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J Biomol Screen. 2011 Feb;16(2):174-82. doi: 10.1177/1087057110392996. Epub 2011 Jan 18.

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4.

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  • 1Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, and Conrad Prebys Center for Chemical Genomics, La Jolla, CA 92037, USA.


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 Lopacâ„¢ and 2000 for Spectrumâ„¢ 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.

[PubMed - indexed for MEDLINE]
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