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

Wellcome Centre for Anti Infectives Research Division of Biological Chemistry and Drug Discovery School of Life Sciences University of Dundee Dundee UK

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Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity