FlipGFP protease assay for evaluating in vitro inhibitory activity against SARS-CoV-2 Mpro and PLpro

Summary FlipGFP assay characterizes the intracellular drug target engagement to Mpro and PLpro and can be performed in the biosafety level 1/2 settings. Here, we provide the detailed protocol for the cell-based FlipGFP assay to identify and characterize SARS-CoV-2 Mpro and PLpro inhibitors. We describe steps for cell passage and seeding, transfection, addition of compounds, and their incubation and timing. We then detail the quantification of the fluorescence signal of the assay For complete details on the use and execution of this protocol, please refer to Ma et al.1

characterize their cellular target engagement. Herein, we introduce the FlipGFP protease assay for characterizing M pro and PL pro inhibitors. FlipGFP protease assay can predict the antiviral activity of M pro and PL pro inhibitors in biosafety level (BSL) 1/2 settings without using infectious SARS-CoV-2 virus, which requires a BSL-3 facility. FlipGFP assay has the additional advantage of ruling out compounds with cellular toxicity or poor membrane permeability. 14,16 In the FlipGFP protease assay, HEK-293T cells are transfected with two plasmids: one expressing the protease (M pro or PL pro ) and another expressing reporter flipped green fluorescence protein (FlipGFP) (Figure 1). The reporter FlipGFP is initially in an inactive conformation. Upon protease digestion, a conformational change in the reporter FlipGFP leads to restoring the GFP signal. Whereas in the presence of protease inhibitors, GFP signal restoration is inhibited.
We have been using FlipGFP protease assay to characterize and validate M pro and PL pro inhibitors in the cellular context. 1,[13][14][15]17,18 In this study, we describe the detailed protocols of the FlipGFP protease assay. The plasmid construction, HEK293T cell transfection, and incubation time have been optimized. The assay has been validated with the M pro inhibitors GC376, nirmatrelvir, and the PL pro inhibitor GRL0617.

Protease expression plasmid resources & construction
This section describes the resources and construction of M pro & PL pro expression plasmid. For the construction of M pro expression plasmid, the SARS-CoV-2 M pro sequence is obtained from NCBI (NCBI: YP_009725301.1) and codon optimized for mammalian cell expression. M pro sequence is constructed in pLVX expression plasmid by Addgene. For the construction of PL pro expression plasmid, SARS-CoV-2 PL pro sequence is obtained from NCBI (NCBI: YP_009742610.1) and codon optimized for mammalian cell expression. PL pro sequence is constructed into pcDNA 3.1 expression plasmid by Genescript.

Reporter plasmid construction
Timing: 1 month (for steps 1 to 17) This section describes the construction of M pro FlipGFP reporter plasmid using starting plasmid obtained from Addgene. Three PCR reactions are performed to construct the reporter plasmid. PL pro FlipGFP reporter plasmid is constructed using the same method with different primers in step 2.  5. Separate the PCR product of PCR-1 (and PCR-2) using 1% agarose gel electrophoresis. The size of the PCR-1 product is 381 bp, and 199 bp for the PCR-2 product. 6. Collect the gel containing PCR product, and perform purification using Wizard SV Gel and PCR clean-up System. 7. Perform PCR-3.
8. Separate the PCR product of PCR-3 using 1% agarose gel electrophoresis. The size of the PCR-3 product is 556 bp. 9. Collect the gel containing PCR-3 product, and perform purification using Wizard SV Gel and PCR clean-up System. 10. Perform endonuclease digestion for starting plasmid pcDNA3-TEV-flipGFP-T2A-mCherry.
a. Add 1mL of endonuclease SacI and HindIII. b. Mix well and briefly spin down. c. Incubate at 37 C for 2 h. d. Heat-inactivate the endonuclease digestion by placing the sample on a heat block at 80 C for 20 min. 11. Separate the digested starting plasmid pcDNA3-TEV-flipGFP-T2A-mCherry using 1% agarose gel electrophoresis. The size is 7000 bp. 12. Collect the gel containing digested starting plasmid pcDNA3-TEV-flipGFP-T2A-mCherry, perform purification using Wizard SV Gel and PCR clean-up System. 13. Perform endonuclease digestion for purified PCR-3 product.
a. Add 1mL of SacI and HindIII to the PCR-3 product.

PCR-2 cycling conditions
Steps

MATERIALS AND EQUIPMENT
Note: To dilute 103 PBS to 13, add 100 mL of 103 PBS to 900 mL of ddH 2 O with complete mixing. Autoclave to sterilize prior to cell culture applications.
CRITICAL: Prepare complete medium under aseptic conditions in the biosafety cabinet.

