Induction of apoptosis promoted by Bang52; a small molecule that downregulates Bcl-xL

Article abstract of DOI:10.1016/j.bmcl.2009.03.067

Cancer cells evade death by over-producing specific proteins that inhibit apoptosis. One such group of proteins is the Bcl-2 family, of which Bcl-x_L is an important member. This protein binds and inhibits BAK, another protein that promotes apop

Full text of DOI:10.1016/j.bmcl.2009.03.067

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2429–2434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Induction of apoptosis promoted by Bang52; a small molecule
that downregulates Bcl-xL
Matteo Rossi a, Jeong-kyu Bang a, Sharlyn Mazur a, Jaclyn A. Iera b, Darren C. Phillips c,
Gerard P. Zambetti c, Daniel H. Appella b,
*
a Laboratory of Cell Biology, NCI, NIH, Bethesda, MD 20892, United States
b Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD 20892, United States
c Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN 38105, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer cells evade death by over-producing specific proteins that inhibit apoptosis. One such group of
proteins is the Bcl-2 family, of which Bcl-xL is an important member. This protein binds and inhibits
BAK, another protein that promotes apoptosis. While the development of chemical inhibitors that block
Bcl-xL–BAK association have been the focus of intense research efforts, we demonstrate in this manu-
script an alternative strategy to downregulate Bcl-xL. We have identified a small molecule (Bang52) that
induces apoptosis in a lymphoblast-derived cell line by lowering levels of Bcl-xL. Since Bang52 bears no
resemblance to any chemical binder of Bcl-xL we believe that degradation of the protein is stimulated by
a new type of pathway. These findings highlight a novel approach to the development of small molecules
that promote apoptosis.
Received 12 February 2009
Accepted 17 March 2009
Available online 21 March 2009
Keywords:
Cancer
Apoptosis
Bcl-xL
Ó 2009 Elsevier Ltd. All rights reserved.
Approaches in the development of new anticancer therapeu-
tics are focusing on the selective induction of tumor cell death
through the activation of apoptotic pathways. Apoptosis is a
highly controlled cellular process, that is, essential for the main-
tenance of normal tissue homeostasis and embryonic develop-
ported to selectively induce apoptosis in cancer cells that possess
mutant p53.8–11 Two of the most studied molecules in this area
are PRIMA-1 and the related mono-methyl ether derivative PRI-
MA-1Met (Fig. 1).12–19 Both PRIMA molecules are reported to re-
store the proper function to many different mutant forms of p53,
although the exact targets of these molecules is not clear. In our
own research, we previously reported a very simple chemical scaf-
fold to probe for reactivation of mutant p53 and we identified a
molecule (1) that was able to induce apoptosis in cells that had
been engineered to express different forms of mutant p53.20 While
these results served as a model for selectively targeting cells with
mutant p53, we later found that 1 did not reactivate p53 in a hu-
man B-lymphoblast-derived cell line that expresses a specific form
of mutant p53, M237I.
ment. However,
a failure to appropriately induce apoptosis
often results in the accumulation of defective cells that are
symptomatic of disease states such as cancer.1,2 Therefore, small
molecules that selectively induce apoptosis in tumor cells are of
considerable interest in the clinical management of cancer. The
identification of several key protein targets that regulate apopto-
sis has resulted in the development of molecules that bind to
one (or more) of these proteins and activate apoptotic path-
ways.3 At the same time, cell-based screens have also identified
interesting compounds that induce apoptosis. Some of these
studies have reported molecules with the unique ability to re-
store activity to mutant forms of the protein p53.4
To test whether the original molecular scaffold could be modi-
fied to achieve reactivation of p53, we developed a new synthetic
p53 functions as an important guardian of cellular function. If a
healthy cell is damaged, p53 is able to inhibit cell growth or induce
apoptosis. Mutated forms of p53 lose these functions, and there-
fore allow the uncontrolled growth of damaged cells. Mutant forms
of p53 are present in about half of all cancers.5–7 Restoration of
activity to mutant p53 by small molecules has been a recent dis-
covery, and by now, a small collection of molecules have been re-
O
O
O
HCl H2N
HCl H2N
AcO
Ph
O
Ph
N
F
OR
N
H
F
OH
PRIMA-1, R = H
1
Bang52
PRIMA-1Met, R = Me
* Corresponding author. Tel.: +1 3014511052; fax: +1 3014804977.
