Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B
| Autor: | Nag S. Kumar, Thanh G. Nguyen, Suzana Kovacic, Lubomir Vezenkov, Tom A. Pfeifer, Damien Bosc, Nicolette S. Honson, Robert N. Young |
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| Rok vydání: | 2015 |
| Předmět: |
Steric effects
Recombinant Fusion Proteins Molecular Sequence Data Clinical Biochemistry Autophagy-Related Proteins Pharmaceutical Science Peptide Cysteine Proteinase Inhibitors Cleavage (embryo) Biochemistry Drug Discovery Small peptide Autophagy Fluorescence Resonance Energy Transfer Humans Amino Acid Sequence Molecular Biology Fluorescent Dyes chemistry.chemical_classification Organic Chemistry Fluorescence Cysteine protease Cysteine Endopeptidases Enzyme chemistry Proteolysis Molecular Medicine Biological Assay Peptides Microtubule-Associated Proteins |
| Zdroj: | Bioorganic & Medicinal Chemistry. 23:3237-3247 |
| ISSN: | 0968-0896 |
| DOI: | 10.1016/j.bmc.2015.04.064 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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