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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
M. Izquierdo, . Lin, S. O'Neill, M. Zoltner, L. Webster, A. Hope, DW. Gray, MC. Field, J. González-Bacerio
Language English Country United States
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
203134/Z/16/Z
Wellcome Trust - United Kingdom
204697/Z/16/
Wellcome Trust - United Kingdom
- MeSH
- Chagas Disease diagnosis enzymology parasitology MeSH
- Mass Spectrometry MeSH
- Kinetics MeSH
- Leucyl Aminopeptidase genetics isolation & purification MeSH
- Humans MeSH
- High-Throughput Screening Assays * MeSH
- Amino Acid Sequence genetics MeSH
- Substrate Specificity MeSH
- Trypanosoma cruzi enzymology isolation & purification pathogenicity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
Centre for Protein Studies Faculty of Biology University of Havana La Habana Cuba
Department of Biochemistry Faculty of Biology University of Havana La Habana Cuba
Department of Parasitology Faculty of Science Charles University BIOCEV Vestec Czech Republic
Drug Discovery Unit Wellcome Centre for Anti Infectives Research University of Dundee Dundee UK
References provided by Crossref.org
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