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. 2022 Jul 13;17(7):e0271112.
doi: 10.1371/journal.pone.0271112. eCollection 2022.

Epigallocatechin gallate (EGCG) attenuates severe acute respiratory coronavirus disease 2 (SARS-CoV-2) infection by blocking the interaction of SARS-CoV-2 spike protein receptor-binding domain to human angiotensin-converting enzyme 2

Tomokazu Ohishi  1 , Takayuki Hishiki  2 , Mirza S Baig  3 , Sajjan Rajpoot  3 , Uzma Saqib  4 , Tomohiko Takasaki  2 , Yukihiko Hara  5
Affiliations

Epigallocatechin gallate (EGCG) attenuates severe acute respiratory coronavirus disease 2 (SARS-CoV-2) infection by blocking the interaction of SARS-CoV-2 spike protein receptor-binding domain to human angiotensin-converting enzyme 2

Tomokazu Ohishi et al. PLoS One. .

Abstract

The outbreak of the coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 triggered a global pandemic where control is needed through therapeutic and preventive interventions. This study aims to identify natural compounds that could affect the fusion between the viral membrane (receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein) and the human cell receptor angiotensin-converting enzyme 2. Accordingly, we performed the enzyme-linked immunosorbent assay-based screening of 10 phytochemicals that already showed numerous positive effects on human health in several epidemiological studies and clinical trials. Among these phytochemicals, epigallocatechin gallate, a polyphenol and a major component of green tea, could effectively inhibit the interaction between the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein and the human cell receptor angiotensin-converting enzyme 2. Alternately, in silico molecular docking studies of epigallocatechin gallate and angiotensin-converting enzyme 2 indicated a binding score of -7.8 kcal/mol and identified a hydrogen bond between R393 and angiotensin-converting enzyme 2, which is considered as a key interacting residue involved in binding with the severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain, suggesting the possible blocking of interaction between receptor-binding domain and angiotensin-converting enzyme 2. Furthermore, epigallocatechin gallate could attenuate severe acute respiratory syndrome coronavirus 2 infection and replication in Caco-2 cells. These results shed insight into identification and validation of severe acute respiratory syndrome coronavirus 2 entry inhibitors.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Chemical structures of phytochemicals.
1: Epigallocatechin gallate (EGCG); 2: epigallocatechin (EGC); 3: epicatechin gallate (ECG); 4: epicatechin (EC); 5: chlorogenic acid; 6: genistein; 7: quercetin; 8: curcumin; 9: resveratrol; 10: sulforaphane.
Fig 2
Fig 2. Epigallocatechin gallate (EGCG) inhibits the interaction between the spike receptor-binding domain (RBD) and human cell receptor angiotensin-converting enzyme 2 (ACE2).
(A) Schematic of the ELISA-based screening of phytochemical inhibitory activity on ACE2 and SARS-CoV-2 spike RBD binding. Upper scheme: When the compound does not block the ACE2 and spike RBD binding. Lower scheme: When the compound blocks the ACE2 and spike RBD binding. (B) ELISA results of phytochemical inhibition of the ACE2 and spike RBD binding. Low-intensity HRP signals indicate that the compound successfully blocked the ACE2 and spike RBD binding. Values are presented as the mean ± SD. Asterisks indicate the significant difference compared with the DMSO-treated control (**p < 0.01, *p < 0.05). (C) ELISA results of serially diluted EGCG (3.125–100 μM) inhibition of ACE2 and spike RBD binding b. IC50 was calculated using the IC50 calculator (https://www.aatbio.com/tools/ic50-calculator).
Fig 3
Fig 3. Docking interaction of epigallocatechin gallate (EGCG) with human angiotensin-converting enzyme 2 (ACE2).
(A) Interacting residues determined within a 5-Å region at the interface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) and human ACE2 from their crystal structure (6M17). (B & C) Molecular docking of human ACE2 and EGCG. (B) Three-dimensional representation of EGCG binding with the human ACE2 peptidase domain (left) and magnified image (right). (C) Two-dimensional interaction analysis of ACE2 and EGCG, highlighting the ACE2 residues that interact with EGCG and the types and lengths of their bonds. Residues are color coded according to the type of interaction: dark green, conventional hydrogen bonds; light green, van der Waals forces; dark pink, Pi–Pi stacked interactions; and light pink, Pi–alkyl interactions.
Fig 4
Fig 4. Effects of epigallocatechin gallate (EGCG) treatment for 2 h on the growth of angiotensin-converting enzyme 2 (ACE2)-receptor expressing Caco-2 cells.
(A) Images of 293FT and Caco-2 cells grown on 100-mm plates taken under a phase-contrast microscope. Scale bar: 100 μm. (B) Cropped image of western blot performed with ACE2 and Glyceraldehyde 3-phosphate dehydrogenase antibodies. (C) Outline of the experimental procedure. Caco-2 cells were treated with or without EGCG for 2 h. After treatment, the cells were washed with PBS thrice, and fresh medium was added. After 22 h of incubation, cell growth was determined. (D) Effects of EGCG on Caco-2 cell growth. Cell growth was assessed by the cell proliferation assay and expressed as a percentage of dimethyl sulfoxide (DMSO)-treated control cells. Asterisks indicate the significant difference compared with the DMSO-treated control (**p < 0.01).
Fig 5
Fig 5. Epigallocatechin gallate (EGCG) inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and replication in Caco-2 cells.
(A) Schematic outline of the experimental procedure. SARS-CoV-2 (MOI 0.5)-infected Caco-2 cells were co-cultured with EGCG at a final concentration of 50 or 100 μM. At 2 hpi, the cells were washed with phosphate-buffered saline thrice, and fresh medium was added. At 24 hpi, the cell culture supernatant and cell lysate were collected, and the amount of viral RNA was analyzed by real-time reverse transcription-polymerase chain reaction (RT-qPCR). (B) Measurement of the SARS-CoV-2 growth inhibition by EGCG determined by RT-qPCR of the nucleocapsid region of the SARS-CoV-2 genome using the supernatant (left) or cells (right). Values are presented as a percentage of control (mean ± SD). Asterisks indicate the significant difference compared with the dimethyl sulfoxide–treated control (**p < 0.01, *p < 0.05).
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Grants and funding

This research was supported in part by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant numbers 18K08693 (T.O.), 22K06783 (T.O.), and 20K07532 (T.H.) and by the Japan Agency for Medical Research and Development (AMED) under grant number JP21am0401013 (T.O.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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