This work was supported by UK Medical Research Council core funding to the MRC-UCL University Unit Grant Ref MC_U12266B, MRC Dementia Platform Grant UK MR/M02492X/1, Pancreatic Cancer UK (grant reference 2018RIF_15), and the UCL Therapeutic Acceleration Support scheme, supported by funding from MRC Confidence in Concept 2020 UCL MC/PC/19054. The plasmid encoding the ActinLC3dNGLUC (pMOWS-ActinLC3dNGLUC) was obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin).

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).
<ol>
	<li>Culture HEK293T cell...

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

<ol>
	<li>Kocaturk, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+as+a+molecular+target+for+cancer+treatment.">Autophagy as a molecular target for cancer treatment.</a> European Journal of Pharmaceutical Sciences. 134, 116-137 (2019).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+ATG4+in+cancer+therapy.">Targeting ATG4 in cancer therapy.</a> Cancers. 11 (5), 649 (2019).</li><li>Towers, C. G., Thorburn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+autophagy.">Therapeutic targeting of autophagy.</a> EBioMedicine. 14, 15-23 (2016).</li><li>Agrotis, A., Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+ATG4B+as+drug+target+for+treatment+of+solid+tumours-the+knowns+and+the+unknowns.">On ATG4B as drug target for treatment of solid tumours-the knowns and the unknowns.</a> Cells. 9 (1), 53 (2019).</li><li>Levy, J. M. M., Towers, C. G., Thorburn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+autophagy+in+cancer.">Targeting autophagy in cancer.</a> Nature Reviews Cancer. 17 (9), 528-542 (2017).</li><li>Kimmelman, A. C., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+tumor+metabolism.">Autophagy and tumor metabolism.</a> Cell Metabolism. 25 (5), 1037-1043 (2017).</li><li>Tran, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+role+of+ATG4B+as+target+for+autophagy+inhibition+in+prostate+cancer+therapy.">Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy.</a> Biochemical and Biophysical Research Communications. 441 (4), 726-731 (2013).</li><li>Pengo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+kinases+and+phosphatases+that+regulate+ATG4B+activity+by+siRNA+and+small+molecule+screening+in+cells.">Identification of kinases and phosphatases that regulate ATG4B activity by siRNA and small molecule screening in cells.</a> Frontiers in Cell and Developmental Biology. 6, 148 (2018).</li><li>Kauffman, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delipidation+of+mammalian+Atg8-family+proteins+by+each+of+the+four+ATG4+proteases.">Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.</a> Autophagy. 14 (6), 992-1010 (2018).</li><li>Tanida, I., Sou, Y. -. S., Minematsu-Ikeguchi, N., Ueno, T., Kominami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg8L/Apg8L+is+the+fourth+mammalian+modifier+of+mammalian+Atg8+conjugation+mediated+by+human+Atg4B,+Atg7+and+Atg3.">Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3.</a> The FEBS Journal. 273 (11), 2553-2562 (2006).</li><li>Agrotis, A., Pengo, N., Burden, J. J., Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundancy+of+human+ATG4+protease+isoforms+in+autophagy+and+LC3/GABARAP+processing+revealed+in+cells.">Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells.</a> Autophagy. 15 (6), 976-997 (2019).</li><li>Nguyen, T. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg8+family+LC3/GABARAP+proteins+are+crucial+for+autophagosome-lysosome+fusion+but+not+autophagosome+formation+during+PINK1/Parkin+mitophagy+and+starvation.">Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.</a> The Journal of Cell Biology. 215 (6), 857-874 (2016).</li><li>Ketteler, R., Tooze, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATG4:+More+than+a+protease.">ATG4: More than a protease.</a> Trends in Cell Biology. 31 (7), 515-516 (2021).</li><li>Zhang, L., Li, J., Ouyang, L., Liu, B., Cheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+roles+of+Atg4+proteases+from+autophagy+modulation+to+targeted+cancer+therapy.">Unraveling the roles of Atg4 proteases from autophagy modulation to targeted cancer therapy.</a> Cancer Letters. 373 (1), 19-26 (2016).</li><li>Fern&#225;ndez, &. #. 1. 9. 3. ;. F., L&#243;pez-Ot&#237;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+pathologic+relevance+of+autophagy+proteases.">The functional and pathologic relevance of autophagy proteases.</a> The Journal of Clinical Investigation. 125 (1), 33-41 (2015).</li><li>Maruyama, T., Noda, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy-regulating+protease+Atg4:+structure,+function,+regulation+and+inhibition.">Autophagy-regulating protease Atg4: structure, function, regulation and inhibition.</a> The Journal of Antibiotics. 71 (1), 72-78 (2017).</li><li>Ketteler, R., Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+autophagy+by+luciferase+release+assay.">Quantitation of autophagy by luciferase release assay.</a> Autophagy. 4 (6), 801-806 (2008).</li><li>Ketteler, R., Sun, Z., Kovacs, K. F., He, W. -. W., Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathway+sensor+for+genome-wide+screens+of+intracellular+proteolytic+cleavage.">A pathway sensor for genome-wide screens of intracellular proteolytic cleavage.</a> Genome Biology. 9 (4), 64 (2008).</li><li>Luft, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Gaussia+luciferase+in+bicistronic+and+non-conventional+secretion+reporter+constructs.">Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs.</a> BMC Biochemistry. 15, 14 (2014).</li><li>Ketteler, R., Glaser, S., Sandra, O., Martens, U. M., Klingm&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+transgene+expression+in+primitive+hematopoietic+progenitor+cells+and+embryonic+stem+cells+efficiently+transduced+by+optimized+retroviral+hybrid+vectors.">Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors.</a> Gene Therapy. 9 (8), 477-487 (2002).</li></ol>

