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Updated: Mar 24, 2026
Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells
Published on: June 30, 2023
Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform
Danqing Xu1, Zhiheng Xu1, Li Han1
11 Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China.
Abstract:
Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.
Insights
Researchers developed a novel assay to screen for compounds that inhibit ATG4B, a key protein in autophagy. This led to the discovery of a potent inhibitor, advancing autophagy research and drug discovery for diseases linked to its malfunction.
Area of Science:
- Cellular Biology
- Biochemistry
- Drug Discovery
Background:
- Autophagy is a critical cellular process for maintaining homeostasis, involving the degradation of damaged components via autophagosomes.
- Dysfunctional autophagy is implicated in various diseases, including cancer, viral infections, and neurodegenerative disorders.
- Targeting autophagy requires specific modulators, but their availability has been limited, hindering disease mechanism studies.
Purpose of the Study:
- To develop and validate a novel assay for measuring the protease activity of ATG4B, a key regulator of autophagy.
- To establish a high-throughput screening (HTS) platform for identifying ATG4B inhibitors.
- To discover and characterize novel ATG4B inhibitors for potential therapeutic applications and further research into autophagy.
Main Methods:
- Development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to quantify ATG4B protease activity.
- Validation of the TR-FRET assay and adaptation for high-throughput screening (HTS) using a library of 57,000 compounds.
- Hit confirmation, counterscreening to eliminate interfering compounds, and subsequent chemical modification for structure-activity relationship (SAR) studies.
Main Results:
- A novel TR-FRET assay for ATG4B protease activity was successfully developed and validated.
- HTS identified 267 confirmed hits (0.49% hit rate), with 65 compounds showing IC50 < 50 µM.
- A lead compound mimicking the LC3 peptide substrate was optimized through SAR, yielding a 10-fold more potent inhibitor that forms a covalent bond with ATG4B and shows cellular activity.
Conclusions:
- The developed TR-FRET assay coupled with a counterscreen provides a robust platform for identifying ATG4B inhibitors.
- The discovery of a potent, covalently binding ATG4B inhibitor offers a valuable tool for autophagy research.
- These findings facilitate the elucidation of autophagy mechanisms and present new avenues for drug discovery targeting autophagy-related diseases.
