Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Summary

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Abstract

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

Introduction

Autophagy is a conserved metabolic process that allows cells to keep intracellular homeostasis and respond to stress by degrading aged, defective, or unnecessary cellular contents via the lysosomes^1,2,3. Under some pathophysiologic conditions, this process acts as a crucial cellular response to nutrient and oxygen deprivation, resulting in recycled nutrients and lipids, allowing the cells to adapt to their metabolic needs^2,3,4. Autophagy has also been identified as a cellular stress response related to several diseases, such as neurodegenerative disorders, pathogen infection, and various types of cancer. The function of autophagy in cancer is complex and dependent on the type, stage, and status of the tumor. It can suppress tumorigenesis through autophagic degradation of damaged cells, but can also promote the survival of advanced tumors by improving cell survival during stressful conditions, such as hypoxia, nutrient deprivation, and cytotoxic damage^2,4,5,6.

Several studies have shown that autophagy inhibition provides a benefit as an anticancer strategy. Thus, the inhibition of critical steps, such as autophagosome formation or its fusion with the lysosome, could be an effective method for cancer control^2,4,5,6. Growing evidence has shown that ATG4B is involved in certain pathological conditions, and it has gained attention as a potential anticancer target^2,3,4. For instance, it was observed that colorectal cancer cells and human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells had significantly higher ATG4B expression levels than adjacent normal cells^2,4. In prostate cancer cells, inhibition of ATG4B resulted in a cell line-specific susceptibility to chemotherapy and radiotherapy⁷. Recently, strong evidence has emerged that pancreatic ductal adenocarcinoma (PDAC) is particularly vulnerable to ATG4B inhibition. For instance, in a genetically engineered mouse model, it was shown that intermittent loss of ATG4B function reduces PDAC tumor growth and increases survival^3,4. Overall, ATG4B is highly overexpressed in some cancer types, is related to the progression of tumor, and is linked to cancer therapy resistance^2,4,8.

The ATG4 cysteine proteases in mammals have four family members, ATG4A-ATG4D. These proteins exhibit some target selectivity toward the LC3/GABARAP (ATG8) family of proteins^9,10,11 and may have additional functions not linked to their protease activity^12,13. Furthermore, ATG4 functions in regulating a novel type of post-translational modification, the ATG8-ylation of proteins^11,12. While ATG4B and its main substrate LC3B are the most widely studied, a picture is emerging that suggests a complex role for each subfamily member in the regulation of autophagic and non-autophagic processes. This is further corroborated by a complex network of post-translational modifications that regulate ATG4B activity via phosphorylation, acetylation, glycosylation, and nitrosylation^{9,10,11,12,13}.

Several known ATG4B inhibitors have been published^2,4,14,15. While these are suitable as research tools, their pharmacodynamic profile, selectivity, or potency have yet precluded them from development as preclinical candidates^4,16. Overall, there is an urgent need to identify more potent and selective compounds. Often, the compounds are good biochemical inhibitors of protein function, yet their efficacy in cell-based assays is poor. There are multiple assays to monitor ATG4B activity, including biochemical methods and cell-based assays⁴. We have previously developed a simple, luminescence-based, high-throughput assay for monitoring ATG4B activity in cells^8,17. This assay utilizes a luciferase protein from Gaussia princeps (GLUC) that is stable and active in the extracellular milieu and can be inducibly released from cells in response to ATG4B proteolytic activity^18,19.

In this reporter construct, dNGLUC is linked to the actin cytoskeleton of cells. A protease-specific linker can be introduced between the β-actin anchor and dNGLUC, turning the secretion dependent on cleavage of the linker. We used the full-length open reading frame of LC3B between β-actin and dNGLUC, to be able to monitor LC3B cleavage^17,18,19. Although the secretion mechanism of dNGLUC is poorly understood, it is specific for monitoring ATG4B activity, does not depend on overall autophagy as it occurs in ATG5 knockout cells, and is mediated by non-conventional mechanisms that do not require a classical signal peptide^4,18,19. We have successfully used this reporter to screen small molecules and siRNA libraries, and have identified novel regulators of ATG4B activity, such as the Akt protein kinases⁸. This paper describes a detailed protocol for the use of this luciferase reporter in a semi-automated, high-throughput screening format.

Protocol

NOTE: The assay process is outlined in Figure 1. See the Table of Materials for details related to all materials, reagents, and equipment used in this protocol.

