title: Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease 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 4h 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.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Lubomir
authors.initials: L
authors.lastname: Vezenkov
authors.affiliation: Centre for Drug Research and Development, Vancouver, BC, Canada.
authors.forename: Nicolette S
authors.initials: NS
authors.lastname: Honson
authors.affiliation: Centre for Drug Research and Development, Vancouver, BC, Canada.
authors.forename: Nag S
authors.initials: NS
authors.lastname: Kumar
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Damien
authors.initials: D
authors.lastname: Bosc
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Suzana
authors.initials: S
authors.lastname: Kovacic
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: Thanh G
authors.initials: TG
authors.lastname: Nguyen
authors.affiliation: Centre for Drug Research and Development, Vancouver, BC, Canada.
authors.forename: Tom A
authors.initials: TA
authors.lastname: Pfeifer
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada. Electronic address: robert_young@sfu.ca.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D001681:Biological Assay; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000071183:Autophagy-Related Proteins; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
keywords: ATG4B; Autophagy; Cysteine protease; Fluorogenic peptide; Inhibitor; LC3; Screen; Substrate