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 i....

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 cell.......

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