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 ..
BioActive Compounds: 33
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.
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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
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)