Pyrogallol-Type Catechins Inhibit SARS-CoV-2 by Disrupting Spike-ACE2 Binding

Pyrogallol-Type Catechins Inhibit SARS-CoV-2 by Disrupting Spike-ACE2 Binding | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pyrogallol-Type Catechins Inhibit SARS-CoV-2 by Disrupting Spike-ACE2 Binding Futaba Matsumoto, Satomi Nagai, Nanako Ikeda, Kanji Ishimaru, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6766643/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Severe acute respiratory syndrome coronavirus 2 infects host cells through binding of the spike protein receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 receptor. In this study, the antiviral activity of 14 catechin derivatives was evaluated using a pseudovirus assay that emulates spike-mediated cell fusion. Among these, gallocatechin gallate, epigallocatechin gallate, epigallocatechin 3-(3''-O-methyl) gallate, and epigallocatechin notably exhibited strong inhibitory effects on infection. A structural comparison of the compounds revealed that catechins with a pyrogallol-type B-ring configuration exhibited greater inhibitory effects than their catechol-type counterparts. Docking simulations demonstrated that the hydroxyl group at the 5-position of the B-ring forms a hydrogen bond with Gln493 on the spike RBD, thereby facilitating additional stabilizing interactions with adjacent residues, such as Tyr453. Although catechin bioavailability is low, the results of this study suggest that regular consumption or gargling may offer localized antiviral activity at mucosal surfaces, such as those found in the oral or nasal cavity, because the catechin concentrations used in the cell assays are similar to those observed in green tea (100 µM). This study underscores the potential of pyrogallol-type catechins to act as antiviral agents. Biological sciences/Microbiology/Virology Biological sciences/Biochemistry Health sciences/Diseases/Infectious diseases/Viral infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In 2020, the World Health Organization declared the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a global pandemic. The development of effective preventive and therapeutic strategies against SARS-CoV-2 remains a critical component of preparedness for current and future pandemics. SARS-CoV-2 enters host cells through two primary routes: (1) an endocytic pathway and (2) a non-endocytic mechanism involving direct membrane fusion at the cell surface, activated by the transmembrane serine protease 2 (TMPRSS2). 1 , 2 Regardless of the entry route, the first step of infection is the binding of the spike protein receptor-binding domain (RBD) to the host angiotensin-converting enzyme 2 (ACE2) receptor to form a complex. 3 , 4 Therefore, inhibiting the RBD-ACE2 interaction represents a promising strategy to block viral entry and prevent infection. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has demonstrated broad-spectrum antiviral activity in vitro , including the inhibition of multiple viruses such as influenza, human immunodeficiency, hepatitis B and C, chikungunya, vaccinia, herpes simplex, and vesicular stomatitis viruses, adenovirus, and reovirus. 5 – 10 EGCG has also shown potential in inhibiting SARS-CoV-2 infection. 11 – 15 This antiviral activity is attributed to EGCG’s ability to interact with various biomolecules, including proteins, lipids, and carbohydrates. 16 – 18 Accordingly, multiple mechanisms have been proposed to explain its antiviral activity. For instance, EGCG has been shown to bind to heparan sulfate on the surface of host cells and inhibit viral entry, as demonstrated using a lentiviral pseudoparticle system. 12 , 19 In addition, catechins have been reported to inhibit the activity of recombinant SARS-CoV-2 main protease (Mpro), 20 suggesting a possible mechanism for suppressing viral replication. Furthermore, in silico analyses have indicated direct interactions between catechins and the SARS-CoV-2 spike protein RBD. 21 This has been experimentally supported by pull-down assays, which demonstrated that EGCG disrupts binding between the RBD and ACE2. 22 Considering that the RBD-ACE2 interaction represents a critical initial step for viral entry, we focused our investigation on this interaction to evaluate the inhibitory effects of various catechins on SARS-CoV-2 infection. Tea is broadly categorized into non-fermented green tea, semi-fermented oolong tea, fermented black tea, and tea fermented by microbial processes, with each containing different catechins. 23 Catechins consist of two benzene rings (A-ring and B-ring) and a dihydropyran heterocycle (C-ring). They are classified according to three factors: (1) the stereochemical configuration at the C2 position of the C-ring, (2) the number and distribution of hydroxyl groups on the B-ring, and (3) the presence or absence of a galloyl group (D-ring) at the C3 position of the C-ring (Fig. 1 ). During tea processing, isomerization reactions occur at the C2 position of the C-ring, resulting in the existence of (-)-catechin (C) and (-)-epicatechin (EC) 24 , 25 . Catechins/epicatechins with hydroxyl groups at positions 3 and 4 of the B-ring are classified as catechol-type, whereas those with an additional hydroxyl group at position 5 are classified as pyrogallol-type. These include gallocatechin (GC) and epigallocatechin (EGC). In addition, esterification of (−)-catechin by gallic acid, a plant-derived organic compound, produces galloylated catechins, such as ECG and EGCG. Methylated catechins, such as ECGMe and EGCGMe, are derivatives in which the 3-position of the galloyl group in ECG and EGCG is methylated, leading to improved chemical stability and membrane permeability. As a result, these compounds may exhibit enhanced biological activity compared with their non-methylated counterparts. Theaflavins are catechin dimers formed by enzymatic oxidative condensation between catechol- (EC, ECG) and pyrogallol-type catechins (EGC, EGCG). Theaflavins are classified into four types: theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate (TFDG). Teadenol A (TA) and teadenol B (TB) are catechin derivatives specifically detected in post-fermented teas produced through fermentation by Aspergillus species, with TA derived from EGCG and TB from GCG. 26 In this study, we investigated whether catechins inhibit the formation of the spike RBD-ACE2 complex using a newly developed cell-based pseudovirus assay system. Furthermore, we analyzed the structure-activity relationships (SARs) of structurally diverse catechins to identify the key molecular features responsible for their antiviral effects. Results Establishment of a cell-based SARS-CoV-2 infection assay Several polyphenols, including catechins and curcumin, have been proposed as potential inhibitors of SARS-CoV-2 infection. 27 The present study was undertaken to evaluate the antiviral potential of catechins. To this end, a cell-based SARS-CoV-2 infection assay was developed. This assay uses a cell-cell fusion mechanism that is mediated by the spike protein of the virus. An overview of the assay system is shown in Fig. 2 A. In this system, 293T cells expressing the SARS-CoV-2 spike protein were transfected with a T7 polymerase expression vector (pCAG-T7pol) to create pseudovirus cells. The target cells were generated by transfecting VeroE6/TMPRSS2 cells with the pT7-IRES-Luc2 vector, which enabled luciferase expression under the control of a T7 promoter. The interaction between spike-expressing pseudovirus cells and ACE2-expressing target cells leads to cell fusion, enabling T7 polymerase to initiate luciferase expression in the target cells. Luminescence intensity has been shown to serve as a quantitative indicator of infection. The initial step in validating the experimental system involved co-transfecting 293T cells with pT7-IRES-luc2 and pCAG-T7pol and measuring luciferase luminescence. Increased luciferase activity was observed in transfected cells compared with the control cells devoid of T7 polymerase (Fig. 2 B). This finding serves as a validation of the T7 promoter-dependent reporter system. The establishment of a cell-cell interaction-based experimental system was achieved by generating 293T cells that stably express the SARS-CoV-2 spike protein (293T-Spike-C9 cells). Expression of the spike protein was confirmed by western blotting (Fig. 3 A). Subsequently, cells were subjected to transfection with pT7-IRES-Luc2 to generate pseudovirus cells. These cells were then co-cultured with target cells to assess the efficiency of infection. After co-culture for 20 h, luminescence intensity was approximately 260-fold higher than that in the control, which included mock-transfected pseudovirus cells (Fig. 3 B). Microscopic analysis revealed the presence of large multinucleated cells, indicating spike-ACE2-mediated cell fusion (Fig. 3 C, red arrow). To verify that the fused cells were the result of a spike-ACE2 interaction, the experiment was repeated using the pT7-IRES-EmGFP reporter instead of the pT7-IRES-Luc2 reporter. The reporter drives expression of the fluorescent protein EmGFP in the presence of T7 polymerase. Strong green fluorescent protein (GFP) fluorescence was observed in multinucleated cells (Fig. 3 D, white arrowhead). This observation confirms that fusion was dependent on the interaction between spike and ACE2. Collectively, these results substantiate the reliability of our assay in recapitulating spike-mediated infection and facilitating quantitative assessment of inhibitory compounds. Inhibitory effects of catechins in a