Bookmark and Share
BioAssay: AID 602404

SAR analysis of small molecule UBC13 Polyubiquitin Inhibitors via a TR-FRET Assay - 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 ..
_
   
 Tested Compounds
 Tested Compounds
All(76)
 
 
Active(33)
 
 
Inactive(44)
 
 
 Tested Substances
 Tested Substances
All(78)
 
 
Active(34)
 
 
Inactive(44)
 
 
AID: 602404
Data Source: Burnham Center for Chemical Genomics (SBCCG-A810-UBC13-DryPowder-TR-FRET-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 ):           Active    All
Target
BioActive Compounds: 33
Depositor Specified Assays
AIDNameTypeComment
485273uHTS identification of UBC13 Polyubiquitin Inhibitors via a TR-FRET Assayscreening
485343Summary of small molecule inhibitors of UBC13 PolyubiquitinsummarySummary AID.
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.

The goal of this screen is to measure the biochemical activity of Ubc13 in vitro, based on the principal of time-resolved fluorescence resonance energy transfer (TR-FRET). Ubiquitination reactions contain purified recombinant proteins (E1, Ubc13 and Uev1a) mixed with labeled ubiquitins (terbium- and fluorescein-labeled) and ATP-regenerating system to trigger polyubiquitin chain synthesis in vitro. The reaction product is mixed chains of terbium-(fluorescence donor) and fluorescein-(fluorescene acceptor) conjugated Ubiquitin, thus creating the basis for robust TR-FRET signals.

This dose response assay is developed and performed to confirm hits originally identified in "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 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
Assay Materials:
Assay buffer: 50 mM Hepes (pH 7.5), 0.1mM DTT, 0.005% Empigen, 0.1% BSA, 1.25 mM MgCl2
E1: Produced at the Sanford-Burnham Medical Research Institute's protein production core facility
Fl-Ub: Invitrogen
Tb-Ub: Invitrogen
ATP: Sigma
Ubc13: Produced at the Sanford-Burnham Medical Research Institute's protein production core facility
Uev1a: Produced at the Sanford-Burnham Medical Research Institute's protein production core facility
Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)

I. Compound Addition:
1. Dose response curves contained 10 concentrations of compounds obtained using a twofold serial dilution. Compounds were serially diluted in 100 % DMSO (1% final DMSO concentration). Using LabCyte Echo, transfer 40 nL compounds in triplicate into assay plate columns 5 - 48. Transfer 40 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 Ubc13 assay:
4. Prepare assay buffer
5. E2: E2 is prepared in two steps. First, prepare E2 stock from Ubc13 and Uev1a at 10 uM. This E2 stock will form a heterodimer after 2 hour incubation at 4 degrees C. After 2 hour incubation at 4 degrees C, dilute E2 stock in assay buffer to make 2 X intermediate solution at 250 nM.
6. E1/Ub/ATP: 30 minutes before the end of E2 stock incubation at 4 degrees C, prepare 2X intermediate E1/Ub/ATP solution with E1 at 200 nM, Fl-Ub at 150 nM, Tb-Ub at 12 nM, and ATP at 2 mM.

III. Reagent Addition:
7. Add 2 ul assay buffer to columns 1&2
8. Add 2 ul 2X intermediate E2 to columns 3 48
9. Add 2ul of 2X intermediate E1/Ub/ATP to all columns
10. Incubate at RT for 90 mins

IV. Reading plates:
11. After 90 minutes of incubation, plates are read
12. Read plates using BMG PHERAstar FS using Lanthascreen protocol.
Comment
Compounds that demonstrated an IC50_Mean <20 uM are defined as inhibitors of the reaction.

