Inhibitors of USP1/UAF1: Hit Validation in Primary 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 ..
BioActive Compounds: 25
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. This assay was used to confirm the hits identified from the qHTS.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030552-01
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
3 microliters of reagents (buffer in columns 3 and 4 as negative control and 1nM USP1/UAF1 complex in columns 1, 2, and 5-48) are dispensed into Greiner black solid bottom 1536-well assay plates. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10nl slotted pins, V&P Scientific, San Diego, CA). Following an incubation step of 15 min at room temperature, 1ul of Ub-Rho substrate (150nM final concentration) is added to initiate the reaction. The plates are immediately centrifuged at 1000 rpm for 15 seconds, and subsequently transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein kinetic measurements of fluorescence are acquired using 480 nm excitation/540 nm emission filter set (6 reads every 60 seconds, see Table 1). All reagents are diluted in an assay buffer consisting of 50mM HEPES (pH 7.8), 0.5mM EDTA, 0.1 mg/ml BSA, 1mM TCEP, and 0.01% Tween-20.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)