Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1
One arm of apoptosis is regulated 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 conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in tertiary structure. more ..
BioActive Compounds: 237
University of New Mexico Assay Overview:
Assay Support: NIH 1X01 MH079850-01
HTS to identify small molecule regulators of Bcl-2 family protein interactions
PI: Larry Sklar, Ph.D.
Assay Implementatiion: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter MS
Assay Background and Significance:
One arm of apoptosis is regulated 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 conserved motifs designated as three Bcl-2 homology (BH) regions, BH1, BH2, and BH3 [Reed, et al. 2004]. These domains form a hydrophobic cleft in 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. Overexpression of pro-apoptotic BH3 peptides has been shown to increase apoptosis of leukemia cells in in vitro and animal model studies [Holinger, et al. 1999; Wang et al. 2000; Walensky et al. 2004]. 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. This is 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).
Each component of the multiplex assay consists of a glutathione labeled bead, a GST Bcl-fusion protein target (six total, supplied by project collaborator), and a fluorescent peptide probe, F-Bim (FITC-Axh-DMRPEIWIAQELRRIGDEFNAYYAR-OH; Commonwealth Biotech, USA). Bead sets are coated with individual GST-conjugated Bcl-2 proteins in HPSMTB buffer (30mM HEPES, 100mM KCl, 20mM NaCl, 1mM MgCl(2), 0.01% Tween-20, 0.1% BSA) and incubated overnight at 4 degrees C.
The mulitplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in HPSMTB buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The bead sets (ThermoFisher Scientific product numbers XPR-1687-XPR-1696), have similar size (~ 4 micron diameter) and are distinguished by distince emission characteristics at 665 +/-10 nm with excitation at 635 nm. Thus, GST-Bfl-1 might be noncovalently coated onto red level 1 beads, GST-Bcl-XL onto red level 2 beads, etc. The 6 bead sets (each with bound protein) and uncoated beads (see below) are first centrifuged separately, then combined and centrifuged again, and finally diluted just before loading into 384-well plates to minimize bead-protein dissociation before the assay begins.
The assay is conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100nM F-Bim in HPSMTB). Test compound concentration is 10 micromolar. Controls, which contain bead mixture and F-Bim but no test compound, are located in columns 1 and 2 on each plate. Plates are placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C.
A glutathione-only bead set control (no associated GST-protein) is incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. Specificity of F-Bim binding is determined with a Positive Control using a block of the F-Bim Fluor with a non-fluoresceinated F-Bim peptide. The F-Bim blocking control is run daily as a separate single tube assay using F-Bim at 5 micromolar.
Sample analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis is performed using time-resolved acquisition into a single data file and analysis using IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the median fluorescence per bead population is calculated.
In order to get a significant measurement of the effect by a compound on a particular protein, 25 beads with that particular protein bound is the minimum number of beads to be collected from a well. When less than 25 beads are counted, the result for that protein is considered missing. Compounds from missing wells is given PUBCHEM_ACTIVITY_OUTCOME = 4, and are automatically assigned a PUBCHEM_ACTIVITY_SCORE of 0. In this set of 60,660 compounds, for Bfl-1 there are 17 missing compounds.
When the measured emission is potentially attributed to the innate fluorescence of the compound, these compounds are flagged as "Possible Green Fluorescent Compound" (see column titled PUBCHEM_ASSAYDATA_COMMENT) and the results from a fluorescent compound is considered to be 'inconclusive' (PUBCHEM_ACTIVITY_OUTCOME = 3, PUBCHEM_ACTIVITY_SCORE = 0). Assessment of fluorescent compound is made by comparing the influence of compound fluorescence on all the proteins in one well. Difference between Sample fluorescence and Negative Control fluorescence are calculated for all the different proteins in a well. The following equation describes this difference (CompoundFLinBfl-1) for Bfl-1;
CompoundFLinBfl-1 = Bfl-1SampleFL - NCntrlBfl-1FL
with Bfl-1SampleFL being the Sample fluorescence and NCntrlBfl-1FL the Positive Control fluorescence of Bfl-1 coated beads. Next, the coefficient of variation (CV) of all these CompoundFL from the different protein beads (Bfl-1, Bcl-XL, Bcl-2, Bcl-W, Bcl-B, Mcl-1,) in the same well are calculated. If the CV was less than 30%, meaning the compound attributed fluorescence was very similar between all the different proteins, then the compound was flagged as a potential fluorescent compound. In this set of 60,660 compounds, for Bfl-1 there are only 36 fluorescent compounds.
Due to potential systematic trends in data over the entire plate (whole plate trends), normalization is utilized to calculate the percent inhibition of the test compound. Whole plate trends of the negative controls are evaluated by linear regression. Then due to the plate location of a sample, a calculated Negative Control value (notated with LinFit) is utilized for calculating % inhibition normalized by whole plate:
% Inhibition = 100 x (LinFitNCntrl - SampleFL)/(LinFitNCntrl - BlockCntrl)
where all variables are Median Fluorescence Intensity associated with the bead set bound with a specific protein. SampleFL is for beads in wells containing test compound, LinFitNCntrl is the calculated value based on whole plate linear regression of wells without test compounds, and BlockCntrl is for measurement in presence 5 micromolar non-fluorescent Bim. Baseline of % Inhibition is 0%.
Maximum PUBCHEM_ACTIVITY_SCORE of 100 was given for the primary screening and it corresponded directly with %Inhibition.
A compound was considered "Active" if the change in %Inhibition was greater than 40%. PUBCHEM_ACTIVITY_OUTCOME is indicated as 2 for "Active" and 1 for "Non-active".
Average Z prime for the 190 plates tested was 0.70 +/- 0.09.
Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, Bcl, Bim, Bfl-1,Bcl-XL, Bcl-2, Bcl-W, Bcl-B, Mcl-1 multiplex bead-based screening
** Test Concentration.
Data Table (Concise)