols evaporate easily, and sodium hypochlorite has a short half-life, making then unsuitable for long-term disinfection. Titanium oxide (TiO2) is used for sustained bacterial and virus inactivation2. TiO2 continuously generates hydroxyl radicals in response to UV light and exhibits strong oxidizing effects2. This mechanism primarily contributes to the inactivation of bacteria and viruses. Furthermore, TiO2-containing substances can deactivate viruses even under visible light irradiation3. These substances are used as coating agents for the long-term deactivation of bacteria and viruses. However, the antiviral effects of such photocatalysts are reduced in the dark3. Therefore, development of disinfectants that do not weaken in the dark and maintain their anti-bacterial and antiviral effects is critical to prevent future pandemics. Here, we focused on the nitroxyl radicals, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) and 2-azaadamantane N-oxyl (AZADO).

Nitroxyl radicals are commonly used as catalysts for alcohol oxidations in both laboratories and industries. Nitroxyl radicals generate their corresponding oxoammonium ions as the catalytically active species for alcohol oxidation and can be isolated as salts in the presence of appropriate counteranions4. Nitroxyl-radical-catalyzed alcohol oxidation proceeds smoothly with various terminal oxidants, including molecular oxygen in air5,6,7. Alcohol oxidation is not light dependent; hence it can be used for sustained oxidation in the dark. A nitroxyl radical promotes peptide cleavage under specific conditions, possibly mediated by the oxidation of alcohol groups of serine residues8. Therefore, we hypothesized that nitroxyl radicals sustain the oxidation reactions even in dark conditions. Although some TEMPO-adduct compounds exert antibacterial effects9, the action mechanisms and active species remain unknown. Furthermore, the antiviral effects of AZADO remain unclear. Therefore, in this study, we evaluated the antiviral effects of the oxoammonium salts corresponding to TEMPO and AZADO (TEMPO-Oxo and AZADO-Oxo, respectively), the isolatable active species of TEMPO- and AZADO-catalyzed alcohol oxidation, on coronaviruses and investigated their action mechanisms.

SARS-CoV2 enter into cells through the binding of its spike proteins to ACE2 receptors10,11. To evaluate the effects of TEMPO-Oxo and AZADO-Oxo on the binding of the SARS-CoV2 spike protein receptor-binding domain (S-RBD) to ACE2, we conducted flowcytometric analysis as previously reported by Tai et al.11, using recombinant S-RBD and ACE2 293T cells, which are HEK293T cells stably transfected with human ACE2. Chemical structures of TEMPO, AZADO, TEMPO-Oxo, and AZADO-Oxo are shown in Fig. 1a. S-RBD exhibited stronger binding to ACE2 293T cells than non-transfected HEK293T cells (Supplementary Fig. 1). Preincubation of S-RBD with TEMPO-Oxo (Fig. 1b) and AZADO-Oxo (Fig. 1c) decreased the binding of S-RBD to ACE2 293T cells in a molar ratio-dependent manner. Based on the median of fluorescent intensity (MFI), we quantitatively compared the effects of TEMPO-Oxo and AZADO-Oxo on the ACE2-binding activity of S-RBD. Both oxoammonium salt treatments significantly inhibited ACE2-binding activity of S-RBD when the molar ratio of oxoammonium salts/S-RBD was above 5 (Fig. 1d). Further, the inhibitory effect of AZADO-Oxo at the molar ratio of 5 was almost the same as that of TEMPO-Oxo at the molar ratio of 50 (Fig. 1d), indicating that AZADO-Oxo is approximately 10-fold more potent than TEMPO-Oxo. Ascorbic acid, an antioxidant, completely cancelled this inhibitory effect of AZADO-Oxo (Supplementary Fig. 2).