1. Retrovirus production

NOTE: The plasmid encoding the ActinLC3dNGLUC is pMOWS-ActinLC3dNGLUC²⁰. Use a low-passage number of cells for high-titer virus production (ideally less than P20).

1. Culture HEK293T cells in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C in a humidified incubator, with an atmosphere of 5% CO₂ until they are 80%-90% confluent before seeding for transfection.
2. The day before transfection, seed the cells into a 6-well plate at a density of 1 × 10⁶ cells/well in 2 mL/well of complete growth medium. Incubate the plate overnight in a humidified incubator with an atmosphere of 5% CO₂.
   NOTE: Follow the manufacturer's instructions if using a liposomal-based transfection reagent. This protocol describes the use of a non-liposomal-based reagent. Before starting the transfection, allow the DNA transfection reagent, DNA, and media to equilibrate to room temperature (~15 min).
3. For each transfection, add 200 µL of serum-free medium to 1.5 mL microcentrifuge tubes. In each tube, add the following amounts of plasmids: 1,000 ng of pMOWS-ActinLC3dNGLUC; 900 ng of GagPol; 100 ng of VSV-G.
   NOTE: The amount of plasmids and volume of media listed here are for a 12-well plate. If using a different plate type, adjust the media volume and plasmid amounts to reach the final concentration of 5 ng/µL of transfer plasmid, 4.5 ng/µL of packing plasmid, and 0.5 ng/µL of envelope plasmid. Use any packing plasmid that contains gag- and pol-expressing genes, and any envelope plasmid that contains the VSV-G expressing gene.
4. Vortex the DNA transfection reagent vial for 30 s. Pipette 4 µL of the transfection reagent directly into the medium containing the diluted DNA. Mix gently by gently tipping the tube.
   NOTE: Do not touch the walls of the plastic tubes with the tips. Do not pipette up and down or vortex.
5. Incubate the reaction for 15 min at room temperature.
6. While incubating the reaction, remove the old medium from the 6-well plate and replace it with 2 mL/well of fresh serum-free media.
7. Add each transfection complex to each well in a dropwise manner.
8. Gently shake or swirl the plate to ensure even distribution over the entire well surface.
9. Incubate the plate overnight at 37 °C in a humidified incubator with an atmosphere of 5% CO₂.
10. After 24 h, replace the old medium with fresh complete medium (2 mL/well) and return the cells back to the humidified incubator with an atmosphere of 5% CO₂ for 72 h.
11. After 72 h, harvest the supernatant in a 50 mL conical tube and centrifuge for 10 min at 4,000 × g at 4 °C to remove dead cells and debris. For further purification, use a large 60 mL syringe to pass the supernatant through a 0.20 µm filter. Immediately prepare single-use aliquots of 300 µL and store at -80 °C.
    ​NOTE: Avoid a freeze-thaw cycle to maintain maximum product activity.

2. Retroviral transduction

1. The day before transducing the cells, seed the target cells at a medium density (PANC1 at 1 × 10⁵ cells/well) in a 12-well plate in 1 mL/well of medium supplemented with 10% FBS and 1% P/S. Incubate the plate overnight at 37 °C in a humidified incubator with an atmosphere of 5% CO₂.
   NOTE: The best cell density has to be established in advance, as different cell types have different attachment abilities. Ideally, use a cell density to obtain 40%-50% confluency.
2. Remove the frozen retrovirus aliquots from the -80 °C freezer and thaw on ice before each use.
   NOTE: Do not refreeze unused aliquots.
3. In a 50 mL conical tube, prepare a mixture of viral supernatant and polybrene at a final concentration of 8 µg/mL.
   NOTE: The subsequent volumes apply to the transduction of a 12-well plate containing a final volume of 500 µL/well. The final viral supernatant volume can be estimated by testing a range of viral dilutions in the presence of polybrene. Higher or lower dilutions can be used depending on the desired expression levels of the transgene and the size of the vessel used.
4. Remove the old medium from the 12-well plate and add 500 µL of the mixture to each well. Incubate the cells with the viral supernatant overnight at 37 °C in a humidified incubator with an atmosphere of 5% CO₂.
   NOTE: Keep two wells with complete medium (no viral supernatant) to use as a control for selection.
5. Replace the virus-containing medium with fresh complete growth medium (1 mL/well). Place the cells back in the humidified incubator with an atmosphere of 5% CO₂ for 48 h.

