High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity

Jamie L. Arnst, Christopher W. Davies, Srikumar M. Raja, Chittaranjan Das, Amarnath Natarajan

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (associated molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent down-regulation of activated cell surface receptors via the endolysosomal degradative pathway. Therefore, small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently, there are no known selective inhibitors of AMSH or high-throughput compatible assays for their identification. We report the development and optimization of a novel fluorescence resonance energy transfer (FRET)-based add-and-read AMSH DUB assay in a 384-well format. In this format, the optimal temperature for a high-throughput screen (HTS) was determined to be 30 °C, the assay tolerates 5% dimethyl sulfoxide (DMSO), and it has a Z-score of 0.71, indicating HTS compatibility. The assay was used to show that AMSH selectively cleaves Lys63-linked diubiquitin over Lys48-and Lys11-linked diubiquitin. The IC50 value of the nonspecific small molecule DUB inhibitor N-ethylmaleimide was 16.2 ± 3.2 μM and can be used as a qualitative positive control for the screen. We conclude that this assay is high-throughput compatible and can be used to identify novel small molecule inhibitors of AMSH.

Original languageEnglish (US)
Pages (from-to)71-77
Number of pages7
JournalAnalytical Biochemistry
Volume440
Issue number1
DOIs
StatePublished - Jan 1 2013

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Fluorescence Resonance Energy Transfer
Assays
Throughput
Molecules
Cell Surface Receptors
Ubiquitin
Ethylmaleimide
src Homology Domains
Corrosion inhibitors
Dimethyl Sulfoxide
Inhibitory Concentration 50
Down-Regulation
Deubiquitinating Enzymes
Degradation
Temperature
Proteins

Keywords

  • AMSH
  • Deubiquitinase assay
  • FRET
  • HTS

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity. / Arnst, Jamie L.; Davies, Christopher W.; Raja, Srikumar M.; Das, Chittaranjan; Natarajan, Amarnath.

In: Analytical Biochemistry, Vol. 440, No. 1, 01.01.2013, p. 71-77.

Research output: Contribution to journalArticle

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