Computational and experimental evidence of the anti‐COVID‐19 potential of honeybee propolis ingredients, caffeic acid phenethyl ester and artepillin c (2023)

Honeybee products such as honey, propolis, beeswax and royal jelly have been reported to have antiviral, anticancer and immunomodulatory potential (Cornara, Biagi, Xiao, & Burlando, 2017; Pasupuleti, Sammugam, Ramesh, & Gan, 2017). Several studies have shown that propolis has antiviral properties for influenza and the human rhinovirus (Bhargava et al., 2021; Kwon, Shin, Perumalsamy, Wang, & Ahn, 2020; Shimizu et al., 2008). The antiviral properties of propolis have been assigned to its polyphenolic components, including kaempferol, coumaric acid, caffeic acid phenethyl ester (CAPE), and others (Erdemli, Akyol, Armutcu, & Akyol, 2015; Kumar, Dhanjal, Kaul, Wadhwa, & Sundar, 2021; Schnitzler et al., 2010). Although the antiviral properties of propolis and its active constituent are known, the molecular mechanism of action of these compounds against SARS-CoV-2 has not yet been reported. In the present study, the molecular interactions and anti-COVID-19 activity of two main components, CAPE and ARC (artepillin c) of New Zealand and Brazil propolis, respectively, have been investigated through in silico and in vitro experiments. The 3D structures of main protease (Mpro), Angiotensin-converting enzyme2 (ACE2)/spike protein and ACE2 bound with its competitive inhibitor MLN4760 were downloaded from protein data bank (PDB; IDs 6 LU7, 6LZG and 1R4L, respectively). The experimental 3D crystal structure of Transmembrane Serine protease 2 (TMPRSS2) was not available in the PDB, therefore, homology modelled structure was retrieved from Swiss model repository (O15393) (2022). The ligands, namely, ARC (CID 5472440) and CAPE (CID 5281787), were retrieved from PubChem. The pre-processing of proteins and ligands, the molecular docking and classical MD simulations (100 ns) were performed to analyze the binding interactions and stability of the ligands against the protein targets using the Schrodinger Maestro suite as described earlier (Kumar et al., 2022; Dhanjal et al., 2021; Kalra et al., 2022; Kumar, Singh, Hasnain, & Sundar, 2021; Schrödinger Release, 2020). The effect of CAPE and ARC on the expression level of ACE2 and TMPRSS2 was also examined by Western blotting (WB), immunocytochemistry (ICC) using specific antibodies. Finally, the nontoxic doses (as determined by independent cell viability assays) of CAPE and ARC were used to determine their anti-SARS-CoV-2 (USA-WA1/2020 strain) in Vero E6 cells by qRT-PCR-based antiviral activity assay (Dhanjal et al., 2021). Both CAPE and ARC showed unstable binding at ACE2-spike interface. Further, these compounds along with positive control (MLN4760) were docked and simulated at ACE2 active site. The docking result showed that the two compounds could bind with ACE2 active site with considerable binding energy involving 2–3 hydrogen bonds as shown in Figure 1a–c and Table S1 . When these complexes were simulated further, they showed stable interactions involving Arg 273 and Tyr515 as critical interacting residues making hydrogen bond and water-mediated interactions similar to MLN4760 (Figure S1). In the ACE2-ARC complex, Tyr515 was involved in hydrogen bonding and His374, Glu375, His378, Glu402 and Zn803 were involved in ionic interaction for a significant amount of simulation time (Figure S1). In ACE2-CAPE complex, Arg514, Tyr515 and Zn were involved in the hydrophobic interactions (Figure S1). To investigate the binding affinities of the ligands towards the ACE2, the MM/GBSA binding free energy was calculated. The binding free energy showed that MLN4760 (−24 ± 1.04 kcal/mol) had the highest binding energy, followed by CAPE (−15.93 ± 7.3 kcal/mol) and ARC (−4.76 ± 2.29 