Inhibitory effect of TEMPO-Oxo and AZADO-Oxo on the binding of S-RBD to ACE2. (a) Chemical structures of TEMPO, AZADO, TEMPO-Oxo, and AZADO-Oxo. (b and c) ACE2 293T cells were incubated with TEMPO-Oxo (b)- and AZADO-Oxo (c)-treated S-RBD and the binding of S-RBD to ACE2 was analyzed by flowcytometry. TEMPO-Oxo and AZADO-Oxo treatments were performed at the indicated molar ratios for 30 min. Representative histograms with FITC fluorescence intensity on the x-axis and cell counts on the y-axis. (d) MFI of each group was determined by flowcytometry. **P < 0.01 vs. non-AZADO-Oxo-treated group (AZADO-Oxo/S-RBD = 0) and ##P < 0.01 vs. non-TEMPO-Oxo-treated group (TEMPO-Oxo/S-RBD = 0). All data are represented as the mean ± S.E.M. (n = 3).

To clarify whether TEMPO-Oxo and AZADO-Oxo denature the S-RBD via their oxidative activities, changes in the amount of S-RBD were analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue (CBB) staining. Treatment of S-RBD with TEMPO-Oxo or AZADO-Oxo at the indicated molar ratios for 30 min decreased the amount of S-RBD at the correct molecular weight (Fig. 2a and b). Decrease in the intensity of S-RBD bands was significant when the molar ratio of TEMPO-Oxo/S-RBD was > 15 and that of AZADO-Oxo/S-RBD was > 5 (Fig. 2c). The reduction in the S-RBD band intensity with an AZADO-Oxo/S-RBD molar ratio of 5 was almost the same as that with a TEMPO-Oxo/S-RBD molar ratio of 15 or 50 (Fig. 2c). Notably, only 1 min treatment with AZADO-Oxo at the molar ratio 50 was enough to denature the S-RBD (Fig. 2d).

Denaturation of S-RBD by TEMPO-Oxo and AZADO-Oxo. (a and b) Representative CBB-stained gel images of TEMPO-Oxo (a)- and AZADO-Oxo (b)-treated S-RBD samples. TEMPO-Oxo and AZADO-Oxo treatments were performed at the indicated molar ratios for 30 min. The S-RBD band was observed 35–40 kDa. (c) Band intensity of TEMPO-Oxo- and AZADO-Oxo-treated S-RBD. Each band intensity was quantified using the ImageJ software and the intensity ratio was calculated. **P < 0.01 vs. non-AZADO-Oxo-treated group (AZADO-Oxo/S-RBD = 0) and ##P < 0.01 vs. non-TEMPO-Oxo-treated group (TEMPO-Oxo/S-RBD = 0). All data are represented as the mean ± S.E.M. (n = 3).

To confirm the effects of AZADO-Oxo in the dark, we treated S-RBD with AZADO-Oxo in the dark. No difference in AZADO-Oxo activity was observed in the dark or not (Fig. 3a). Moreover, S-RBD denaturation induced by AZADO-Oxo in the dark was almost the same as that not in the dark (Fig. 3b).

Effect of AZADO-Oxo activity against S-RBD in the dark. (a and b) AZADO-Oxo treatment was performed as the indicated molar ratios for 30 min in the dark or not. (a) MFI of each group was determined by flowcytory. (b) Band intensity was determined by CBB staining and quantification using the ImageJ software. *P < 0.05, **P < 0.01 vs. non-AZADO-Oxo-treated group not in the dark and #P < 0.05, ##P < 0.01 vs. non-AZADO-Oxo-treated group in the dark. All data are represented as the mean ± S.E.M. (n = 3).

To clarify whether AZADO-Oxo cleaves S-RBD, S-RBD peptides were treated with AZADO-Oxo and their degradation was analyzed using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). S-RBD contains ACE2- and heparan sulfate-binding sites11,12,13. Heparan sulfate enhances the binding of S-RBD to ACE214,15. RFASVYAWNR and SKVGGNYNYL contains the ACE2- and heparan sulfate-binding sites of the S-RBD of wild-type SARS-CoV2, respectively. SKVSGNYNYL is based on the S-RBD of the SARS-COV2 omicron variant. Here, each S-RBD partial peptide peak disappeared, and only some peaks were detected after treatment with AZADO-Oxo (Fig. 4a and b). A fragment peak of RFASV (m/z 579 [M + H]+) was detected in the reaction sample treated with RFASVYAWNR and AZADO-Oxo (Fig. 4c). The fragment peaks of SKVGGN (m/z 561 [M + H]+) and SKVGGNYN (m/z 420 [M + 2 H]2+) were detected in the reaction sample treated with SKVGGNYNYL and AZADO-Oxo (Fig. 4d). Moreover, fragment peaks of SKVSGN (m/z 591 [M + H]+) and SKVSGNYN (m/z 435 [M + 2 H]2+) were detected in the reaction sample treated with SKVSGNYNYL and AZADO-Oxo (Fig. 4e). From MS/MS analysis of AZADO-Oxo treated-SKVSGNYNYL peptide, fragment ion peaks including oxidized tyrosine were detected (Supplementary Fig. 3). Further, we confirmed a catechol was obtained from the reaction of tyrosine residue with AZADO-Oxo. (Supplementary Fig. 4). These results suggest that AZADO-Oxo cleaves peptides on the N-terminal side of tyrosine residues through tyrosine oxidation (Fig. 4f).