cell-based SARS-CoV-2 infection assay Using the established cell-based infection assay, we evaluated the inhibitory effects of 14 catechin derivatives (C, CG, GCG, EC, ECG, EGC, EGCG, ECGMe, EGCGMe, TF, TF3G, TFDG, TA, and TB). Pseudovirus and target cells were pre-incubated with each catechin (100 µM) for 30 min at 37°C, followed by co-culture in the continued presence of catechins for 20 h. Camostat mesylate was utilized as a control to assess the efficacy of potential infection inhibitors. The initial phase of the SARS-CoV-2 infection is marked by the binding of the spike protein RBD to ACE2, which is followed by viral entry via two distinct pathways: the TMPRSS2-mediated membrane fusion pathway and the TMPRSS2-independent endocytic pathway. Camostat mesylate selectively inhibits the former pathway by targeting TMPRSS2 and led to 50% inhibition of the infection (Fig. 4 A). Luminescence analysis revealed that eight catechins (GCG, EGCG, EGCGMe, EGC, TF3G, TFDG, TF, and ECGMe) and TB significantly reduced luciferase activity relative to the vehicle control (dimethyl sulfoxide), indicating strong inhibition of cell-cell fusion between pseudovirus cells and target cells (Fig. 4 A) In particular, four pyrogallol-type catechins (EGC, EGCG, EGCGMe, and GCG) showed significant inhibition. The findings indicate that these catechins exhibited a substantial inhibitory effect on the process of cell–cell fusion between pseudovirus cells and target cells, suggesting a potential for these catechins to impede SARS-CoV-2 infection. In order to ascertain whether this inhibition targeted the spike protein or ACE2, we pre-incubated catechins either with pseudovirus cells or target cells separately before co-culturing with the other cell type. Interestingly, camostat mesylate, which is known to inhibit TMPRSS2 activity in target cells, inhibited infection only when pre-incubated with the target cells, demonstrating approximately 50% inhibition (Fig. 4 C). By contrast, pre-incubating pseudovirus cells with camostat mesylate had little to no effect on infection (Fig. 4 B). These findings, where camostat mesylate's efficacy is dependent on target cell pre-incubation, align with its known mechanism as a TMPRSS2 inhibitor that acts on host cells. This demonstrates that the assay system can accurately reflect the inhibitor's mode of action, thereby confirming its effectiveness and appropriateness. When treating pseudovirus cells only, GCG, EGCGMe, and EGC demonstrated significant inhibitory effects (Fig. 4 B). However, all eight catechins and TB showed significant inhibitory effects when used to treat target cells only (Fig. 4 C). These results suggest that catechins act on both viral and host cell components, including the spike protein and ACE2. Several studies have reported direct interactions between catechins and the spike protein RBD. 28 The present study focused on these interactions to identify the structural features of catechins that are critical for inhibitory activity. A subsequent analysis focusing on the B-ring structure revealed that catechins with a catechol-type configuration bearing hydroxyl groups at the 3- and 4-positions, such as C, EC, CG, and ECG, did not demonstrate significant inhibition. However, pyrogallol-type catechins with an additional hydroxyl group at the 5-position, such as EGC, GCG, and EGCG, exhibited significant inhibitory activity (Fig. 4 A). Notably, the three catechins that interacted with the spike protein (EGCG, GCG, and EGC) all share a common feature: a pyrogallol-type B-ring structure (Fig. 1 A and Fig. 4 B). These findings indicate that the pyrogallol-type B-ring is a critical structural determinant for catechin-mediated inhibition of SARS-CoV-2 infection. A comparison between epi- and non-epi-type catechins, which differ in stereochemistry at the C2 position of the C-ring (Fig. 1 A), revealed that both EGCG, which are important catechins found in green tea, effectively inhibited infection when pre-incubated with either spike-expressing pseudovirus cells or ACE2-expressing target cells (Fig. 4 A). However, when incubated with spike protein alone, only GCG exhibited significant inhibition (Fig. 4 B), suggesting that the non-epi configuration may facilitate more effective interaction with the spike protein. Docking simulation of catechins with spike RBD In silico docking simulations of selected catechins with the RBD of the SARS-CoV-2 spike protein were performed using AutoDock Vina to elucidate the molecular basis for the observed inhibition. The docking poses of both catechol-type (A–C, green) and pyrogallol-type (D–F, blue) catechins are shown in Fig. 5 . The pink regions represent 10 key amino acid residues predicted in silico to be critical for the interaction between the RBD and ACE2. 29 The importance of three of these amino acid residues for actual binding was confirmed by in vitro pull-down assays using recombinant RBD and ACE2. 22 A remarkable observation of the present study is that all catechins were shown to bind to the cavity of the RBD located around the critical amino acids for binding to ACE2 (Fig. 5 ), which occurred in a variety of binding modes. The results of these simulations were analyzed to extract catechin pairs that share the same binding mode by comparing catechol-type molecules (EC, CG, ECG) with their corresponding pyrogallol-type counterparts (EGC, GCG, EGCG; Fig. 5 G–I, light blue). The energies for these dockings, which share the same binding mode, are presented in Table 1. The pyrogallol-type catechins exhibited stronger binding (lower docking energies) than the catechol-type catechins (Table 1). These results indicate that pyrogallol-type catechins, despite having equivalent binding modes, exhibit an increased affinity for the RBD compared with their catechol-type counterparts. Binding mode 4, which is listed in Table 1 as a common binding conformation for CG and GCG, is shown in an enlarged view in Fig. 6 . In GCG, the oxygen at the 5-position of the B-ring forms a hydrogen bond with Gln493 (3.0 Å; Fig. 6 B), whereas CG lacks a hydroxyl group at this position and therefore cannot establish the same interaction (Fig. 6 A). The presence of an additional hydrogen bond in GCG appears to have a significant impact on molecular orientation, thereby facilitating the formation of a new hydrogen bond between the hydroxyl group at the 3-position of the B-ring and the oxygen atom of Tyr453, with a distance of 3.0 Å. Additionally, a notable shortening in distance is observed among the hydrogen bonds involving the oxygen and hydroxyl group at the 4-position of the B-ring, suggesting greater strength in these interactions. These effects synergistically enhance the overall binding strength, and findings indicate that the presence of a hydroxyl group at the 5-position of the B-ring significantly enhances the interaction with the RBD, even though binding occurs within the same region. The stereoisomeric nature of epi- and non-epi-type catechins means that their three-dimensional structures exhibit significant differences, which complicates the identification of a shared binding mode. Binding energies for the catechins with the highest values obtained in docking simulations are presented in Table 2. Overall, non-epi-type catechins (C, CG, GCG) exhibited stronger binding to the spike protein RBD than their epi-type counterparts (EC, ECG, EGCG). This phenomenon can be attributed to the trans-configuration of non-epi-type catechins, which allows for an increased contact area with the protein surface and positions the hydroxyl group at the 5-position of the B-ring in a favorable orientation for binding. This in turn enhances inhibitory activity. Collectively, the docking simulation results support our in vitro findings, demonstrating that catechins with pyrogallol-type B-rings and non-epicatechin stereochemistry exhibit stronger and more versatile binding to the spike RBD than their catechol-type counterparts. These structural features are likely to act synergistically to inhibit the RBD-ACE2 interaction. Discussion In this study, we investigated the inhibitory effects of structurally diverse catechins on the interaction between the SARS-CoV-2 spike protein RBD and the human ACE2 receptor using a cell-based pseudovirus assay. The findings of this study demonstrate that catechins with a pyrogallol-type B-ring exhibit significantly greater inhibitory activity than their catechol-type counterparts. Additionally, it is thought that the non-epi stereochemistry may also contribute to these inhibitory effects to some extent. These conclusions were consistently supported by both experimental infection assays and in silico docking simulations. Previous computational studies have identified amino acid residues within the RBD (specifically Tyr449, Tyr453, Leu455, Asn487, Tyr489, Gln493, Gln498, Thr500, Asn501, and Tyr505) as being critical for interactions with ACE2. In this study, the importance of Tyr453, Gln493, and Asn501 was experimentally validated by performing pull-down assays. 