SARS-CoV-2 3CLpro and PLpro Luminescent Assays
Promega Corporation
Publication Date: December 2020
Luminescent Protease Assays
Luminescent protease assays use peptide aminoluciferin (peptide-aLuc) substrates in a homogeneous format that is ideally configured in multiwell plates and used for screening and characterizing protease inhibitors. In a first reaction, a protease cleaves a substrate peptide moiety to release aminoluciferin (aLuc) that accumulates and drives a second reaction with a luciferase, producing light in proportion to protease activity (Figure 1). The system uses a highly stabilized luciferase (Ultra-Glo™ Luciferase) in a Luciferin Detection Reagent that produces glow-type luminescence with a typical half-life ≥ 2 hours.
Figure 1. Luminescent protease assay scheme. Protease and test article or vehicle are combined in an opaque white multi-well plate and the reaction is initiated by addition of a peptide-aLuc substrate. The reaction is stopped, and luminescence is initiated by adding Luciferin Detection Reagent. Signal is recorded on a plate-reading luminometer. Inhibitors are identified as test articles that reduce light output.
SARS CoV-2 3CLpro and PLpro Luminescent Assays
Figure 4. SARS-CoV-2 3CLpro enzyme assay with luminogenic substrate. 6His-3CLpro (SignalChem, Cat.# C19CL-G241H) was combined with 20µM Ac-TSTKLQ-aLuc, 50mM HEPES (pH 7.2), 10mM DTT, and 0.1mM EDTA in a 50µl reaction volume in an opaque white 96-well plate and incubated for 1 hour at 37°C. Reactions were then terminated by adding 50µl of Luciferin Detection Reagent (Cat.# V8920) and after 20 minutes at room temperature (20–25°C) luminescence was recorded on a GloMax® luminometer (Cat.# GM2000). See step-by-step test compound screening protocol below.
Figure 5. SARS-CoV-2 PLpro enzyme assay with luminogenic substrate. GST-PLpro (R&D Systems, Cat.# E611-050) was combined with 20µM Z-RLRGG-aLuc, 50mM HEPES (pH 7.2), 10mM DTT, and 0.1mM EDTA in a 50µl reaction volume in an opaque white 96-well plate and incubated for 30 minutes at 20–25°C. Reactions were then terminated by adding 50µl of Luciferin Detection Reagent (Cat.# V8920) and after 20 minutes at room temperature (20–25°C) luminescence was recorded on a GloMax® luminometer (Cat.# GM2000). See step-by-step test compound screening protocol below.
Figure 6. Detection of SARS-CoV-2 PLpro inhibition with luminescent assay. 0.5nM SARS-CoV-2 GST-PLpro (R&D Systems, Cat.# E-611-050) was combined with 20µM Z-RLRGG-aLuc, the PLpro inhibitor HY-17542 (MedChemExpress, diluted from 100mM stock DMSO solution), 50mM HEPES (pH 7.2), 10mM DTT, and 0.1mM EDTA in a 50µl reaction volume in an opaque white 96-well plate, and incubated for 30 minutes at 20–25°C. Reactions were then terminated by adding 50µl of Luciferin Detection Reagent (Cat.# V8920) and after 20 minutes at room temperature luminescence was recorded on a GloMax® luminometer (Cat.# GM2000). See step-by-step test compound screening protocol, below.
Compound Screening: SARS-CoV-2 PLpro Assay Protocol
Materials Required
- The PLpro substrate is supplied at 4mM in 0.5M HEPES, pH 7.2, and is available as a custom material from Promega. Please Enquire.
- Assay buffer: 50mM HEPES pH 7.2, 10mM DTT, and 0.1mM EDTA (prepared by user)
- Luciferin Detection Reagent (Cat.# V8920 or V8921)
- Recombinant PLpro sources: R&D Systems Cat.# E-611-050; AcroBiosystems Cat# PAE-C5148
- Opaque white 96 well plates (e.g., Corning Cat.# 3912)
- Plate reading luminometer (e.g., GloMax® Navigator, Cat.# GM2000)
Reagent Preparation
- Prepare 2X PLpro substrate solution at room temperature. Dilute PLpro substrate to 40µM in assay buffer.
- Prepare 4X PLpro enzyme solution on ice. Dilute PLpro recombinant enzyme to 2nM in assay buffer (see Figure 5 to consider enzyme concentration adjustments).
- Prepare 4X test compound solutions in assay buffer at room temperature.
- Prepare Luciferin Detection Reagent at room temperature. This reagent is supplied as two components—a lyophilized preparation and a reconstitution buffer. Add the entire contents of the reconstitution buffer to the Luciferin Detection Reagent lyophilized cake. Mix thoroughly but gently to avoid forming bubbles. Store unused portion at –20°C)
Assay Protocol
- Dispense 25µl 2X PLpro substrate solution into an opaque white 96-well plate.
- Add 12.5µl 4X test compound solutions to substrate solution in the 96-well plate.
- Initiate reactions by adding 12.5µl 4X PLpro solution to substrate solution in the 96-well plate.
- Mix plate and incubate for 30 minutes at room temperature (20–25°C).
- Add 50µl Luciferin Detection Reagent to each well to stop reactions and initiate luminescent signals. Allow 10 minutes for signal stabilization.
- Read luminescence on a plate-reading luminometer.
Compound Screening: SARS-CoV-2 3CLpro Assay Protocol
Materials
- The 3CLpro substrate is supplied at 4mM in 0.5M HEPES, pH 7.2 and is available as a custom material from Promega. Please Enquire.
- Assay buffer: 50mM HEPES pH 7.2, 10mM DTT, and 0.1mM EDTA (prepared by user)
- Luciferin Detection Reagent (Cat.# V8920 or V8921)
- Recombinant 3CLpro (SignalChem, Cat.# C19CL-G241H)
- Opaque white 96-well plates (e.g., Corning, Cat.# 3912)
- Plate reading luminometer (e.g., GloMax® Navigator, Cat.# GM2000)
Reagent Preparation
- Prepare 2X 3CLpro substrate solution at RT. Dilute 4mM 3CLpro substrate to 40µM in assay buffer.
- Prepare 4X 3CLpro enzyme solution on ice. Dilute 3CLpro recombinant enzyme to 0.16µg/µl in assay buffer (see Figure 4 to consider enzyme concentration adjustments).
- Prepare 4X test compound solutions in assay buffer at room temperature.
- Prepare Luciferin Detection Reagent at room temperature. This reagent is supplied as two components—a lyophilized preparation and a reconstitution buffer. Add the entire contents of the reconstitution buffer to the Luciferin Detection Reagent lyophilized cake. Mix thoroughly but gently to avoid forming bubbles. Store unused portion at –20°C.
Assay Protocol
- Dispense 25µl 2X 3CLpro substrate solution into an opaque white 96-well plate.
- Add 12.5µl 4X test compound solutions to substrate solution in the 96-well plate.
- Initiate reactions by adding 12.5µl 4X 3CLpro enzyme solution to substrate solution in the 96-well plate.
- Mix plate and incubate for 1 hour at 37°C.
- Add 50µl Luciferin Detection Reagent to each well to stop reactions and initiate luminescent signals. Allow 20 minutes for signal stabilization.
- Read luminescence on a plate reading luminometer.