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Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.
Izquierdo, Maikel; Lin, De; O'Neill, Sandra; Zoltner, Martin; Webster, Lauren; Hope, Anthony; Gray, David W; Field, Mark C; González-Bacerio, Jorge.
Afiliação
  • Izquierdo M; Centre for Protein Studies, Faculty of Biology, University of Havana, La Habana, Cuba.
  • Lin; Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK.
  • O'Neill S; Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK.
  • Zoltner M; Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, UK.
  • Webster L; Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic.
  • Hope A; Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK.
  • Gray DW; Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK.
  • Field MC; Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, University of Dundee, Dundee, UK.
  • González-Bacerio J; Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, UK.
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Trypanosoma cruzi / Doença de Chagas / Ensaios de Triagem em Larga Escala / 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
Texto completo
Adicionar na Minha BVS
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XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Trypanosoma cruzi / Doença de Chagas / Ensaios de Triagem em Larga Escala / 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