3. Pooled population selection and maintenance

1. Replace the growth medium with selection medium (complete growth medium with a final concentration of 1 µg/mL puromycin). Monitor the growth of the cells and change the selection media every 2-3 days. At confluence, expand into a 6-well dish, and then into a 10 cm diameter tissue culture dish.
2. Keep the cells in selection medium for at least as long as it takes for the control (untransduced) cells to completely die.
   NOTE: For successful results, it is recommended that optimal concentrations of puromycin be determined prior to initiating the experimental project. For this, generate a puromycin kill curve to determine the minimum concentration required to kill untransduced cells between 3 and 10 days.
3. Once the cells are growing in the selection medium, expand the cells on complete growth media and freeze stock aliquots for the experimental project.
   NOTE: Record the passage number and avoid working with pooled populations from frozen stock with passage numbers higher than five. Check regularly for mycoplasma contamination prior to performing the assay. Ideally, use a fresh cell batch before each assay to obtain the maximum signal of luciferase.
4. Maintain the cells in selection medium and seed enough cells to reach the desired density the day before the assay.

4. Compound addition

NOTE: The Selleckchem small molecule library consists of approximately 4,000 compounds arranged in eight rows and 10 columns in fifty 96-well plates at a stock concentration of 10 mM in dimethyl sulfoxide (DMSO).

1. Aliquot 30 µL of compound into the appropriate well of a source plate that is compatible with a nanoscale acoustic liquid dispenser. Use a 384-well polypropylene plate (384PP plate). Keep these plates sealed and stored at -20 °C.
   NOTE: The screening protocol described here is for a total of 10 assay plates per day; a fixed concentration of 10 µM was used as the final assay concentration along with a 24 h incubation period.
2. Thaw the compound library plates at room temperature.
   NOTE: Make sure the plate is completely thawed and equilibrated to room temperature. A temperature gradient across the plate may affect liquid handling.
3. Dispense 50 nL/well into a 384-well assay plate using a nanoscale acoustic liquid dispenser.
   NOTE: Ensure the selected source and destination plates in the program matches the ones used for this step.
4. Create a dispensing program on a spreadsheet.
5. Open the software.
6. Open a new protocol. From the Protocol tab, select the following options: Sample plate format, 384PP; Sample plate type, 384PP_DMSO2; and Destination plate type, CellCarrier-384 Ultra PN (Figure 2A).
7. Under Pick List, select the import option (Figure 2B) to import the spreadsheet containing the dispensing program (Figure 2C).
8. Select the Run protocol option (Figure 2D), verify if the displayed information is correct, and click Run.
9. On the new window called Run status (Figure 2E), click on Start and follow the steps displayed on the prompt windows (Figure 2F,G).

5. Cell seeding

1. Trypsinize the cells from a cell culture flask or cell culture Petri dishes and neutralize the trypsin by adding FBS-containing media.
2. Transfer the cell suspension to a 50 mL conical tube and centrifuge at 390 × g for 5 min at room temperature. Then, gently remove the supernatant and resuspend the cell pellet in 10 mL of complete growth media.
3. Perform cell counting.
4. Prepare the cell suspension with 4.6 × 10⁷ cells in 230 mL of complete culture media.
   NOTE: This volume and cell density is for 10x 384-well assay plates plus a dead volume of two 384-well plates.
5. Dispense 50 µL of cell suspension into each well of a 384-well assay plate, prepared in section 4.
   NOTE: Cell seeding can be done either manually or by using a bulk dispenser.
6. Incubate the cells at 37 °C in a humidified incubator with an atmosphere of 5% CO₂ for 24 h.

6. Harvesting the cellular supernatant

NOTE: The liquid handling robotic platform used here performs liquid handling with a multichannel arm for 96-tips. If no liquid-handling automation is available, the protocol can be adapted to low-throughput format by using multichannel pipettes.

