title: Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.
abstract: 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.
authors.affiliation: Multidisciplinary Neuroprotection Laboratories, Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA. wei.yang@duke.edu
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Liangli
authors.initials: L
authors.lastname: Wang
authors.forename: Wulf
authors.initials: W
authors.lastname: Paschen
mesh_terms: D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D012680:Sensitivity and Specificity; D025841:Small Ubiquitin-Related Modifier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D058207:Sumoylation / Q000187:drug effects; D014452:Ubiquitins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011480:Protease Inhibitors; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins
keywords: AlphaScreen technology; SUMO; SUMO proteases; assay development; inhibitor