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
Show more
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, IC50_1. 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**)% inhibition at the test concentrationFloat%
9% Activity at 50 uM_first_point (50μM**)% inhibition at the test concentrationFloat%
10% Activity at 25 uM_first_point (25μM**)% inhibition at the test concentrationFloat%
11% Activity at 12.5 uM_first_point (12.5μM**)% inhibition at the test concentrationFloat%
12% Activity at 6.25 uM_first_point (6.25μM**)% inhibition at the test concentrationFloat%
13% Activity at 3.125 uM_first_point (3.125μM**)% inhibition at the test concentrationFloat%
14% Activity at 1.5625 uM_first_point (1.5625μM**)% inhibition at the test concentrationFloat%
15% Activity at 0.78125 uM_first_point (0.78125μM**)% inhibition at the test concentrationFloat%
16% Activity at 0.390625 uM_first_point (0.390625μM**)% inhibition at the test concentrationFloat%
17% Activity at 0.1953125 uM_first_point (0.195312μM**)% inhibition 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**)% inhibition at the test concentrationFloat%
20% Activity at 50 uM_second_point (50μM**)% inhibition at the test concentrationFloat%
21% Activity at 25 uM_second_point (25μM**)% inhibition at the test concentrationFloat%
22% Activity at 12.5 uM_second_point (12.5μM**)% inhibition at the test concentrationFloat%
23% Activity at 6.25 uM_second_point (6.25μM**)% inhibition at the test concentrationFloat%
24% Activity at 3.125 uM_second_point (3.125μM**)% inhibition at the test concentrationFloat%
25% Activity at 1.5625 uM_second_point (1.5625μM**)% inhibition at the test concentrationFloat%
26% Activity at 0.78125 uM_second_point (0.78125μM**)% inhibition at the test concentrationFloat%
27% Activity at 0.390625 uM_second_point (0.390625μM**)% inhibition at the test concentrationFloat%
28% Activity at 0.1953125 uM_second_point (0.195312μM**)% inhibition 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**)% inhibition at the test concentrationFloat%
31% Activity at 50 uM_third_point (50μM**)% inhibition at the test concentrationFloat%
32% Activity at 25 uM_third_point (25μM**)% inhibition at the test concentrationFloat%
33% Activity at 12.5 uM_third_point (12.5μM**)% inhibition at the test concentrationFloat%
34% Activity at 6.25 uM_third_point (6.25μM**)% inhibition at the test concentrationFloat%
35% Activity at 3.125 uM_third_point (3.125μM**)% inhibition at the test concentrationFloat%
36% Activity at 1.5625 uM_third_point (1.5625μM**)% inhibition at the test concentrationFloat%
37% Activity at 0.78125 uM_third_point (0.78125μM**)% inhibition at the test concentrationFloat%
38% Activity at 0.390625 uM_third_point (0.390625μM**)% inhibition at the test concentrationFloat%
39% Activity at 0.1953125 uM_third_point (0.195312μM**)% inhibition at the test concentrationFloat%
40IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. 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**)% inhibition at the test concentrationFloat%
46% Activity at 50 uM_fourth_point (50μM**)% inhibition at the test concentrationFloat%
47% Activity at 25 uM_fourth_point (25μM**)% inhibition at the test concentrationFloat%
48% Activity at 12.5 uM_fourth_point (12.5μM**)% inhibition at the test concentrationFloat%
49% Activity at 6.25 uM_fourth_point (6.25μM**)% inhibition at the test concentrationFloat%
50% Activity at 3.125 uM_fourth_point (3.125μM**)% inhibition at the test concentrationFloat%
51% Activity at 1.5625 uM_fourth_point (1.5625μM**)% inhibition at the test concentrationFloat%
52% Activity at 0.78125 uM_fourth_point (0.78125μM**)% inhibition at the test concentrationFloat%
53% Activity at 0.390625 uM_fourth_point (0.390625μM**)% inhibition at the test concentrationFloat%
54% Activity at 0.1953125 uM_fourth_point (0.195312μM**)% inhibition 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**)% inhibition at the test concentrationFloat%
57% Activity at 50 uM_fifth_point (50μM**)% inhibition at the test concentrationFloat%
58% Activity at 25 uM_fifth_point (25μM**)% inhibition at the test concentrationFloat%
59% Activity at 12.5 uM_fifth_point (12.5μM**)% inhibition at the test concentrationFloat%
60% Activity at 6.25 uM_fifth_point (6.25μM**)% inhibition at the test concentrationFloat%
61% Activity at 3.125 uM_fifth_point (3.125μM**)% inhibition at the test concentrationFloat%
62% Activity at 1.5625 uM_fifth_point (1.5625μM**)% inhibition at the test concentrationFloat%
63% Activity at 0.78125 uM_fifth_point (0.78125μM**)% inhibition at the test concentrationFloat%
64% Activity at 0.390625 uM_fifth_point (0.390625μM**)% inhibition at the test concentrationFloat%
65% Activity at 0.1953125 uM_fifth_point (0.195312μM**)% inhibition 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**)% inhibition at the test concentrationFloat%
68% Activity at 50 uM_sixth_point (50μM**)% inhibition at the test concentrationFloat%
69% Activity at 25 uM_sixth_point (25μM**)% inhibition at the test concentrationFloat%
70% Activity at 12.5 uM_sixth_point (12.5μM**)% inhibition at the test concentrationFloat%
71% Activity at 6.25 uM_sixth_point (6.25μM**)% inhibition at the test concentrationFloat%
72% Activity at 3.125 uM_sixth_point (3.125μM**)% inhibition at the test concentrationFloat%
73% Activity at 1.5625 uM_sixth_point (1.5625μM**)% inhibition at the test concentrationFloat%
74% Activity at 0.78125 uM_sixth_point (0.78125μM**)% inhibition at the test concentrationFloat%
75% Activity at 0.390625 uM_sixth_point (0.390625μM**)% inhibition at the test concentrationFloat%
76% Activity at 0.1953125 uM_sixth_point (0.195312μM**)% inhibition at the test concentrationFloat%

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

Data Table (Concise)
Classification
PageFrom: