期刊
BIOORGANIC & MEDICINAL CHEMISTRY
卷 23, 期 13, 页码 3237-3247出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmc.2015.04.064
关键词
Autophagy; ATG4B; Cysteine protease; Fluorogenic peptide; Substrate; Screen; Inhibitor; LC3
资金
- Canadian Institutes for Health Research (CIHR) [10216]
- Centre for Drug Research and Development (CDRD)
- Canadian Foundation for Innovation (CFI)
- Natural Sciences and Research Council of Canada (NSERC)
An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B. Substrate cleavage was readily monitored and was linear for up to 4 h but enzyme concentrations of about ten-fold higher were required compared to assays using protein substrate LC3 or analogs thereof (such as FRET-LC3). Several known inhibitors of ATG4B were evaluated using the small peptide substrate and gave IC50 values 3-7 fold higher than previously obtained values using the FRET-LC3 substrate. (C) 2015 Elsevier Ltd. All rights reserved.
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