Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.
SLAS Discov
; 25(9): 1064-1071, 2020 10.
Article
em En
| MEDLINE
| ID: mdl-32400260
Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Trypanosoma cruzi
/
Doença de Chagas
/
Ensaios de Triagem em Larga Escala
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Leucil Aminopeptidase
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
SLAS Discov
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Cuba
País de publicação:
Estados Unidos