E-mail address: appellad@niddk.nih.gov (D.H. Appella).
Figure 1. Molecules tested for p53 reactivation.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.067

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M. Rossi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2429–2434
approach to make a small library of molecules. The ability of each
molecule to induce apoptosis was investigated in three cell-lines
that differ in their p53 status (cells were either p53 positive, neg-
ative, or expressed the M237I mutant). In this manuscript, we de-
scribe the chemistry to make the library, the identification of an
active molecule (Bang 52) from this library, and the effects of
Bang52 on apoptosis in comparison to PRIMA-1. Surprisingly, both
molecules kill cells independently of the p53 status, but Bang52 in-
duces apoptosis in a unique manner involving reduction of the pro-
survival factor Bcl-xL.
Chemistry. The initial goal for modification of 1 was to increase
the stability in cells. To achieve this, we replaced the ester in the
sidechain of 1 with an amide. The synthesis of these molecules
(i.e., Bang52 and its analogs) was developed on a solid support so
that numerous derivatives could be rapidly prepared (Fig. 2). First,
the Weinreb amide derivative of Alloc-protected diaminopropionic
acid (Alloc-Dap) was coupled to p-nitrophenylcarbonate Wang re-
sin. Reaction with an excess of a phenyl Grignard reagent was then
performed, followed by Alloc deprotection and acylation of the pri-
mary amine. Cleavage from the resin then afforded the crude prod-
ucts, which were purified by HPLC. Using this procedure, 54
different analogs were prepared with variations at the aromatic
position (Ar) and the sidechain (R1). Ultimately, Bang52 was iden-
tified as the only molecule from the library that induces apoptosis
in our new cell-based assay. For comparison to Bang52, PRIMA-1
was also prepared by following published procedures in the
literature.
Biological results. In the studies described below, the activities of
Bang52 and PRIMA-1 were compared across three human B lym-
phoblast cell lines: TK6 (wild-type p53), NH-32 (null for p53),21
and WTK1 (M237I mutant p53).22,23 These cell lines were derived
from the same progenitor, WIL2,21,24,25 and differ only in their
p53 status. Therefore, results obtained across these three cell lines
directly show whether p53 is a crucial component for induction of
apoptosis. As a control, doxorubicin (DOX) was also included in the
studies because there is a clear mechanism of action for this mol-
ecule that involves activation of p53.
The effect of p53 status oncellular responses to Bang52, PRIMA-1,
and DOX was examined in TK6, NH-32, and WTK1 cells by flow cyto-
metric analysis of DNA cell cycle profiles.26,27 An indication of the
amount of cell death was obtained by analysis of the sub-G0/G1
DNA content of each cell cycle histogram. As shown in Figure 3, all
a large fraction of cells with sub-G0/G1 DNA content in the TK6 cells,
which express wild-type p53. This effect was abrogated in NH-32
and WTK1 cells, which are null for p53 or contain a mutant p53,
respectively. In contrast to DOX, Bang 52 and PRIMA-1 induced sig-
nificant amounts of sub-G0/G1 DNA in all three cells lines (Fig. 3).
The requirement for fully functional p53 in the B lymphoblast
cell lines was confirmed by Western Blot analysis using antibodies
to detect: p53 expression, p53 activation via phosphorylation of
Ser15, and p53 transcriptional activity through detection of
p21Cip1/Waf1. In TK6 cells, which express wild-type p53, phospho-
p53 (at Ser15) and p21 expression was observed following DOX
treatment. In the WTK1 cells, which express mutant p53, DOX pro-
motes formation of phospho-p53 without induction of p21. This
result is consistent with loss of p53 transcriptional activity in this
cell line. In contrast with DOX, Bang52 and PRIMA-1 did not induce
phospho-p53 or p21 expression. Collectively, these data indicate
that Bang52 and PRIMA-1 induce apoptosis in the B lymphoblast
cells lines independently of the p53 status (Fig. 4).
Several additional methods were used to examine the mecha-
nism of PRIMA-1 or Bang52-induced cell death. Apoptosis is
accompanied by loss of organization in the plasma membrane,
and a marker of this event is the translocation of the phospholipid
phosphatidylserine (PS) from the inner to the outer portion of the
membrane. Annexin V is a PS-binding protein that can be conju-
gated to fluorescent groups and used in FACS analysis to determine
cell viability. Annexin V is typically used in conjunction with
7-amino-actinomycin (7-ADD), a fluorescent molecule that is
impermeable to cells with intact membranes but permeable to
dead cells. In the FACS analysis of untreated TK6 cells (Fig. 5A),
the majority of cells appeared negative for staining with Annexin
V and 7-ADD, indicating that the cells were healthy. Upon treat-
ment with Bang52 for 8 h, FACS analysis showed significant shifts
in the cell population. Specifically, the percentage of healthy cells
decreased significantly from 68% to 24% and, in addition, two
new populations of cells were observable. The most significant
new population of cells was positive for both Annexin V and 7-
ADD, indicating these cells had already died either by necrosis or
apoptosis. An additional cell population that is more positive for
Annexin V than for 7-ADD was also observable, indicating that
these cells were undergoing apoptosis. Under the same conditions,
PRIMA-1 had no effect on the cells populations compared to the
untreated cells.
three cell lines treated with 25
considerable increases in sub-G0/G1 phase DNA after 72 h as com-
pared to untreated cells. As a control, the same cells were treated
with 0.1 lM of DOX. Since DOX is known to induce DNA damage
and stimulate apoptosis through a p53-mediated pathway, results
with this molecule demonstrated how each cell line responds
according to its p53 status. As expected, DOX treatment produced
l
M of Bang52 or PRIMA-1 showed
The mitochondria act as a point of convergence for numerous
apoptotic signaling pathways. While the precise mechanisms of
mitochondrial involvement in apoptosis are still the subject of ongo-
ingresearch, thelossof theinnermitochondrial membranepotential
is associated with the release of pro-apoptotic factors, such as cyto-
chrome c, into the cytosol. Furthermore, loss of mitochondrial func-
tion is linked to the activation of caspases and caspase-dependent
nucleases that induce DNA fragmentation, which are key features
of apoptosis.28,29 Consistent with the previous results, a probe for
mitochondrial depolarization showed that Bang52 significantly im-
pactsthemitochondria. Thereleaseofpro-apoptoticfactorsfromthe
mitochondria is often associated with mitochondrial depolarization.
The cationic dye JC-1 selectively accumulates in mitochondria such
that its fluorescence properties shift from green (JC-1 monomer,
FL1H) to orange (JC-1 aggregates in the mitochondria, FL2H). Mito-
chondrial depolarization reduces JC-1 concentration within the cell,
resulting in decreased orange fluorescence.30,31 In the treatment of
TK6 cells with Bang52, significant depolarization of the mitochon-
dria is observed after 8 h relative to a positive control (carbonyl cya-
nide3-chlorophenylhydrazone(CCCP)). In contrast, PRIMA-1caused
no observable depolarization under the same conditions (Fig. 6).
The presence of additional apoptotic indicators were examined
for all three cell lines under conditions where the cells were trea-
ted with Bang52, PRIMA-1, or DOX. During apoptosis, several cas-
NHAlloc
NHAlloc
Ar
Me
O
O
ArMgX
N
O
O
OMe
N
H
N
H
THF, 0 C
Wang
Resin
O
O
O
H
N
O
H
N
R1
O
R1
Ar
1. Pd[(PPh3)]4
Ar
H2N
2. R1COOH, HBTU
3. TFA
O
N
O
H
O
Bang52 and derivatives
Figure 2. Synthetic strategy to make Bang52 and associated derivatives. The
synthesis was performed on solid support.

M. Rossi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2429–2434
2431
TK6
CTR
Bang52
PRIMA-1
dox
Sub-G1
G1
S
G2
0%
20%
40%
60%
80%
100%
NH-32
CTR
Bang52
PRIMA-1
dox
Sub-G1
G1
S
G2
0%
20%
40%
60%
WTK1
80%
100%
CTR
Bang52
PRIMA-1
dox
Sub-G1
G1
S
G2
0%
20%
40%
60%
80%
100%
Figure 3. Bang52 and PRIMA-1 induced apoptosis independently of p53 function in human B lymphoblast cell lines. TK6 (WTp53), NH32 (p53À/À) and WTK1 (MTp53) cells
treated with Vehicle (lane 1), Bang 52 (25 M; lane 2), PRIMA-1 (25 M; lane 3) or Doxorubicin (0.1 M; lane 4) for 72 h. Cells were harvested, washed twice in ice cold PBS
and resuspended in hypotonic fluorochrome solution (50 g/ml propidium iodide, 25 g/ml RNase, 0.1% sodium citrate, 0.1% Triton X-100). Cell samples were incubated 1.5 h
l
l
l
l
l
at 4 °C in the dark prior to DNA cell cycle analysis by flow cytometry. The percentage of cells in each phase of the cell cycle and the percentage of apoptotic cells was
subsequently determined from 10,000 events per histogram.
pase proteins are converted from the inactive (pro-form) to the
active form. Treatment of the three cell lines with Bang52, pro-
duced a strong increase in the level of cleaved caspase-3, and a
simultaneous decrease in the level of pro-caspase-3 (Fig. 7). Cleav-
age of poly(ADP-ribose) polymerase (PARP) by activated caspases
is another indicator of apoptosis. Consistent with caspase-3
activation, enhanced PARP cleavage was also observed. In contrast,
PRIMA-1 induced only modest changes in the levels of pro-cas-
pase-3, caspase-3, and PARP under the same conditions.
wanted to determine the impact of small alterations in Bang52
on the activity of the molecule and also probe whether degradation
of Bang52 could account for the activity of the parent molecule.
The importance of the difluoroacetamide group is highlighted by
comparing Bang3, 53, and 44 (Fig. 8). Replacement of the two fluo-
rines with hydrogens (Bang3) eliminated the ability of the molecule
to induce cell death, whereas monofluoro or trifluoroacetamide
derivatives exhibited diminished activity. The difluoroacetamide
group is not the only portion of the molecule responsible for the
activity. Removal of the phenyl ketone or the primary amine (as seen
in Bang56-57) completely eliminated all activity, resulting in mole-
Several additional derivatives were examined for their ability to
induce cell death in a manner similar to Bang52. In general, we

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M. Rossi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2429–2434
cules that are inactive and non-toxic (See Supplementary data for
results of cellular assays).
TK6
NH32
WTK1
1 2 3 4 1 2 3 4
1 2 3 4
Bcl-2 and Bcl-xL are anti-apoptotic members of the Bcl-2 family
that maintain mitochondrial integrity by preventing the release of
pro-apoptotic factors.28,29 Cancer cells expressing elevated levels of
Bcl-2 and/or Bcl-xL possess a survival advantage and are in some
cases ‘addicted’ to these oncogenes for survival. Therefore, loss of
functions associated with these proteins leads to apoptosis.32 Fur-
thermore, enhanced expression of Bcl-2 and Bcl-xL are commonly
associated with drug resistance and poor prognosis in several types
of cancer.1,2,33–35 Strategies that antagonize the function of Bcl-2
proteins are therefore the focus of therapeutic development.36–38
Western blot analysis showed that Bang52 significantly reduced
the amount of Bcl-xL in all three cell types of this study (Fig. 9).
Similarly, PRIMA-1 also reduced the amount of this protein,
Phospho-p53 (Ser15)
Total p53
p21Cip1/Waf1
β-actin
Figure 4. Bang52 does not induce p53 phosphorylation or mediate p53-dependent
transcription. TK6, NH32 and WTK1 cells were treated with Vehicle (lane 1), Bang 52
(25 lM; lane 2), PRIMA-1 (25 lM; lane 3) or Doxorubicin (0.1 lM; lane 4) for 8 h prior
toproteinextractionandWesternBlotanalysisasdescribedinSupporting Information.
