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Biology

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

Published: June 30th, 2023

DOI:

10.3791/65464

1Laboratory for Molecular Cell Biology, University College London, 2Department of Human Medicine, Medical School Berlin

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 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.

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 i....

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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 cell.......

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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

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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. .......

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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).

....

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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

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