Inhibition of Bfl-1 with N-aryl maleimides

Article abstract of DOI:10.1016/j.bmcl.2010.09.046

High-throughput screening of 66,000 compounds using competitive binding of peptides comprising the BH3 domain to anti-apoptotic Bfl-1 led to the identification of 14 validated 'hits' as inhibitors of Bfl-1. N-Aryl maleimide 1 was among the validated 'hits'. A chemical library encompassing over 280 analogs of 1 was prepared following a two-step synthesis. Structure-activity studies for inhibition of Bfl-1 by analogs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development candidates for anti-cancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2010.09.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6560–6564
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of Bfl-1 with N-aryl maleimides
John R. Cashman a, , Mary MacDonald a, Senait Ghirmai a, Karl J. Okolotowicz a, Eduard Sergienko b,
⇑
Brock Brown b, Xochella Garcia b, Dayong Zhai b, Russell Dahl b, John C. Reed b
a Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, United States
b Sanford-Burnham Institute for Medical Research, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2010
Revised 5 September 2010
Accepted 8 September 2010
Available online 21 September 2010
High-throughput screening of 66,000 compounds using competitive binding of peptides comprising the
BH3 domain to anti-apoptotic Bfl-1 led to the identification of 14 validated ‘hits’ as inhibitors of Bfl-1.
N-Aryl maleimide 1 was among the validated ‘hits’. A chemical library encompassing over 280 analogs
of 1 was prepared following a two-step synthesis. Structure–activity studies for inhibition of Bfl-1 by ana-
logs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring
and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development
candidates for anti-cancer therapeutics.
Keywords:
Bfl-1 inhibitors
N-Aryl maleimides
High-throughput screening
Anti-cancer agents
Ó 2010 Elsevier Ltd. All rights reserved.
The B-cell lymphoma/leukemia-2 (Bcl-2) family of proteins plays
a prominent role in regulating programmed cell death (apoptosis).
In humans, proteins within the Bcl-2 family having anti-apoptotic
activity include Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bcl-B, and Bfl-1.1 Mem-
bers of this group of proteins are commonly over-expressed in
human cancers thereby promoting tumorigenesis.2,3 Additional
groups of proteins within the Bcl-2 family have pro-apoptotic
activity and include Bak, Bax, Bad, Bim, and Bid. Anti-apoptotic
and pro-apoptotic proteins can dimerize, either activating, or antag-
onizing each other.4
Bcl-2 proteins, mimicking BH3 peptides to block their cytoprotec-
tive effects. Some small molecules inhibit a broad range of the
anti-apoptotic Bcl-2 proteins while others are more selective.
Bfl-1 is a NF-jB-inducible member of the Bcl-2 family, that is
highly expressed in immune cells.15 Defective apoptosis has been
implicated in several autoimmune diseases, thus suggesting a ther-
apeutic opportunity for agents that neutralize Bfl-1. In addition,
Bfl-1 is highly expressed in some cancers accounting for resistance
to selective Bcl-2/Bcl-XL inhibitors that fail to neutralize this anti-
apoptosis member of the Bcl-2 family. Based on these results, we
decided to focus on small molecule inhibitors of Bfl-1 that interact
at the BH3 binding site.
Heterodimerization occurs at a hydrophobic pocket on the
surface of anti-apoptotic proteins and a region of pro-apoptotic
proteins called the BH3 region to neutralize the cytoprotective
function of anti-apoptotic Bcl-2 family proteins. The BH3 region is
Herein, we describe the process used to identify and synthesize
promising lead compounds for inhibition of the anti-apoptotic
protein, Bfl-1. A high-throughput fluorescence polarization (FP)
assay was developed to detect inhibition of Bfl-1 by small mole-
cules.14 In this assay, inhibitors were incubated with glutathione-
S-transferase (GST)-Bcl-2 anti-apoptotic fusion proteins.16 FP was
measured after addition of an N-terminal fluoroscein isothiocya-
nate (FITC) BH3 peptide (i.e., FITC-Ahx-EDIIRNIARHLAQVGDSM
DR-CO2H).5,17 Inhibitory potency was confirmed using a Lantha-
Screen time-resolved fluorescence resonance energy transfer
(TR-FRET) assay that examined the same molecular interaction
between the FITC-peptide and GST-tagged protein.18 An initial
‘hit’ was selected, and exploratory libraries based on the ‘hit’ were
generated and tested to identify structural features necessary for
inhibition of Bfl-1.