22 The docking simulations showed that a significant proportion of the catechins exhibited a binding preference for regions close to these residues (Fig. 5 , pink), confirming their ability to inhibit RBD-ACE2 complex formation. Whereas prior studies have primarily focused on EGCG because of its abundance in green tea, our study highlights that pyrogallol-type (GCG, EGC, and EGCGMe) and non-epi-type catechins (GCG) may offer superior inhibition of SARS-CoV-2 entry. This study represents the first SAR investigation of catechins, with a particular focus on comparing their potency in relation to B-ring hydroxylation and C-ring stereochemistry. Notably, catechin epimerization occurs during tea processing, resulting in increased levels of non-epi-type catechins in ready-to-drink bottled teas. 24 Although these epimers are present in lower concentrations in freshly brewed tea, they may contribute substantially to the efficacy of the antiviral properties of tea. The bioavailability of catechins, particularly EGCG, has been investigated. Only a small proportion of orally ingested EGCG is absorbed in the small intestine, with peak plasma concentrations typically reaching no more than 1 µM. 30 As demonstrated by the in vitro experiments, which showed inhibitory effects on RBD-ACE2 binding occurring with ~ 100 µM EGCG, it is improbable that similar effects would occur systemically at physiological concentrations. Considering the catechin concentration of ~ 100 µM in green tea 31 and the localization of SARS-CoV-2 infection to mucosal surfaces, frequent consumption or gargling may allow these compounds to exert localized antiviral effects, despite limited systemic bioavailability. Although the direct binding of individual catechins to the RBD is relatively weak, our docking data suggest that multiple molecules can bind in overlapping but independent binding poses in diverse modes, collectively masking critical ACE2-binding regions. Given the propensity of the virus to undergo genetic mutations that alter its RBD structure, the capacity of catechins to engage the RBD through multiple independent binding modes has the potential to provide a robust antiviral mechanism that is less susceptible to evasion by viral mutation. Methods Materials Teadenols A and B were obtained from the Japanese post-fermented tea “Kippuku-cha” as previously described. 26 , 32 Catechins and theaflavins were purchased from Nagara Science Co. Ltd. (Gifu, Japan). Unless otherwise noted, other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan) and Nakalai Tesque (Kyoto, Japan). All reagents used were of the highest available analytical grade. Plasmid design Genes encoding Luc2 and EmGFP were amplified by polymerase chain reaction from pGL4.23 (Promega) and pcDNA6.2/N-EmGFP-DEST (ThermoFisher Scientific, Waltham, MA, USA), respectively. These fragments were cloned into the pT7-IRES His-C vector (Takara Bio, Ohtsu, Japan) downstream of the internal ribosome entry site (IRES) using the In-Fusion cloning system (Takara Bio), resulting in the reporter plasmids pT7-IRES-Luc2 and pT7-IRES-EmGFP. The SARS-CoV-2 spike protein expression vector, pBA-spike-C9, was constructed by amplifying the spike gene, including a C-terminal C9 tag, from pcDNA3.1-SARS2-spike and cloning it into the BamHI site of the pBApo-EF1α Pur vector (Takara Bio) using In-Fusion cloning. The plasmid pcDNA3.1-SARS2-spike was a gift from Fang Li 22 (Addgene plasmid #145032). The T7 RNA polymerase expression vector, pCAG-T7pol, was a gift from Dr. Ian Wickersham (Addgene plasmid #59926). Cell culture VeroE6/TMPRSS2 cells and 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin, at 37°C in a humidified 5% CO 2 incubator. VeroE6/TMPRSS2 cells 33 (JCRB1819) were obtained from JCRB Cell Bank (Osaka, Japan). 293T cells were provided from RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Transfection of DNA Transfections were performed using polyethyleneimine Max (PEI; Polysciences, Inc., Warrington, PA, USA) as previously described. 34 Briefly, 4 µg plasmid DNA was mixed with 250 µL Opti-MEM and incubated at 25ºC for 5 min. Separately, 10 µg PEI was diluted in 250 µL Opti-MEM and was also incubated for 5 min. The PEI and DNA solutions were then combined and incubated for a further 20 min. The mixture was added to a six-well plate containing 5 × 10⁵ cells in 2 mL DMEM with 5% FBS. After 4 h, the medium was replaced with fresh DMEM containing 5% FBS and the cells were incubated overnight. To generate spike-expressing cells, 293T cells were transfected with pBA-Spike-C9 and selected with 2 µg/mL puromycin. Clonal selection was performed by limiting dilution. Clones expressing high levels of spike protein were identified by western blotting as previously described 34 using an anti-rhodopsin antibody (Rho 1D4, Sigma) recognizing the C9 tag. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Cell-based SARS-CoV-2 infection assay Pseudovirus cells were generated by transfecting 293T-Spike-C9 cells with pCAG-T7pol using PEI as described above. Target cells were generated by transfecting VeroE6/TMPRSS2 cells with pT7-IRES-Luc2 (or pT7-IRES-EmGFP for fluorescence assays). VeroE6/TMPRSS2 cells highly express ACE2, the receptor for SARS-CoV-2 entry. 35 , 36 Both cell types were washed with saline and detached using TrypLE Express (ThermoFisher Scientific) for 3 min at 37°C. They were resuspended in DMEM with 5% FBS. Pseudovirus and target cells (2 × 10⁴ each) were pre-incubated with catechins (final concentration: 100 µM) or vehicle (DMSO) for 30 min, then co-seeded and co-cultured for 20 h at 37°C. The next day, luciferase activity was quantified using the PicaGene™ luciferase assay system (Toyo Ink, Tokyo, Japan), and luminescence was measured with a Luminoskan™ Ascent (ThermoFisher Scientific). Docking simulation The crystal structure of the SARS-CoV-2 RBD-ACE2 complex (PDB ID: 6M0J) was downloaded from the Protein Data Bank ( https://www.rcsb.org/ ). Using PyMOL, all water molecules, ligands, ions, and the ACE2 portion were removed. Polar hydrogens and charges were added in AutoDock Vina (version 1.5.7, https://ccsb.scripps.edu/mgltools/ ), and the structure was converted to PDBQT format. In accordance with previous studies, 10 RBD residues critical for ACE2 interaction (Tyr449, Tyr453, Leu455, Asn487, Tyr489, Gln493, Gln498, Thr500, Asn501, andTyr505) 29 , 36 were designated as flexible side chains. Separate PDBQT files were prepared for flexible and rigid regions. Catechin structures were obtained from PubChem ( https://pubchem.ncbi.nlm.nih.gov ) in SDF format, energy-minimized using the Merck Molecular Force Field 94 (MMFF94), and converted to PDBQT format. The grid box was configured to encompass the entire RBD. Docking simulations were conducted in AutoDock Vina by assigning each catechin as a ligand with rotatable bonds, following standard procedures. 27 , 37 , 38 Abbreviations CH, (-)-Catechin hydrate; EC, (-)-epicatechin; CG, (-)-catechin gallate; ECG, (-)-epicatechin gallate; ECGMe, (-)-epicatechin 3-(3''-O-methyl) gallate; GC, (-)-gallocatechin; EGC, (-)-epigallocatechin; GCG, (-)-gallocatechin gallate; EGCG, (-)-epigallocatechin gallate; EGCGMe, (-)-epigallocatechin 3-(3''-O-methyl) gallate; TF, theaflavin; TF3G, theaflavin 3-gallate; TFDG, theaflavin 3,3’-di-O-gallate; TA, teadenol A; TB, teadenol B ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain. Declarations Author Contribution F.M., S.N., and N.I. conducted the experiments. K.I., K.S., and T.M. provided the resources and methodology. Y.H. validated and supervised the experiments. S.M. was responsible for conceptualization, manuscript writing, supervision, and project administration. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research (C) (Grant No. 23K05130) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We also gratefully acknowledge funding from the 2020 Advanced Research Promotion Program of the United Graduate School of Agricultural Sciences, Kagoshima University. We thank Professor Yukio Nagano of Saga University for his invaluable support in obtaining government approval for experimental procedures involving the SARS-CoV-2 spike gene during the COVID-19 pandemic. We would like to express our gratitude to Dr. M. Horitani and Ms. S. Tsuji (Saga University) for their valuable technical guidance in the docking simulations. The authors also thank all laboratory members for their technical assistance and helpful discussions throughout the course of this study. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl. Acad. Sci. U S A 117 , (2020). Hoffmann, M. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181 , 271–280e8 (2020). Stäb, S. et al. Up, up, down, down: the structural biology of the SARS-CoV-2 spike protein and how it cheats the immune system. Crystallogr. Rev. 30 , 74–117 (2024). Jackson, C. B., Farzan, M., Chen, B. & Choe, H. Mechanisms of SARS-CoV-2 entry into cells. 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Catechin and curcumin interact with S protein of SARS-CoV2 and ACE2 of human cell membrane: insights from computational studies. Sci Rep 11 , (2021). Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581 , 221–224 (2020). Botten, D., Fugallo, G., Fraternali, F. & Molteni, C. Structural Properties of Green Tea Catechins. J. Phys. Chem. B . 119 , 12860–12867 (2015). Seto, R., Nakamura, H., Nanjo, F. & Hara, Y. Preparation of Epimers of Tea Catechins by Heat Treatment . Biosci Biotech. Biochcm 61 (1997). https://academic.oup.com/bbb/article/61/9/1434/5947385 Xu, J. Z., Leung, L. K., Huang, Y. & Chen, Z. Y. Epimerisation of tea polyphenols in tea drinks. J Sci. Food Agric 83 , (2003). Wulandari, R. A. et al. New phenolic compounds from Camellia sinensis L. leaves fermented with Aspergillus sp. J Nat. Med 65 , (2011). Jena, A. B., Kanungo, N., Nayak, V., Chainy, G. B. N. & Dandapat, J. Catechin and curcumin interact with S protein of SARS-CoV2 and ACE2 of human cell membrane: insights from computational studies. Sci Rep 11 , (2021). Liu, J. et al. Epigallocatechin gallate from green tea effectively blocks infection of SARS-CoV-2 and new variants by inhibiting spike binding to ACE2 receptor. Cell Biosci 11 , (2021). Shahbazi, B., Mafakher, L. & Teimoori-Toolabi, L. Different compounds against Angiotensin-Converting Enzyme 2 (ACE2) receptor potentially containing the infectivity of SARS-CoV-2: an in silico study. J Mol. Model 28 , (2022). Chow, H. H. S. et al. Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals. Clinical Cancer Research 9 , (2003). Maruyama, K. et al. Simultaneous Determination of Catechins and Caffeine in Green Tea-Based Beverages and Foods for Specified Health Uses. Food Nutr. Sci. 08 , 316–325 (2017). Nagasawa, T., Ishimaru, K., Higashiyama, S., Hama, Y. & Mitsutake, S. Teadenol A in microbial fermented tea acts as a novel ligand on GPR120 to increase GLP-1 secretion. Food Funct. 11 , 10534–10541 (2020). Nao, N. et al. Consensus and variations in cell line specificity among human metapneumovirus strains. PLoS One . 14 , e0215822 (2019). Nagasawa, T. et al. Phytosphingosine is a novel activator of GPR120. J. Biochem. 164 , 27–32 (2018). Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426 , (2003). Drosten, C., Preiser, W., Günther, S., Schmitz, H. & Doerr, H. W. Severe acute respiratory syndrome: Identification of the etiological agent. Trends in Molecular Medicine vol. 9 Preprint at (2003). https://doi.org/10.1016/S1471-4914(03)00133-3 Akishino, M. et al. Red algae-derived isofloridoside activates the sweet taste receptor T1R2/T1R3. Food Biosci 50 , (2022). Nagasawa, T. et al. The molecular mechanism of phytosphingosine binding to FFAR4/GPR120 differs from that of other fatty acids. FEBS Open. Bio . 11 , 3081–3089 (2021). Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.pdf Table 1 Comparison of binding affinity between catechol-types and pyrogallol-type catechins toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) at the common binding mode Table2.pdf Table 2 Comparison of the five strongest binding modes between epicatechin-type and non-epicatechin-type catechins Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers agreed at journal 18 Jun, 2025 Reviewers invited by journal 18 Jun, 2025 Editor assigned by journal 18 Jun, 2025 Editor invited by journal 10 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 28 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6766643","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472953625,"identity":"4f11e44f-33c8-49e0-b660-bdc5ecda5115","order_by":0,"name":"Futaba Matsumoto","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Futaba","middleName":"","lastName":"Matsumoto","suffix":""},{"id":472953626,"identity":"787e3582-eb9d-4320-a506-44899ec9d9be","order_by":1,"name":"Satomi Nagai","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Satomi","middleName":"","lastName":"Nagai","suffix":""},{"id":472953627,"identity":"62eab731-1df7-477e-a639-434ff2b9cb3c","order_by":2,"name":"Nanako Ikeda","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Nanako","middleName":"","lastName":"Ikeda","suffix":""},{"id":472953628,"identity":"a334dbf8-ced3-4dde-8de4-4d7742052e45","order_by":3,"name":"Kanji Ishimaru","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Kanji","middleName":"","lastName":"Ishimaru","suffix":""},{"id":472953629,"identity":"e2ab46d3-f738-4aec-8bd6-950c0a218797","order_by":4,"name":"Kozue Sakao","email":"","orcid":"","institution":"Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Kozue","middleName":"","lastName":"Sakao","suffix":""},{"id":472953630,"identity":"5d9bd214-b04c-497a-b32d-c7606bacf932","order_by":5,"name":"Takeshi Miyata","email":"","orcid":"","institution":"Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Miyata","suffix":""},{"id":472953631,"identity":"d9b6dabb-381b-4a17-b2c7-8be70d74fcbc","order_by":6,"name":"Yoichiro Hama","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Yoichiro","middleName":"","lastName":"Hama","suffix":""},{"id":472953632,"identity":"1df17d63-62a2-40f8-9949-66932c512b2c","order_by":7,"name":"Susumu Mitsutake","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBADHn40ATbCWiQbSNXCYHCAWAfJz0h/JnWzbZuM8fHDDxh/1NTKM0gkMH74wcCXh9PwGzlm0rltt3nMzqQZMPMcO27YIJHALNnDwFaMU4tEDhtEyw0eBmYGtmOM+28kMEgD/ZLYgEMLyGFgLcYzeBgYf/w7Zg+y5Tc+LQw3EiAOM5DgYWDgbatJBGphw2uLwZk3xtY5527zSAD9cpi370ByA8/DNsseA9x+kW9Pf3g7p+y2PX/74YcPf3yrs21gTz5840fFMZwhhgIOMDAcBlKMQCcZHEsgSgsQ1MEYNURrGQWjYBSMgmEPAK9AUE8BDLzNAAAAAElFTkSuQmCC","orcid":"","institution":"Saga University","correspondingAuthor":true,"prefix":"","firstName":"Susumu","middleName":"","lastName":"Mitsutake","suffix":""}],"badges":[],"createdAt":"2025-05-28 09:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6766643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6766643/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-41170-6","type":"published","date":"2026-02-28T15:59:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85026934,"identity":"4931e80d-d37b-4b7f-9bae-238e366ed439","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":505494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatechins and the derivatives examined in this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCatechins are composed of A- and B-ring benzene structures and a C-ring heterocycle, and are classified on the basis of C-ring stereochemistry, B-ring hydroxylation pattern, and the presence of a galloyl group at C3. (-)-Catechin (CH, CID: 107957), (−)-epicatechin (EC, CID: 72276), (−)-catechin gallate (CG, CID: 6419835), (−)-epicatechin gallate (ECG, CID: 107905), and (−)-epicatechin 3-(3''-O-methyl) gallate (ECGMe, CID: 467296) are classified as catechol-type catechins. (−)-Gallocatechin (GC, CID: 9882981), (−)-epigallocatechin (EGC, CID: 72277), (−)-gallocatechin gallate (GCG, CID: 199472), (−)-epigallocatechin gallate (EGCG, CID: 65064), and (−)-epigallocatechin 3-(3''-O-methyl) gallate (EGCGMe, CID: 467296) are categorized as pyrogallol-type catechins. Theaflavin (TF, CID: 134503798), theaflavin 3-gallate (TF3G, CID: 169167), and theaflavin 3,3’-di-O-gallate (TFDG, CID: 58252602) are generated through tea leaf fermentation. Teadenol A (TA, CID: 68196392) and teadenol B (TB, CID: 68196394) are through tea produced by fermentation caused by microbes.\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/f8ca07a6fd1be552c216f821.png"},{"id":85026932,"identity":"5e1222ad-84ee-4764-bf8d-8b34cb9cdcae","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the cell-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe established a pseudovirus infection assay system on the basis of cell fusion induced by the interaction between the SARS-CoV-2 spike protein receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). The overall scheme is illustrated in \u003cem\u003eA\u003c/em\u003e. Cells expressing the SARS-CoV-2 spike protein and T7 polymerase were used as pseudovirus cells, while target cells were engineered to express ACE2 and carry a luciferase reporter gene (Luc2) under the regulation of the T7 promoter. Upon binding of the spike RBD to ACE2 and subsequent cell fusion, T7 polymerase from the pseudovirus cells activates the expression of Luc2 in the target cells. By adding the luciferase substrate, infection can be quantitatively measured on the basis of luminescence intensity. The quantification of luciferase activity in 293T cells co-transfected with pT7-IRES-Luc2 and pCAG-T7pol is shown in \u003cem\u003eB\u003c/em\u003e. Detailed experimental procedures are described in the Methods. Luciferase activity is shown as a fold increase compared with control cells lacking pCAG-T7pol transfection. Data were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test and error bars represent standard deviation (SD); *P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.21.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/e3574830857ce734aaf962c2.png"},{"id":85026938,"identity":"2a539bae-b3f6-4b57-81e6-cb13ca3abd0c","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":817160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of the cell-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Expression of the spike protein in the established 293T-Spike-C9 cells was confirmed by western blotting using an anti-rhodopsin antibody targeting the C9 peptide fused to the C-terminus of the spike protein. GAPDH was detected as a loading control using an anti-GAPDH antibody. Details are described in the Methods. (B) Pseudovirus cells were co-cultured with target cells for 20 h and Luc2 expression was quantified. Luciferase activity is shown as the fold increase relative to the control, where mock pseudovirus cells (not transfected with pCAG-T7) were co-cultured with target cells. Data were analyzed using Student’s \u003cem\u003et\u003c/em\u003e-test, and error bars represent standard deviation (SD); *P \u0026lt; 0.01. (C) Microscopic image captured after 20 h co-culture with pseudovirus and target cells. Arrows indicate large multinucleated cells formed by cell fusion. (D) Fluorescence and phase contrast images following co-culture of pseudovirus and target cells in which the reporter plasmid was changed from pT7-IRES-Luc2 to pT7-IRES-EmGFP. \u003cem\u003ea\u003c/em\u003eshows the phase contrast image, and \u003cem\u003eb\u003c/em\u003e shows the corresponding green fluorescent protein (GFP) fluorescence. Arrowheads indicate large GFP-positive cells. The scale bars in \u003cem\u003eC\u003c/em\u003e-\u003cem\u003eD\u003c/em\u003e represent 100 μm.\u003c/p\u003e","description":"","filename":"Fig.31.