STEP-BY-STEP METHOD DETAILS
In the FlipGFP protease assay, two plasmids are transfected to HEK-239T cells. One expressing the protease (M pro or PL pro ) is denoted as the protease plasmid. The other expressing the reporter FlipGFP is denoted as the reporter plasmid. The reporter plasmid encodes three components, GFP b1-9, b10-11, and the mCherry ( Figure 1). The b10-11 is engineered in the parallel orientation via the K5/E5 coiled coil. This orientation blocks its association with b1-9. Expression of the protease plasmid produces the M pro or PL pro , which cleaves the M pro or PL pro substrate linker between K5 and b11. The cleavage shifts b10-11 into the antiparallel configuration, which can associate with b1-9 and thus restore the GFP signal. The mCherry is included to normalize the transfection efficacy and indicate the cytotoxicity of testing compounds. The assay quantifies the fluorescence signal of GFP and mCherry. The ratio of GFP to mCherry correlates with the M pro or PL pro enzymatic activity. Effective inhibitors have been shown to decrease the value of GFP/mCherry ratio in a dose-dependent manner. 14

Cell passage & seeding
Timing: 1 h This section describes the subculturing and seeding of the HEK293T cell line (Figure 2A).
Note: all procedures should be performed under aseptic conditions in the biosafety cabinet.  b. Add 10 mL prewarmed (to room temperature) complete culture medium containing DMEM (with 4.5 g/L glucose, L-glutamine, and sodium pyruvate), 10% FBS, and 1% penicillin-streptomycin to the flask. c. Gently disperse the medium with pipetting using a serological pipette over the cell monolayer to recover 95% of the cells. d. Mix the cell suspension by pipetting up and down. 4. Transfer cell suspension to a 15 mL centrifuge tube and centrifuge at 500 3 g for 5 min at room temperature (20 C-30 C). 5. Aspirate the supernatant and resuspend the cell pellet with 10 mL of complete culture medium. 6. Seed the cell to a 96-well plate with 100 mL per well and a 0.8-3.0 3 10 5 cells/mL density. 7. Incubate the flask in a cell culture incubator (Humidified, 5% CO 2 /95% air, 37 C) overnight (16)(17)(18) h) (Figure 2A).
Note: HEK293T cells are suitable for FlipGFP assay within 25 passages. The transfection efficiency is reduced beyond 25 passages.
CRITICAL: it is essential to ensure cells reach 70%-80% of confluence at the time of transfection to achieve the most efficient transfection. More than 95% confluence may result in low efficiency of transfection.

Transfection
Timing: 2 h (for steps 25 to 31) HEK293T cells are transfected with two plasmids: one expressing the protease and another expressing the FlipGFP reporter ( Figure 2B).
This section describes the plasmid transfection procedure in the FlipGFP assay.
8. On the second day after cell seeding, check the cell confluency in the T75 flask using a phasecontrast inverted microscope. 70-80% confluence gives the most efficient transfection. 9. Dilute GFP reporter plasmid and protease plasmid to 500 ng/mL using autoclaved nuclease-free water. 10. To assemble the transfection complex, mix 9 mL of Opti-MEM, 0.1 mL of 500 ng/mL reporter plasmid, 0.1 mL of 500 ng/mL protease plasmid, and 0.3 mL of transIT-293 for each well of a 96-well plate ( Figure 2B).
Note: master mix for multiple wells is recommended.
11. Gently vortex the transfection complex and incubate at room temperature for 30 min. 12. Add 9.5 mL of transfection complex to each well of the 96-well plate, such that 50 ng of GFP reporter plasmid and 50ng of protease plasmid are aliquoted to each well. 13. Mix the transfection complex in each well by shaking the plate on a shaker for 5 min. 14. Incubate the plate in a cell culture incubator (humidified, 5% CO 2 /95% air, 37 C) for transfection and protein expression. Incubate for 3 h.
Note: To validate the FlipGFP assay, control experiments with matching or mismatching protease-FlipGFP reporter pairs need to be performed ( Figure 3A). In addition, control compounds should be tested. Positive control (a compound with potent protease inhibitory activity and without cytotoxicity) should show dose-dependent inhibition of the GFP signal with a consistent EC 50 value. Negative control (a compound without protease inhibitory activity and

EXPECTED OUTCOMES
To calibrate the specificity of the FlipGFP assay, cells are transfected with either the FlipGFP reporter plasmid alone or with the matching or mismatching protease plasmid ( Figure 3A). Transfection of cells with FlipGFP M pro or PL pro reporter plasmid alone should only produce mCherry signal but not GFP signal ( Figure 3A first two columns). Similarly, cells transfected with mismatching pairs (FlipGFP M pro reporter plasmid + PL pro plasmid; FlipGFP PL pro reporter plasmid + M pro plasmid) should also only produce mCherry signal but not GFP signal ( Figure 3A third and  FlipGFP PL pro reporter plasmid + PL pro plasmid) produce both mCherry and GFP signals ( Figure 3A last two columns).
In the presence of a potent protease inhibitor, dose-dependent inhibition of GFP signal should be observed ( Figure 3B first, second, fourth rows, 4A top row). For the negative control compound, GFP signal is constant with all testing concentrations ( Figure 3B third row, 4A bottom row).