comprised of an amphipathic a-helix of roughly 16–25 amino acids
that binds to the hydrophobic region of the anti-apoptotic Bcl-2
family proteins.1 Agents that mimic the BH3 region of pro-apoptotic
proteins may be used to antagonize anti-apoptotic Bcl-2 proteins by
binding to the hydrophobic pocket thus promoting cell death. Syn-
thetic peptides mimicking the BH3 region have been shown to bind
to the anti-apoptotic Bcl-2 proteins and initiate apoptosis.5,6
Small molecule inhibitors of anti-apoptotic Bcl-2 members have
been reported.7 Some natural product inhibitors include gossypol8
and epigallocatechine (EGCG).9 Some synthetic and semisynthetic
inhibitors include ABT-737,10 GX15-070,11 apogossypol,12 and the
commercially available thioxothiazolidine, BH3I-1.13,14 These
agents act by binding to the hydrophobic pocket of anti-apoptotic
The NIH Roadmap Small Molecule Library (at the time, repre-
senting approximately 66,000 compounds from BioFocus, San
Francisco, CA) was screened for inhibition of GST-Bfl-1. Twenty
⇑
Corresponding author. Tel.: +1 858 458 9305; fax: +1 858 458 9311.
E-mail address: JCashman@hbri.org (J.R. Cashman).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.046

J. R. Cashman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6560–6564
6561
compounds were identified in the initial FP assay to inhibit
GST-Bfl-1 with potency of 20 M or less. Of those 20 ‘hits’, 14
Because the original ‘hit’ contained 3,4-dichloro substituents in the
N-aryl ring, maleimides with different permutations of mono- and
dichloro-substituted N-aryl rings were synthesized. Additional
compounds with N-aryl methyl, and nitro substituents were pre-
pared to examine possible electronic substituent effects of the N-
aryl ring on GST-Bfl-1 inhibition. From among compounds 1 and
8–16 (Table 1) the original ‘hit’ 1 was the most potent GST-Bfl-1
l
showed similar GST-Bfl-1 inhibition in the TR-FRET assay. Overall,
there was reasonably good agreement between the FP and TR-FRET
assay for potency of Bfl-1 inhibition on the basis of IC50 values
(shown in Tables 1 and 2). The most potent ‘hit’ was compound
1 (Fig. 1) with submicromolar potency in both assays.
‘Hit’ 1 was amenable to structure–activity relationship (SAR)
studies. Three regions of the molecule were examined in an itera-
tive process of chemical synthesis and in both FP and TR-FRET
in vitro binding assays to define Bfl-1 inhibition SAR potency.
The regions for SAR studies included: (1) the chloro substituent(s)
and unsaturation of the maleimide ring, (2) substituents on the
N-aryl ring, and (3) the amine functionality of the maleimide ring
(Fig. 1). More than 280 analogs of 1 were prepared to optimize
selectivity and potency for inhibition of Bfl-1.
We first investigated the necessity of the chloride substitution
and the requirement of unsaturation of the maleimide ring system
for potency of Bfl-1 inhibition. Two commercially available analogs
of 1 were purchased (ChemBridge, San Diego, CA) and tested as
inhibitors of GST-Bfl-1 (Fig. 2). Compound 2 did not have the
maleimide chloride substituent but retained the maleimide ring
double bond while compound 3 possessed neither the chloride
nor the ring double bond. Both compounds were not potent (i.e.,
inhibitor in both the FP and TR-FRET assays (IC50 = 0.6 0.1
and 0.8 0.1 M, respectively). Of the dichlorophenyl compounds
examined, both the 3,5- (8) and 2,4-dichlorophenyl (9) substitu-
ents afforded IC50 values of 2.4 0.4 M and 2.6 0.2 M, respec-
lM
l
l
l
tively, and had similar activities but were threefold less potent
than the original ‘hit’ 1. Other disubstituted dichloro compounds
(i.e., 10) had much lower inhibitory potency (i.e., IC50 values of
8.7
chloro monosubstituted compounds (i.e., 12 and 13, with IC50 val-
ues of 3.3 0.3 M and 2.1 0.2 M, respectively) were not signif-
l
M).20 Compared to compounds 8 and 9, the 2-chloro or 3-
l
l
icantly different in the inhibition potency of GST-Bfl-1.
