Bcl-2 family members Fluorescence polarization assay with Set1 of powder compounds
Apoptosis is regulated in part by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of three conserved motifs designated as Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2000]. These domains form a hydrophobic cleft in the tertiary structure. Pro-apoptotic more ..
Depositor Specified Assays
University of New Mexico Assay Overview:
Assay Support: 1X01 MH079850-01
Project Title: HTS for BCI-2 Family Multiplex
Assay Provider: Larry Sklar/John C. Reed
UNMScreening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist at Screening Center: Peter Simons PhD
Chemistry Center/ PI: Sanford-Burnham Center for Chemical Genomics / John C. Reed
Chemistry Center Lead: Thomas Chung Ph.D., Robert Ardecky Ph.D., Anton Cheltsov Ph.D.
Assay Implementation: Daiyong Zhai Ph.D.
Assay Background and Significance:
Apoptosis is regulated in part by the balance of anti-apoptotic and pro-apoptotic Bcl-2 family members. In humans, six genes have been identified that encode anti-apoptotic proteins characterized by the presence of three conserved motifs designated as Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2000]. These domains form a hydrophobic cleft in the tertiary structure. Pro-apoptotic family members contain BH3 regions that form an amphipathic helix, and these helices bind in the clefts of the anti-apoptotic proteins. The binding of fluorochrome-conjugated BH3 peptides (including Bim) to Bcl-2 family members thus provides the basis for construction of fluorescence-based assays amenable to flow cytometry high throughput screening for small molecule regulators of these interactions (Zhai et al 2006). The high throughput screen was a multiplexed assay to identify small molecule regulators of protein interactions between the BH3 peptide of Bim and the following six Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1, and Mcl-1 and Bcl-2 (the eponymous founding member of the Bcl-2 family). A selective probe candidate scaffold has now emerged as an inhibitor of the interaction of Bcl-B with BIM peptide.
Overall, the screen identified three compounds as specific inhibitors of Bcl-B but not the other Bcl-2 family members; fluorescence polarization and isothermal titration calorimetry data corroborated the flow cytometry findings. 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.
Panel set1 of fluorescence polarization data for Bcl2 family proteins - Panel set1 of fluorescence polarization data for Bcl2 family proteins
§ Panel component ID.
1. GST-Bcl-2 family proteins are all expressed in E. coli & purified at the Prof. John Reed's laboratory (SBMRI)
a. Bcl-2 family proteins are GST-fusion protein lacking C-terminal transmembrane domains (~20 amino acids)
b. FITC-TR3-R8 (FITC-Ahx-FSRSLHSLL-GX-R8 peptide); Ahx = N-aminohexanoic acid linker; X = N-aminocaproic acid; R8 = 8 residues of D-arginine
2. Assay Buffer: 25 mM HEPES-KOH,20 mM N-glycerophosphate, 0.005% Tween-20, pH 7.5, 1 mM TCEP.
3. 44.4 nM GST-Bcl-2 working solution in assay buffer, freshly prepared and kept on ice prior to use.
4. 44.4 nM : FITC-TR3-R8 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 3-22 of Greiner 384-well black small-volume plates (784076).
3. Columns 1-2 and 23-24 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-B working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Columns 23-24 represent negative control wells.
6. Plates were incubated for 1h at 4 degree Celsius.
7. 9 uL of freshly prepared FITC-TR3 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 sigmoidal dose-response equation through non-linear regression using GraphPad Prism (GraphPad Software, San Diego, California). Resulting in estimates of IC50 values.
Active Compounds have a measured fluorescence difference greater than 25. Individual panel member scores were calculated by the following equation:
Individual Score = 100 *(1-IC50/10)
And Total Score was a summation of all the individual scores.
** Test Concentration. § Panel component ID.