Article

High-Throughput Screen for the Chemical Inhibitors of Antiapoptotic Bcl-2 Family Proteins by Multiplex Flow Cytometry

Computational Chemistry Group, Romanian Academy Institute of Chemistry, Timisoara, Romania.
Assay and Drug Development Technologies (Impact Factor: 1.53). 05/2011; 9(5):465-74. DOI: 10.1089/adt.2010.0363
Source: PubMed

ABSTRACT

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

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Available from: Cristian Bologa
High-Throughput Screen for the Chemical Inhibitors
of Antiapoptotic Bcl-2 Family Proteins
by Multiplex Flow Cytometry
Ramona F. Curpan,
1
Peter C. Simons,
2
Dayong Zhai,
3
Susan M. Young,
2
Mark B. Carter,
2
Cristian G. Bologa,
4
Tudor I. Oprea,
4
Arnold C. Satterthwait,
3
John C. Reed,
3
Bruce S. Edwards,
2
and Larry A. Sklar
2
1
Computational Chemistry Group, Romanian Academy Institute
of Chemistry, Timisoara, Romania.
2
New Mexico Center for Molecular Discovery, Department
of Pathology;
4
Division of Biocomputing, Department
of Biochemistry and Molecular Biology; University of New Mexico,
Albuquerque, New Mexico.
3
Sanford-Burnham Medical Research Institute,
La Jolla, California.
ABSTRACT
The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B,
Bcl-W, Bcl-X
L
, Bfl-1, and Mcl-1) and many proapoptotic members,
wherein a balance between the two determines cell life or death in many
physiological and disease contexts. Elevated expression of various anti-
apoptotic Bcl-2 members is commonly observed in cancers, and chemical
inhibitors of these proteins have been shown to promote apoptosis of
malignant cells in culture, in animal models, and in human clinical trials.
All six antiapoptotic members bind a helix from the proapoptotic family
member Bim, thus quenching Bim’s apoptotic signal. Here, we describe
the use of a multiplex, high-throughput flow cytometry assay for the
discovery of small molecule modulators that disrupt the interaction be-
tween the antiapoptotic members of the Bcl-2 family and Bim. The six
antiapoptotic Bcl-2 family members were expressed as glutathione-S-
transferase fusion proteins and bound individually to six glutathione bead
sets, with each set having a different intensity of red fluorescence. A
fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim
was employed as a universal ligand. Flow cytometry measured the
amount of green peptide bound to each bead set in a given well, with
inhibitory compounds resulting in a decrease of green fluorescence on one
or more bead set(s). Hits and cheminformatically selected analogs were
retested in a dose–response series, resulting in three ‘active’ compounds
for Bcl-B. These three compounds were validated by fluorescence polar-
ization and isothermal titration calorimetry. We discuss some of the
lessons learned about screening a chemical library provided by the
National Institutes of Health Small Molecule Repository (*195,000
compounds) using high-throughput flow cytometry.
INTRODUCTION
T
he Molecular Libraries Initiative of the National Institutes of
Health (NIH) in the United States supports high-throughput
screening (HTS) for important target proteins and protein
families, which are carried out by dedicated screening cen-
ters against a compound collection provided by the NIH Small
Molecule Repository, presently consisting of *350,000 compounds.
Multiplexing by flow cytometry gained prominence when commer-
cial polystyrene bead sets with different fluorescence intensities,
covalently attached to different antibodies to cytokines, were com-
bined in suspension to simultaneously quantify 15 cytokines using
a multiplexed flow cytometric assay.
1
Glutathione-S-transferase
fusion proteins (GST-fusion proteins) are widely used in cell biology
because a small laboratory can easily construct a DNA sequence for
the fusion protein, express the protein in bacteria, and partially
purify the protein using commercial glutathione (GSH) affinity
beads. Although GST-fusion proteins noncovalently bind to GSH
with a fairly weak dissociation constant of about 10 nM, making it
improbable that the bound proteins would stay on the bead during 1 h
of incubation, in fact the fusion proteins dissociate from such beads
at a slow rate, perhaps facilitated by dimerization of the proteins at
the bead surface where high GSH densities reside (USP 7,785,900).
2,3
When amino polystyrene bead sets of different red fluorescent in-
tensities became commercially available, we envisioned opportuni-
ties for devising HTS assays wherein the bead surface would be
covalently coated with GSH, allowing noncovalent binding of vari-
ous GST-fusion proteins, which could then be used for quantitative
measurement of the binding of various fluorochrome-conjugated
analytes that associated with the immobilized GST-fusion proteins,
such as peptides, nucleotides, and steroid hormones.
The antiapoptotic members of the Bcl-2 family have been found to
be elevated in various cancers and/or become elevated during
ABBREVIATIONS: BH3, Bcl-2 homology region 3; CID, PubChem compound accession identifier number; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; F-Bim,
fluoresceinated Bim; FP, fluorescence polarization; GSH, glutathione; GST, glutathione-S-transferase; HTS, high-throughput screening; ITC, isothermal titration calorimetry;
MLSMR, Molecular Libraries’ Small Molecule Repository; NIH, National Institutes of Health; SMCC, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate.