Monosubstituted N-aryl maleimides with either methyl (14) or ni-
tro (15) groups afforded GST-Bfl-1 inhibitors with much greater
IC50 values (i.e., 12.6 2.3 lM and 6.8 1.4 lM, respectively) indi-
cating that alkyl groups or polar groups on the N-aryl portion of the
maleimide did not increase GST-Bfl-1 inhibition potency. Addition
of a 3-methyl group to the 2,6-dichloro aryl group (11) further de-
creased potency 2–3-fold. Pyridine rings in place of the aryl ring
also had decreased potency (16). Based on these results, the 3,4-
dichlorophenyl group was chosen as the optimal substituent of
the N-phenyl ring for further SAR studies on the piperazine portion
of the molecule.
An additional sublibrary of compounds was prepared that re-
tained the 3,4-dichlorophenyl substituent in the N-aryl portion of
the molecule while the amine group on the maleimide ring was
varied (17–26). Thus, 3,4-dichloro-1-(3,4-dichlorophenyl)-1H-pyr-
role-2,5-dione 6 was treated with amines to examine the effect of
substitution on the maleimide ring itself. Amines used included
morpholines, piperidines, pyrrolidines, cyclohexylamine, ethanol-
amine, benzylamine, substituted piperazines, piperazinone, and
dimethylamine. Dimethylamino 21, morpholino 22, and 3-
methyl-piperazino 24 afforded compounds equipotent to original
hit 1. The remainder of the amine substituents examined (17–20,
23, and 25–26) afforded poorly potent compounds. N-terminal
piperazines with a center of chirality was tested to examine the
stereoselectivity of Bfl-1 inhibition (Table 2).
IC50 >10 lM) in both the FP and TR-FRET assays showing the neces-
sity of each moiety for potent inhibition of GST-Bfl-1.
N-Aryl maleimide analogs were prepared using a convenient
two-step process starting from commercially available starting
materials (Scheme 1).19 In the first step, substituted aniline 4
was heated with 1 equiv of dichloromaleic anhydride 5 in acetic
acid for 1 h. After extraction and purification by silica gel chroma-
tography, N-aryl dichloromaleimide 6 was isolated. Monosubstitu-
tion of the dichloromaleimide with amines was done in a medium
throughput format. Dichloromaleimides 6 dissolved in dioxane
were combined with amines dissolved in tetrahydrofuran at room
temperature to afford 3-chloro-1-aryl-4-amino-1H-pyrrole-2,5-
dione analogs of 7. Reaction progress was monitored by TLC and
reactions were heated if necessary. Products 7 were purified by
preparative TLC and verified by electrospray mass spectrometry.
Reactions were conducted on a 5 mg scale. Amounts of product iso-
lated ranged from 3 to 5 mg.
Initially, SAR studies of Bfl-1 inhibition were conducted with
compounds of structure 7 where the N-methylpiperazine of the
side chain amine was kept constant and substituents on the N-phe-
nyl ring were varied. A total of 10 anilines (4) were used (Table 1).
Compounds 24 and 25 have an unsubstituted nitrogen atom but
include a methyl group in the
a position resulting in a center of
Table 1
Effect of N-aryl maleimide ring substitution on GST-Bfl-1 inhibition
O
Cl
R1
N
N
N
O
Compound #
R1
Bfl-1 FPa IC50
(l
M)
Bfl-1 TR-FRETa IC50
(lM)
1
8
9
10
11
12
13
14
15
16
3,4-Dichloro-phenyl
3,5-Dichloro-phenyl
2,4-Dichloro-phenyl
2,3-Dichloro-phenyl
2,6-Dichloro-3-methyl-phenyl
2-Chloro-phenyl
3-Chloro-phenyl
3-Methyl-phenyl
3-Nitro-phenyl
3-Chloro-pyridin-4yl
0.6 0.1
2.4 0.4
2.6 0.2
8.7 1.7
23.9 3.9
3.3 0.3
2.1 0.2
12.6 2.3
6.8 1.4
29 3.9
0.8 0.1
1.9 0.2
1.4 0.1
2.6 0.1
11.1 2.3
1.0 0.1
1.2 0.1
10.8 0.9
3.9 0.4
35.6 3.9
a
Data are the mean SEM.

