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plpro inhibitor grl0617  (R&D Systems)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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sars cov 2 plpro  (TargetMol)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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inhibitors targeting both plpro and mpro of sars-cov-2  (Therapeutic Innovations)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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plpro inhibitor jun12682  (Pfizer Inc)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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plpro inhibitor pf-0795472  (Pfizer Inc)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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non-covalent inhibitors of sars-cov-2 papain-like protease (plpro)  (Kiira Motors Corporation)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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plpro positive control inhibitor  (Selleck Chemicals)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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plpro inhibitor program  (Pfizer Inc)
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor <t>GRL0617</t> were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.
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VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor GRL0617 were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.

Journal: medRxiv

Article Title: A Cell-Based Papain-like Protease (PLpro) Activity Assay for Rapid Detection of Active SARS-CoV-2 Infections and Antivirals

doi: 10.1101/2024.05.21.24307588

Figure Lengend Snippet: VeroE6 cells were seeded in a 24-well plate and incubated at 37 °C for 12 hours. Various dilutions of the inhibitor GRL0617 were prepared in MEM without FCS, added to the cells, and incubated for 2 hours at 37 °C. Control cells were uninfected and infected cells treated with medium only and uninfected cells treated with medium plus DMSO. SARS-CoV-2 (0.01 MOI) was added for 24 hours at 37 °C and lysates prepared. PLpro substrate Pun74 was added in 2x assay buffer and 25µl added to wells in a 96-well plate for a final concentration of 50nM. A) The increase in relative fluorescence units (RFU) from Pun74 cleavage in x2 replicates was monitored every 15 minutes for 30 minutes at room temperature in a fluorescence plate reader using excitation and emission wavelengths of ʎ ex =485nm and ʎ em =528nm. B) The mean RFU after 30 minutes in lysates from cells pretreated with GRL0617 and Max (medium plus virus) and Min (medium without virus) values were plotted and the IC 50 calculated using GraphPad Prism, nonlinear regression, variable slope. Some error bars are within the symbols.

Article Snippet: The well-characterized PLpro inhibitor GRL0617 (R&D Systems) was diluted in MEM without fetal calf serum to achieve concentrations between 2μM-100μM and 100μl was added to near confluent VeroE6 cells in wells of a 24-well plate.

Techniques: Incubation, Control, Infection, Concentration Assay, Fluorescence, Virus