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

6 Citations (Scopus)
58 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

Fingerprint

Ubiquitin-Protein Ligases
Drug Discovery
Ubiquitin
Ionization
Assays
Desorption
Lasers
Screening
Libraries
Enzymes
Ubiquitin-Conjugating Enzymes
High-Throughput Screening Assays
Preclinical Drug Evaluations
Throughput
Fluorescent Dyes
Mass Spectrometry
Mass spectrometry
Labels
Pharmaceutical Preparations
Testing

Keywords

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

Cite this

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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.",
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author = "{De Cesare}, Virginia and Clare Johnson and Victoria Barlow and Charles Hastie and Axel Knebel and Matthias Trost",
note = "This work was funded by Medical Research Council UK (MC_UU_12016/5), the pharmaceutical companies supporting the Division of Signal Transduction Therapy (DSTT) (Boehringer Ingelheim, GlaxoSmithKline and Merck KGaA).",
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The MALDI TOF E2/E3 ligase assay as universal tool for drug discovery in the ubiquitin pathway. / De Cesare, Virginia (Lead / Corresponding author); Johnson, Clare; Barlow, Victoria; Hastie, Charles; Knebel, Axel; Trost, Matthias (Lead / Corresponding author).

In: Cell Chemical Biology, Vol. 25, No. 9, 20.09.2018, p. 1117-1127.4.

Research output: Contribution to journalArticle

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