TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors.
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.
Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Charitha
C
Madiraju
Kate
K
Welsh
Michael P
MP
Cuddy
Paulo H
PH
Godoi
Ian
I
Pass
Tram
T
Ngo
Stefan
S
Vasile
Eduard A
EA
Sergienko
Paul
P
Diaz
Shu-Ichi
S
Matsuzawa
John C
JC
Reed
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D025821:Polyubiquitin / Q000096:biosynthesis* / Q000737:chemistry; D054852:Small Molecule Libraries; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors*; D054875:Ubiquitination
D054852:Small Molecule Libraries; D025821:Polyubiquitin; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes