743255 1 NCGC usp1-uaf1-p1 Inhibitors of USP1/UAF1: Primary Screen 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. 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 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. Compound Ranking: 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. 504878 Summary AID 12917691 15996926 16951176 17558397 7398 603478 9606 http://www.ncgc.nih.gov 1 Phenotype Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. 4 254 2 Potency Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed. 1 5 3 Efficacy Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves. 1 15 4 Analysis Comment Annotation/notes on a particular compound's data or its analysis. 4 254 5 Activity_Score Activity score. 2 254 6 Curve_Description A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control. 4 254 7 Fit_LogAC50 The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). 1 254 8 Fit_HillSlope The Hill slope from a fit of the data to the Hill equation. 1 254 9 Fit_R2 R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. 1 254 10 Fit_InfiniteActivity The asymptotic efficacy from a fit of the data to the Hill equation. 1 15 11 Fit_ZeroActivity Efficacy at zero concentration of compound from a fit of the data to the Hill equation. 1 15 12 Fit_CurveClass Numerical encoding of curve description for the fitted Hill equation. 1 254 13 Excluded_Points Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. 4 254 14 Max_Response Maximum activity observed for compound (usually at highest concentration tested). 1 15 15 Activity at 0.457 uM % Activity at given concentration. 1 15 0.456999987363815 5 1 16 Activity at 2.290 uM % Activity at given concentration. 1 15 2.28999996185303 5 1 17 Activity at 11.40 uM % Activity at given concentration. 1 15 11.3999996185303 5 1 18 Activity at 57.10 uM % Activity at given concentration. 1 15 57.0999984741211 5 1 19 Compound QC NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' 4 254 1 USP1 protein [Homo sapiens] 118600387 1 Homo sapiens human taxon 9606 2 1 CR Plot label 1 Concentration Response 0 DA030552 2