1. Configure the deck of the lab automation workstation as shown in Figure 3.
2. Place the disposable 96-tip stack on position P1 (Figure 3A).
   NOTE: Each 96-tip stack is formed of eight disposable racks. When using a multichannel arm for 96-tips, the 384-well plate is divided in four quadrants. Thus, each tip stack is enough to transfer the supernatant from two assay plates into two empty, solid-black, 384-well plates. The entire tip stack needs to be replaced after each run of two plates.
3. Place the assay plates on positions P2 and P4 (Figure 3A).
4. Place the empty, solid-black, 384-well plates on positions P3 and P5 (Figure 3A).
   NOTE: This flexible and capable robotic platform has been adapted for this assay and a special program was written (Figure 3B).
5. Get the tips from position P1.
6. Aspirate 10 µL of supernatant from the plate on position P2 and transfer into the empty, solid-black plate on position P3.
   NOTE: The tips should be positioned at an appropriate depth inside the wells to aspirate the supernatant without disturbing the cell monolayer at the bottom of the well.
7. Drop the tips in waste on position P6 (Figure 3A).
8. Repeat steps 6.5-6.7 for the remaining wells of the plate on position P2, and then repeat the same steps to transfer the supernatant from position P4 to position P5.
   NOTE: Be sure to collect the supernatant and dispense on the corresponding wells into the empty, solid-black plate (Figure 3C,D). As secreted dNGLUC is very stable in cell culture medium, the plates can be sealed and kept for up to 7 days at 4 °C in the dark.

7. Luciferase assay

NOTE: The dNGLUC used in the reporter exhibits flash kinetics with rapid signal decay. Due to the rapid decay of luminescence after adding substrate (coelenterazine), the plate reader should be set to measure the luminescence signal in the supernatants; inject the substrate to a well and read that well after a few seconds. For this reason, use a plate reader that is capable of monitoring luminescence and equipped with a substrate injector to ensure the time between the injection and read steps will be uniform for all samples. The settings used on the plate reader can be found in Figure 4.

1. Prepare native coelenterazine as a 1 mg/mL stock solution in acidified methanol (10 µL of 3 M HCl to 1 mL of methanol).
   NOTE: Follow local health and safety guidance regarding the handling of methanol in the lab and avoid contact with skin. Prepare the fresh working substrate solution before starting the assay.
2. Initialize the injector pump (Figure 5A).
3. Rinse the tubing with deionized water (Figure 5B-D).
4. Rinse the tubing with methanol.
5. While rinsing the tubing, prepare the working substrate solution by diluting the substrate 1:100 (for one 384-well plate, add 220 µL from the substrate stock solution into 21.8 mL of 1x phosphate-buffered saline [PBS]).
6. Rinse the tubing with the substrate working solution (Figure 5B-D).
7. Load the plate into the reader and start the measurement using the settings described in Figure 4.
8. Repeat steps 7.6-7.7 for all assay plates.
9. Once finished with all the assay plates, rinse the tubing with methanol.
10. Rinse the tubing with deionized water.
    ​NOTE: At the end of this step, raw luciferase values are obtained that are correlative to the cellular ATG4B activity. For normalization to cell numbers, the next steps are necessary by counting the cell number in each well by fluorescence microscopy.

8. Cell fixation and staining

NOTE: This step can be performed manually with the aid of a multichannel pipette or by using a bulk dispenser.

1. Fix the cells with 4% paraformaldehyde (in 1x PBS) for 15 min.
   NOTE: Follow local health and safety guidance regarding the handling of paraformaldehyde in the lab and avoid contact with skin. Perform this step in a safety hood if possible.
2. Wash three times with 1x PBS.
3. Stain the nuclei with Hoechst 33342 diluted 1:5,000 in 1x PBS for 15 min.
4. Wash three times with 1x PBS.

9. Image acquisition

NOTE: Perform image acquisition using an automated microscope. As an alternative to image acquisition to determine number of cells, the intracellular luciferase activity can also be determined. There are advantages and disadvantages with regards to whether one normalizes to cell numbers or to intracellular luciferase activity, which is discussed below. We find that determining cell numbers is less invasive and results in lower variability than determining intracellular luciferase values.

1. Launch the microscope operating software (Figure 6).
2. In the Setup tab, choose the correct predefined plate type. If the plate type is not preset, enter the plate dimensions manually.
3. Load the plate into the microscope by clicking on Eject and Load option.
4. Then, choose the 20x Air (numerical aperture [NA]: 0.4) objective.
   NOTE: Ensure the objective collar is set to the correct value, allowing proper focus with different plate types.
5. Under Channel Selection, choose Hoechst 33342.
   NOTE: The channel settings for time, power, and height have to be optimized according to the plate type used.
6. On Define Layout, select all wells from the plate and four fields from the well.
7. On Online Jobs, select the corresponding folder to transfer the data to the analysis software.

