Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Maikel Izquierdo, De Lin, Sandra O'Neill, Martin Zoltner, Lauren Webster, Anthony Hope, David W. Gray, Mark C. Field, Jorge González-Bacerio (Lead / Corresponding author)

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Number of pages8
JournalSLAS Discovery
Early online date13 May 2020
DOIs
Publication statusE-pub ahead of print - 13 May 2020

Keywords

  • arphamenine
  • bestatin
  • high-throughput screening
  • M17-leucyl aminopeptidase
  • RapidFire mass spectrometry

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