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 &#946;-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. ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 &#181;L Disposable Tips - Non-filtered, Pure, Nested 8 Stack (Passive Stack)</td>
			<td>Tecan</td>
			<td>30038609</td>
			<td>Disposable 96-tip rack</td>
		</tr>
		<tr>
			<td>BioTek MultiFlo</td>
			<td>BioTek</td>
			<td></td>
			<td>bulk dispenser</td>
		</tr>
		<tr>
			<td>Coelenterazine</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-205904</td>
			<td>substrate</td>
		</tr>
		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Version 2.9.1</td>
			<td>image analysis software</td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Echo Qualified 384-Well Polypropylene Microplate, Clear, Non-sterile</td>
			<td>Beckman Coulter</td>
			<td>001-14555</td>
			<td>384PP plate</td>
		</tr>
		<tr>
			<td>EnVision II</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>luminescence plate reader</td>
		</tr>
		<tr>
			<td>Express pick Library (96-well)-L3600-Z369949-100&#181;L</td>
			<td>Selleckchem</td>
			<td>L3600</td>
			<td>Selleckchem</td>
		</tr>
		<tr>
			<td>FMK9A</td>
			<td>MedChemExpress</td>
			<td>HY-100522</td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner FLUOTRAC 200 384 well plates</td>
			<td>Greiner Bio-One</td>
			<td>781076</td>
			<td>solid-black 384-well plates</td>
		</tr>
		<tr>
			<td>Harmony Imaging software</td>
			<td>Perkin Elmer</td>
			<td>Version 5.1</td>
			<td>imaging software</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>Hoechst 33342</td>
		</tr>
		<tr>
			<td>Labcyte Echo 550 series with Echo Cherry Pick software</td>
			<td>Labcyte/Beckman Coulter</td>
			<td></td>
			<td>nanoscale acoustic liquid dispenser</td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td></td>
			<td></td>
			<td>deionized water</td>
		</tr>
		<tr>
			<td>Opera Phenix High-Content Screening System</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>automated microscope</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>PhenoPlate 384-well, black, optically clear flat-bottom, tissue-culture treated, lids</td>
			<td>Perkin Elmer</td>
			<td>6057300</td>
			<td>CellCarrier-384 Ultra PN</td>
		</tr>
		<tr>
			<td>pMOWS-ActinLC3dNGLUC</td>
			<td></td>
			<td></td>
			<td>Obtained from Dr. Robin Ketteler (Department of Human Medicine, Medical School Berlin)</td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Merck</td>
			<td>TR-1003-G</td>
			<td>polybrene</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride, 98%, Thermo Scientific Chemicals</td>
			<td>ThermoFisher</td>
			<td>J61278.ME</td>
			<td>Puromycin</td>
		</tr>
		<tr>
			<td>Tecan Freedom EVO 200 robot</td>
			<td>Tecan</td>
			<td></td>
			<td>liquid handling robotic platform</td>
		</tr>
		<tr>
			<td>X-tremeGENE HP DNA Transfection Reagent Roche</td>
			<td>Merck</td>
			<td>6366244001</td>
			<td>DNA transfection reagent</td>
		</tr>
	</tbody>
</table>

cell-based drug screening for inhibitors of autophagy related 4b cysteine peptidase

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.

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.

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</...

Watch this Scientific Journal Video about A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells at JoVE.com

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 ...

cell-based-drug-screening-for-inhibitors-autophagy-related-4b