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

Materials

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.

Cell culture

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.

Treatment of S-RBD with TEMPO-Oxo and AZADO-Oxo

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.

Evaluation of the binding of S-RBD to ACE2 293T cells by flowcytometry

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.

Evaluation of S-RBD denaturation by SDS-PAGE and CBB stain

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.

Evaluation of AZADO-Oxo-treated S-RBD partial peptides by LC/ESI-MS/MS

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.

Evaluation of the antiviral efficacies of TEMPO-Oxo and AZADO-Oxo

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.

Evaluation of the effect of AZADO-Oxo on the TCID50 of FCoV

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.

Statistical analysis

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.