Identification of AZADO-Oxo cleavage site in S-RBD peptides. Total ion current chromatogram (TICC) of (a) non-AZADO-Oxo or (b) AZADO-Oxo-treated mixture of RFASVYAWNR (top), SKVGGNYNYL (middle), and SKVSGNYNYL (bottom) for 24 h. Extracted ion chromatogram (EIC) and MS/MS of (c) RFASV (m/z 579 [M + H]+), (d) SKVGGN (m/z 560 [M + H]+) and SKVGGNYN (m/z 420 [M + 2 H]2+), (e) SKVSGN (m/z 590 [M + H]+), and SKVSGNYN (m/z 435 [M + 2 H]2+). (f) Cleavage sites of the S-RBD partial peptides treated with AZADO-Oxo.

To evaluate the antiviral activities of TEMPO-Oxo and AZADO-Oxo, we infected Crandell-Rees Feline Kidney (CRFK) cells with FCoV. First, we evaluated the concentration-dependence of TEMPO-Oxo and AZADO-Oxo on the viability and rate of cytopathic effect (CPE) of FCoV-infected cells. As shown in Fig. 5a, FCoV infection for three days decreased the cell viability by approximately 50%. Treatment with 10 mM TEMPO-Oxo or 1 mM AZADO-Oxo restored the cell viability to that in the virus-free group. More than 1 mM TEMPO-Oxo significantly suppressed the CPE of FCoV (Fig. 5b). AZADO-Oxo suppressed the CPE in a concentration-dependent manner at lower concentrations than TEMPO-Oxo (Fig. 5b). In the photograph of AZADO-Oxo treated cells, the dots caused by the CPEs of FCoV completely disappeared (Fig. 5c). Consistently, treatment with 1 mM AZADO-Oxo for 2 h significantly reduced the 50% tissue culture infectious dose (TCID50) of FCoV by less than 10− 3 times (Fig. 5d).

Antiviral effects of TEMPO-Oxo and AZADO-Oxo against FCoV. (a) Viability of FCoV-treated CRFK cells. FCoV solution was incubated with the indicated concentrations of TEMPO-Oxo and AZADO-Oxo for 2 h before FCoV infection. FCoV-infected CRFK cells were cultured for three days, and their viability was measured using cell counting kit-8. Results are shown as a percentage of viability compared to that of the virus-free group set at 100%. **P < 0.01 vs. non-AZADO-Oxo-treated FCoV-infected group and ##P < 0.01 vs. non-TEMPO-Oxo-treated FCoV-infected group. All data are represented as the mean ± S.E.M. (n = 24). (b) Rate of CPE in each FCoV-treated group. TEMPO-Oxo and AZADO-Oxo treatments were performed as mentioned above. *P < 0.05, **P < 0.01 vs. non-AZADO-Oxo-treated FCoV-infected group and ##P < 0.01 vs. non-TEMPO-Oxo-treated FCoV-infected group. All data are represented as the mean ± S.E.M. (n = 3). (c) Representative image of CPE after FCoV infection. (d) Change of TCID50 of FCoV after AZADO-Oxo treatment. FCoV solution with or without 1 mM AZADO-Oxo treatment for 2 h was used for the experiment. TCID50 of FCoV was calculated using Behrens–Karber method.

In this study, for the first time, we revealed that oxoammonium salts corresponding to TEMPO and AZADO denature the S-RBD of SARS-CoV2 and exert antiviral effects against coronavirus. These results provide valuable information for the practical applications of active form of nitroxyl radicals.

TEMPO and AZADO catalyze the oxidiation of alcohols using oxygen in the air6,8. The ability to catalytically exhibit antiviral activity using oxygen in the air is necessary to exert sustained antiviral effects in the living environment. In this study, we evaluated the effects of the oxoammonium salts to gain insights into the antiviral effect of nitroxyl radicals. TEMPO-Oxo and AZADO-Oxo inhibited the binding of S-RBD via their oxidizing ability, possibly via the cleavage of S-RBD, and exhibited antiviral activity. Our findings suggest that TEMPO-Oxo and AZADO-Oxo exert antiviral effects by denaturing the virus surface proteins. Notably, the effects of AZADO-Oxo were approximately 10-fold greater than those of TEMPO-Oxo. Moreover, AZADO exhibits less steric hindrance at the active site and much higher catalytic activity for alcohol oxidation than TEMPO15. As proteins have complex three-dimensional structures, we hypothesized that the low steric hindrance of AZADO-Oxo is responsible for its higher antivirus activity than that of TEMPO-Oxo. The next step should be to establish a method for efficiently producing oxoammonium ions from nitroxyl radicals using oxygen in the air in our living environment.

Here, treatment with 20 pmol of S-RBD and 1 nmol of AZADO-Oxo for 30 min completely inhibited the binding to ACE2 protein. SARS-CoV2 is an enveloped virus with a positive-sense single-stranded RNA genom16. The diameter of the virus particles is approximately 100–200 nm, and S-protein present on the particle surface is composed of the S1 and S2 subunits17. S1 subunit has one ACE2 receptor-binding domain. The S-protein on the virus surface exists as a homotrimer, with one RBD per S-protein monomer facing the extracellular side17. The binding of RBD to ACE2 receptors on the cell surface triggers the SARS-CoV2 infection10. Approximately 25–50 S-protein trimers, containing approximately 75–150 RBDs, exist in a single virus particle17. Hence, 20 pmol of RBD is equivalent to the number of RBDs in 0.8–1.6 × 1011 virions. Sender et al. have reported that 109-1011 SARS-CoV2 virions are present in the lung tissues of SARS-CoV2-infected individuals18. They also revealed that 109 to 1011 SARS-CoV2 virions have virus titers of 105 to 107 TCID50. These reports suggest that 1 nmol of AZADO-Oxo potentially inactivates 105 to 107 TCID50 of SRAS-CoV2 virus. This antiviral potential is comparable to that of TiO2-coating substances19 and is sufficient for use as an antiviral agent.

We found that AZADO-Oxo-induced peptide cleavage occurred selectively at N-terminal side of the tyrosine residues. We found tyrosine residue oxidized to a catechol by AZADO-Oxo. L-dopa, bearing a similar catechol moiety, is known to give dihydroxyindole via a cyclization under oxidative conditions20,21. From this insight, we proposed a plausible mechanism of AZADO-Oxo induced peptide cleavage via a catechol (Supplementary Fig. 4). Our study is the first to report the peptide degradation site of AZADO-Oxo. Seki Y. et al. have reported that keto-ABNO, another nitroxyl radical, cleaves peptides around serine residues8. The amino acid residues targeted for cleavage differ depending on the type of nitroxyl radical. Alternatively, the nitroxyl radical species may exhibit different amino acid residue reactivities depending on the peptide sequence. Analyses using a wide range of proteins and nitroxyl radicals are required in the future. In any case, the amino acid-selective cleavage of peptides by nitroxyl radical species is a useful method for modifying middle molecules and biomolecules and for the structural analysis of proteins.

TEMPO-Oxo and AZADO-Oxo induce the denaturation of S-RBD, a viral surface protein, and exhibit antiviral activity against FCoV, which uses feline aminopeptidase N as a receptor22. These compounds denature proteins on the surface of various viruses, including non-enveloped viruses, such as rotavirus, which cannot be inactivated by alcohol or quaternary ammonium salts. We further confirmed the inhibitory effect of AZADO-Oxo on the binding of mutated S-RBD (Omicron variant and Y508H variant) to ACE2 receptors (Supplementary Fig. 5). Here, we confirmed that AZADO-Oxo exhibited significant bactericidal activity against Escherichia coli (see Supplementary Fig. 6). Some compounds containing nitroxyl radicals exert anti-bacterial effects for Pseudomonas aeruginosa and Staphylococcus epidermidis9,23. AZADO-Oxo also denatured ovalbumin and green fluorescent protein (Supplementary Fig. 7). Oxoammonium salts denature various types of proteins; therefore, they will be able to target variety of environmental allergens, viruses and bacteria.

It is important as a future candidate of disinfectant to consider toxicity of oxoammonium salts to living cell and tissues. ACE2 293T cells showed significant cytotoxicity when treated with 1 mM or more of AZADO-Oxo (Supplementary Fig. 8), which is similar to the concentration at which antiviral activity is observed against FCoV. Given that AZADO-Oxo acts on a variety of proteins as targets, it is reasonable that cytotoxic concentration is similar to the concentration expressing antiviral activity. It will be necessary to appropriately select the concentration and the carriers when applying the nitroxyl radical or its oxoammonium salts in the future.

In this study, AZADO-Oxo inhibited the binding of S-RBD to ACE2 and degraded S-RBD, even in the dark. This activity was equal to that observed under visible-light irradiation. Although some TiO2-containing substances exert antiviral effects under both UV and visible light, their effect in the dark is reduced to less than 1/10 of that in the presence of light3. Therefore, nitroxyl radicals can effectively be used as antiviral agents in the dark. In combination with technologies facilitating their sustained antiviral effects, nitroxyl radicals can be used to eliminate viruses in environments where TiO2 is ineffective. We are currently investigating the sustainability of the antiviral effects of AZADO.

To the best of our knowledge, this study is the first to demonstrate the potential of oxoammonium salts corresponding to TEMPO and AZADO as useful antiviral and anti-bacterial agents. The antiviral activity of AZADO-Oxo was more potent than that of TEMPO-Oxo and not suppressed even in the dark. Furthermore, the protein denaturation mechanism of AZADO-Oxo involved the cleavage of peptides through tyrosine oxidation. As a conclusion, active form of nitroxyl radicals, oxoammonium salts, are new antiviral candidate materials effectively using even in the dark. By establishing the optimal conditions for sustained antiviral effects in future studies, nitroxyl radicals can be used as effective tools to prevent future pandemics.

Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Sigma (St. Louis, MO, USA) and penicillin G potassium and streptomycin sulfate were obtained from Meiji Seika Co. (Tokyo, Japan). TEMPO-Oxo and AZADO-Oxo were prepared as previously described24. Histidine-tagged recombinant S-RBD (RP-87678) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Quick CBB PLUS was purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). FITC-conjugated Anti-6X His tag antibody (AD1.1.10) was also purchased from Thermo Fisher Scientific. Cell counting kit-8 was purchased from DOJINDO Laboratories (Kumamoto, Japan). Protein LoBind tubes (0.5 mL) were purchased from Eppendorf (Hamburg, Germany). S-RBD partial peptides were purchased from GenScript (Piscataway, NJ, USA). All sequences are shown in Fig. 4.

ACE2 293T cells were purchased from Takara Bio Inc. (Shiga, Japan). CRFK cells were kindly provided by Dr. Kodama (Tohoku University, Japan). These cells were cultured in DMEM supplemented with 10%FBS, 18 µg/mL of penicillin G potassium, and 50 µg/mL streptomycin sulfate, and maintained at 37 °C, 5% CO2, and 95% relative humidity. FCoV (WSU 79-1683) was kindly provided by Dr. Kodama. FCoV was grown in confluent CRFK cells. The collected FCoV culture medium was used as the FCoV stock medium. TCID50 of the FCoV stock medium was calculated using the Behrens–Karber method.

S-RBD (20 pmol) was treated with TEMPO-Oxo or AZADO-Oxo in phosphate-buffered saline (PBS) at 20 to 25 °C for 1 to 30 min under conditions in which the molar ratio of S-RBD and TEMPO-Oxo or AZADO-Oxo was 1:5, 1:15 and 1:50. Total reaction volume of S-RBD solution was set as 20 µL.