QUANTIFICATION AND STATISTICAL ANALYSIS
This section uses FlipGFP PL pro assay as an example for data analysis. The readout of FlipGFP assay includes the GFP readings of testing compounds and the control compound at different concentrations, the mCherry readings of the testing compounds and the control compound at different concentrations.
The ratio of GFP readout to mCherry readout (GFP/mCherry) is calculated and plotted against logscale concentration to determine the half maximal effective concentration (EC 50 ).

GFP Signal Readout mCherry Signal Readout
The mCherry readout is plotted against log-scale concentration to determine the half maximal cytotoxic concentration (CC 50 ).
Both GFP/mCherry ratio and mCherry alone should be normalized prior to EC 50 and CC 50 plotting.
For the normalization of EC 50 analysis, the GFP/mCherry value of the DMSO treated group is defined as 100%. The GFP/mCherry value of the control compound at 60 mM (highest concentration) is defined as the cut-off of 0% for the control compound itself and all testing compounds. A control compound should be included in each experiment. In CC 50 analysis, the mCherry readout of the DMSO-treated group is defined as 100%. The mCherry readout of the non-transfected group is the background of the assay and is defined as 0%.    Figure 3C and 4B).
Note: GFP/mCherry value will not be dose-dependent for compounds with cytotoxicity at high concentrations. This is reflected by the decrease of mCherry signal at higher drug concentrations due to cytotoxicity. In this situation, discard the GFP/mCherry values at toxic concentrations and re-plot the lower concentrations.

LIMITATIONS
The FlipGFP assay has the advantage of predicting the antiviral activity in the cellular context in the biosafety level 1/2 facilities. Compared to FRET enzymatic assay, FlipGFP assay also rule out compounds with poor membrane permeability and cellular cytotoxicity. Nevertheless, FlipGFP assay has several limitations. Since it is a cell-based assay requiring the delicate operation of transfection, it is not suitable for high-throughput screening (HTS). Furthermore, compounds with fluorescence interference properties may give false positive results. Therefore, FRET and binding assays must be performed to validate the results.
An efficient antiviral drug discovery pipeline should start with FRET-based high-throughput screening. Next, FlipGFP assay can be applied to characterize their cellular protease inhibitory activity and rule out compounds with poor membrane permeability or cytotoxic. With these assays, potent candidates can be efficiently identified for the next step of antiviral assay and in vivo animal model studies.  Cytotoxicity is also a cause for no signal, and this issue usually occurs when compound concentration is above the toxic threshold. Concentrations below the cytotoxic threshold should produce GFP and mCherry signals if the transfection and expression are successful.

Problem 2
Low fluorescence signal.

Potential solutions
Low fluorescence signal is usually caused by inefficient transfection. Check and adjust the cell confluence at the time of transfection (Step 8 in step-by-step method details). More than 95% of confluence may result in low efficiency of transfection and hence result in low fluorescence signal. The optimal confluency for transfection is 70-80%. Besides, subculturing HEK-293T cell for more than 25 passages also result in reduced transfection efficiency and low signal. If the cell line is more than 25 subcultures, discard the cell, and use the HEK-293T cell lines with fewer subculture cycles.

Potential solutions
Cell detachment is usually caused by contamination and improper handling. For contamination, discard the cell and initiate new HEK-293T from stock. After two cycles of subculturing, the newly initiated cell line is ready for assay. To avoid HEK-293T detachment, transfection complex and compound solution should be added gently to the edge of the well of the culture plate.

Problem 4
Failure of control compounds.
Potential solutions GC376 is used as the positive control for FlipGFP M pro assay, and GRL0617 is used as a positive control for FlipGFP PL pro assay. When the control compounds fail to show dose-dependent inhibition, first discard the Opti-MEM. Use unexpired, clean Opti-MEM TM I Reduced Serum Medium. Then double-check if the cell line is healthy, not contaminated, and within 25 subcultures. Finally, doublecheck and repeat the compound dilution and transfection complex to ensure the correct compound concentration, plasmid concentration, and transfection reagent volume (Step 10 in step-by-step method details).

Problem 5
Large variations.

Potential solutions
Large variations are typically caused by cell detachment, insufficient mixing, and usage of edge wells of the culture plate. For cell detachment, please refer to the solutions in problem 3. For insufficient mixing of transfection complex and compound, shake the plate on a shaker immediately after addition at 100 rpm for 8 min (Steps 13 and 16 in step-by-step method details). Finally, avoid using the edge wells on the 96-well plate, as solutions in these wells have evaporation issues.

Materials availability
This study did not generate new reagents. Data and code availability This study did not use any database and code.