The MALDI TOF E2/E3 ligase assay as universal tool for drug discovery in the ubiquitin pathway

Virginia De Cesare (Lead / Corresponding author), Clare Johnson, Victoria Barlow, Charles Hastie, Axel Knebel, Matthias Trost (Lead / Corresponding author)

Research output: Contribution to journalArticle

18 Citations (Scopus)
188 Downloads (Pure)

Abstract

Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high throughput assays. Here we report the first label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI TOF) mass spectrometry. The MALDI TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared to standard biochemical assays.
Original languageEnglish
Pages (from-to)1117-1127.4
Number of pages11
JournalCell Chemical Biology
Volume25
Issue number9
Early online date12 Jul 2018
DOIs
Publication statusPublished - 20 Sep 2018

Keywords

  • Ubiquitin
  • E3 ligase
  • E2 enzyme
  • MALDI TOF
  • mass spectrometry
  • drug discovery
  • high-throughput
  • assay
  • MDM2
  • HOIP
  • ITCH
  • MALDI-TOF
  • high-throughput assay
  • ubiquitin

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