We conducted a flowcytometric analysis of S-RBD binding to ACE2, as previously reported, with some modifications11. Briefly, ACE2 293T cells were resuspended at 1 × 106 cells/mL in 100 µL of 1% bovine serum albumin (BSA)-PBS. Four microliters of vehicle, TEMPO-Oxo, or AZADO-Oxo-treated Histidine-tagged recombinant S-RBD solution were added to ACE2 293T resuspended solution. After incubation for 30 min on ice, 800 µL of 1% BSA-PBS was added to cell suspension, and then centrifuged at 400 × g, 4 °C for 4 min. Cell pellets were resuspended by 100 µL of 1% BSA-PBS and incubated with 10 µL of FITC-conjugated Anti-6X His tag antibody for 30 min in the dark. After 30 min, Cells were washed by 1 mL of 1% BSA-PBS and resuspended by 300 µL of 1% BSA-PBS. The fluorescence intensity of each cell was measured using CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using CytExpert software.

Untreated, TEMPO-Oxo-treated, and AZADO-Oxo-treated S-RBD solutions (16 µL) were mixed with 4 µL of 5 × sample buffer and denatured at 95 °C for 5 min. Then, 10 µL of denatured samples were subjected to 10% (w/v) SDS-PAGE. Gels were stained using Quick CBB PLUS, according to the manufacturer’s protocol. Finally, band intensity was quantified using the GelAnalyzer function of ImageJ software25.

All reactions were performed in Protein LoBind tubes. Digestion was performed at an S-RBD partial peptide: AZADO-Oxo ratio of 1:10. The samples were analyzed by LC/ESI-MS and MS/MS using the LC system. A mixture of S-RBD partial peptides (1.0 mM, 10 µL), AZADO-Oxo (10 mM, 10 µL), and water (80 µL) was incubated at 20 to 25 °C for 24 h. Control samples were mixed with water instead of AZADO-Oxo. LC was carried out at the following conditions: Column, Cosmosil 5C18-AR-II (octadecylsilyl) column (150 × 2.0 mm i.d., 5 μm, 120 Å; Nacalai Tesque, Inc.); mobile phases (A) 0.1% (v/v) FA in H2O, (B) 0.1% (v/v) FA in MeCN; flow rate 0.2 mL/min; and column temperature, 40 °C. Chromatography was carried out using the Ultimate 3000 LC system (Thermo Fisher Scientific) equipped with an SRD-3600 degasser, DGP-3600 MB pump, FLM-3100B (nano, 2 × 2P-10P) flow manager, and WPS-3000TBPL (nano, CAP) autosampler was used with the following linear gradient: 0 min, 1% B; 100 min, 50% B; 101 min, 100% B; 111 min, 100% B; 112 min, 1% B; 127 min, 1% and 0 min, 1% B; 100 min, 100% B; 110 min, 100% B; 111 min, 1% B; 126 min, 1% B. An aliquot of the solution (10 µL) was injected into the system. The eluate obtained between 5 and 100 min was introduced into the MS system. MS system used the LTQ Orbitrap Velos hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an ESI source in positive ion mode with the following parameters: Analyzer, Fourier transform-MS; heated capillary, 275 °C; spray voltage, 3.0 kV; resolution, 60,000; scan rate, normal (33,000 amu/s); sheath gas flow rate, 50 arb; and auxiliary gas flow rate. Full scanning analyses were performed in the range of m/z 300–2000. Helium was used at 1.5 mTorr as the collision gas in the collision-induced dissociation experiments coupled with MS/MS. The relative collision energy was set to 35%. Data were processed using the Xcalibur software (version 2.2. SR2; Themo Fisher Scientific). Proteome Discoverer (version 1.3) (Thermo Fisher Scientific, Inc.) was used to identify peptide fragments, using the following parameters. All the identified peptide fragments were confirmed by checking their MS/MS spectra. For S-RBD partial peptides: minimum precursor mass, 100 Da; maximum precursor mass, 5000 Da; enzyme, no-enzyme (unspecific); precursor mass tolerance, 2 Da; fragment mass tolerance, 0.8 Da; dynamic modification, oxidation (tyrosine, serine, tryptophan); target false discovery rate (strict), 0.01; and target false discovery rate (relaxed), 0.05.

