Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 μM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.

Ginkgolic acids (GA), a group of alkyl phenols found in crude extracts of Ginkgo biloba leaves, are known to have anticancer activity, but their mode of action is not well understood. Our aim in this study was to investigate the anti-migratory activity of seven GA against breast cancer cells and to determine the molecular mechanism behind this activity. All seven GA and their mixture inhibited wound healing in MCF-7 and MDA-MB 231 breast cancer cells. None of the compounds nor the mixture showed cytotoxicity towards the two cell lines, if tested by the resazurin assay. C13:0 inhibited NF-κB activity in the HEK Blue Null 1 reporter cell line. Furthermore, C13:0 inhibited degradation of nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor α (IκBα). Sumoylation assay revealed that GA inhibited sumoylation of NF-κB essential modulator (NEMO). Molecular docking on SUMO-activating enzyme E1 showed that the seven GA bound to the active adenylation site with high calculated affinities ranging from -10.28 to -12.27 kcal/mol. Quantitative RT-PCR using C15:0, C13:0 and the mixture showed a significant down-regulation of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinase 9 (MMP-9). We conclude that GA revealed considerable anti-migratory activity at non-cytotoxic concentrations, indicating anti-metastatic activity with low toxicity. This effect can be explained by the inhibition of NEMO sumoylation leading to inhibition of IκBα degradation and consequently a reduction of NF-κB activity, leading to the down-regulation of metastasis related genes including uPA, PAI-1, CXCR4, and MMP-9.

Background and Purpose Celastrol exhibits anti‐arthritic effects in rheumatoid arthritis (RA), but the role of celastrol‐mediated Ca 2+ mobilization in treatment of RA remains undefined. Here, we describe a regulatory role for celastrol‐induced Ca 2+ signalling in synovial fibroblasts of RA patients and adjuvant‐induced arthritis (AIA) in rats. Experimental Approach We used computational docking, Ca 2+ dynamics and functional assays to study the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase pump (SERCA). In rheumatoid arthritis synovial fibroblasts (RASFs)/rheumatoid arthritis fibroblast‐like synoviocytes (RAFLS), mechanisms of Ca 2+ ‐mediated autophagy were analysed by histological, immunohistochemical and flow cytometric techniques. Anti‐arthritic effects of celastrol, autophagy induction, and growth rate of synovial fibroblasts in AIA rats were monitored by microCT and immunofluorescence staining. mRNA from joint tissues of AIA rats was isolated for transcriptional analysis of inflammatory genes, using siRNA methods to study calmodulin, calpains, and calcineurin. Key Results Celastrol inhibited SERCA to induce autophagy‐dependent cytotoxicity in RASFs/RAFLS via Ca 2+ /calmodulin‐dependent kinase kinase‐β–AMP‐activated protein kinase–mTOR pathway and repressed arthritis symptoms in AIA rats. BAPTA/AM hampered the in vitro and in vivo effectiveness of celastrol. Inflammatory‐ and autoimmunity‐associated genes down‐regulated by celastrol in joint tissues of AIA rat were restored by BAPTA/AM. Knockdown of calmodulin, calpains, and calcineurin in RAFLS confirmed the role of Ca 2+ in celastrol‐regulated gene expression. Conclusion and Implications Celastrol triggered Ca 2+ signalling to induce autophagic cell death in RASFs/RAFLS and ameliorated arthritis in AIA rats mediated by calcium‐dependent/‐binding proteins facilitating the exploitation of anti‐arthritic drugs based on manipulation of Ca 2+ signalling.

The outbreak of SARS-CoV-2 virus caused more than 80,155,187 confirmed COVID-19 cases worldwide, which has posed a serious threat to global public health and the economy. The development of vaccines and discovery of novel drugs for COVID-19 are urgently needed. Although the FDA-approved SARS-CoV-2 vaccines has been launched in many countries recently, the strength of safety, stringent storage condition and the possibly short-term immunized efficacy remain as the major challenges in the popularity and recognition of using vaccines against SARS-CoV-2. With the spike-receptor binding domain (RBD) of SARS-CoV-2 being responsible for binding to human angiotensin-converting enzyme 2 receptor (hACE2), ACE2 is identified as the receptor for the entry and viral infection of SARS-CoV-2. In this study, molecular docking and biolayer interferometry (BLI) binding assay were adopted to determine the direct molecular interactions between natural small-molecule, 1,2,3,4,6-Pentagalloyl glucose (PGG) and the spike-RBD of the SARS-CoV-2. Our results showed that PGG preferentially binds to a pocket that contains residues Glu 340 to Lys 356 of spike-RBD with a relatively low binding energy of -8 kcal/mol. BLI assay further confirmed that PGG exhibits a relatively strong binding affinity to SARS-CoV-2-RBD protein in comparison to hACE2. In addition, both ELISA and immunocytochemistry assay proved that PGG blocks SARS-CoV-2-RBD binding to hACE2 dose dependently in cellular level. Notably, PGG was confirmed to abolish the infectious property of RBD-pseudotyped lentivirus in hACE2 overexpressing HEK293 cells, which mimicked the entry of wild type SARS-CoV-2 virus in human host cells. Finally, maximal tolerated dose (MTD) studies revealed that up to 200 mg/kg/day of PGG was confirmed orally safe in mice. Our findings suggest that PGG may be a safe and potential antiviral agent against the COVID-19 by blockade the fusion of SARS-CoV-2 spike-RBD to hACE2 receptors. Therefore, PGG may be considered as a safe and natural antiviral agent for its possible preventive application in daily anti-virus hygienic products such as a disinfectant spray or face mask.