6562
J. R. Cashman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6560–6564
Table 2
SAR for analogs of ‘hit’ 1 with varied amines
O
Cl
Cl
N
R
Cl
O
Compound #
R
Bfl-1 FPa IC50
(lM)
Bfl-1 TR-FRETa IC50
(lM)
N
17
18
>50
>50
>50
>50
OH
N
N
N
19
>50
>50
20
21
22
>50
>50
N
N
0.3 0.2
0.8 0.1
0.4 0.1
0.45 0.03
O
N
N
N
23
24
25
1.7 0.2
0.6 0.3
2.2 0.4
1.0 0.1
0.8 0.1
1.1 0.1
N
NH
NH
O
26
N
N
N
5.6 0.4
2.0 0.4
NH
27
28
10.5 1.7
3.3 0.3
N
N
0.24 0.03
0.62 0.03
OH
O
N
N
N
N
N
N
N
29
30
31
32
33
34
35
0.3 0.2
4.9 0.8
9.6 2.0
1.2 0.4
>10
0.40 0.04
4.1 0.5
6.2 0.3
5.0 0.8
8.3 2.1
>10
N
N
N
N
N
OH
Ph
Ph-o-OMe
Ph-m-OMe
Ph-p-OMe
Ph-m-Me
Ph-m-CF3
>10
N
N
>10
>10
N
N
36
37
2.4 0.2
4.1 0.4
1.3 0.1
2.1 0.2
N
N
O
O
N
38
0.25 0.05
0.69 0.03
N
a
Data are the mean SEM.

J. R. Cashman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6560–6564
6563
tively, from the FP assay). Compared to ‘hit’ 1, carbon chains
longer that methyl on the piperazine terminal nitrogen decreased
chloro group
and unsaturation
O
Cl
N
potency (i.e., 20 and 21 with 1.7 0.2
pectively). Compared to 1, N-phenylpiperazine (i.e., 24 with IC50
4.9 0.8 M) had decreased potency and substitution around the
terminal phenyl ring in the ortho-, meta-, and para-positions (i.e.,
31, 33–35 with IC50 >9 M) showed lower potency in the FP assay.
Meta-Methoxy phenyl was the exception 32 showing a moderate
potency (i.e., IC50 (FP) value 1.2 0.4 M) but much improved
compared to the m-methyl (33) and m-CF3 (34) equivalents. Both
compounds (i.e., 27 and 28) had IC50 values >10 M for GST-Bfl-1
inhibition in the FP assay. The conclusion was that a hydrophilic
pocket was present in GST-Bfl-1 or a hydrogen bonding interaction
was occurring with GST-Bfl-1 and 32 between the meta-position to
increase inhibitory potency. Compared with 1, maleimides with N-
terminal piperazine benzyl or 3,4-methylenedioxybenzyl substitu-
lM and 10.5 1.7 lM, res-
Cl
N
substituents
l
other amines and
on N-phenyl
Cl
O
different N-substituted
N
piperazines
l
1
IC50 (µM)
0.6 0.1
l
Bfl-1 FP
Bfl-1 TR FRET 0.8 0.1
l
Figure 1. Chemical structure of ‘hit’ 1 from a high-throughput screen for inhibition
of GST-Bfl-1 and regions of ‘hit’ amenable to SAR work.
O
O
ents (i.e., 35 and 36, IC50 values of 2.4 0.2 lM and 4.1 0.4 lM,
respectively) were less potent GST-Bfl-1 inhibitors by two- and
four-fold, respectively. Maleimide 38 with a cinnamyl group on
the N-terminal piperazine was a potent GST-Bfl-1 inhibitor and
comparable to compounds 28 and 29 (i.e., IC50 values of
Cl
N
Cl
N
N
N
O
Cl
O
N
N
2
3
0.25 0.05
TR-FRET assay were similar for all three compounds (0.4–
0.69 M). It is postulated that the BH3 region of Bfl-1 where the
lM vs 0.3 0.2 lM and 0.3 0.2 lM). Data from the
Figure 2. Analogs of 1 without the maleimide chloro atom and double bond
moieties necessary for potent GST-Bfl-1 inhibition.
l
amine functionality of the inhibitor resides is large enough to
accommodate larger groups on the maleimide ring. A cinnamyl
group may induce additional pi–pi aromatic interactions with
Bfl-1 to increased inhibitory potency.