DOI: 10.1089/adt.2010.0363 ª MARY ANN LIEBERT, INC. VOL. 9 NO. 5 OCTOBER 2011 ASSAY and Drug Development Technologies 465
Page 1
treatment. New chemical scaffolds that modulate Bcl-2 functions are
attractive targets for chemical screening. In humans, the Bcl-2 family
consists of many proapoptotic and six antiapoptotic members (Bcl-2,
Bcl-B, Bcl-W, Bcl-X
L
, Mcl-1, and Bfl-1), where the balance between
the two decides at what level of damage or stress a cell will undergo
apoptosis.
4
For this reason, antiapoptotic Bcl-2 family proteins are
important players in cancer cell survival and are recognized as rel-
evant targets in cancer treatment. Pro and antiapoptotic Bcl-2 family
proteins exert their cellular functions at least in part by physically
interacting with each other to either suppress or promote apoptosis.
The best documented mode of interaction among Bcl-2 family pro-
teins involves the association of an a-helical segment, called Bcl-2
homology region 3 (BH3), provided by proapoptotic proteins, with a
complementary surface crevice found on the antiapoptotic proteins.
5
Synthetic BH3 peptides mimic the cellular activities of many of the
proapoptotic Bcl-2 family proteins, serving as antagonistic ligands
for the antiapoptotic members Bcl-2, Bcl-X
L
, Mcl-1, Bcl-W, Bfl-1,
and Bcl-B. Thus, it has been shown that simple HTS assays can be
constructed, in which chemical libraries are screened for compounds
that displace BH3 peptides from antiapoptotic Bcl-2 family proteins,
providing a starting point for generation of chemical antagonists that
might be useful as cancer therapeutics.
6
BH3 domains of proapoptotic Bcl-2 family proteins can be either
selective or broad spectrum in their interactions with antiapoptotic
Bcl-2 family proteins. For example, though many proapoptotic Bcl-2
family proteins such as Bcl-G show preferential binding to particular
antiapoptotic members of the family, some proapoptotic members of
the family such as Bim possess BH3 domains that interact with all six
antiapoptotic family members with high affinity.
7
By analogy to BH3
peptides, synthetic nonpeptidyl chemical antagonists of antiapoptotic
Bcl-2 family proteins can be either selective or broad spectrum in terms
of their binding to and neutralization of these cytoprotective proteins.
From a therapeutic standpoint, however, the ideal spectrum of binding
activity of chemical antagonists to antiapoptotic Bcl-2 family proteins
is debatable and probably varies depending on the clinical indication.
On one hand, broad-spectrum inhibitors that neutralize all six family
members might be preferred for many types of cancer to ensure cy-
totoxic activity, but could also confer unacceptable toxicities. On the
other hand, highly selective inhibitors that only neutralize specific
antiapoptotic members of the Bcl-2 family might be better tolerated,
but also could be limited in their efficacy only to those tumors where
the relevant members are expressed in isolation without other re-
dundant members of the family. Indeed, examples of resistance to
selective inhibitors have been documented for chemical antagonists of
Bcl-2 and Bcl-X
L
, where high levels of either Mcl-1 or Bfl-1 have been
demonstrated to negate cytotoxic activity against cancer and leukemia
cells.
8
Still other applications of Bcl-2 antagonists can be envisioned
for diseases beyond cancer, such as autoimmunity, wherein anti-
apoptotic Bcl-2 family proteins are believed to play an important role
in allowing the survival of destructive, autoreactive lymphocytes. For
example, Bcl-B is highly expressed in plasma cells,
9
which suggests
that it might serve as a target for eradication of long-lived autoanti-
body-producing cells.
To identify chemical inhibitors with different spectrums of activity
against the six antiapoptotic Bcl-2 family members, it would be ideal
to have an HTS assay method for efficient and simultaneous analysis
of all six targets in one well. Herein, we describe such a multiplexed
assay using high-throughput flow cytometry. We present a combined
approach of biological screening and cheminformatics data mining
to identify small molecule modulators of the antiapoptotic members
of Bcl-2 family within a large collection of compounds provided by
the National Institute of Health’s Molecular Libraries’ Small Mole-
cules Repository (MLSMR). The MLSMR is an important part of the
NIH Roadmap initiative, which comprises a diverse collection of
small molecules that generally display drug-like properties.
10
By
conducting a multiplexed assay for competitive displacement of
fluorochrome-conjugated Bim BH3 peptide to each of the six anti-
apoptotic family members, we have been able to identify a number of
different unrelated classes of compounds as candidate modulators of
antiapoptotic Bcl-2 proteins. As an example, we focus on inhibitors
of Bcl-B, which were validated by fluorescence polarization (FP) and
isothermal calorimetry.
MATERIALS AND METHODS
General
Unless otherwise noted, all reagents were of analytical grade and
purchased from Sigma-Aldrich. The six GST-fusion proteins were
constructed, in which the Bcl-2 moieties lacked only an *20-amino
acid hydrophobic tail; these were bacterially expressed and affinity
purified as previously described.