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/f4c0e2fbd522b5681e694c0e.png"},{"id":85026936,"identity":"56d58671-91f6-44e9-b486-f819515fbd2f","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of catechins on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using a cell-based assay\u003c/strong\u003e\u003cbr\u003e\n(A) Pseudovirus cells and target cells were each incubated with catechins (final concentration: 100 μM) for 30 min, followed by co-culture for 20 h in the continued presence of catechins to evaluate their inhibitory effect on viral infection. (B) Pseudovirus cells were pre-incubated with catechins (final concentration: 100 μM) for 30 min, then co-cultured for 20 h with target cells in the absence of catechins to assess the effects of catechins on the spike protein. (C) Target cells were pre-incubated with catechins (final concentration: 100 μM) for 30 min, then co-cultured with pseudovirus cells in the absence of catechins for 20 h to evaluate the effects of catechins on target cells, including angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Data are presented as relative luciferase activity, with the vehicle control (DMSO) set to 100%. Data represent mean ± standard deviation (SD) and were compared using an analysis of variance (ANOVA); *P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"FIg.41.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/57c3e336bc866e363c35d6c0.png"},{"id":85026933,"identity":"71afde51-4584-41b7-bb6d-bd595d98e865","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1894018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDocking models of catechins with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD)\u003c/strong\u003e\u003cbr\u003e\nPanels A–C show the superimposed docking poses of catechol-type catechins (epicatechin [EC], catechin gallate [CG], epicatechin gallate [ECG]) with the SARS-CoV-2 spike RBD, while panels D–F show the corresponding pyrogallol-type catechins (epigallocatechin [EGC], gallocatechin gallate [GCG], epigallocatechin gallate [EGCG]). Panels G–I illustrate the representative binding modes shared between each catechol-type catechin and its pyrogallol-type counterpart to the SARS-CoV-2 spike RBD. The calculated binding affinities of these pairs exhibiting common binding modes are summarized in Table 1. The complex structures of the SARS-CoV-2 spike RBD and the angiotensin-converting enzyme 2 (ACE2) receptor were obtained from the Protein Data Bank. The three-dimensional structures of all catechins were retrieved from PubChem. After structural optimization, docking simulations were conducted using AutoDock Vina. Detailed experiments are described in the Methods section.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/8dcaf794fa43defa61fae3c9.png"},{"id":85027080,"identity":"aa327d8f-4b58-418a-95ae-fa24858002f5","added_by":"auto","created_at":"2025-06-20 06:34:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":943221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetailed comparison of CG and GCG binding to the spike receptor-binding domain (RBD) in the common binding mode\u003c/strong\u003e\u003cbr\u003e\nDocking mode 4 (Table 1) was identified as a shared binding conformation between catechin gallate (CG) (A) and gallocatechin gallate(GCG) (B) and was visualized using PyMOL to illustrate the hydrogen bonding patterns with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike RBD. The red regions of the catechin structures indicate the oxygen atoms at positions 3, 4, and 5 of the B-ring (only 3 and 4 in the case of CG). Yellow dashed lines represent predicted hydrogen bonds between the oxygens of catechins and key residues on the spike RBD (Gln493, Tyr453, and Tyr505), indicating stable interactions within the common binding pocket. Interatomic distances for these hydrogen bonds (\u0026lt;3.0 Å) are also shown, with shorter distances suggesting stronger interactions. The docking scores for CG and GCG in this mode were −6.3 and −7.4 kcal/mol, respectively, indicating that GCG showed stronger binding affinity than CG in the same binding conformation.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/6e79e898a22c841e4ba2f385.png"},{"id":103765635,"identity":"4a0e4053-83da-4e71-b5b7-dcc425628c2f","added_by":"auto","created_at":"2026-03-02 16:06:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5482824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/fe649e93-676d-44e3-9b32-5ed08f14a766.pdf"},{"id":85026931,"identity":"ee4791b7-5f44-48af-8b0f-f06335f6525e","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":81826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1 Comparison of binding affinity between catechol-types and pyrogallol-type catechins toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) at the common binding mode\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/d2faa1bf4e2e70330915daa8.pdf"},{"id":85026937,"identity":"56e00e8b-1972-4612-a6dc-9b71bc0559bb","added_by":"auto","created_at":"2025-06-20 06:26:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":65839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2 Comparison of the five strongest binding modes between epicatechin-type and non-epicatechin-type catechins\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766643/v1/4c1d17dcf6c9b464c09b0341.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePyrogallol-Type Catechins Inhibit SARS-CoV-2 by Disrupting Spike-ACE2 Binding\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2020, the World Health Organization declared the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a global pandemic. The development of effective preventive and therapeutic strategies against SARS-CoV-2 remains a critical component of preparedness for current and future pandemics. SARS-CoV-2 enters host cells through two primary routes: (1) an endocytic pathway and (2) a non-endocytic mechanism involving direct membrane fusion at the cell surface, activated by the transmembrane serine protease 2 (TMPRSS2).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Regardless of the entry route, the first step of infection is the binding of the spike protein receptor-binding domain (RBD) to the host angiotensin-converting enzyme 2 (ACE2) receptor to form a complex.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Therefore, inhibiting the RBD-ACE2 interaction represents a promising strategy to block viral entry and prevent infection. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has demonstrated broad-spectrum antiviral activity \u003cem\u003ein vitro\u003c/em\u003e, including the inhibition of multiple viruses such as influenza, human immunodeficiency, hepatitis B and C, chikungunya, vaccinia, herpes simplex, and vesicular stomatitis viruses, adenovirus, and reovirus. \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e EGCG has also shown potential in inhibiting SARS-CoV-2 infection.\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e This antiviral activity is attributed to EGCG\u0026rsquo;s ability to interact with various biomolecules, including proteins, lipids, and carbohydrates.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Accordingly, multiple mechanisms have been proposed to explain its antiviral activity. For instance, EGCG has been shown to bind to heparan sulfate on the surface of host cells and inhibit viral entry, as demonstrated using a lentiviral pseudoparticle system.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e In addition, catechins have been reported to inhibit the activity of recombinant SARS-CoV-2 main protease (Mpro),\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e suggesting a possible mechanism for suppressing viral replication. Furthermore, \u003cem\u003ein silico\u003c/em\u003e analyses have indicated direct interactions between catechins and the SARS-CoV-2 spike protein RBD.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e This has been experimentally supported by pull-down assays, which demonstrated that EGCG disrupts binding between the RBD and ACE2.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Considering that the RBD-ACE2 interaction represents a critical initial step for viral entry, we focused our investigation on this interaction to evaluate the inhibitory effects of various catechins on SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003eTea is broadly categorized into non-fermented green tea, semi-fermented oolong tea, fermented black tea, and tea fermented by microbial processes, with each containing different catechins.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Catechins consist of two benzene rings (A-ring and B-ring) and a dihydropyran heterocycle (C-ring). They are classified according to three factors: (1) the stereochemical configuration at the C2 position of the C-ring, (2) the number and distribution of hydroxyl groups on the B-ring, and (3) the presence or absence of a galloyl group (D-ring) at the C3 position of the C-ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). During tea processing, isomerization reactions occur at the C2 position of the C-ring, resulting in the existence of (-)-catechin (C) and (-)-epicatechin (EC) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Catechins/epicatechins with hydroxyl groups at positions 3 and 4 of the B-ring are classified as catechol-type, whereas those with an additional hydroxyl group at position 5 are classified as pyrogallol-type. These include gallocatechin (GC) and epigallocatechin (EGC). In addition, esterification of (\u0026minus;)-catechin by gallic acid, a plant-derived organic compound, produces galloylated catechins, such as ECG and EGCG. Methylated catechins, such as ECGMe and EGCGMe, are derivatives in which the 3-position of the galloyl group in ECG and EGCG is methylated, leading to improved chemical stability and membrane permeability. As a result, these compounds may exhibit enhanced biological activity compared with their non-methylated counterparts. Theaflavins are catechin dimers formed by enzymatic oxidative condensation between catechol- (EC, ECG) and pyrogallol-type catechins (EGC, EGCG). Theaflavins are classified into four types: theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3\u0026prime;-gallate (TF3\u0026prime;G), and theaflavin-3,3\u0026prime;-digallate (TFDG). Teadenol A (TA) and teadenol B (TB) are catechin derivatives specifically detected in post-fermented teas produced through fermentation by \u003cem\u003eAspergillus\u003c/em\u003e species, with TA derived from EGCG and TB from GCG.