Other combinations of anilines and amines were prepared and
tested in order to look at structural synergistic effects (see Table 3
in Supplementary data): 3-methoxy, 4-nitro, 3-nitro, 4-methyl,
3-trifluoro, 4-trifluoro anilines in combination of the amines used
for compounds from Table 2. None of them showed submicromolar
potencies.
O
O
Cl
Cl
Cl
Cl
R1
R1
AcOH, 80 °C
+
O
N
NH2
O
O
4
5
6
O
N
A potent agent (i.e., 1) and two less potent agents (i.e., 19 and
21) were examined in cell-based viability studies or assays using
cancer cell lines or other mammalian cell lines.21 For compound
1, inhibition of human H69AR small cell lung tumor cell growth
Cl
NR2R3
R1
amine, THF, dioxane
O
7
was observed at a concentration of 10
not inhibitory to cell viability in a mammalian cell line but com-
pound 19 decreased cell viability at 15 g/mL. Thus, as a class it
lM. Compound 21 was
Scheme 1. Synthesis of maleimides following a two-step procedure.
l
does not appear that the compounds possess universal toxicity
but depending on the structure, certain N-aryl maleimide Bfl-1
inhibitors can decrease cancer cell viability or cause toxicity to
other mammalian cell lines.
chirality. Compound 24 with R stereochemistry at C-3 of pipera-
zine showed considerable Bfl-1 inhibitory potency (i.e., IC50 values
for FP and TR-FRET assays of 0.6 0.3
l
M and 0.8 0.1
l
M, respec-
In summary, more than 280 substituted maleimides were pre-
pared in a medium throughput format from readily available
starting materials. SAR analysis revealed the effects of substitu-
tion on the N-phenyl ring and variation of amines on the malei-
mide ring system, and the necessity of a chloro substituent and
a double bond in the maleimide ring for inhibition of GST-Bfl-1.
The N-3,4-dichloroaryl moiety of the original ‘hit’ 1 provided
the optimal substitution pattern on the N-aryl ring. Optimal
amines for substitution of one maleimide chloride atom included
hydrophilic amines or amines that could participate in hydrogen
bonding or pi–pi interactions. Submicromolar IC50 values for inhi-
bition of Bfl-1 were observed for maleimides substituted with
dimethylamine, N-methylpiperazine, and piperazines (i.e., 21,
22, 24, 28, 29, and 38) containing water-soluble groups or a cin-
namyl group on the terminal nitrogen atom. For one subset of
piperazines possessing a center of chirality, considerable stereose-
lectivity of Bfl-1 inhibition was observed (i.e., 24 > 25). The SAR
studies reported herein provide valuable information for the
structural requirements for inhibition of Bfl-1 by maleimides
and may provide insight into development candidates for anti-
cancer therapeutics.
tively). In contrast, compound 25 with the S stereochemistry had
considerably less GST-Bfl-1 inhibitory potency (i.e., fourfold great-
er IC50 value for the FP assay and approximately the same IC50 va-
lue for the TR-FRET assay).
Accordingly, another sublibrary of compounds based on
compound 1 was prepared that incorporated the 3,4-dichloro-
phenyl moiety and modification of substituted piperazines to
the maleimide structure. Modifications of the 4-N-substituent
of the piperazine would allow for property adjustment such
as solubility, and metabolic stability in further studies. A com-
plete list of 4-N-substituents tested is available in the Supple-
mentary data. Compounds 27–38 (Table 2) are the most
representatives of the SAR trends for this region of the mole-
cule. N-substitution included alkyls, hydroxyl, amine and ether
groups, non-substituted and substituted phenyl, benzyl, and
cinnamyl groups.
