Journal: Cell & Bioscience

Article Title: Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors

doi: 10.1186/s13578-021-00720-3

Figure Lengend Snippet: Preparation and characterization of SARS-CoV-2 main protease (Mpro). a Expression and purification of SARS-CoV-2 Mpro. The highly active Mpro was expressed in E. coli Rosetta (DE3) cells as the soluble protein after induction with 0.2 mM IPTG at 30 °C for 8 h. A HisTrap TM chelating column was used to purify polyhistidine-tagged Mpro from E. coli cell extracts, and then the purity of purified Mpro was analyzed using SDS-PAGE. M: protein marker; 1: total cell extracts after IPTG induction; 2: supernatant of the cell lysate; 3: precipitated Mpro in 25% saturated ammonium sulfate solution; 4: purified Mpro band (34 kDa). b Reaction buffer optimization. The reaction mixture containing 0.25 µM Mpro and 10 µM FRET substrate was incubated in the indicated three buffers, and the change of RFU value was continuously recorded by a microplate reader (BioTek) using a FRET assay. The initial velocity (V I ) of the proteolytic activity was calculated by a linear regression for the first 30 s of the kinetic progress, and the curve was plotted in GraphPad Prism 5.0. c Plotting the MCA standard curve to convert RFU value to the amount of the cleaved FRET substrate (pmol) using the derived equation. MCA: 7-methoxycoumarin-4-acetic acid. d Calculation of Mpro kinetic constant K m and k cat values in the FP assay buffer. The Michaelis-Menten equation of Mpro proteolytic kinetics was plotted using GraphPad Prism 5.0 according to the V I , and then K m , V max and k cat values were calculated

Article Snippet: The IC 50 value of dieckol was analyzed using GraphPad Prism 5.0.

Techniques: Expressing, Purification, SDS Page, Marker, Incubation, Activity Assay, Derivative Assay, FP Assay

Journal: Cell & Bioscience

Article Title: Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors

doi: 10.1186/s13578-021-00720-3

Figure Lengend Snippet: The primary screening of a natural product library. a A general view for the primary screening. The red dashed line in the figure indicated 50% inhibition, and the inhibitory activity of candidate compound was higher than this defined red line. b The chemical structure of dieckol, a natural phlorotannin component extracted from Ecklonia cava . c The inhibitory activity of dieckol in the FP screening assay. The inhibitory analysis of dieckol in the absence or presence of 1 mM DTT was performed as described for GC-376. The IC 50 value of dieckol was analyzed using GraphPad Prism 5.0. All assays were performed in triplicate. d Analysis of interaction between dieckol and Mpro using the SPR assay. The binding kinetics of dieckol (0, 6.25, 12.5, 25 µM) to immobilized Mpro was plotted according to the change of RIU value

Article Snippet: The IC 50 value of dieckol was analyzed using GraphPad Prism 5.0.

Techniques: Inhibition, Activity Assay, Screening Assay, SPR Assay, Binding Assay