autophagy protease tag-compound addition and cellular supernatant harvest for drug screening

High-Throughput Screening Analysis for 4B Cysteine Peptidase Autophagy Inhibitor

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 lysosomes1,2,3. 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 needs2,3,4. 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 damage2,4,5,6.
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 control2,4,5,6. Growing evidence has shown that ATG4B is involved in certain pathological conditions, and it has gained attention as a potential anticancer target2,3,4. 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 cells2,4. 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 survival3,4. Overall, ATG4B is highly overexpressed in some cancer types, is related to the progression of tumor, and is linked to cancer therapy resistance2,4,8.
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 proteins9,10,11 and may have additional functions not linked to their protease activity12,13. Furthermore, ATG4 functions in regulating a novel type of post-translational modification, the ATG8-ylation of proteins11,12. 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 nitrosylation9,10,11,12,13.
Several known ATG4B inhibitors have been published2,4,14,15. While these are suitable as research tools, their pharmacodynamic profile, selectivity, or potency have yet precluded them from development as preclinical candidates4,16. 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 cells8,17. 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 activity18,19.
In this reporter construct, dNGLUC is linked to the actin cytoskeleton of cells. A protease-specific linker can be introduced between the &#946;-actin anchor and dNGLUC, turning the secretion dependent on cleavage of the linker. We used the full-length open reading frame of LC3B between &#946;-actin and dNGLUC, to be able to monitor LC3B cleavage17,18,19. 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 peptide4,18,19. 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.

Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells 

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

The main aim of research in my lab is to understand the regulation of autophagy and in particular of the ATG4 cysteine protean and use this knowledge to develop small molecule inhibitors that target this pathway in particular for diseases such as pancreatic cancer. We use high content, high throughput screening technologies to identify drug targets as well as drug candidates and we focus on the ATG4 cysteine protease family because we think it's a very good drug target for various forms of cancer. We have been studying the ATG4 family members for quite some time now.We made significant discoveries into dysfunction of this protein family and cells. We've also identified novel post-translational mechanisms of regulation of the ATG4 family members and currently we are developing small molecule inhibitors and also activators of the ATG4 families for diseases such as cancer and neuro degeneration. There are not many assays to monitor ATG4 protease activity in cells.So we were one of the first to develop a luciferase based assay to monitor this activity. And our assay is quite unusual because it uses secreted, let's say phrase, therefore we don't need to license the cells and we can adapt this protocol easily to high throughput screening methods and lab automation. We have focused on the ATG4 B protein protease for quite some time now.We have generated knockout cell lines of the ATG4 family members. We have identified novel post-translational regulation mechanisms of ATG4, and ultimately we have also identified several novel small molecule compounds that could inhibit and even activate the ATG4 B protease. In the future, we'd like to continue our work on the ATG4 family members.We think that's quite a lot that is currently unknown and still remains to be uncovered. For example, how these proteins regulate autophagy in neurons is currently completely not understood. We'd also like to focus our work on validation of these small molecule compounds that we've identified and potentially to use activators in neurodegenerative diseases.

Watch this Scientific Journal Video about Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening at JoVE.com

Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening  

Begin by thawing the Selleckchem Small Molecule Compound Library plates at room temperature. Open the software and click on New Protocol. On the Protocol tab, click on Sample Plate Format, followed by 384PP.Then on Sample Plate Type, select 384PP_DMSO2. Next, choose Cell Carrier 384 Ultra PN as the Destination Plate Type. Click on Pick List and select the import options to import the spreadsheet containing the dispensing program.Click on the Run Protocol option to verify the accuracy of the displayed information and then click on Run. Next press Start on the new window titled Run status, and then follow the steps displayed on the prompt windows. Use a nanoscale acoustic liquid dispenser and dispense 50 nanoliters per well of the Small Molecule Compound Library into a 384 well assay plate.To begin cellular supernatant harvesting, start by configuring the deck of the lab automation workstation. Place a disposable 96 tip rack on the P1 position and place the assay plates on positions P2 and P4.Finally, place the empty, solid black 384 well plates at positions P3 and P5.Using the tips from P1, aspirate 10 microliters of the supernatant from the plate at P2 and then transfer it into the empty solid black plate on P3.Discard the tips at position P6.Repeat the transfer of supernatant from P4 to P5.