11
Beads were delivered from a
384-well plate (Catalog No. 784201 from Greiner Bio-One) using an
autosampler and a peristaltic pump (HyperCyt, available from In-
telliCyt). The details of this particle delivery system have been pre-
viously described.
12,13
Briefly, the autosampler dips into a well for
900 ms, then travels through the air between wells for 0.5 s, and then
samples the next well. These 384 clusters of beads are delivered to a
CyAn flow cytometer (Dako Corporation, now Becton-Dickinson)
and appear as 384 groups of beads or groups of ‘events’ in a single
time file. The flow cytometry file is analyzed using a commercial
software, HyperView (Intellicyt), which can determine the mean
green fluorescence on each bead set for each cluster (well) of beads.
Synthesis of GSH Beads
Amino polystyrene beads, 4 mm in diameter, were dyed with a
proprietary red fluorophore to different intensities by Duke Scientific
(now Thermo Fisher Scientific) and supplied at 1.4 · 10
8
particles/mL.
The bifunctional chemical crosslinker succinimidyl 4-[N-mal-
eimidomethyl]-cyclohexane-1-carboxylate (SMCC) was used to co-
valently react with the amino groups on the beads and then to GSH
added later. About 3.6 mL of beads (5 · 10
8
beads) were spun at
1,500 · g for 2 min and resuspended in 400 mL of 50 mM sodium
phosphate buffer (pH 7.5) containing 0.01% Tween-20. Eight mi-
croliters of 100 mM SMCC in dimethyl sulfoxide (DMSO) was added
and the beads were mixed gently for 30 min. The beads were spun and
resuspended in 360 mL of fresh buffer and then 40 mL of 200 mM GSH
(pH 7) and 4 mL of 100 mM EDTA (pH 7–8) were added. Nitrogen was
CURPAN ET AL.
466 ASSAY and Drug Development Technologies OCTOBER 2011
Page 2
bubbled slowly through the suspension for 5 min, the tube was
capped to exclude oxygen, and the beads were gently mixed for
30 min. The beads were washed four times and stored at
1.4 · 10
8
beads/mL in 30 mM HEPES (pH 7.5), 100 mM KCl, 20 mM
NaCl, 1 mM EDTA, and 0.02% NaN
3
at 4C.
Assay for Inhibitors of Fluoresceinated Bim BH3 Peptide
Binding to Bcl-2 Family Proteins
This assay is described in detail in another report
14
and an over-
view is given here. A schematic diagram of our strategy is shown as
Figure 1. The assay buffer was 30 mM HEPES (pH 7.5), 100 mM KCl,
20 mM NaCl, 1 mM EDTA, 0.1% BSA, and 0.01% Tween-20. The six
antiapoptotic Bcl-2 family members (Bcl-B, Bcl-X
L
, Bcl-2, Bcl-W,
Bfl-1, and Mcl-1) were separately expressed as GST-fusion proteins,
affinity purified, cleared of GSH, and mixed separately with the six
GSH bead sets having different red intensities, and a seventh set of
beads was left without any protein as a control for nonspecific
binding.
Currently, HTS flow cytometry screening of 100 plates per day
is performed with 4 people and three to four cytometers in our
center. However, in this report, for screening 24 plates per day with a
single operator and cytometer, 140 mL of each GSH bead set was
added to 340 mL of assay buffer and mixed twice over 20 min at
room temperature, giving seven suspensions of surface-passivated
beads. An average of 20 m g of each GST-Bcl-2 family member
was added to the passivated bead set and mixed twice on ice over
15 min, and then the seven suspensions were left on ice overnight
for use the next day. The bead sets were resuspended, and for each
batch of four 384-well plates, 75 mL of each coated bead set was spun
down in a separate tube. The sets were then combined in 200 mLof
fresh buffer and spun once more. The beads were diluted to 8.5 mL
and transferred to the four 384-well assay plates in 5 mL of buffer
per well.
The compounds to be tested were supplied as 1 mM DMSO stock
solutions in 384-well plates and were added to the assay plates using
a BioMek FX
P
robot (Beckman Coulter) equipped with a 384 pin tool
optimized to transfer 0.1 mL of the sample. The fluoresceinated Bim
(F-Bim) BH3 peptide was then added to all wells in 5 mL of buffer per
well, the plates were mixed for 15 s at 2,000 rpm, and the beads were
gently rotated end over end for 1 h at 4C (surface tension keeps the
suspensions in their wells). The plates were mixed again and deliv-
ered by the HyperCyt system to a flow cytometer for interrogation.
The flow cytometer was set to acquire forward scatter, side scatter,
FL1 (fluorescein filter set, 488 nm excitation, 530 20 nm emission),
FL8 (635 nm excitation, 665 10 nm emission), and time. Figure 1C
shows the seven bead sets from one assay plate resolved from each
other in red fluorescence and gated, following which the green
fluorescence on each bead set is easily obtained. An arbitrary cutoff
of 40% inhibition was taken to indicate a screening ‘hit’ or possibly
active compound. This was a deliberate use of low stringency and
assumed that further analysis would be done on many false-positive
hits. The assay can also be performed at a series of compound con-
centrations to form a dose–response curve (Fig. 3). Table 1 diagrams a
step-by-step protocol for this assay.
