Inhibitors of USP1/UAF1: Hit Validation in USP2 Diubiquiting Gel Assay
Deubiquitinating enzymes (DUBs) are a class of enzymes that can cleave isopeptide bond formed between the C-terminal carboxylate of ubiquitin and a lysine side-chain amino group on the target protein. Among them, ubiquitin specific proteases (USPs) constitute the largest DUB family. Human USPs are emerging as promising targets for pharmacological intervention because of their connection to a more ..
Deubiquitinating enzymes (DUBs) are a class of enzymes that can cleave isopeptide bond formed between the C-terminal carboxylate of ubiquitin and a lysine side-chain amino group on the target protein. Among them, ubiquitin specific proteases (USPs) constitute the largest DUB family. Human USPs are emerging as promising targets for pharmacological intervention because of their connection to a number of human diseases, including prostate, colon and breast cancer (1, 2), pediatric acute lymphoblastic leukemia (3), and familial cylindromatosis (4). The advantage of inhibiting USPs lies in the potential specificity of therapeutic intervention that can lead to better efficacy and reduce nonspecific side effects.
In a collaboration between the University of Delaware and the NIH Chemical Genomics Center, a high-throughput screen assay was developed to screen for USP1/UAF1 inhibitors. This miniaturized assay has a fluorescent read-out and is used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) in order to identify a small molecule that inhibits USP1. Follow-up assays have also been developed to further test the hits from the primary screen. This assay validates the hits identified from the primary screen using a USP2 diubiquiting gel assay.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030552
Assay Submitter (PI): Zhihao Zhuang, University of Delaware
1. Priolo, C., et al. (2006) The isopeptidase USP2a protects human prostate cancer from apoptosis. Cancer Res 66, 8625-8632
2. Popov, N., et al. (2007) The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 9, 765-774
3. De Pitta, C., et al. (2005) A leukemia-enriched cDNA microarray platform identifies new transcripts with relevance to the biology of pediatric acute lymphoblastic leukemia. Haematologica 90, 890-898
4. Kovalenko, A., et al. (2003) The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424, 801-805
The inhibition assay contains 20 nM USP2, 2 microM K63 linked diubiquitin, and inhibitors at varied concentrations (0.08 microM - 114 microM). The reaction was allowed for 1 hour at 37 degrees C, and quenched by the addition of Laemmli sample buffer. The reaction product was separated on a 20% denaturing SDS-PAGE gel and stained with Coomassie Blue. The intensity of the individual diubiquitin and monoubiquitin band was quantified using Quantity One 4.3.1 (Biorad, Hercules, CA). The percentage of the conversion was determined and used to calculate the reaction rate. The rate was plotted against inhibitor concentrations and fitted to determine the IC50 values using GraphPad Prism (GraphPad Software, Inc. La Jolla, CA).
Compounds with an IC50 =< 75 uM are considered "active" and are assigned a score of 90.
Compounds with an IC50 between > 75 uM and < 100 uM are considered "inconclsuive" and are assigned a score of 50.
Compounds with an IC50 => 100 uM are considered "inactive" and are assigned a score of 10.
Data Table (Concise)