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In this study, we investigated whether catechins inhibit the formation of the spike RBD-ACE2 complex using a newly developed cell-based pseudovirus assay system. Furthermore, we analyzed the structure-activity relationships (SARs) of structurally diverse catechins to identify the key molecular features responsible for their antiviral effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of a cell-based SARS-CoV-2 infection assay\u003c/h2\u003e \u003cp\u003eSeveral polyphenols, including catechins and curcumin, have been proposed as potential inhibitors of SARS-CoV-2 infection.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The present study was undertaken to evaluate the antiviral potential of catechins. To this end, a cell-based SARS-CoV-2 infection assay was developed. This assay uses a cell-cell fusion mechanism that is mediated by the spike protein of the virus. An overview of the assay system is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. In this system, 293T cells expressing the SARS-CoV-2 spike protein were transfected with a T7 polymerase expression vector (pCAG-T7pol) to create pseudovirus cells. The target cells were generated by transfecting VeroE6/TMPRSS2 cells with the pT7-IRES-Luc2 vector, which enabled luciferase expression under the control of a T7 promoter. The interaction between spike-expressing pseudovirus cells and ACE2-expressing target cells leads to cell fusion, enabling T7 polymerase to initiate luciferase expression in the target cells. Luminescence intensity has been shown to serve as a quantitative indicator of infection. The initial step in validating the experimental system involved co-transfecting 293T cells with pT7-IRES-luc2 and pCAG-T7pol and measuring luciferase luminescence. Increased luciferase activity was observed in transfected cells compared with the control cells devoid of T7 polymerase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This finding serves as a validation of the T7 promoter-dependent reporter system. The establishment of a cell-cell interaction-based experimental system was achieved by generating 293T cells that stably express the SARS-CoV-2 spike protein (293T-Spike-C9 cells). Expression of the spike protein was confirmed by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Subsequently, cells were subjected to transfection with pT7-IRES-Luc2 to generate pseudovirus cells. These cells were then co-cultured with target cells to assess the efficiency of infection. After co-culture for 20 h, luminescence intensity was approximately 260-fold higher than that in the control, which included mock-transfected pseudovirus cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Microscopic analysis revealed the presence of large multinucleated cells, indicating spike-ACE2-mediated cell fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, red arrow). To verify that the fused cells were the result of a spike-ACE2 interaction, the experiment was repeated using the pT7-IRES-EmGFP reporter instead of the pT7-IRES-Luc2 reporter. The reporter drives expression of the fluorescent protein EmGFP in the presence of T7 polymerase. Strong green fluorescent protein (GFP) fluorescence was observed in multinucleated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, white arrowhead). This observation confirms that fusion was dependent on the interaction between spike and ACE2. Collectively, these results substantiate the reliability of our assay in recapitulating spike-mediated infection and facilitating quantitative assessment of inhibitory compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInhibitory effects of catechins in a cell-based SARS-CoV-2 infection assay\u003c/h3\u003e\n\u003cp\u003eUsing the established cell-based infection assay, we evaluated the inhibitory effects of 14 catechin derivatives (C, CG, GCG, EC, ECG, EGC, EGCG, ECGMe, EGCGMe, TF, TF3G, TFDG, TA, and TB). Pseudovirus and target cells were pre-incubated with each catechin (100 \u0026micro;M) for 30 min at 37\u0026deg;C, followed by co-culture in the continued presence of catechins for 20 h. Camostat mesylate was utilized as a control to assess the efficacy of potential infection inhibitors. The initial phase of the SARS-CoV-2 infection is marked by the binding of the spike protein RBD to ACE2, which is followed by viral entry via two distinct pathways: the TMPRSS2-mediated membrane fusion pathway and the TMPRSS2-independent endocytic pathway. Camostat mesylate selectively inhibits the former pathway by targeting TMPRSS2 and led to 50% inhibition of the infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Luminescence analysis revealed that eight catechins (GCG, EGCG, EGCGMe, EGC, TF3G, TFDG, TF, and ECGMe) and TB significantly reduced luciferase activity relative to the vehicle control (dimethyl sulfoxide), indicating strong inhibition of cell-cell fusion between pseudovirus cells and target cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) In particular, four pyrogallol-type catechins (EGC, EGCG, EGCGMe, and GCG) showed significant inhibition. The findings indicate that these catechins exhibited a substantial inhibitory effect on the process of cell\u0026ndash;cell fusion between pseudovirus cells and target cells, suggesting a potential for these catechins to impede SARS-CoV-2 infection. In order to ascertain whether this inhibition targeted the spike protein or ACE2, we pre-incubated catechins either with pseudovirus cells or target cells separately before co-culturing with the other cell type. Interestingly, camostat mesylate, which is known to inhibit TMPRSS2 activity in target cells, inhibited infection only when pre-incubated with the target cells, demonstrating approximately 50% inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). By contrast, pre-incubating pseudovirus cells with camostat mesylate had little to no effect on infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings, where camostat mesylate's efficacy is dependent on target cell pre-incubation, align with its known mechanism as a TMPRSS2 inhibitor that acts on host cells. This demonstrates that the assay system can accurately reflect the inhibitor's mode of action, thereby confirming its effectiveness and appropriateness. When treating pseudovirus cells only, GCG, EGCGMe, and EGC demonstrated significant inhibitory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, all eight catechins and TB showed significant inhibitory effects when used to treat target cells only (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results suggest that catechins act on both viral and host cell components, including the spike protein and ACE2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral studies have reported direct interactions between catechins and the spike protein RBD.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The present study focused on these interactions to identify the structural features of catechins that are critical for inhibitory activity. A subsequent analysis focusing on the B-ring structure revealed that catechins with a catechol-type configuration bearing hydroxyl groups at the 3- and 4-positions, such as C, EC, CG, and ECG, did not demonstrate significant inhibition. However, pyrogallol-type catechins with an additional hydroxyl group at the 5-position, such as EGC, GCG, and EGCG, exhibited significant inhibitory activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, the three catechins that interacted with the spike protein (EGCG, GCG, and EGC) all share a common feature: a pyrogallol-type B-ring structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings indicate that the pyrogallol-type B-ring is a critical structural determinant for catechin-mediated inhibition of SARS-CoV-2 infection. A comparison between epi- and non-epi-type catechins, which differ in stereochemistry at the C2 position of the C-ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), revealed that both EGCG, which are important catechins found in green tea, effectively inhibited infection when pre-incubated with either spike-expressing pseudovirus cells or ACE2-expressing target cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, when incubated with spike protein alone, only GCG exhibited significant inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that the non-epi configuration may facilitate more effective interaction with the spike protein.\u003c/p\u003e\n\u003ch3\u003eDocking simulation of catechins with spike RBD\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e docking simulations of selected catechins with the RBD of the SARS-CoV-2 spike protein were performed using AutoDock Vina to elucidate the molecular basis for the observed inhibition. The docking poses of both catechol-type (A\u0026ndash;C, green) and pyrogallol-type (D\u0026ndash;F, blue) catechins are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The pink regions represent 10 key amino acid residues predicted \u003cem\u003ein silico\u003c/em\u003e to be critical for the interaction between the RBD and ACE2.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The importance of three of these amino acid residues for actual binding was confirmed by \u003cem\u003ein vitro\u003c/em\u003e pull-down assays using recombinant RBD and ACE2.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e A remarkable observation of the present study is that all catechins were shown to bind to the cavity of the RBD located around the critical amino acids for binding to ACE2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which occurred in a variety of binding modes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of these simulations were analyzed to extract catechin pairs that share the same binding mode by comparing catechol-type molecules (EC, CG, ECG) with their corresponding pyrogallol-type counterparts (EGC, GCG, EGCG; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;I, light blue). The energies for these dockings, which share the same binding mode, are presented in Table\u0026nbsp;1. The pyrogallol-type catechins exhibited stronger binding (lower docking energies) than the catechol-type catechins (Table\u0026nbsp;1). These results indicate that pyrogallol-type catechins, despite having equivalent binding modes, exhibit an increased affinity for the RBD compared with their catechol-type counterparts. Binding mode 4, which is listed in Table\u0026nbsp;1 as a common binding conformation for CG and GCG, is shown in an enlarged view in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In GCG, the oxygen at the 5-position of the B-ring forms a hydrogen bond with Gln493 (3.0 \u0026Aring;; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), whereas CG lacks a hydroxyl group at this position and therefore cannot establish the same interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The presence of an additional hydrogen bond in GCG appears to have a significant impact on molecular orientation, thereby facilitating the formation of a new hydrogen bond between the hydroxyl group at the 3-position of the B-ring and the oxygen atom of Tyr453, with a distance of 3.0 \u0026Aring;. Additionally, a notable shortening in distance is observed among the hydrogen bonds involving the oxygen and hydroxyl group at the 4-position of the B-ring, suggesting greater strength in these interactions. These effects synergistically enhance the overall binding strength, and findings indicate that the presence of a hydroxyl group at the 5-position of the B-ring significantly enhances the interaction with the RBD, even though binding occurs within the same region. The stereoisomeric nature of epi- and non-epi-type catechins means that their three-dimensional structures exhibit significant differences, which complicates the identification of a shared binding mode. Binding energies for the catechins with the highest values obtained in docking simulations are presented in Table\u0026nbsp;2. Overall, non-epi-type catechins (C, CG, GCG) exhibited stronger binding to the spike protein RBD than their epi-type counterparts (EC, ECG, EGCG). This phenomenon can be attributed to the trans-configuration of non-epi-type catechins, which allows for an increased contact area with the protein surface and positions the hydroxyl group at the 5-position of the B-ring in a favorable orientation for binding. This in turn enhances inhibitory activity. Collectively, the docking simulation results support our \u003cem\u003ein vitro\u003c/em\u003e findings, demonstrating that catechins with pyrogallol-type B-rings and non-epicatechin stereochemistry exhibit stronger and more versatile binding to the spike RBD than their catechol-type counterparts. These structural features are likely to act synergistically to inhibit the RBD-ACE2 interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the inhibitory effects of structurally diverse catechins on the interaction between the SARS-CoV-2 spike protein RBD and the human ACE2 receptor using a cell-based pseudovirus assay. The findings of this study demonstrate that catechins with a pyrogallol-type B-ring exhibit significantly greater inhibitory activity than their catechol-type counterparts. Additionally, it is thought that the non-epi stereochemistry may also contribute to these inhibitory effects to some extent. These conclusions were consistently supported by both experimental infection assays and \u003cem\u003ein silico\u003c/em\u003e docking simulations. Previous computational studies have identified amino acid residues within the RBD (specifically Tyr449, Tyr453, Leu455, Asn487, Tyr489, Gln493, Gln498, Thr500, Asn501, and Tyr505) as being critical for interactions with ACE2. In this study, the importance of Tyr453, Gln493, and Asn501 was experimentally validated by performing pull-down assays.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The docking simulations showed that a significant proportion of the catechins exhibited a binding preference for regions close to these residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, pink), confirming their ability to inhibit RBD-ACE2 complex formation. Whereas prior studies have primarily focused on EGCG because of its abundance in green tea, our study highlights that pyrogallol-type (GCG, EGC, and EGCGMe) and non-epi-type catechins (GCG) may offer superior inhibition of SARS-CoV-2 entry. This study represents the first SAR investigation of catechins, with a particular focus on comparing their potency in relation to B-ring hydroxylation and C-ring stereochemistry.\u003c/p\u003e \u003cp\u003eNotably, catechin epimerization occurs during tea processing, resulting in increased levels of non-epi-type catechins in ready-to-drink bottled teas. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Although these epimers are present in lower concentrations in freshly brewed tea, they may contribute substantially to the efficacy of the antiviral properties of tea. The bioavailability of catechins, particularly EGCG, has been investigated. Only a small proportion of orally ingested EGCG is absorbed in the small intestine, with peak plasma concentrations typically reaching no more than 1 \u0026micro;M.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e As demonstrated by the \u003cem\u003ein vitro\u003c/em\u003e experiments, which showed inhibitory effects on RBD-ACE2 binding occurring with ~\u0026thinsp;100 \u0026micro;M EGCG, it is improbable that similar effects would occur systemically at physiological concentrations. Considering the catechin concentration of ~\u0026thinsp;100 \u0026micro;M in green tea\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and the localization of SARS-CoV-2 infection to mucosal surfaces, frequent consumption or gargling may allow these compounds to exert localized antiviral effects, despite limited systemic bioavailability.\u003c/p\u003e \u003cp\u003eAlthough the direct binding of individual catechins to the RBD is relatively weak, our docking data suggest that multiple molecules can bind in overlapping but independent binding poses in diverse modes, collectively masking critical ACE2-binding regions. Given the propensity of the virus to undergo genetic mutations that alter its RBD structure, the capacity of catechins to engage the RBD through multiple independent binding modes has the potential to provide a robust antiviral mechanism that is less susceptible to evasion by viral mutation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eTeadenols A and B were obtained from the Japanese post-fermented tea \u0026ldquo;Kippuku-cha\u0026rdquo; as previously described.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Catechins and theaflavins were purchased from Nagara Science Co. Ltd. (Gifu, Japan). Unless otherwise noted, other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan) and Nakalai Tesque (Kyoto, Japan). All reagents used were of the highest available analytical grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid design\u003c/h3\u003e\n\u003cp\u003eGenes encoding \u003cem\u003eLuc2\u003c/em\u003e and \u003cem\u003eEmGFP\u003c/em\u003e were amplified by polymerase chain reaction from pGL4.23 (Promega) and pcDNA6.2/N-EmGFP-DEST (ThermoFisher Scientific, Waltham, MA, USA), respectively. These fragments were cloned into the pT7-IRES His-C vector (Takara Bio, Ohtsu, Japan) downstream of the internal ribosome entry site (IRES) using the In-Fusion cloning system (Takara Bio), resulting in the reporter plasmids pT7-IRES-Luc2 and pT7-IRES-EmGFP. The SARS-CoV-2 spike protein expression vector, pBA-spike-C9, was constructed by amplifying the spike gene, including a C-terminal C9 tag, from pcDNA3.1-SARS2-spike and cloning it into the BamHI site of the pBApo-EF1α Pur vector (Takara Bio) using In-Fusion cloning. The plasmid pcDNA3.1-SARS2-spike was a gift from Fang Li \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e(Addgene plasmid #145032). The T7 RNA polymerase expression vector, pCAG-T7pol, was a gift from Dr. Ian Wickersham (Addgene plasmid #59926).