Hydrophilic hydroxy ethyl groups at the terminal piperazine
nitrogen atom were associated with the most potent GST-Bfl-1
inhibitory functional activity in this series (i.e., compounds 28
and 29 with IC50 values 0.24 0.03 lM and 0.3 0.2 lM, respec-

6564
J. R. Cashman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6560–6564
17. Day, C. L.; Chen, L.; Richardson, S. J.; Harrison, P. J.; Huang, D. C. S.; Hings, M. G.
J. Biol. Chem. 2005, 280. High-throughput FP assay: Chemical inhibitors were
dissolved in 10% DMSO at 100 M stock concentration. From this solution, 4
were incubated with 8 L of Bfl-1 working solution (7.4 nM GST-Bfl-1 fusion
Acknowledgments
l
lL
This work was funded by grant 1 X01 MH077632-01 and UO1-
CA-113318 to J.C.R. We thank Dr. Ying Su for his help with the
work. We thank Alyssa M. Morgosh for her help in providing ana-
lytical data for the manuscript. We thank Dr. Marion Lanier for her
careful editing of the manuscript.
l
protein in 25 mM Bis-Tris pH 7.0, 1 mM TCEP, 0.005% Tween 20) at room
temperature away from direct sunlight in 384-well black plates (Greiner Bio-
One, Monroe, NC). After 1 h, FITC-Bid BH3 peptide (8 lL of 5.6 nM in 25 mM
Bis-Tris pH 7.0, 1 mM TCEP, 0.005% Tween 20) was added, and plates were
incubated at room temperature away from direct sunlight. Fluorescence
polarization was measured using an Analyst GT plate reader (Molecular
Devices, Inc., Sunnyvale, CA) using fluorescein filters (excitation filter 485 nm,
emission filter 530 nm, dichroic mirror # 505 nm). Data analysis was done
using CBIS software (ChemInnovations, Inc., San Diego, CA). Dose–response
confirmation: Chemical inhibitors were serially diluted in DMSO then in water
so final concentration was in 10% DMSO. Ten concentrations of each compound
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.046.
were used. Each inhibitor (4
(Greiner Bio-One, Monroe, NC) followed by GST-Bfl-1 solution described above
(8 L). Plates were incubated for 1 h at 4 °C. FITC-Bid BH3 peptide solution
described above (8 L) was added, and plates were incubated for 4 h at room
lL) was transferred to 384-well black plates
l
References and notes
l
temperature. Fluorescence polarization was measured at described above. Data
analysis was performed using sigmoidal dose–response equation through non-
linear regression.
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18. Peptide Synthesis: FITC-Bid BH3 peptide was synthesized on the ACT 350
Multiple Peptide Synthesizer using Fmoc chemistry on Wang resin. FITC was
coupled to the N-terminus while the peptide was still on the resin. Peptide was
cleaved from the resin using 94% TFA/2.5% H2O/2.5% EDT/1% TIS for 2 h at room
temperature. Crude peptide was purified on a Gilson HPLC with 0.1% TFA
water/acetonitrile and characterized by mass spectroscopy on an Applied
Biosystems Voyager System 6264 using MALDI-TOF ionization. TR-FRET Assay:
Dose–response curves contained 10 concentrations of compounds obtained
using twofold serial dilution. Compounds were serially diluted in 100% DMSO,
and then diluted with water to 10% final DMSO concentration. Compounds in
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10% DMSO (4
white small-volume plates (784075). Each compound concentration was
assayed in duplicate wells. Columns 1–2 and 23–24 contained 4 L of 10%
DMSO. Assay buffer (8 L of 25 mM Bis-Tris, pH 7.0, 1 mM TCEP, 0.005% Tween
20) was added to columns 1–2, which were reserved for positive controls,
using a WellMate bulk dispenser (Matrix). Bfl-1 working solution (8 L of
7.4 nM GST-Bfl-1 in assay buffer) was added to columns 3–24 using WellMate
bulk dispenser (Matrix). Columns 23–24 represent negative control wells.
Plates were incubated for 1 h at +4 °C. Freshly prepared FITC-Bid/Tb-Ab
lL) were transferred into columns 3–22 of Greiner 384-well
l
l
l
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was added to the whole plate using WellMate bulk dispenser (Matrix). Plates
were incubated for 4 h at room temperature protected from direct light.
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