Fig 1: FXa inhibits wild-type SARS-CoV-2 infection by targeting viral particles.(a and b) FX protein levels in lungs (a) or serum (b) of COVID-19 patients vs. non-COVID-19 donors, using IHC (a) and ELISA (b), respectively. (c) FX concentrations in serum of COVID-19 patients as shown post diagnosis of infection were measured by ELISA. (d and e) HEK293T cells co-transfected with ACE2 and FXa or an EV in the absence (d) or presence (e) of TMPRSS2 were infected by VSV-SARS-CoV-2, and quantified by flow cytometry at 16, 24, 36, and 48 hpi. (f) MA104 cells transduced with FXa (MA104-FXa) or EV (MA104-EV) were infected by VSV-SARS-CoV-2 and imaged at 16, 24, 36, and 48 hpi by fluorescence microscopy. (g and h) VSV-SARS-CoV-2 was preincubated with FXa at indicated concentrations 1 hour before infection. Cells were imaged at 16, 24, 36, and 48 hpi by fluorescence microscopy (g) and the corresponding infectivity was measured by flow cytometry (h). (i and j). MA104 and Vero E6 cells were infected with live wild-type SARS-CoV-2. At 24 hpi, infectivity was measured by immune-plaque assay (i). (j) The summary data of (i). Experiments in i are representative of two independent experiments with similar data, and the other data are representative of at least three independent experiments. For all panels, error bars indicate standard deviation (SD), and statistical analyses were performed by Student’s t tests (b-e) and linear mixed model (h). *P = 0.05; **P = 0.01; ***P = 0.001; ****P = 0.0001; n.s, not significant.
Fig 2: HT (Hesperetin) and HD (Hesperidin) suppressed the protein expressions of ACE2 and TMPRSS2 in normal and malignant lung cells. Beas 2B (A) and H460 (B) cell lines were treated with HT and HD in a dose-dependent manner for 2 days followed by the examination of protein expression in Western blotting with indicated antibodies. The quantitative results of ACE2 and TMPRSS2 expressions in Beas 2B and H460 were normalized with a level of actin and were shown below to Western blot images. ? is shown as the mean of every independent experiment. Data are shown as mean ± SEM from 3 independent experiments with triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS, no significance.
Fig 3: HT (Hesperetin) decreased the interaction of ACE2 and the spike protein. FRET assay was performed to determine the interaction between human receptor ACE2 and S protein (A). The in vitro enzymatic activity of TMPRSS2 (B), PLpro (C), and 3CLpro (D) was determined after 1 hr incubation with HT and HD (Hesperidin). Data are shown as mean ± SEM from 3 independent experiments with triplicates. * p < 0.05.
Fig 4: FXa suppresses viral entry by binding to and cleaving the SARS-CoV-2 S protein.(a) The binding affinity of FXa with full-length wild-type S protein, subunit S1, subunit S2, and RBD was quantified by ELISA. (b and c) The interaction between FXa protein and full-length S protein was examined by pull-down assay (b). The binding affinity at indicated concentrations of FXa was measured by ELISA (c). (d) The cleavage of S protein by furin, TMPRSS2, and FXa was analyzed by immunoblotting. (e). Schema of the cleavage sites for furin, TMPRSS2 and FXa on the full-length S protein. (f) 293 T cells were co-transfected with a S protein expression plasmid and various amounts of an FXa expression plasmid. S protein cleavage by FXa inside of cells was analyzed by immunoblotting. All data are representative of at least three independent experiments. Experiments in b and d are representative of three independent experiments with similar data. For all panels, error bars indicate SD, and statistical analyses were performed by one-way ANOVA models. ***P = 0.001.
Fig 5: Molecular docking pose visualization for the interaction of ACE2/TMPRSS2 and HT/HD. Compound–protein interaction between HT (PDB code: 5JDC; red structure)/HD (PDB code: 6CCF; blue structure) and ACE2 protein (PDB code: 3D0G) in 3D (A–D) and 2D (E,F). Compound–protein interaction between HT/HD and TMPRSS2 protein (PDB code: 1Z8A) in 3D (G–J) and 2D (K,L).
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