autophagy-protease-tag-compound-addition-cellular-supernatant-harvest

Research

JoVE Journal

Biology

94 Views.  University College London. Begin by thawing the Selleckchem Small Molecule Compound Library plates at room temperature. Open the software and click on New Protocol. On the Protocol tab, click on Sample Plate Format, followed by 384PP.Then on Sample Plate Type, select 384PP_DMSO2. Next, choose Cell Carrier 384 Ultra PN as the Destination Plate Type. Click on Pick List and select the import options to import the spreadsheet containing the dispensing program.Click on the Run Protocol option to verify the acc...

Video: Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening  

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.

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

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...

Luciferase Assay for the Detection of Autophagy Protease ATG4B

Begin by opening the program and initialize the injector pump by clicking on the Dispenser Control, and select Pump 2, and then clicking on Init. To rinse the tubing with the ionized water, click on Rinse, then press Next. Select Pump 2 and set the tip mount to the desired tip.Then click on Next again. Click Start to begin tube rinsing. Repeat rinsing with methanol.Meanwhile, prepare a working substrate solution by diluting the substrate stock by 1 to 100. Finally, rinse the tubing with the working dilution of the substrate stock in PBS. Load the plate into the plate reader.And then, on Measurement settings, choose the aperture to 384 plate US luminescence aperture, followed by the distance between plate and detector to 0.6 millimeters, and the measurement time as 0.5 seconds. Once the measurement settings have been settled, on the Protocol-General settings tab, select the plate type as 384 Nunc, and choose the By columns bi-directional option as the measurement mode. Input 1 into the number of assay repeats tab.Now, on the Dispense tab, select the desired dispense measurement. Then set the measurement time for each well as one second and select the desired used pump. Dispensing speed of 200 microliters per second.And the dispensing volume to 50 microliters. Choose Dispensing volume as the syringe filling volume for Pump 2. And then set the Start dispensing at measurement number to 1.On the Well selection tab, use the Add/remove samples dropdown menu and choose the desired sample type, and select the wells to be measured. After that, start the measurement by clicking Run. The basal luminescence signal from DMSO is seen in column 1, and the decrease of luminescence signal on the presence of ATG4B inhibitor, FMK-9a, is seen in column 24.

Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay

Begin by launching the microscope operating software. Under the setup tab, choose the correct predefined plate type. Press eject to access the plate stage to position the plate and then press load to load the plate into the microscope.Next, choose the 20 times air objective with a numerical aperture of 0.4. Adjust the correction collar of the 20x air objective for the plate thickness of the chosen plate type. Set the channel selection to Hoechst 33342, and adjust the imaging parameters for the Hoechst channel.Test the chosen settings by selecting a representative well within the plate. On defined layout, select all plate wells and for fields. Then choose the corresponding folder under online jobs to transfer the data to the online analysis software.The experiment settings are saved. Under the run experiment tab, the plate which is going to be imaged is named, and the imaging process is started. To begin image analysis, first launch the software, choose the images for analysis, and then click on the image analysis tab to start image segmentation.Next, click on the plus sign in the input image tab to add a new building block. From the list, click on find nuclei. Choose the most appropriate segmentation method after inspecting the segmented objects on the image.Adjust the detection parameters within the segmentation method. Click on define results and choose standard output as the method. Click on save analysis to database to save the analysis pipeline.Under the batch analysis tab, click on the saved pipeline which will appear under the method option. Then select the data to be analyzed. Start the analysis by clicking on start analysis to generate the dataset for cell numbers.The described cell-based assay relies on a reporter readout that indirectly reflects changes in the ATG4B activity, enabling the detection of both inhibitors and activators of ATG4B activity. The ratio of the ATG4B activity of each compound to its cell viability is used as a cutoff value for the identification of ATG4B inhibitors. Ratios greater than one indicate possible ATG4B activators and ratios less than one indicate possible ATG4B inhibitors.