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BioAssay: AID 602401

SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Caspase-3 counterscreen - Set 4

Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) are a family of adapter proteins that bind an unusual ubiquitin-conjugating enzyme, Ubc13, which produces polyubiquitin chains linked at lysine 63 of ubiquitin. These lysine 63-linked ubiquitin polymers trigger changes in protein activity. Ubiquitination by Ubc13 of TRAFs and the various protein kinases to which TRAFs bind is recognized as more ..
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AID: 602401
Data Source: Burnham Center for Chemical Genomics (SBCCG-A809-UBC13-Caspase3-CTR-DryPowder-Assay-4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-03-15
Hold-until Date: 2012-10-16
Modify Date: 2012-10-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeComment
485273uHTS identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET AssayScreeningdepositor-specified cross reference
485343Summary of small molecule inhibitors of UBC13 PolyubiquitinSummarydepositor-specified cross reference: Summary AID.
488856Single concentration confirmation of UBC13 Polyubiquitin Inhibitors using a Caspase-3 counter screenScreeningsame project related to Summary assay
488859Single concentration confirmation of uHTS for the identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET AssayScreeningsame project related to Summary assay
493155Dose Response confirmation of uHTS for the identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET AssayConfirmatorysame project related to Summary assay
493182Dose Response confirmation of uHTS for the identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay reconfirmConfirmatorysame project related to Summary assay
504680SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Caspase-3 counterscreenConfirmatorysame project related to Summary assay
504685SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Bfl-1 counterscreenConfirmatorysame project related to Summary assay
504687SAR analysis of small molecule UBC13 Polyubiquitin Inhibitors via a TR-FRET AssayConfirmatorysame project related to Summary assay
504689Dose Response confirmation of UBC13 Polyubiquitin Inhibitors using a Bfl-1 counterscreenConfirmatorysame project related to Summary assay
588375SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Caspase-3 counterscreen - Set 2Confirmatorysame project related to Summary assay
588376SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Bfl-1 counterscreen - Set 2Confirmatorysame project related to Summary assay
588390SAR analysis of small molecule UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay - Set 2Confirmatorysame project related to Summary assay
602205SAR analysis of small molecule UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay - Set 3Confirmatorysame project related to Summary assay
602206SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Bfl-1 counterscreen - Set 3Confirmatorysame project related to Summary assay
602207SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Caspase-3 counterscreen - Set 3Confirmatorysame project related to Summary assay
602400SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Bfl-1 counterscreen - Set 4Confirmatorysame project related to Summary assay
602404SAR analysis of small molecule UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay - Set 4Confirmatorysame project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R03 MH085677-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA

Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) are a family of adapter proteins that bind an unusual ubiquitin-conjugating enzyme, Ubc13, which produces polyubiquitin chains linked at lysine 63 of ubiquitin. These lysine 63-linked ubiquitin polymers trigger changes in protein activity. Ubiquitination by Ubc13 of TRAFs and the various protein kinases to which TRAFs bind is recognized as a critical step in signaling by TNFRs, TLRs, NLRs, and T-cell and B-cell antigen receptors (TCR/BCR) during innate and acquired immune responses. Since aberrant signaling by these receptor systems is linked to a wide variety of autoimmune, inflammatory, and infectious diseases; compounds that neutralize Ubc13 may prove useful as a novel type of immunosuppressive or anti-inflammatory agent.

This assay uses a Caspase-3 sulfhydryl screen: Caspase-Glo(R) 3/7 Assay (Promega), a luminescent assay, as a counter screen for TR-FRET. The purpose of this assay is to confirm hits from "uHTS identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay", AID 485273 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds active in both the TR-FRET and Caspase-3 assays are considered to be false positives. Samples are either acquired from commercial sources or synthesized internally.

