Ace2 sars cov 2 spike inhibitor screening assay kit

MG was purchased from ChemFaces (Wuhan, China). 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 procured for biolayer interferometry. HEK293T cells stably expressing human ACE2 and TMPRSS2 cells were obtained from GeneCopoeia (Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (Biotechnics Research, Lake Forest, CA, USA) and 1% penicillin/streptomycin (Cellgro, Manassas, VA, USA) at 37 °C in a 5% CO₂ incubator. Further, wild-type (WT) and mutant (D614G) SARS-CoV-2 spike pseudotyped viruses were purchased from GeneCopoeia (Rockville, MD, USA). Vero cells were purchased from American Type Culture Collection (Manassas, VA, USA). SARS-CoV-2 (βCoV/Korea/KCDC03/2020) was provided by Korea Centers for Disease Control and Prevention (Cheongju, Korea).

The SARS-CoV-2 Spike-ACE2 Inhibitor Screening Assay Kit (Cat: #79931) was purchased from BPS Biosciences (San Diego, CA, USA). Nickel-coated 96-well white microplates were coated with 1 μg/mL ACE2-His-tag protein following the protocol of the manufacturer. Briefly, 1 ng/uL SARS-CoV-2 RBD was added to ACE2-His-tag coated test wells in the presence of 0, 50 or 100 μM solution of tested compound. Wells without the compounds and SARS-CoV-2 RBD protein were set as blank control. ELISA ECL substrate solution containing anti-His horseradish peroxidase was added into the microplate wells followed by the chemiluminescence measurement using the luminometer (Tecan, Männedorf, Switzerland). The data were then analyzed using Prism 7.0 (GraphPad, San Diego, CA, USA) and IC₅₀ was calculated according to the following equation: $$Y=Bottom+\frac{Top-Bottom}{1+\frac{X}{I{C}_{50}}}$$
Concentration of tested compound is reported on the X and response on the Y axes. “Top” and “Bottom” indicate plateaus in the units of the Y axis, representing maximum and minimum measured activity values. Quantification bars were obtained from the results of 3 independent experiments.

KC was purchased from ChemFaces (Wuhan, China). 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. Human ACE2/TMPRSS2-overexpressing HEK293T stable cell line was purchased from GeneCopoeia (Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (Biotechnics Research, Lake Forest, CA, USA) and 1% penicillin/streptomycin (Cellgro, Manassas, VA, USA) at 37 °C in a 5% CO₂ incubator. WT and mutant (D614G) SARS-CoV-2 spike pseudotyped viruses (cat. no. SP101-100 and SP103-100) were purchased from GeneCopoeia (Rockville, MD, USA). Vero cells were purchased from American Type Culture Collection (Manassas, VA, USA). A clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) was provided by Korea Centers for Disease Control and Prevention (Cheongju, Korea).