TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

J Biomol Screen. 2012 Feb;17(2):163-76. doi: 10.1177/1087057111423417. Epub 2011 Oct 27.

Abstract

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Resonance Energy Transfer / methods
  • High-Throughput Screening Assays / methods*
  • Polyubiquitin / biosynthesis*
  • Polyubiquitin / chemistry
  • Small Molecule Libraries
  • Ubiquitin-Conjugating Enzymes / antagonists & inhibitors*
  • Ubiquitination

Substances

  • Small Molecule Libraries
  • Polyubiquitin
  • UBE2N protein, human
  • Ubiquitin-Conjugating Enzymes