10. Image analysis

NOTE: Any image analysis software can be used to segment and count cell nuclei from the acquired images. Here, we describe the steps to use a specific online software that is compatible to multiple automated microscopes files.

1. Launch the image analysis software.
2. Go to the Image Analysis tab to start the image segmentation (Figure 7A).
3. In the Input Image tab, click on the + sign to add a new building block (Figure 7A).
4. From the list, select the Find Nuclei option (Figure 7A) and select Hoechst 33342 as the channel option (Figure 7B).
5. Visually inspect the segmented objects on the image and select the most accurate segmentation method.
   NOTE: For this experiment, we used method C (Figure 7C). Each method option has subcategories that can be adjusted to obtain the best segmentation possible.
6. Then, click on the Define Results tab (Figure 7C) and select Standard Output as the Method option.
7. From the subcategory, select Nuclei-number of objects and Object count (Figure 7C).
8. Save the analysis pipeline using the Save analysis to Disk or Save analysis to Database option.
9. Under the Batch Analysis tab, select the data to be analyzed from the tree.
10. Under Method, select the analysis pipeline saved in step 10.8.
    NOTE: It is also possible to upload a script file from outside the referenced software or upload an existing analysis saved to the database.
11. Click on Run Analysis to start the analysis (Figure 7D). At the end of this workflow, two datasets are generated: raw luciferase values from the supernatants and the cell number in each well. Use both to normalize the luciferase value per cell.

Results

In a previous publication⁸, we successfully used this assay to screen small molecule and siRNA libraries and identified novel regulators of ATG4B. Here, we describe the protocol and representative results of this luciferase reporter in a semi-automated, high-throughput screening format. Figure 8 shows an example of the raw data analysis for both cell nuclei and luminescence. A typical result of a luminescence measurement is depicted in Figure 8A

Discussion

This protocol describes a cell-based reporter-gene assay for the identification of ATG4B inhibitors. The identification of primary hits is based on luciferase activity upon the treatment of cells expressing the full-length open reading frame of LC3B between β-actin and dNGLUC. Some advantages of this assay are that it is sensitive, highly quantitative, and noninvasive, as it can detect dNGLUC without lysing the cells. This paper presents a detailed protocol for generating a stable cell line and a primary screening. ...

Materials

Name	Company	Catalog Number	Comments
50 µL Disposable Tips - Non-filtered, Pure, Nested 8 Stack (Passive Stack)	Tecan	30038609	Disposable 96-tip rack
BioTek MultiFlo	BioTek		bulk dispenser
Coelenterazine	Santa Cruz Biotechnology	sc-205904	substrate
Columbus Image analysis software	Perkin Elmer	Version 2.9.1	image analysis software
DPBS (1x)	Gibco	14190-144
Echo Qualified 384-Well Polypropylene Microplate, Clear, Non-sterile	Beckman Coulter	001-14555	384PP plate
EnVision II	Perkin Elmer		luminescence plate reader
Express pick Library (96-well)-L3600-Z369949-100µL	Selleckchem	L3600	Selleckchem
FMK9A	MedChemExpress	HY-100522
Greiner FLUOTRAC 200 384 well plates	Greiner Bio-One	781076	solid-black 384-well plates
Harmony Imaging software	Perkin Elmer	Version 5.1	imaging software
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water	ThermoFisher	H3570	Hoechst 33342
Labcyte Echo 550 series with Echo Cherry Pick software	Labcyte/Beckman Coulter		nanoscale acoustic liquid dispenser
Milli-Q water			deionized water
Opera Phenix High-Content Screening System	Perkin Elmer		automated microscope
Paraformaldehyde solution 4% in PBS	Santa Cruz Biotechnology	sc-281692
PhenoPlate 384-well, black, optically clear flat-bottom, tissue-culture treated, lids	Perkin Elmer	6057300	CellCarrier-384 Ultra PN
pMOWS-ActinLC3dNGLUC			Obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin)
Polybrene Infection / Transfection Reagent	Merck	TR-1003-G	polybrene
Puromycin dihydrochloride, 98%, Thermo Scientific Chemicals	ThermoFisher	J61278.ME	Puromycin
Tecan Freedom EVO 200 robot	Tecan		liquid handling robotic platform
X-tremeGENE HP DNA Transfection Reagent Roche	Merck	6366244001	DNA transfection reagent