SAR analysis of selective Bcl-B inhibitors using a Fluorescence Polarization Bcl-XL/Bim-BH3 Assay
Bcl-B is an anti-apoptotic member of the Bcl-2 family that is prominently expressed in plasma and multiple myeloma cells. Prior experiments have suggested that Bcl-B is important for the survival of plasma cell malignancies [Luciano et al., 2007] and several types of solid tumors show pathological elevation of Bcl-B protein, sometimes correlating with poor prognosis [Kajewska et al., 2008]. A specific Bcl-B inhibitor would be useful as a probe for defining the mechanism by which Bcl-B acts and also potentially as a lead compound for therapy. ..more
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network(MLPCN)
Grant Number: 1X01 MH079850-01
Assay Provider: John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA
Bcl-B is an anti-apoptotic member of the Bcl-2 family that is prominently expressed in plasma and multiple myeloma cells. Prior experiments have suggested that Bcl-B is important for the survival of plasma cell malignancies [Luciano et al., 2007] and several types of solid tumors show pathological elevation of Bcl-B protein, sometimes correlating with poor prognosis [Kajewska et al., 2008]. A specific Bcl-B inhibitor would be useful as a probe for defining the mechanism by which Bcl-B acts and also potentially as a lead compound for therapy.
This assay performed in the laboratory of the assay provider is to evaluate the cross reactivity of the compounds tested in "SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay" (AID pending). Compounds are either acquired from commercial sources or synthesized internally.
Krajewska, M.; Kitada, S.; Winter, J. N.; Variakojis, D.; Lichtenstein, A.; Zhai, D.; Cuddy, M.; Huang, X.; Luciano, F.; Baker, C. H.; Kim, H.; Shin, E.; Kennedy, S.; Olson, A. H.; Badzio, A.; Jassem, J.; Meinhold- Heerlein, I.; Duffy, M. J.; Schimmer, A. D.; Tsao, M.; Brown, E.; Sawyers, A.; Andreeff, M.; Mercola, D.; Krajewski, S.; Reed, J. C. Bcl-B Expression in Human Epithelial and Nonepithelial Malignancies. Clin. Cancer Res. 2008, 14, 3011-3021.
Luciano, F.; Krajewska, M.; Ortiz-Rubio, P.; Krajewski, S.; Zhai, D.; Faustin, B.; Bruey, J. M.; Bailly-Maitre, B.; Lichtenstein, A.; Kolluri, S. K.; Satterthwait, A. C.; Zhang, X. K.; Reed, J. C. Nur77 Converts Phenotype of Bcl- B, an Antiapoptotic Protein Expressed in Plasma Cells and Myeloma. Blood 2007, 109, 3849-3855
1. Bcl-XL was expressed in E. coli & purified at the Prof. John Reed's laboratory (SBMRI)
a. Bcl-XL was GST-fusion protein lacking C-terminal transmembrane domains (~20 amino acids)
b. FITC-Bim (FITC-Ahx- DMRPEIWIAQELRRIGDEFNAYYAR peptide); Ahx = N-aminohexanoic acid linker; X = N-aminocaproic acid;
2. Assay Buffer: 25 mM HEPES-KOH, 0.005% Tween-20, pH 7.5, 1 mM TCEP.
3. 40 nM GST-Bcl-2 working solution in assay buffer, freshly prepared and kept on ice prior to use.
4. 20 nM : FITC-Bim peptide working solution in assay buffer
1. Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration.
2. 2 uL compounds in 10% DMSO were transferred into columns 5-24 of Greiner 384-well black small-volume plates (784076).
3. Columns 1-2 and 3-4 contained 4 uL of 10% DMSO.
4. Columns 1-2 were reserved for positive controls and 9 uL of assay buffer were added to them using WellMate bulk dispenser (Matrix).
5. 9 uL of Bcl-XL working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Columns 3-4 represent negative control wells.
6. Plates were incubated for 20 min at 4 degree Celsius.
7. 9 uL of freshly prepared FITC-Bim working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
8. Plates were incubated for 10 min at room temperature protected from direct light.
9. Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein optics (ex/em - 485/530 nm, dichroic mirror - 505 nm). The signal for each well was acquired for 100 ms.
10. Data analysis was performed using CBIS software (ChemInnovations, Inc) using sigmoidal dose-response equation through non-linear regression.
Compounds with an IC50 <= 20 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)