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Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.

Publication ,  Journal Article
Yang, W; Wang, L; Paschen, W
Published in: J Biomol Screen
June 2013

Small ubiquitin-like modifier (SUMO1-3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high-throughput screening (HTS)-compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z' factor was 0.83 ± 0.04, confirming the suitability for HTS applications.

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Published In

J Biomol Screen

DOI

EISSN

1552-454X

Publication Date

June 2013

Volume

18

Issue

5

Start / End Page

621 / 628

Location

United States

Related Subject Headings

  • Ubiquitins
  • Sumoylation
  • Substrate Specificity
  • Small Ubiquitin-Related Modifier Proteins
  • Sensitivity and Specificity
  • Protease Inhibitors
  • Mutant Proteins
  • Models, Biological
  • Medicinal & Biomolecular Chemistry
  • Humans
 

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Yang, W., Wang, L., & Paschen, W. (2013). Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases. J Biomol Screen, 18(5), 621–628. https://doi.org/10.1177/1087057113479971
Yang, Wei, Liangli Wang, and Wulf Paschen. “Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.J Biomol Screen 18, no. 5 (June 2013): 621–28. https://doi.org/10.1177/1087057113479971.
Yang, Wei, et al. “Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.J Biomol Screen, vol. 18, no. 5, June 2013, pp. 621–28. Pubmed, doi:10.1177/1087057113479971.
Journal cover image

Published In

J Biomol Screen

DOI

EISSN

1552-454X

Publication Date

June 2013

Volume

18

Issue

5

Start / End Page

621 / 628

Location

United States

Related Subject Headings

  • Ubiquitins
  • Sumoylation
  • Substrate Specificity
  • Small Ubiquitin-Related Modifier Proteins
  • Sensitivity and Specificity
  • Protease Inhibitors
  • Mutant Proteins
  • Models, Biological
  • Medicinal & Biomolecular Chemistry
  • Humans