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Updated: Jun 5, 2026
Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase
Published on: June 30, 2023
High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4
Chih-Wen Shu1, Charitha Madiraju, Dayong Zhai
1Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, and Conrad Prebys Center for Chemical Genomics, La Jolla, CA 92037, USA.
Abstract:
Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.
Insights
Researchers developed a novel assay to measure autophagin-1 (Atg4B) activity, aiding the search for autophagy modulators. This tool helps investigate autophagy
Area of Science:
- Biochemistry
- Cell Biology
- Drug Discovery
Background:
- Autophagy is a crucial cellular process for degrading damaged components.
- Dysregulation of autophagy is linked to diseases like cancer and neurodegeneration.
- Limited availability of chemical modulators hinders research into autophagy's disease relevance.
Purpose of the Study:
- To develop a novel fluorescence-based assay for measuring autophagin-1 (Atg4B) activity.
- To enable high-throughput screening (HTS) for Atg4B inhibitors.
- To provide a tool for studying autophagy's role in disease.
Main Methods:
- Developed a reporter substrate (LC3B-PLA(2)) for Atg4B protease activity.
- Validated a high-throughput screening (HTS) assay with a Z' factor >0.7.
- Employed counterscreens (PLA(2), caspase-3) to ensure specificity.
Main Results:
- The LC3B-PLA(2) assay demonstrated robustness for over 5 hours.
- Pilot screens identified confirmed hit rates of 0.23% and 0.70% for compound libraries.
- The assay is suitable for screening large chemical libraries.
Conclusions:
- The LC3B-PLA(2) reporter assay is a validated platform for Atg4B inhibitor screening.
- This assay facilitates the identification of specific Atg4B inhibitors.
- These inhibitors can serve as tools to explore autophagy's role in disease models.