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eVeroE6/TMPRSS2 cells and 293T cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin, at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. VeroE6/TMPRSS2 cells\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (JCRB1819) were obtained from JCRB Cell Bank (Osaka, Japan). 293T cells were provided from RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransfection of DNA\u003c/h2\u003e \u003cp\u003eTransfections were performed using polyethyleneimine Max (PEI; Polysciences, Inc., Warrington, PA, USA) as previously described. \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Briefly, 4 \u0026micro;g plasmid DNA was mixed with 250 \u0026micro;L Opti-MEM and incubated at 25\u0026ordm;C for 5 min. Separately, 10 \u0026micro;g PEI was diluted in 250 \u0026micro;L Opti-MEM and was also incubated for 5 min. The PEI and DNA solutions were then combined and incubated for a further 20 min. The mixture was added to a six-well plate containing 5 \u0026times; 10⁵ cells in 2 mL DMEM with 5% FBS. After 4 h, the medium was replaced with fresh DMEM containing 5% FBS and the cells were incubated overnight. To generate spike-expressing cells, 293T cells were transfected with pBA-Spike-C9 and selected with 2 \u0026micro;g/mL puromycin. Clonal selection was performed by limiting dilution. Clones expressing high levels of spike protein were identified by western blotting as previously described\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e using an anti-rhodopsin antibody (Rho 1D4, Sigma) recognizing the C9 tag. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell-based SARS-CoV-2 infection assay\u003c/h2\u003e \u003cp\u003ePseudovirus cells were generated by transfecting 293T-Spike-C9 cells with pCAG-T7pol using PEI as described above. Target cells were generated by transfecting VeroE6/TMPRSS2 cells with pT7-IRES-Luc2 (or pT7-IRES-EmGFP for fluorescence assays). VeroE6/TMPRSS2 cells highly express ACE2, the receptor for SARS-CoV-2 entry.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Both cell types were washed with saline and detached using TrypLE Express (ThermoFisher Scientific) for 3 min at 37\u0026deg;C. They were resuspended in DMEM with 5% FBS. Pseudovirus and target cells (2 \u0026times; 10⁴ each) were pre-incubated with catechins (final concentration: 100 \u0026micro;M) or vehicle (DMSO) for 30 min, then co-seeded and co-cultured for 20 h at 37\u0026deg;C. The next day, luciferase activity was quantified using the PicaGene\u0026trade; luciferase assay system (Toyo Ink, Tokyo, Japan), and luminescence was measured with a Luminoskan\u0026trade; Ascent (ThermoFisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDocking simulation\u003c/h2\u003e \u003cp\u003eThe crystal structure of the SARS-CoV-2 RBD-ACE2 complex (PDB ID: 6M0J) was downloaded from the Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using PyMOL, all water molecules, ligands, ions, and the ACE2 portion were removed. Polar hydrogens and charges were added in AutoDock Vina (version 1.5.7, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccsb.scripps.edu/mgltools/\u003c/span\u003e\u003cspan address=\"https://ccsb.scripps.edu/mgltools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the structure was converted to PDBQT format. In accordance with previous studies, 10 RBD residues critical for ACE2 interaction (Tyr449, Tyr453, Leu455, Asn487, Tyr489, Gln493, Gln498, Thr500, Asn501, andTyr505)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e were designated as flexible side chains. Separate PDBQT files were prepared for flexible and rigid regions. Catechin structures were obtained from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in SDF format, energy-minimized using the Merck Molecular Force Field 94 (MMFF94), and converted to PDBQT format. The grid box was configured to encompass the entire RBD. Docking simulations were conducted in AutoDock Vina by assigning each catechin as a ligand with rotatable bonds, following standard procedures.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCH, (-)-Catechin hydrate; EC, (-)-epicatechin; CG, (-)-catechin gallate; ECG, (-)-epicatechin gallate; ECGMe, (-)-epicatechin 3-(3''-O-methyl) gallate; GC, (-)-gallocatechin; EGC, (-)-epigallocatechin; GCG, (-)-gallocatechin gallate; EGCG, (-)-epigallocatechin gallate; EGCGMe, (-)-epigallocatechin 3-(3''-O-methyl) gallate; TF, theaflavin; TF3G, theaflavin 3-gallate; TFDG, theaflavin 3,3’-di-O-gallate; TA, teadenol A; TB, teadenol B\u003c/p\u003e\n\u003cp\u003eACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.M., S.N., and N.I. conducted the experiments. K.I., K.S., and T.M. provided the resources and methodology. Y.H. validated and supervised the experiments. S.M. was responsible for conceptualization, manuscript writing, supervision, and project administration.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported in part by a Grant-in-Aid for Scientific Research (C) (Grant No. 23K05130) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We also gratefully acknowledge funding from the 2020 Advanced Research Promotion Program of the United Graduate School of Agricultural Sciences, Kagoshima University. We thank Professor Yukio Nagano of Saga University for his invaluable support in obtaining government approval for experimental procedures involving the SARS-CoV-2 spike gene during the COVID-19 pandemic. We would like to express our gratitude to Dr. M. Horitani and Ms. S. Tsuji (Saga University) for their valuable technical guidance in the docking simulations. The authors also thank all laboratory members for their technical assistance and helpful discussions throughout the course of this study. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShang, J. et al. Cell entry mechanisms of SARS-CoV-2. \u003cem\u003eProc Natl. Acad. Sci. U S A\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmann, M. et al. 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Bio\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, 3081\u0026ndash;3089 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6766643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6766643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSevere acute respiratory syndrome coronavirus 2 infects host cells through binding of the spike protein receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 receptor. In this study, the antiviral activity of 14 catechin derivatives was evaluated using a pseudovirus assay that emulates spike-mediated cell fusion. Among these, gallocatechin gallate, epigallocatechin gallate, epigallocatechin 3-(3''-O-methyl) gallate, and epigallocatechin notably exhibited strong inhibitory effects on infection. A structural comparison of the compounds revealed that catechins with a pyrogallol-type B-ring configuration exhibited greater inhibitory effects than their catechol-type counterparts. Docking simulations demonstrated that the hydroxyl group at the 5-position of the B-ring forms a hydrogen bond with Gln493 on the spike RBD, thereby facilitating additional stabilizing interactions with adjacent residues, such as Tyr453. Although catechin bioavailability is low, the results of this study suggest that regular consumption or gargling may offer localized antiviral activity at mucosal surfaces, such as those found in the oral or nasal cavity, because the catechin concentrations used in the cell assays are similar to those observed in green tea (100 \u0026micro;M). This study underscores the potential of pyrogallol-type catechins to act as antiviral agents.\u003c/p\u003e","manuscriptTitle":"Pyrogallol-Type Catechins Inhibit SARS-CoV-2 by Disrupting Spike-ACE2 Binding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 06:26:09","doi":"10.21203/rs.3.rs-6766643/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T06:03:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T10:55:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103186931160714512163343027146661821033","date":"2025-10-05T11:47:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321604040865525425150074020841711463404","date":"2025-08-11T17:55:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T17:56:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38973578635638682428967404744635716017","date":"2025-06-26T11:48:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69481287093903786439408651956864426279","date":"2025-06-18T06:18:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T06:13:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T06:12:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-10T09:04:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T09:20:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-28T09:32:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"423c02c5-ffc5-4650-a566-6478adfca309","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50329947,"name":"Biological sciences/Microbiology/Virology"},{"id":50329948,"name":"Biological sciences/Biochemistry"},{"id":50329949,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2026-03-02T16:02:40+00:00","versionOfRecord":{"articleIdentity":"rs-6766643","link":"https://doi.org/10.1038/s41598-026-41170-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-28 15:59:02","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-06-20 06:26:09","video":"","vorDoi":"10.1038/s41598-026-41170-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41170-6","workflowStages":[]},"version":"v1","identity":"rs-6766643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6766643","identity":"rs-6766643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}