REFERENCES
1. Fukushima, T., Matsuzawa, S., Kress, C. L., Bruey, J. M., Krajewska, M., Lefebvre, S., Zapata, J. M., Ronai, Z., and Reed, J.C. (2007) Proc Natl Acad Sci USA 104(15), 6371-6376 (PMC1851032)
2. Yamamoto, M., Okamoto, T., Takeda, K., Sato, S., Sanjo, H., Uematsu, S., Saitoh, T., Yamamoto, N., Sakurai, H., Ishii, K. J., Yamaoka, S., Kawai, T., Matsuura, Y., Takeuchi, O., and Akira, S. (2006) Nat Immunol 7, 962-970
3. Yamamoto, M., Sato, S., Saitoh, T., Sakurai, H., Uematsu, S., Kawai, T., Ishii, K. J., Takeuchi, O., and Akira, S. (2006) J Immunol 177, 7520-7524
4. Akira, S., Takeda, K., and Kaisho, T. (2001) Nat Immunol 2, 675-680
5. Chen, Z. J. (2005) Nat Cell Biol 7, 758-765
6. Karin, M., and Ben-Neriah, Y. (2000) Ann Rev Immunol 18, 621-663
7. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z. J. (2000) Cell 103, 351-361
8. Wooff, J., Pastushok, L., Hanna, M., Fu, Y., and Xiao, W. (2004) FEBS Lett 566, 229-233
9. Wu, H., and Arron, J. R. (2003) Bioessays 25, 1096-1105
10. McKenna, S., Hu, J., Moraes, T., Xiao, W., Ellison, M. J., and Spyracopoulos, L. (2003) Biochemistry 42, 7922-7930
11. Hau, D. D., Lewis, M. J., Saltibus, L. F., Pastushok, L., Xiao, W., and Spyracopoulos, L. (2006) Biochemistry 45, 9866-9877
Protocol
Materials:
Caspase-Glo(R) 3/7 Assay: Promega (Catalogue #: G8092)
Assay buffer: 150 mM Hepes (pH 7.8), 4.0 mM DTT, 1.0 mM EDTA, 0.01% Tween, 0.1% BSA
Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Protocol:
I. Compound Addition:
1. Using LabCyte Echo, transfer 20 nL from a 10 mM Echo qualified plate containing test compounds into assay plate columns 5 - 48 (final concentration of test compounds is 100 microM, 1.0 % DMSO). Transfer 20 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.
3. Seal the plates and leave them at RT.
Note: Compounds are added to the plates before reagent addition
II. Set up of Caspase-3 assay:
4. Prepare assay buffer
5.Caspase 3: Dilute Caspase 3 in assay buffer to make 2X intermediate solution at 0.046 nM
6. Caspase Glo substrate: Dilute 1X substrate solution in assay buffer to make 2X intermediate solution at 0.75X
III. Reagent Addition:
7. Add 1 ul assay buffer to columns 1 & 2
8. Add 1 ul 2X intermediate Caspase 3 to columns 3 - 48
9. Add 1 ul 2X intermediate Caspase Glo substrate to all columns
10. Incubate at RT for 60 mins
IV. Reading plates:
11. Read plates using PerkinElmer ViewLux using SENP Luminescence 60 sec protocol.
Comment
Compounds that demonstrated activity of <100 uM are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 100 uM_first_point (100μM**)% Activity at the test concentrationFloat%
9% Activity at 50 uM_first_point (50μM**)% Activity at the test concentrationFloat%
10% Activity at 25 uM_first_point (25μM**)% Activity at the test concentrationFloat%
11% Activity at 12.5 uM_first_point (12.5μM**)% Activity at the test concentrationFloat%
12% Activity at 6.25 uM_first_point (6.25μM**)% Activity at the test concentrationFloat%
13% Activity at 3.125 uM_first_point (3.125μM**)% Activity at the test concentrationFloat%
14% Activity at 1.5625 uM_first_point (1.5625μM**)% Activity at the test concentrationFloat%
15% Activity at 0.78125 uM_first_point (0.78125μM**)% Activity at the test concentrationFloat%
16% Activity at 0.390625 uM_first_point (0.390625μM**)% Activity at the test concentrationFloat%
17% Activity at 0.1953125 uM_first_point (0.195312μM**)% Activity at the test concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 100 uM_second_point (100μM**)% Activity at the test concentrationFloat%
20% Activity at 50 uM_second_point (50μM**)% Activity at the test concentrationFloat%
21% Activity at 25 uM_second_point (25μM**)% Activity at the test concentrationFloat%
22% Activity at 12.5 uM_second_point (12.5μM**)% Activity at the test concentrationFloat%
23% Activity at 6.25 uM_second_point (6.25μM**)% Activity at the test concentrationFloat%
24% Activity at 3.125 uM_second_point (3.125μM**)% Activity at the test concentrationFloat%
25% Activity at 1.5625 uM_second_point (1.5625μM**)% Activity at the test concentrationFloat%
26% Activity at 0.78125 uM_second_point (0.78125μM**)% Activity at the test concentrationFloat%
27% Activity at 0.390625 uM_second_point (0.390625μM**)% Activity at the test concentrationFloat%
28% Activity at 0.1953125 uM_second_point (0.195312μM**)% Activity at the test concentrationFloat%
29Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
30% Activity at 100 uM_third_point (100μM**)% Activity at the test concentrationFloat%
31% Activity at 50 uM_third_point (50μM**)% Activity at the test concentrationFloat%
32% Activity at 25 uM_third_point (25μM**)% Activity at the test concentrationFloat%
33% Activity at 12.5 uM_third_point (12.5μM**)% Activity at the test concentrationFloat%
34% Activity at 6.25 uM_third_point (6.25μM**)% Activity at the test concentrationFloat%
35% Activity at 3.125 uM_third_point (3.125μM**)% Activity at the test concentrationFloat%
36% Activity at 1.5625 uM_third_point (1.5625μM**)% Activity at the test concentrationFloat%
37% Activity at 0.78125 uM_third_point (0.78125μM**)% Activity at the test concentrationFloat%
38% Activity at 0.390625 uM_third_point (0.390625μM**)% Activity at the test concentrationFloat%
39% Activity at 0.1953125 uM_third_point (0.195312μM**)% Activity at the test concentrationFloat%
40IC50_Qualifier_2This qualifier is to be used with the next TID, . If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
41IC50_2IC50 value determined using a sigmoidal dose response equationFloatμM
42Std.Err(IC50)_2Standard Error of the IC50 valueFloatμM
43nH_2Hill coefficient determined using sigmoidal dose response equationFloat
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 100 uM_fourth_point (100μM**)% Activity at the test concentrationFloat%
46% Activity at 50 uM_fourth_point (50μM**)% Activity at the test concentrationFloat%
47% Activity at 25 uM_fourth_point (25μM**)% Activity at the test concentrationFloat%
48% Activity at 12.5 uM_fourth_point (12.5μM**)% Activity at the test concentrationFloat%
49% Activity at 6.25 uM_fourth_point (6.25μM**)% Activity at the test concentrationFloat%
50% Activity at 3.125 uM_fourth_point (3.125μM**)% Activity at the test concentrationFloat%
51% Activity at 1.5625 uM_fourth_point (1.5625μM**)% Activity at the test concentrationFloat%
52% Activity at 0.78125 uM_fourth_point (0.78125μM**)% Activity at the test concentrationFloat%
53% Activity at 0.390625 uM_fourth_point (0.390625μM**)% Activity at the test concentrationFloat%
54% Activity at 0.1953125 uM_fourth_point (0.195312μM**)% Activity at the test concentrationFloat%
55Excluded_Points_fifth_pointFlags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
56% Activity at 100 uM_fifth_point (100μM**)% Activity at the test concentrationFloat%
57% Activity at 50 uM_fifth_point (50μM**)% Activity at the test concentrationFloat%
58% Activity at 25 uM_fifth_point (25μM**)% Activity at the test concentrationFloat%
59% Activity at 12.5 uM_fifth_point (12.5μM**)% Activity at the test concentrationFloat%
60% Activity at 6.25 uM_fifth_point (6.25μM**)% Activity at the test concentrationFloat%
61% Activity at 3.125 uM_fifth_point (3.125μM**)% Activity at the test concentrationFloat%
62% Activity at 1.5625 uM_fifth_point (1.5625μM**)% Activity at the test concentrationFloat%
63% Activity at 0.78125 uM_fifth_point (0.78125μM**)% Activity at the test concentrationFloat%
64% Activity at 0.390625 uM_fifth_point (0.390625μM**)% Activity at the test concentrationFloat%
65% Activity at 0.1953125 uM_fifth_point (0.195312μM**)% Activity at the test concentrationFloat%
66Excluded_Points_sixth_pointFlags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
67% Activity at 100 uM_sixth_point (100μM**)% Activity at the test concentrationFloat%
68% Activity at 50 uM_sixth_point (50μM**)% Activity at the test concentrationFloat%
69% Activity at 25 uM_sixth_point (25μM**)% Activity at the test concentrationFloat%
70% Activity at 12.5 uM_sixth_point (12.5μM**)% Activity at the test concentrationFloat%
71% Activity at 6.25 uM_sixth_point (6.25μM**)% Activity at the test concentrationFloat%
72% Activity at 3.125 uM_sixth_point (3.125μM**)% Activity at the test concentrationFloat%
73% Activity at 1.5625 uM_sixth_point (1.5625μM**)% Activity at the test concentrationFloat%
74% Activity at 0.78125 uM_sixth_point (0.78125μM**)% Activity at the test concentrationFloat%
75% Activity at 0.390625 uM_sixth_point (0.390625μM**)% Activity at the test concentrationFloat%
76% Activity at 0.1953125 uM_sixth_point (0.195312μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03 MH085677-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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