High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4.
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.
Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, and Conrad Prebys Center for Chemical Genomics, La Jolla, CA 92037, USA.
Chih-Wen
CW
Shu
Charitha
C
Madiraju
Dayong
D
Zhai
Kate
K
Welsh
Paul
P
Diaz
Eduard
E
Sergienko
Renata
R
Sano
John C
JC
Reed
D001343:Autophagy; D000071183:Autophagy-Related Proteins; D053148:Caspase 3 / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000378:metabolism*; D023061:Gene Order; D057166:High-Throughput Screening Assays; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries; D013050:Spectrometry, Fluorescence
D000071183:Autophagy-Related Proteins; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; C474025:ATG4A protein, human; D053148:Caspase 3; D003546:Cysteine Endopeptidases