The green tea catechin epigallocatechin gallate inhibits SARS-CoV-2 infection

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and more than a million deaths. The infection causes COVID-19, a disease of the respiratory system of divergent severity. No treatment exists. Epigallocatechin-3-gallate (EGCG), the major component of green tea, has several beneficial properties, including antiviral activities. Therefore, we examined whether EGCG has antiviral activity against SARS-CoV-2. EGCG blocked not only the entry of SARS-CoV-2, but also MERS- and SARS-CoV pseudotyped lentiviral vectors and inhibited virus infections in vitro. Mechanistically, inhibition of the SARS-CoV-2 spike-receptor interaction was observed. Thus, EGCG might be suitable for use as a lead structure to develop more effective anti-COVID-19 drugs.

Keywords: EGCG; Green tea; MERS-CoV, SARS-CoV; SARS-CoV-2; pseudotype.

Fig. 1.

EGCG inhibits transduction with SARS-CoV-2-pseudotyped lentiviral vectors. (a) EGCG or EC were added at the indicated concentrations to SARS-CoV-2-pseudotyped vectors encoding luciferase, incubated for 30 min at 37 °C and then added to HEK293T-ACE2 cells. Gene transfer into HEK293T-ACE2 cells was analysed after 24 and 48 h by measurement of luciferase activity indicated as % of the untreated control. The values for EGCG are depicted in black and grey, and those for EC in blue and light blue. The 24 h incubation was done in triplicate. The values for the 48 h incubation are mean values of three experiments done in triplicate. Significant differences between the untreated control and EGCG treatment for 48 h at the indicated concentrations are shown by the p-values, which were calculated using Student’s t-test. The -values for EGCG concentrations lower than 5 µg ml⁻¹ were not significant. (b) Toxicity of EGCG and EC on HEK293T-ACE2 and Huh7 cells was tested by monitoring ATP with a luminescence assay. Cell viability (as relative light units) after 24 h incubation with EGCG is depicted in grey and viability after 48 h in black for 293T-ACE2 cells and in dark blue and light blue for Huh7 cells. The values for both assays are mean values of three experiments done in triplicate (Huh7 cells: one experiment in triplicate). (c) Gene transfer by SARS-CoV-, NL63-,and VSV-G-pseudotyped vectors into HEK293T-ACE2 cells and MERS-CoV vectors into Huh7 cells, was analysed in triplicate after 48 h by measurement of luciferase activity in five independent experiments. The corresponding mean IC₅₀ values and their standard deviations are depicted and were calculated using the GraphPad Prism 7.04 software (La Jolla, CA, USA).

Fig. 2.

EGCG inhibits coronavirus infections. (a) Vero cells were infected with SARS-CoV-2, MERS- or SARS-CoV in the presence of EGCG or EC. The values are mean values of three experiments (MERS-CoV n=4) performed in duplicate. Significant differences between EC and EGCG treatments are indicated as p-values 0.0009 for SARS-CoV-2,<0.0001 for MERS-CoV and 0.0023 for SARS-CoV. P-values were calculated with Student’s t-test, using the GraphPad Prism 7.04 software (La Jolla, CA, USA). (b) Toxicity of EGCG and EC on Vero cells was tested by monitoring ATP with a luminescence assay. Cell viability (as relative light units) after 24 h incubation with EGCG is depicted in grey and viability after 48 h in black. The values for both assays are mean values of two experiments done in triplicate.

Fig. 3.

EGCG inhibits attachment of SARS-CoV-2 RBD to ACE2. The sVNT assay was performed with the indicated concentrations of EGCG (black) or EC (grey) according to the manufacturer’s protocol (SARS-CoV-2 sVNT Kit; Genscript, Leiden, Netherlands). Mean values and standard deviations of three independent experiments done in duplicate are given. The negative (non-inhibiting) and positive (inhibiting) controls of the sVNT kit were included. Inhibition was calculated following the manufacturer’s protocol using the following equation: Inhibition in % = (1 – OD of sample/OD of negative control) ×100.

Fig. 4.

Time-of-drug-addition experiment. (a) Vero cells were infected with SARS-CoV-2 and 10 µg ml⁻¹ EGCG was added either 1 h before the infection, 1 h after or 6 h after the start of the infection. SARS-CoV-2 was left on the cells for 1 h (black bar), was removed and medium was changed to either medium containing 10 µg ml⁻¹ EGCG or medium only. Bold arrows represent incubation with 10 µg ml⁻¹ EGCG. After 11 h of infection, cells were lysed and analysed using RT-qPCR. (b) Mean values and standard deviation of SARS-CoV-2 –specific RNA copies of three independent experiments at different time points of infection.