High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Figure 1. Substrates, principles of FRET assay. (a) The fluorescent PLpro and NS2B-NS3pro substrates, (b) schematics of the FRET-based proteolytic assay, eGFP is excited with a green light at 488 nm, FRET transfers the excitation to mCherry which emits the light that is detected. After proteolytic cleavage, this FRET signal is abolished, (c,d) the reaction of PLpro substrate with a serial dilution of PLpro enzyme (500–7 nM), (c) schematics of reactions followed in the fluorescent plate reader, (d) these reactions were loaded on SDS-PAGE gel and visualized on a fluorescent scanner (e), where (S) and (P) donate substrate and product bands and full-length gel are shown in Figure S8.

Figure 2. Inhibition of TBEV NS2B–NS3pro by small molecules. Curves derived from the FRET inhibition assays of (a) aprotinin, (c) DTNB, and (d) leupeptin. Each curve is derived from three experiments. (b) Validation by SDS-PAGE analysis showing the successful inhibition by aprotinin (top) and no inhibition by leupeptin (bellow) where (M) is a marker, (SF) is fresh substrate at reaction concentration, (+) is positive control reaction where there was no inhibitor present, (–) is negative control reaction where there was no enzyme present, (S) is the substrate, (P) is the product. The illustrative evaluations of the validation SDS-PAGE gels and more details are shown in Figure S5.

Figure 3. Inhibition of SARS-CoV2 PLpro by small molecules. Curves derived from the FRET inhibition assays of (a) JB24, (c) Antabuse, and (d) 2-MP and 6-MP. Each curve is derived from three experiments. (b) SDS-PAGE gel from one of the titrations with JB24 where (M) is a marker, (+) is a positive control reaction in which there was no inhibitor present, (–) is a negative control reaction where there was no enzyme present, (S) is the substrate, and (P) is the product. The illustrative evaluations of the SDS-PAGE gels resolving the reaction progress and more details are shown in Figure S6.