SAR Analysis of small molecule UBC13 Polyubiquitin Inhibitors using a Bfl-1 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 ..
BioActive Compounds: 6
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 a TR-FRET assay based on FITC-Bid BH3 peptide binding to GST-Bfl-1 in the presence of Terbium-labeled anti-GST antibody. 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 are either acquired from commercial sources or synthesized internally.
This Bfl-1 assay is used as a counter screen assay for UBC13. Active compounds in both UBC13 and Bfl-1 assays are considered false positives.
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Assay buffer: 25 mM Bis-Tris, 1 mM TCEP, 0.005% Tween 20
Bfl-1: Produced at the Sanford-Burnham Medical Research Institute
F-Bid: Produced at the Sanford-Burnham Medical Research Institute
Tb x GST: Invitrogen (Catalogue #: PV3551)
TCEP: Sigma (Catalogue #: 646547)
Assay plate: Corning 1536 Well White Plate (Catalogue #: 3725)
I. Compound Addition:
1. Using LabCyte Echo, transfer 40 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 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 Bfl-1 assay:
4. Prepare assay buffer
5. Bfl-1: Dilute Bfl-1 in assay buffer to make 2X intermediate solution at 10 nM
6. F-Bib/Tb x GST: Dilute F-Bid and Tb x GST in assay buffer to make 2X intermediate solution at the concentration of 8 nM for F-Bid and 5 nM for Tb x GST.
III. Reagent Addition:
7. Add 2 ul assay buffer to columns 1&2
8. Add 2 ul 2X intermediate Bfl-1 to columns 3 - 48
9. Add 2ul of 2X intermediate F-Bid/Tb x GST to all columns
IV. Reading plates:
10. Read plates using BMG PHERAstar FS using Lanthascreen protocol.
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 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)