Epigallocatechin-3-gallate, an active ingredient of Traditional Chinese Medicines, inhibits the 3CLpro activity of SARS-CoV-2

Abstract

SARS-CoV-2 is the etiological agent responsible for the ongoing pandemic of coronavirus disease 2019 (COVID-19). The main protease of SARS-CoV-2, 3CLpro, is an attractive target for antiviral inhibitors due to its indispensable role in viral replication and gene expression of viral proteins. The search of compounds that can effectively inhibit the crucial activity of 3CLpro, which results to interference of the virus life cycle, is now widely pursued. Here, we report that epigallocatechin-3-gallate (EGCG), an active ingredient of Chinese herbal medicine (CHM), is a potent inhibitor of 3CLpro with half-maximum inhibitory concentration (IC50) of 0.874 ± 0.005 μM. In the study, we retrospectively analyzed the clinical data of 123 cases of COVID-19 patients, and found three effective Traditional Chinese Medicines (TCM) prescriptions. Multiple strategies were performed to screen potent inhibitors of SARS-CoV-2 3CLpro from the active ingredients of TCMs, including network pharmacology, molecular docking, surface plasmon resonance (SPR) binding assay and fluorescence resonance energy transfer (FRET)-based inhibition assay. The SPR assay showed good interaction between EGCG and 3CLpro with KD ~6.17 μM, suggesting a relatively high affinity of EGCG with SARS-CoV-2 3CLpro. Our results provide critical insights into the mechanism of action of EGCG as a potential therapeutic agent against COVID-19.

Keywords: Epigallocatechin-3-gallate; SARS-CoV-2; Traditional Chinese Medicine.

Fig. 1

A flow diagram illustrating the research design.

Fig. 2

Dynamic profile of laboratory parameters in 60 patients (mild = 8, moderate = 32, and severe = 20 with COVID-19). Timeline charts illustrate the laboratory parameters taken every other day from the day after the onset of illness. p values indicate differences among different groups (mild, moderate and severe). *p < 0.05 was considered a statistically significant difference.

Fig. 3

Molecular models of the binding of SARS-CoV-2 3CLpro with kaempferol (A), luteolin (B), isorhamnetin (C) and wogonin (D), epigallocatechin-3-gallate (E), naringenin (F), and quercetin (G) shown as 3D diagrams.

Fig. 4

SPR assay of specific binding affinities of several active compounds to immobilized 3CLpro of SARS-CoV-2. Different concentrations of the compounds (1–80 μM) were injected separately on the surface of the ligand chip, and the analyte was sampled at 20 μL/min. The binding time of the analyte to the ligand was 240 s; and the natural dissociation was carried out for 180 s. Data are representative of three independent experiments.

Fig. 5

Inhibitory screening of active compounds against SARS-CoV-2 3CLpro using FRET protease assay. (A) Ebselen. (B) Epigallocatechin-3-gallate. (C) Naringenin. (D) Kaempferol. (E) Quercetin. (F) Luteolin. Dose–response curves for IC50 values were determined by nonlinear regression. All data are shown as mean ± s.e.m., n = 3 biological replicates.

Fig. 6

Thermal Shift Assay (TSA) of SARS-CoV-2 3CLpro stability and interaction with Epigallocatechin-3-gallate (EGCG). A. The experimental system for TSA was validated using quercetin, a compound that was previously reported to alter the thermal stability of 3CLpro by causing destabilization. The melting temperatures (T_m) was identified by plotting the first derivative of the fluorescence emission as a function of temperature (−dF/dT). T_m is represented as the lowest part of the curve. B. The melting temperature (T_m) of 3CLpro with various concentrations of EGCG, showing a dose-dependent trend. C and D. The sigmoidal curves showing the melting temperatures (T_m) of dimethyl sulfoxide (C; negative control) and 62.5 μM EGCG (D). The solid line represents the non-linear fit of the fluorescence curve to Boltzmann Equation.