Time Binning, Data Reduction, and Analysis
The flow cytometry files for each plate were opened
with HyperView software, singlet beads were gated in a
standard forward scatter vs. side scatter dot plot, and then
the individual bead sets were gated in a side scatter versus
FL8 plot as shown in Figure 1C. The library of compounds
(NIH Small Molecule Repository) was supplied in 320 wells
of standard 384-well plates, leaving the first two columns
and last two columns for controls. We chose to leave two
columns free of beads at the end of the plate, so that the
time file presents 16 groups (rows on the plate) of 22
clusters of events (wells with beads) in each row, totaling
352 rather than 384 clusters of events, to allow confidence
in assigning a cluster to a plate position. The software
identified the 352 clusters in about 3 s, and the series of 16
groups of 22 clusters was checked by eye in about 1 min:
as beads are more homogeneous than cells, this check was
rapid. The software then calculated the number of events
and average green fluorescence associated with each bead
for each bead set in each well and exported these reduced
data as comma-separated value files, including the com-
pound identification number. The resulting files were
typically <1% the size of the original flow cytometry file.
Hits were flagged for further testing. Dose–response
curves were fitted using Prism (GraphPad Software).
Table 1. Summary of High-Throughput Screening Flow Cytometry
Multiplex Assay Protocol
Step Parameter Value Description
1 Bead mixture 5 mL Robot with 384 tips
2 Controls 0.1 mL Robot with pin tool; 10 mM
3 Library compounds 0.1 mL Robot with pin tool; 10 mM
4 F-Bim 5 mL Robot with 384 tips; 50 nM
5 Mix, incubate 1–3 h 4C–10C
6 Assay readout FS, SS, FL1, FL8 Flow cytometer
Step Notes
1. Beads coated individually overnight, wash and mix in batches; not Cols 23, 24
2. Cols 1, 23; wash sequence: water, DMSO, EtOH
3. Wash sequence: water, DMSO, EtOH
4. The F-Bim retains 90% fluorescence for 8 h under laboratory fluorescent lights
5. The incubation can be 15 min at room temperature
6. After each plate, back flush and debubble
With 1 person and one cytometer, 24 plates per day containing a total of 7,680 compounds
were screened. With 4 people and four cytometers, 100 plates per day can be screened.
DMSO, dimethyl sulfoxide; F-Bim, fluoresceinated Bim.
HIGH-THROUGHPUT SCREEN FOR THE BCL-2 FAMILY
ª MARY ANN LIEBERT, INC. VOL. 9 NO. 5 OCTOBER 2011 ASSAY and Drug Development Technologies 467
Page 3
Fluorescence Polarization
FP assays were performed similarly to previous reports using
various Bcl-2-family proteins and fluorescein isothiocyanate (FITC)-
conjugated Bim BH3 peptide.
11
Briefly, 50 nM of various recombi-
nant Bcl-2 family proteins were incubated with 10 nM of FITC-Bim
BH3 peptide (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR in
25 mM Bis-Tris buffer containing 1 mM tris(2-carboxyethyl) phos-
phine and 0.005% Tween 20 [pH 7.0]) in the dark. FP was measured
using an Analyst TM AD Assay Detection System (LJL Biosystem)
after 10 min. IC
50
determinations were performed using GraphPad
Prism software (GraphPad). A compound was described as active if it
induced a 40% decrease in FP.
Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was performed on an ITC200
calorimeter from Microcal. Two-microliter aliquots of solution
containing 500 mM compound were injected into 200 mL cells
containing 50 mM GST-Bcl-2 protein. Nineteen injections were
made per titration. The experiments were performed at 23Cin
buffer containing 20 mM HEPES (pH 7.0). Experimental data were
analyzed using Origin software provided by Microcal. A compound
was described as active when it displayed a sigmoidal dose–response
curve, which corresponds to IC
50
<10 mM at the protein concentration
used.
Compound Selection Scheme
Primary screening. The primary screening data were processed
through Microsoft Excel using a template file designed specifically
for this assay. This template segregated data for each protein and the
fluorescence scavenger bead in the multiplex, assembled the desired
statistics and graphs, and flagged possible hits for each molecular
library plate in a batch mode. The analysis and assessment of the
active compounds was made by considering the percentage of inhi-
bition, a value calculated for each compound on each target using a
simple algorithm and the Excel template. In the primary screening, a
compound was considered ‘active’ if the binding of F-Bim was in-
hibited by more than 40%.
Selection criteria for cherry picking the compounds to be tested in
a dose–response assay. In selecting compounds for confirmation in
a multiple concentration–response assay, we used a more relaxed
selection criterion, that is, the percentage of inhibition should be
greater than or equal to the average percentage of inhibition of all
compounds plus three times the standard deviation of the mean.
Cluster generation and analysis. To establish structu re–activity
relationships among the actives, the Mesa Grouping Module was
used to cluster the compounds thatpassedthedefinedcriteria,to
analyze the resul ting families, and to prioritize the clusters (fami-
lies) to be selected for a dose–response assay. Mesa Grouping
Module i s a structure-based clustering and classification software
offered by MESA Analytics & Computing (Santa Fe; www
.mesaac.com). Mesa Software is a suite of programs, and for our
purposes, the Fingerprint M odule was used to generate fingerprints
for structures and the Measures program was employed to generate
asymmetric matrices, using the Tversky measure with a dissimi-
larity threshold cutoff of 0.5. The output of Measures was directed to
the Clustering Module, which uses asymmetric, symmetric, disjoint,
or nondisjoint algorithms to group the compounds. In our case we
used a d isjoint algorithm and 50 different output files were gener-
ated using exclusion region thresholds between 0.01 and 0.5. Al-
though the compounds active for Bcl-B were singletons, a number
of compounds reported for other family members were obtained
using cluster generation.
Dose–response results analysis. We analyzed the results of the
dose–response assay in three steps using Microsoft Excel templates to
select compounds with desirable properties. In the first stage, the
compounds that produced a statistically significant competitive
displacement of the F-Bim BH3 ligand were selected as follows. The
first filter required that the standard error of log EC
50
had to be lower
than the absolute value of log EC
50
. The second filter required that the
standard error of Hill slope had to be smaller than the absolute value
of Hill slope. The third filter required that the biological response
amplitude (span) had to be statistically significant: the bottom plus
three times its standard deviation had to be less than the top minus
three times its standard deviation.
In a second stage of analysis, we identified the compounds that
were active on at least one target. The compounds annotated as
‘active’ had to pass the following filters: first, the computed EC
50
had
to be between 10
- 8
and 10
- 4
M; second, the absolute value of the Hill
coefficient had to be between 0.5 and 2.
The third stage of this analysis was dedicated to the identification
of potentially ‘false hits.’ Some compounds gave an increase in
fluorescence on the GSH (or s cavenger) beads, wh ich would appe ar
as an activator o f binding. A compound was denoted a ‘false acti-
vator hit’ if the span of the compound on GSH beads was greater
than 50% of the span of the appropriate protein beads. Some
compounds gave a decrease in fluorescence in the counter screen
assay (GST to GSH interaction, PubChem AID 1324), in which GSH
beads were coated overnight with 50 nM GST-green fluorescent
protein, as in the coating of the beads with GST-Bcl-2 proteins, and
a dose–response curve of compounds was performed to determine
if GST-green fluorescent protein was competed off the beads.
A compound was denoted a ‘false hit’ if the span of the counter
screen assay was greater than 50% of the span of the appropriate
protein beads. To recover the active compounds and identify the
‘false hits,’ we applied all the filters and selection criteria described
above.
In the final stage of the process, a prioritization scheme was de-
veloped in which the information from the previous steps was
combined with data obtained from querying chemical scaffolds in
different databases to eliminate ‘unattractive’ scaffolds from the list
of active compounds. Scaffolds found to be active in many other
assays from PubChem and the chemical literature were flagged as
undesirable from a chemical perspective. After compounds with
CURPAN ET AL.
468 ASSAY and Drug Development Technologies OCTOBER 2011
Page 4
known reactive groups and undesirable scaffolds were eliminated
from the clusters, the remaining compounds were considered suitable
to consider as candidate Bcl-2 family protein modulators.
RESULTS
Optimizing and Validating a Bead-Based Set of Assays
A diagram of the multiplex assay is shown in Figure 1, in which the
GSH bead sets, consisting of different red fluorescent intensities, were
mixed individually with a GST-fusion protein and then washed and
mixed together with the fluorescent probe (F-Bim). After incubation,
the bead samples were transported to a flow cytometer and resolved
by red fluorescence.
To validate the screening assay, we set out to reproduce a K
d
that
was determined by others and to reproduce an EC
50
value determined
by others using a known competitor, taking experimental differences
into account. Multiplex binding data and inhibition of binding data
are shown in Figure 2, and they additionally demonstrate the stability
of the signal from 1 to 3 h. Binding curves for F-Bim to all six GST-
Bcl-2 family proteins were determined, with results generally
agreeing with the literature that Bim strongly engages all six anti-
apoptotic Bcl-2 proteins.
15
It was fortunate that our K
d
values cov-
ered only a 10-fold range from 6 to 60 nM, which allowed the F-Bim
ligand to be added at a single intermediate concentration of 50 nM,
wherein it displayed only 10-fold more sensitivity to an inhibitor of
the most sensitive protein than to the least sensitive protein. It should
be noted that for any assay describing the binding of one entity to
another, results will differ among laboratories because of differences
in time, temperature, ionic concentrations, source of materials, and
other factors, so that absolute agreement of K
d
values is rarely ob-
tained. Nevertheless, the values obtained here are in general agree-
ment with measures of the K
d
of Bim and other BH3 peptide
interactions with antiapoptotic Bcl-2 family proteins obtained by
other methods.
11,15,16
This binding of F-Bim to each protein was also tested for com-
petitive inhibition by ABT-737, a known Bcl-2 isosteric inhibitor.
5
The concentration of ABT-737 was varied from 10
- 10
to 10
- 4
M, and
the inhibitions obtained agreed with the literature, in particular that
ABT-737 acts only weakly with Mcl-1 or Bfl-1.
11
However, during
the testing of ABT-737, it was observed that the preparation of Bcl-B
had lost almost all activity during a year of storage at - 80C. This is
seen in Figure 2, in which more F-Bim bound to Bcl-B than the other
proteins in the binding curve, whereas less F-Bim bound to Bcl-B in
the competition curves obtained a year later. This figure shows an
advantage of multiplexing: with six individual dose–response series,
one might be tempted to think that not enough F-Bim had been added
to the Bcl-B series, but as all the beads at, for example, 10
- 9
M were
incubated together in the same well, the lack of binding to the Bcl-B
set is not a product of failure to deliver F-Bim to one series.
As specific activities of the individual proteins do not have to be
identical to obtain Z¢ values over 0.5, a fresh sample of Bcl-B
was obtained, in which the binding was similar to that of the other
proteins (Fig. 3A), and used for the dose–response data submitted to
PubChem.
In the primary screening campaign, the MLSMR library of 194,920
samples (194,829 unique compounds) was screened at a single
concentration (10 mM) against GST-Bcl-2, GST-Bcl-B, GST-Bcl-W,
GST-Bcl-X
L
, GST-Bfl-1, and GST-Mcl-1 using an F-Bim BH3 peptide,
as shown in Table 2. The data analysis with user-defined criteria led
to the identification of 518 compounds having a percentage of in-
hibition (response value) of >40%. These data are available from the
PubChem database (http://pubchem.ncbi.nlm.nih.gov) under the
assay identification numbers (AID numbers) presented in Table 2.
The ‘response’ value used in the PubChem database is the percentage
of inhibition of F-Bim binding.
Dose–Response Data Confirm Three Compounds
as Specific Bcl-B Inhibitors
This set of 518 compounds deemed active was subjected to
structure-based clusterization, and 834 compounds covering the
entire Bcl-2 family were obtained as stock solutions in DMSO and
assayed in a dose–response series. The results of the confirmatory
assay (Table 2) have been uploaded in PubChem under the assay
identifier numbers presented in this table and also AID 1324 for the
GST-GSH counter screen, which eliminates inhibition due to dis-
ruption of the GSH to GST binding.
Following the analysis of the dose–response data with the de-
fined criteria and prioritization algorithm, singletons and families
Fig. 1. Diagram of bead coating, mixing, and resolution by cyto-
metry. (A) Six sets of beads are mixed separately with glutathione-
S-transferase (GST)-Bcl-2 family proteins, and a seventh set serves
as a control for nonspecific binding. (B) The seven bead sets are
mixed together and distributed to appropriate wells. (C) The flow
cytometer records red fluorescence for each bead, allowing the
sets to be gated, after which the green fluorescence of beads in
each gate can be measured.
HIGH-THROUGHPUT SCREEN FOR THE BCL-2 FAMILY
ª MARY ANN LIEBERT, INC. VOL. 9 NO. 5 OCTOBER 2011 ASSAY and Drug Development Technologies 469
Page 5
of compounds having members with EC
50
values <10 mMwere
identified. One hundred eight compounds were found as potential
modulators of Bcl-2 f amily of proteins. This number included Bfl-1
hits, which later were found to be false-positives (see Discussion
section).
For follow-up studies, 37 modulators representing the most active
members for each representative scaffold and 10 singletons were
chosen. Forty-two compounds were obtained as dry powders and
were subjected to liquid chromatography–mass spectroscopy anal-
ysis (which passed the quality control criteria that they were >95%
pure by high-performance liquid chromatography and that they
displayed the correct mass by liquid chromatography/mass spec-
trometry and high-resolution mass spectrometry). This set was re-
tested in the dose–response assay and six compounds were
reconfirmed as active against Bcl-B in a dose–response assay, shown
in PubChem bioassay AID 2075.
Figure 3A shows an example of a dose–response curve for one of
these compounds, with PubChem compound accession identifier (CID)
number 650929, in multiplex on all six Bcl-2 family members. The
weak response of the earlier preparation of Bcl-B has been corrected. It
is clear that this compound inhibits the binding of F-Bim to Bcl-B
much more than to any of the other family members. We emphasize
that, for example, all beads with 10
- 5
M compound were in the same
well. When the powders were tested, three of them had log EC
50
values
below - 5(EC
50
<10 mM), which we considered reliably active. They
showed <20% inhibition of the other five antiapoptotic Bcl-2 family
Fig. 2. Binding and inhibition curves. Binding curves were obtained as described, including 1 h of incubation at 4C with end-over-end
mixing before interrogation by the cytometer. As with other data from our laboratory, above 100 nM fluorescein there appears to be
clipping of the signal. The competition curves were obtained as described using 50 nM fluoresceinated Bim (F-Bim) after 1 year of storage
and have been selected to show both the utility of the multiplex assay to demonstrate decreased activity of one protein in the multiplex
(Bcl-B) as well as the stability of the response over 3 h. The proteins correspond to symbols as follows: Bcl-X
L
(-), Bcl-2 (:), Bcl-W (;),
Bcl-B (A), Bfl-1 (), and Mcl-1 (,). All data were obtained in duplicate, with error bars showing the two readings.
