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.

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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

3CLpro (also known as Main Protease or Mpro) is a chymotrypsin-like protease and PLpro a papain-like protease. Both are encoded in the SARS-CoV-2 genome and play essential roles in the lifecycle of the virus.  Luminogenic SARS-CoV-2 protease substrates are available as early access materials from Promega. The aLuc moiety is shown in blue in Figures 2 and 3. The luminogenic 3CLPro Substrate is Ac-TSTKLQ-aLuc and the luminogenic PLpro substrate is Z-RLRGG-aLuc.
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Figure 2.The luminogenic 3CLPro substrate Ac-TSTKLQ-aLuc.
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Figure 3.The luminogenic PLpro substrate Z-RLRGG-aLuc.
Figures 4–6 show data from reactions of either recombinant SARS-CoV-2 3CLpro with Ac-TSTKLQ-aLuc or recombinant SARS-CoV-2 PLpro with Z-RLRGG-aLuc.
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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.

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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.

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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 componentsa 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

  1. Dispense 25µl 2X PLpro substrate solution into an opaque white 96-well plate.
  2. Add 12.5µl 4X test compound solutions to substrate solution in the 96-well plate.
  3. Initiate reactions by adding 12.5µl 4X PLpro solution to substrate solution in the 96-well plate.
  4. Mix plate and incubate for 30 minutes at room temperature (20–25°C).
  5. Add 50µl Luciferin Detection Reagent to each well to stop reactions and initiate luminescent signals. Allow 10 minutes for signal stabilization.
  6. 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

  1. Dispense 25µl 2X 3CLpro substrate solution into an opaque white 96-well plate.
  2. Add 12.5µl 4X test compound solutions to substrate solution in the 96-well plate.
  3. Initiate reactions by adding 12.5µl 4X 3CLpro enzyme solution to substrate solution in the 96-well plate.
  4. Mix plate and incubate for 1 hour at 37°C.
  5. Add 50µl Luciferin Detection Reagent to each well to stop reactions and initiate luminescent signals. Allow 20 minutes for signal stabilization.
  6. Read luminescence on a plate reading luminometer.
The luminogenic SARS-CoV-2 protease substrates are currently available as early access materials.
Please Enquire