Journal: Virology

Article Title: A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors

doi: 10.1016/j.virol.2022.07.006

Figure Lengend Snippet: The inhibitory activity and mechanism of a natural product anacardic acid (AA) against SARS-CoV-2 PLpro in vitro . (A) The 4-step procedure of the FP screening assay for rapid screening of PLpro inhibitors. Step 1: 29 μL PLpro solution (1 μM) was incubated with 1 μL sample of a natural product (2 mg/mL) for 30 min at RT in a black 96-well microplate. Step 2: 20 μL FP probe solution (30 nM) was added and incubated for 30 min at RT. Step 3: The reaction was quenched by adding 10 μL avidin solution (240 nM) followed by 5 min incubation. Step 4: The mP value was measured by a microplate reader (BioTek). (B) The natural product layout in a black 96-well microplate for a large-scale screening. The positive (GRL0617), negative, and background wells were highlighted. (C) The illustration for the primary screening cycle of a natural product library using the FP screening assay. The red dotted line indicates a baseline (equivalent to 50% inhibition) in the primary screening cycle, and 5 candidate compounds were identified. (D) The chemical structure of anacardic acid. (E) Inhibitory effect of anacardic acid on the PLpro activity in the FP screening assay. (F) Inhibitory effect of anacardic acid on the PLpro activity in the FRET screening assay. GRL0617 was used as a positive control. (G) Inhibitory effect of anacardic acid on the Mpro activity in the FRET screening assay. PF-07321332 served as a positive control. (H) Inhibitory effect of anacardic acid on the PLpro isopeptidase activity. The fluorogenic peptide Z -RLRGG-AMC was used as the substrate for inhibition of PLpro isopeptidase activity by anacardic acid in vitro . GRL0617 served as a positive control. (I) The Lineweaver-Burk double-reciprocal plots for inhibition of PLpro activity by anacardic acid in vitro for the FRET substrate. (J) The secondary plots for a K i value.

Article Snippet: The FRET substrate (Dabcyl-FTLKGGAPTKVTE-Edans; λ ex /λ em = 340/500 nm), FP probe (FITC-FTLKGGAPTKVTK-Biotin; λ ex /λ em = 485/535 nm), FITC-FTLKGG peptide, and the fluorogenic peptide Z -RLRGG-AMC (λ ex /λ em = 340/460 nm) were purchased from GL Biochem (Shanghai, China), respectively.

Techniques: Activity Assay, In Vitro, Screening Assay, Incubation, Avidin-Biotin Assay, Inhibition, Positive Control