Papain-like (PLpro) Protease Assay Kit

Background

SARS-CoV-2 PLpro Assay Kit PLpro of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2 PLpro, also referred to as SARS-CoV- 2 Papain-like protease, Nsp3) plays an essential role in processing the polyproteins 1ab for viral replication. Inhibition of this enzyme activity would block viral replication, making it a promising target for anti- coronaviral therapeutic agents. Description The Aurora SARS-CoV-2 PLpro assay kit is a homogeneous assay for screening PLpro Inhibitors. The assay is fast and convenient, and requires just two steps. In the first step, the PLpro enzyme is preincubated with the compound for 30 minutes. The reaction is initiated by adding substrate solution at the second step. Fluorescent intensity is measured with a fluorescent plate reader at the excitation wavelengths of 340-360 nm and emission wavelengths of 460-480 nm. Materials supplied Catalogue Number Item Amount Storage 728205 728252 728256 2X Protease Assay Buffer 0.5 M DTT 5 mM Substrate Recombinant SARS-CoV-2 PLpro Black low binding 96 well plate 20 ml 200 µl 10 µl 5 µg 1 -20°C -20°C -80°C -80°C RT Materials Needed but not supplied A microplate reader capable of measuring fluorescence at excitation wavelengths of 340-360 nm and emission wavelengths of 460-480 nm. Stability 12 months if stored under the indicated conditions.

Assay Protocol

1. Step 1. Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 50 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
2. Step 2. Prepare PLpro solution. Thaw PLpro enzyme on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: PLpro enzyme is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute the PLpro enzyme to 1.25 ng/µl in 1X assay buffer. Add 20 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X assay buffer to each of background well.
3. Step 3. Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 10% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.
4. Step 4. Incubate at room temperature for 30 minutes.
5. Step 5. Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, dilute the 5 mM substrate solution to 20µM in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 5 mM Substrate solution to -80°C. Add 25 µl of diluted substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.
6. Step 6. Incubate at room temperature for 2 hours.
7. Step 7. Measure the fluorescent intensity

Data Analysis

Calculate percentage activity of the enzyme % Activity= (Fp – Fb) – (Fi-Fb) Fp - Fb X 100 Where Fp refers to Fluorescent intensity of the positive control, Fb refers to Fluorescent intensity of background, and Fi refers to Fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test inhibitor. No CPD refers to no compound control (compound vehicle control).