kcal/mol). We next investigated the interactions of CAPE and ARC with the TMPRSS2 catalytic site (Table S1). Although compounds showed stable docking, MD simulation showed that ARC, but not CAPE could interact stably with TMPRSS2 throughout the 100 ns simulation (Figure 1d). The polar and non-polar interaction occupancy of the TMPRSS2 with ARC showed that Gly462 was the only residue that was making crucial interaction (>30% time of the simulation) (Figure S2a). The average MM/GBSA free binding energy of ARC with TMPRSS2 was −28.76 ± 4.65 kcal/mol. In the case of Mpro, both CAPE and ARC could dock at its catalytic site (Figure 1e,f and Table S1). And both CAPE and ARC showed stable interaction with the binding pocket of Mpro throughout the 100 ns of the MD simulation. Simulation interaction occupancy calculation showed His41 and Gln192 were interacting significantly with CAPE and ARC, respectively (Figure S2b,c). The free binding energy showed that CAPE (−43.22 ± 8.67 kcal/mol) and ARC (−36.94 ± 4.17 kcal/mol) had a similar binding affinity. Human oesophageal (T.Tn) and oral squamous (HSC3) cell carcinoma that showed moderate level of ACE2 and TMPRSS2 expression, both at the mRNA and protein, in several independent experiments were selected (Dhanjal et al., 2021; Kalra et al., 2022). As shown in Figure 2, T.Tn cells treated with nontoxic doses of both the compounds, as determined by dose-dependent viability assay (Figures 2a,b), showed a decrease in ACE2 (Figures 2c,d). CAPE-treated cells showed stronger downregulation in ACE2 as compared to the ARC-treated cells, both by WB and ICC assays. mRNA expression of the receptors was next determined by RTqPCR. As shown in Figure 2e, a significant decrease in ACE2 mRNA was detected in cells treated with either ARC or CAPE. Consistent with the WB and ICC data, CAPE-treated cells showed stronger downregulation as compared to the ARC-treated cells. In contrast to the effect of CAPE on both ACE2 and TMPRSS2, ARC caused decrease in ACE2 expression only. TMPRSS2 expression did not show decrease either by WB or ICC (Figures 2c,d) or by RTqPCR in cells treated with ARC (Figure 2e). We also examined the effect of CAPE and ARC on ACE2 and TMPRSS2 expression in HSC3 cells. As shown in Figure S3, CAPE, as compared to ARC, caused stronger downregulation of ACE2 expression. Furthermore, similar to the T.Tn cells, TMPRSS2 expression in HSC3 did not show decrease. We next used nontoxic doses (as determined by independent cell viability assays) of CAPE and ARC to determine their anti-SARS-CoV-2 (USA-WA1/2020 strain) in Vero E6 cells by qRT-PCR-based antiviral activity assay. As shown in Figure 2f, a nontoxic dose of ARC and CAPE caused ~7% and ~33% inhibition in virus replication. These data were consistent with the stronger effects of CAPE as compared to ARC as predicted by the computational and expression assays. The results warrant further laboratory and clinical trials for the therapeutic use of the studied molecules. Vipul Kumar, Anissa Nofita Sari: contributed to computational analysis and manuscript writing; Hazna Noor Meidinna, Ashish Kaul, Brohmomoy Basu: contributed to experimental validation; Yoshiyuki Ishida and Keiji Terao contributed to reagents, resources, and manuscript writing; Sunil C. Kaul, Sudhanshu Vrati contributed to conception, manuscript writing and funding; Durai Sundar, Renu Wadhwa contributed to conception, design, manuscript writing and funding. All authors read and approved the final manuscript. This study was supported by the funds granted by AIST (Japan) and DBT (Government of India) grant no. BT/BI/14/042/2017. The authors declare no conflict of interest. All the data have been provided in the manuscript and in supplementary information. Appendix S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.