Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B. Issue 13 (1st July 2015)

• Record Type: Journal Article
• Authors: Vezenkov, Lubomir; Honson, Nicolette S.; Kumar, Nag S.; Bosc, Damien; Kovacic, Suzana; Nguyen, Thanh G.; Pfeifer, Tom A.; Young, Robert N.
• Is Part Of: Bioorganic & medicinal chemistry. Volume 23:Issue 13(2015)
• Page Start: 3237
• Page End: 3247
• Publication Date: 2015-07-01
• Subjects: VWBVATUTVWMVIR-LJAQVGFWSA-N; ATG4B autophagin 1 -- FRET fluorescence resonance energy transfer -- HTS high throughput screening -- LC3B microtubule-associated protein 1 A/B light chain 3B -- NEM N-ethylmaleimide -- LC–MS liquid chromatography coupled mass spectrometry -- RFU relative fluorescence unit -- PE phosphatidylethanolamine -- TCEP tris-(2-carboxyethyl)phosphine -- Tris tris(hydroxymethyl)aminomethane; Autophagy -- ATG4B -- Cysteine protease -- Fluorogenic peptide -- Substrate -- Screen -- Inhibitor -- LC3; Bioorganic chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Chemistry, Clinical -- Periodicals; Chemistry, Organic -- Periodicals; Chimie bio-organique -- Périodiques; Chimie pharmaceutique -- Périodiques; 615.19
• DOI: 10.1016/j.bmc.2015.04.064
• Languages: English
• ISSNs: 0968-0896

Abstract

Graphical abstract: A small fluorogenic peptide substrate (33 ) has been developed for screening and characterization of inhibitors of the key autophagy-initiating enzyme ATG4B. Abstract: 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.