This report demonstrates a multiplexed, bead-based flow cytometry screen that covers a small family of proteins, requiring <1
mg of each GST-fusion protein per family member, a fluorescent probe, and the GSH bead sets described herein. In general, the capture and display of molecules on microspheres has the potential of reducing the quantities of the captured reagent (the GST-fusion protein) and allows the less-expensive fluorescent reagent (the F-Bim peptide) to be used near its Kd
. By this approach, a screen of a small library of compounds is within the reach of an individual laboratory with a single flow cytometer, but a larger screen would require either a longer duration commitment or multiple flow cytometers operating in parallel. This screen was conducted with two people using one cytometer at a rate of 24 plates per day including analysis and took 26 days to screen the ~195,000 compounds in the library at that time. Biotinylated proteins can also be used on differentially fluorescent streptavidin bead sets for multiplexing.
Our purpose was to find modulators for the Bcl-2 family of proteins, which is a step toward finding small molecules that modulate protein–protein interactions in general. Thus far, small molecule modulators of Bcl-2 family of proteins have been identified through FP assays or NMR-based methods. In this study, we have employed a different type of assay and screening approach, namely high-throughput flow cytometry, to identify potent and selective modulators of antiapoptotic members of Bcl-2 family. A multiplex HTS assay was developed and the MLSMR library (194,920 samples and 194,829 unique compounds) was screened against the six antiapoptotic proteins. A strategy was developed with the aim of identifying new chemotypes that could mimic the interactions between the binding groove of Bcl-2 proteins and BH3 domain of the proapoptotic proteins. The activities of the compounds were confirmed using FP and ITC.
Our approach involved multiple distinct steps, namely the screening of compounds in a primary HTS assay at a single concentration, the confirmation of the primary screening hits in a dose–response assay, run on cherry-picked samples and reordered powders, and follow-up secondary assays. The primary screening assay provided a large amount of data, and the processing of these data to discover active compounds was a challenging task. For this reason, a classification schema was developed to select the compounds from primary screening data to be confirmed in the dose–response analysis. The main idea was to prioritize relevant chemical scaffolds, or chemotypes, taking into account biological and chemical information. The confirmatory assay was aided by a counter screen, whose purpose was to identify potential “false hits,” compounds that could interact with the binding of GST to GSH and give a false biological signal, PubChem Bioassay AID 1324. In this counter screen, the bead sets were coated with 50
nM GST-GFP and assayed for loss of fluorescence when compounds were incubated with them; a loss of >50% fluorescence was defined as a false hit.
From 960 selected compounds, 834 were obtained and actually tested in a dose–response assay against all six proteins. Applying a prioritizing algorithm that took into account the biological responses (EC50
and efficacy) on each particular target, the signal on the GST to GSH interaction (if any), the statistical relevance of data, and the number of members for each family, we identified 19 classes of putatively active compounds and 94 singletons. Because of the problem with the Bfl-1 preparation used in the confirmatory dose response, 385 unique compounds were tested on the other five proteins and reported. The more stringent criteria for confirmation resulted in only 27 of the 385 being declared “active” in the dose–response assay or 7% confirmation of “hits.” This post-HTS analysis revealed an overall “hit” frequency in screening this library of ~0.06% for hits with potency values less than 10
μM, and 0.1% for hits with potency values less than 100
μM. An assay with a Z
′ score greater than 0.5 is considered an excellent assay for screening,17
and our Z
′ values were Bcl-XL
, 0.79±0.11; Bcl-2, 0.81±0.10; Bcl-W, 0.84±0.07; Bcl-B, 0.81±0.08; Bfl-1, 0.81±0.11; and Mcl-1, 0.82±0.12, consistent over the course of the screen. Overall, three selective compounds for Bcl-B were discovered using high-throughput flow cytometry and confirmed via FP and ITC. Although these compounds do not form a chemical series, they nevertheless may serve as a promising starting point for further optimization toward creating potent and biologically active selective inhibitors of the antiapoptotic protein Bcl-B.
The gene encoding Bcl-B was first recognized as the human genome project moved toward completion, with the first publication appearing in 2001. It has no clear ortholog in mice and thus represents an evolutionary progression of the Bcl-2 family in higher organisms. The Bcl-B protein shows selectivity for the BH3-containing proteins with which it interacts. For example, unlike Bcl-2, the Bcl-B protein binds to and neutralizes the proapoptotic protein Bax, but not Bak.18,19
In normal tissues, Bcl-B is highly expressed in B-lymphocytes and especially abundant in plasma cells.20
Prior experiments have suggested that Bcl-B is important for the survival of some plasma cell malignancies.9
Several types of solid tumors show pathological elevation of Bcl-B protein, sometimes correlating with poor patient prognosis.19
Selective inhibitors of Bcl-B, therefore, might find applications for autoimmune diseases, perhaps either by impairing the survival of long-lived autoantibody-producing cells or as a complement to chemical inhibitors of Bcl-2/Bcl-XL
currently in clinical development for cancer.
In summary, this study illustrates a novel approach leading to the identification of chemical modulators of antiapoptotic Bcl-2 family proteins, in which flow cytometry was employed as a platform for simultaneous HTS of six antiapoptotic proteins Bcl-2, Bcl-B, Bcl-W, Bcl-XL, Bfl-1, and Mcl-1 against a large collection of compounds. Several future applications of multiplex flow cytometry can be envisioned for targets in the field of apoptosis, including assays using the BIR domains of IAP family proteins (there are eight IAP family members in humans), many of which are known to bind a tetrapeptide ligand derived from endogenous antagonists (second mitochondria-derived activator of caspase, SMAC, and high temperature requirement A 2, HtrA2, also named OMI) that suppresses their antiapoptotic activity. This multiplex assay method offers the advantage of assessing upfront the differential binding activities of chemicals for members of these multigene families, thus efficiently creating opportunities for identifying starting points for optimization of chemical inhibitors with unique spectrums of activity that may translate into distinct efficacy and toxicity profiles for different clinical indications.