fluorogenic substrate z arg arg 7 amc  (Echelon Biosciences)

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Effects of compounds on Cat L-mediated entry. (A) Vero E6 or Calu-3 cells were inoculated with S-pseudovirus in the presence of compounds as indicated for 48 h. DMSO-treated cells exposed to pseudovirus were used as a control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. (B) Cat L protein was incubated with a fluorogenic substrate (Z-Phe-Arg-7-amido-4-methylcoumarin) in the presence or absence of various concentrations of the test compounds for 1 h in vitro. Luciferase activity of substrate was measured, and relative fluorescence intensity was calculated. (C) hACE2/293T cells were treated with the compounds or Z-FY( t -Bu)-DMK for various periods after S-pseudovirus infection. DMSO-treated cells exposed to pseudovirus were used as the control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. Experiments were performed in triplicate independently. Data are presented as mean ± SEM of three biological replicates.

Article Snippet: The purified recombinant Cathepsin L1 (MCE, NJ, USA) was incubated at 37 °C with 25 μM fluorogenic substrate Z-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-AMC, Eurogentec, Belgium) and assay buffer (20 mM HAc-NaAc, 150 mM NaCl, pH 5.5, filtered) with or without various concentrations of the test compounds at a total reaction volume of 100 μL.

Techniques: Luciferase, Activity Assay, Fluorescence, Incubation, In Vitro, Infection