Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B.
Bioorg Med Chem
; 23(13): 3237-47, 2015 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-25979376
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Bioensaio
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Proteínas Recombinantes de Fusão
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Cisteína Endopeptidases
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Inibidores de Cisteína Proteinase
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Corantes Fluorescentes
Limite:
Humans
Idioma:
En
Revista:
Bioorg Med Chem
Assunto da revista:
BIOQUIMICA
/
QUIMICA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Canadá