Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The inhibition of the interaction between the spike protein S1 receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor by kuwanon C (KC). Spike protein coated on a 96-well plate interacted with a preincubated mixture of the ACE2 receptor and ( A ) anti-SARS-CoV-2 spike S1 antibody as the positive control and ( B ) 0, 3.125, 6.25, 12.5, 25, 50, or 100 μM KC. The inhibition of the spike S1 RBD:ACE2 receptor interaction by KC was determined based on chemiluminescence measurements.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Inhibition, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The global kinetic analysis of KC binding to biotinylated ( A ) spike S1 RBD- and ( B ) ACE2 receptor-immobilized BLI sensors. The kinetics for the binding of KC to the spike S1 RBD or the ACE2 receptor were measured by the association of 0, 50, 200, and 400 μM of KC in PBS containing 1% DMSO with immobilized spike S1 or ACE2 receptor and the subsequent dissociation in PBS containing 1% DMSO.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The binding kinetics of KC to spike S1 RBD and ACE2 receptor.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: In silico docking simulation between KC and the spike protein/ACE2 receptor. KC was docked onto the SARS-CoV-2 spike protein and ACE2 receptor (PDB code: 6M0J) using AutoDock Vina. The pharmacophore of KC with each target proteins was analyzed using BIOVIA Discovery Studio Visualizer.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: In Silico

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC inhibits SARS-CoV-2 lentiviral pseudovirus infection in HEK293T cells stably expressing human ACE2 and TMPRSS2. ( A ) The cytotoxic effect of KC in HEK293T cells stably expressing human ACE2 and TMPRSS2 was determined using the MTT assay. HEK293T cells were cultured in 96-well plates (5 × 10 4 cells/well) for 18 h. ( B ) The ACE2 expression level in HEK293T cells was monitored during KC treatment using real-time quantitative PCR analysis. Then, WT or mutant (D614G) SARS-CoV-2 spike pseudovirus (at a final concentration of 1 × 10 4 TU/mL to each well) were mixed with different concentrations of KC (2 and 20 μM) or anti-SARS-CoV-2 antibody, and the mixtures were incubated at 37 °C for 1 h. Then, these mixtures were added to HEK293T cells. ( C , D ) Green fluorescent protein (GFP) expression levels using flow cytometry were assessed at 72 h after viral infection, scale bar = 100 μm. ( E , F ) The inhibitory effect of SARS-CoV-2 spike pseudovirus infection was determined by measuring GFP expression using flow cytometry and measured under a fluorescence microscope. Bar graph (mean ± SEM) statistics were determined from three experimental data sets using one-way ANOVA with Tukey’s post hoc test, *** p < 0.001, compared with the CON (KC-untreated) samples. ### p < 0.001, compared with the cell-only sample.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Stable Transfection, Expressing, MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC suppresses the infection of a clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) in Vero cells. Vero cells were cultured on 384-well plates (1.2 × 10 4 cells/well) for 24 h. Then, Vero cells were infected with SARS-CoV-2 (MOI 0.0125) immediately after being treated with serially diluted KC and incubated at 37 °C for 24 h. The cells were then stained using anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody, and Hoechst 33342.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: Schematic of the blockade of the SARS-CoV-2 spike S1 RBD:ACE2 receptor interaction by KC.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques:

Journal: medRxiv

Article Title: SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia

doi: 10.1101/2022.12.19.22283645

Figure Lengend Snippet: (A and B) Comparison of SARS-CoV-2 vaccine-elicited spike (A) and RBD (B) ELISA IgG antibody titers in HC (n=30) and CLL (n=95) patients expressed as half-maximal effective concentrations (EC 50 ). (C and D) SARS-CoV-2 vaccine-elicited spike (C) and RBD (D) ELISA IgG antibody titers stratified by CLL disease status: treatment-naïve (Naïve) (n=45), on-therapy (On Tx) (n=34), off-therapy in clinical remission (CR) (n=9), and off therapy and relapsed or refractory (R/R) (n=7). (E-G) Comparison of SARS-CoV-2 vaccine-elicited neutralizing antibody titers for HC (n=30) and CLL patients against (E) D614G (CLL; n=95) and (F) delta (CLL; n=93) spike variants expressed as the reciprocal half-maximal inhibitory dilution (ID 50 ) as determined in an HIV-based pseudovirus neutralization assay or by (G) ACE2/RBD (Wuhan) binding inhibition (CLL; n=95) at a 1:25 dilution. (H-J) SARS-CoV-2 vaccine elicited NAb titers for D614G (H) and delta (I) S variants and ACE2/RBD binding frequencies (J) stratified by CLL disease status. Bars indicate the median with 95% CI. Dotted black lines indicate assay sensitivity cutoffs (EC 50 values of <100 by ELISA, ID 50 values of <20 in the neutralization assay, and >90% ACE2 binding in the RBD-inhibition assay). P values were determined by the Mann-Whitney test (A-B, E-G) or Dunn’s test of multiple comparisons following a Kruskal-Wallis test (C-D, H-J).

Article Snippet: Plates were incubated at room temperature for 1 h, washed 4 times with PBST, and 50 µl of biotinylated goat anti-human ACE2 (R&D) diluted at 0.1 µg/ml in PBST was added to the wells.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Binding Assay, Inhibition, MANN-WHITNEY