CURPAN ET AL.
470 ASSAY and Drug Development Technologies OCTOBER 2011
Page 6
members at the highest concentration tested, 100 mM. Active com-
pounds were also identified for other antiapoptotic family members.
FP Confirms the Flow Cytometry Active Compounds
Two rounds of FP dose–response assays were performed to vali-
date the flow cytometry data, using not just the F-Bim probe, but also
one with a different fluorophore, Cy5-Bim. Figure 3B shows an ex-
ample of an FP dose–response curve for 10 compounds against GST-
Bcl-B with F-Bim, in which only 3 compounds were declared active.
Quantitative results for the three active compounds against Bcl-B are
shown in Table 3, which correspond to the second FP assay in Table 2.
The different fluorophore made little difference in log EC
50
values
obtained by FP, with the values differing only by 0.3 units and not all
in the same direction. The flow cytometric log EC
50
values averaged
Fig. 3. Representative dose–response curves showing active and inactive interactions. (A) Flow cytometry: CID 650929 is active against Bcl-
B(), not against Bfl-1 (-), Bcl-X
L
(;), Bcl-W (A), Mcl-1 (:), or Bcl-2 ( + ); glutathione control beads are (o). (B) Fluorescence
polarization: three compounds are active. The CIDs correspond to symbols as follows: 650929 (,), 1243212 ( + ), and 666339 ( · ). The y-
axis is P · 1,000, where P = (F
k
- F
t
)/(F
k
+ F
t
), F
k
= fluorescence intensity parallel to the excitation plane, and F
t
= fluorescence perpen-
dicular to the excitation plane. (C) Isothermal titration calorimetry: compound accession identifier (CID) 650929 is active against Bcl-B, not
Bcl-X
L
. The fluorescence polarization data (B) were obtained in duplicate, with the error bars showing the two readings. The flow cytometry
(A) and isothermal titration calorimetry (C) data show single-point determinations. CID, PubChem compound accession identifier number.
HIGH-THROUGHPUT SCREEN FOR THE BCL-2 FAMILY
ª MARY ANN LIEBERT, INC. VOL. 9 NO. 5 OCTOBER 2011 ASSAY andDrugDevelopmentTechnologies 471
Page 7
0.3 units higher than the FP data, but agree that all compounds
significantly inhibit F-Bim binding. Multiple tests on different days
showed that the coefficient of variation of flow cytometry log EC
50
values was <10%. Control FP experiments corroborated the speci-
ficity of binding to Bcl-B, with no binding to Bcl-X
L
, Bcl-2, or Bfl-1
(data not shown).
ITC Confirms the Flow Cytometry Active Compounds
Isothermal calorimetry uses a different physical principle to
measure binding and was used to confirm or deny binding of the
three compounds to Bcl-B. Figure 3C shows an example of an ITC
dose–response curve against Bcl-B, in which CID 650929 was de-
clared active, and against Bcl-X
L
, in which the compound was de-
clared inactive. Table 3 shows that the ITC log EC
50
values
demonstrate significant binding of all three compounds to Bcl-B,
making it more plausible that our other measurements were in fact
displaying competitive binding between F-Bim and the compounds
to Bcl-B. The ITC demonstration of binding is particularly instructive,
for the flow cytometry and FP measurements are prone to similar
optical artifacts. The compound CID 650929 appears particularly
strong with respect to Bcl-B binding. Control experiments showed no
binding of the compounds to Bcl-X
L
(data not shown), as a demon-
stration of specificity. The structures of these three active compounds
are shown in Figure 4. PubChem reports many comparisons of the
screening and follow-up data, including the number of assays in
which a compound has been declared active and the number of as-
says in which it has been tested: for CID650929, these numbers are 9/
513 (active/tested); for CID1243212, these are 3/371; and for
CID666339, these are 11/488, making all three compounds attractive
with respect to specificity of interaction.
DISCUSSION
This report demonstrates a multiplexed, bead-based flow cytom-
etry screen that covers a small family of proteins, requiring <1mgof
each GST-fusion protein per family member, a fluorescent probe, and
the GSH bead sets described herein. In general, the capture and dis-
play of molecules on microspheres has the potential of reducing the
quantities of the captured reagent (the GST-fusion protein) and al-
lows the less-expensive fluorescent reagent (the F-Bim peptide) to be
used near its K
d
. 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.