To infect the CRFK cells, 4000-times diluted FCoV stock medium in PBS was used as the untreated FCoV solution. Before treatment with TEMPO-Oxo or AZADO-Oxo, FCoV stock medium was 200-times diluted with PBS. TEMPO-Oxo or AZADO-Oxo was added to 200-times diluted FCoV solution as indicated concentration, and then incubated for 2 h at 25 °C. The TEMPO-Oxo- or AZADO-Oxo-treated FCoV solution was further 20-times diluted with PBS to the same dilution as that of the untreated FCoV solution. This solution was called the TEMPO-Oxo- or AZADO-Oxo-treated FCoV solution.

CRFK cells were seeded at a density of 1 × 104 cells/well in 96 well plates. After 3 days, culture medium was changed to 100 µL of new DMEM containing 2% FBS and further added 100 µL of TEMPO-Oxo- or AZADO-Oxo-treated or untreated FCoV solution. After a day, culture medium was changed to 200 µL of new DMEM containing 2% FBS and cultured for further 2 days. CPE was measured visually, and microscopic images of the CPE were captured using DIGITAL SIGHT DS-L2 (Nikon, Tokyo, Japan). Cell viability was measured using Cell Counting Kit-8.

To infect the CRFK cells, 400-times diluted FCoV stock medium in PBS was used as the untreated FCoV solution. Before treatment with AZADO-Oxo, the FCoV stock medium was diluted 20-times with PBS. AZADO-Oxo was then added to the 20-times diluted FCoV solution at 1 mM final concentration and incubated for 2 h at 37 °C. The AZADO-Oxo-treated FCoV solution was further diluted 20-times with PBS to the same dilution as that of the untreated FCoV solution. Finally, FCoV infection of CRFK cells was conducted as described above, and TCID50 in each group was calculated using the Behrens–Karber method.

Excel statistics (version 7.0; ESUMI, Tokyo, Japan) was used for all statistical analyses. All data are expressed as the mean ± standard error of the mean (S.E.M.). A two-tailed paired Student’s t-test was used to compare the data between two groups. Multiple groups were compared using the analysis of variance followed by Dunnett’s post-hoc test.

All data generated or analyzed in this study are included in this published article and its supplementary information file.

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Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan

Ryosuke Segawa, Yuto Fujisawa & Noriyasu Hirasawa

Laboratory of Synthetic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan

Yusuke Sasano, Shuhei Akutsu & Yoshiharu Iwabuchi

Laboratory of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan

Yusuke Hatakawa & Tomoyuki Oe

Advanced Materials and Processing Laboratory, Research Division, Nissan Motor Co., Ltd, 1 Natsushima-cho, Yokosuka, Kanagawa, 237-8523, Japan

Masanobu Uchimura, Ami Ikura, Kota Matsumoto, Kazuki Sone & Masashi Ito

Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan

Masashi Ito

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R.S., Y.S., M.U., A.I., K.M., K.S., Y.I., M.I., and N.H. designed the concept of this study. R.S. and Y.F. performed in vitro experiments. S.A, Y.S. and Y.I. synthesized oxoammonium salts and performed the experiments about tyrosine oxidation. Y.H. and T.O. performed LC/ESI-MS/MS analysis. R.S., Y.H., and Y.F. wrote the manuscript and prepared the figures. All authors reviewed and approved the manuscript.

Correspondence to Noriyasu Hirasawa.

We received a research grant from Nissan Motor Co., Ltd. Our study relates to the patent (patent applicant is Nissan Motor Co., Ltd., inventor is M.I., A.I., M.U., N.H., Y.S., Y.I., and R.S., application number is PCT/JP2023/031213, patent pending). Other author have no competing interests.

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Segawa, R., Sasano, Y., Hatakawa, Y. et al. Oxoammonium salts exert antiviral effects against coronavirus via denaturation of their spike proteins. Sci Rep 14, 23934 (2024). https://doi.org/10.1038/s41598-024-75097-7

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Received: 04 April 2024

Accepted: 01 October 2024

Published: 13 October 2024

DOI: https://doi.org/10.1038/s41598-024-75097-7

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