Table 2. Primary Screening and Confirmatory Dose–Response Results
Compounds
tested
Number of hits (PubChem AID)
Screening Bcl-2 Bcl-B Bcl-xL Bcl-W Bfl-1 Mcl-1 Total hits
a
Primary 194,829 116 (950) 142 (951) 82 (1007) 48 (952) 237 (1008) 196 (1009) 385
a
Confirmatory
dose response
834 9 (1328) 6
b
(1327) 6 (1322) 18 (1330) 97 (1320) 3 (1329) 27
a
Powder dose
response
42 6 (2075) 9/2
c
(2077) 0 (2084) 5 (2081) 23 (2080) 0 (2086) 9
a
FP (1) 47 3 Not tested Not tested Not tested 3 Not tested 3
FP (2) 10 1 3 0 Not tested 0 Not tested 4
d
ITC 4 Not tested 3 0 Not tested Not tested Not tested 3
Some compounds that did not pass the threshold for being declared as active were nevertheless selected to be tested in follow-up assays.
a
Bfl-1 hits were not included in the number of total hits because the dose–response results from the original Bfl-1 prepara tion were not replicated with a new
preparation in the FP assay dose–response analysis; total hits is thus the number of unique compounds affecting one or more of the other five antiapoptotic Bcl-2 family
members. We hypothesized that redox-based modulation of cysteine 55 in the BH3 binding pocket could account for the discrepancy. However, the C55A mutant of Bfl-1
bound Bim normally and the compounds did not show the activity that they had against the original Bfl-1 preparation (data not shown). We then performed a multiplex
analysis in which the original preparation of Bfl-1, the fresh preparation used in the FP assay, and the mutant protein were bound to individual bead set. These data
recapitulated the activities of the individual preparations, and the fresh preparation set is being uploaded to PubChem to reconcile the earlier results.
b
Compound SID 85203040 was declared inactive in this assay, with an EC
50
= 18.2 mM higher than the chosen threshold (10 mM).
c
Only two Bcl-B hits in this assay were inhibitors; seven other compounds ‘increased’ the binding of the fluorescent peptide and were considered artifacts; compound
SID 85203037 was declared inactive in this assay, with an EC
50
= 16.3 mM higher than the chosen threshold (10 mM).
d
A benzoquinone reactive compound was also identified as hit on all protein targets, but was discarded.
AID, assay identification numbers; BH3, Bcl-2 homology region 3; FP, fluorescence polarization; ITC, isothermal titration calorimetry.
CURPAN ET AL.
472 ASSAY and Drug Development Technologies OCTOBER 2011
Page 8
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 cy-
tometry, 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 con-
centration, 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 ac-
tive compounds was a challenging task. For
this reason, a classification schema was de-
veloped to select the compounds from pri-
mary 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 s ix proteins. Applying a
prioritizing algorithm that took into account the biological re-
sponses (EC
50
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% c onfirmation 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 mM, and 0.1% for
hits with potency values less tha n 100 mM. An assay with a Z¢ score
greater than 0.5 is considered an excellent assay for screening,
17
and our Z¢ values were Bcl-X
L
,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 s creen. 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 B cl-B.
The gene encoding Bcl-B was first recognized as the human ge-
nome project moved toward completion, with the first publication
appearing in 2001. It has no clear ortholog in mice and thus repre-
sents an evolutionary progression of the Bcl-2 family in higher or-
ganisms. 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-lympho-
cytes and especially abundant in plasma cells.
20
Prior experiments
Table 3. Fluorescence Polarization and Isothermal Titration Calorimetry Confirm
the Flow Cytometry Data for Bcl-B
Compound
FP Cy5-Bim
log EC
50
FP F-Bim
log EC
50
Flow F-Bim
log EC
50
ITC compound
log K
d
CID 650929 - 5.7 - 6.0 - 5.3 - 6.7
CID 1243212 - 5.3 - 5.1 - 5.6 - 5.5
CID 666339 - 6.0 - 6.0 - 5.7 - 5.6
Fig. 4. Structures and compound identifier numbers of the three
inhibitors of the binding of F-Bim to Bcl-B.
HIGH-THROUGHPUT SCREEN FOR THE BCL-2 FAMILY
ª MARY ANN LIEBERT, INC. VOL. 9 NO. 5 OCTOBER 2011 ASSAY and Drug Developmen t Technologies 473
Page 9
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-X
L
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-X
L
, 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 spec-
trums of activity that may translate into distinct efficacy and tox-
icity profiles f or different clinical indications.
ACKNOWLEDGMENT
This research was supported by an NIH grant (No. U54MH074425).
AUTHOR DISCLOSURE STATEMENT
L.A.S. and B.S.E. declare competing financial interests as co-
inventors of HyperCyt and cofounders of Intellicyt.
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Address correspondence to:
Larry A. Sklar, PhD
Department of Pathology
UNM Center for Molecular Discovery
University of New Mexico Health Sciences Center
MSC07-4025
Albuquerque, NM 87131
E-mail: lsklar@salud.unm.edu
CURPAN ET AL.
474 ASSAY and Drug Development Technologies OCTOBER 2011
Page 10
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