Samples were probed for p53, phosphor-p53 (Ser15), p21cip1/Waf1 and b-actin.
Figure 5. Bang52 induces apoptosis in TK6 cells. TK6 cells were treated with vehicle, Bang52 (25 lM) or PRIMA-1 (25 lM) for 8 h. Cells were harvested, washed twice with
ice cold PBS and then stained with Annexin-V-PE and 7-ADD as per the manufacturers instructions (BD Biosciences). Samples were analyzed by flow cytometry to determine
Annexin-V and/or 7-ADD positive populations. The flow cytometry histograms are representative of three independent experiments. Numbers represent the percentage of the
total cell population present in each quadrant.
Figure 6. Bang52 induces depolarization of the inner mitochondrial membrane potential (
CCCP (100 M) for 8 h. Twenty minutes prior to the end of the treatment period, cells were loaded with JC-1 (5
and resuspended in 250 l of PBS. JCL-1 fluorescence was determined on dual parameter histograms of FL1H (JC-1 monomers) vs FL2 H (JC-1 aggregates) by flow cytometry.
Data depicts flow cytometry histograms that are representational of three independent experiments.
D
W
m). TK6 cells were treated with vehicle, Bang52 (25
lM), PRIMA-1 (25 lM) or
l
lg/mL). Cells were harvested, washed twice with ice cold PBS
l

M. Rossi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2429–2434
2433
TK6
NH32
WTK1
TK6
NH32
WTK1
1 2 3 4 1 2 3 4 1 2 3 4
1 2 3 4 1 2 3 4 1 2 3 4
Cleaved PARP
Pro-Caspase-3
Bcl-xL
Bcl-2
β-actin
Cleaved Caspase-3
Figure 9. Bang52 induces degradation of Bcl-xL. TK6, NH32 and WTK1 cells treated
with Vehicle (lane 1), Bang 52 (25 M; lane 2), PRIMA-1 (25 M; lane 3) or
Doxorubicin (0.1 M; lane 4) for 8 h prior to protein extraction and Western Blot
l
l
l
β-actin
analysis as described in Supporting Information. Samples were probed for Bcl-xL,
Bcl-2 and b-actin.
Figure 7. Biochemical evidence that Bang52 induced cell death is independent of
p53 status. TK6, NH32 and WTK1 cells treated with Vehicle (lane 1), Bang 52
(25 lM; lane 2), PRIMA-1 (25 lM; lane 3) or Doxorubicin (0.1 lM; lane 4) for 8 h
prior to protein extraction and Western Blot analysis as described in Supporting
Information. Samples were probed for cleaved PARP, caspase-3 and b-actin.
and minimally effective in the NH32 cells. In contrast, Bang52
was highly effective in all three cell lines. The effect seems highly
specific to Bcl-xL because the level of Bcl-2 is unchanged. Quantita-
tive RT-PCR demonstrated that Bcl-xL and Bcl-xS mRNA levels re-
mained
unchanged
following
treatment
with
Bang52
(Supplementary data), indicating that Bang52 does not affect Bcl-
xL at the transcriptional level. Taken together, these results suggest
that Bang52 affects Bcl-xL at the protein level.
O
O
O
HCl H2N
O
HCl H2N
O
HCl H2N
O
Ph
Ph
Ph
To confirm the specificity of action, the effects of Bang52 were
compared in regular HeLa cells and a derivative HeLa cell line that
stably expresses Bcl-xL at elevated levels (called HeLa-XL).39 If
Bang52 targets Bcl-xL for degradation, then over-expression of
Bcl-xL should protect the HeLa-xL cells from death. This effect
was clearly observed, especially at higher concentrations of Bang52
(Fig. 10). Treatment with 100 lM Bang52 substantially increased
the percentage of sub-G1 DNA content in HeLa cells while produc-
ing no effect in HeLa-xL cells.
F
F
F
H3C
N
H
Bang3
N
H
N
H
F
Bang44
Bang53
O
NH2 HCl
Ph
O
O
F
F
N
H
N
H
F
F
Bang56
Bang57
Bcl-xL is a well-known antiapoptotic factor that has been a
target for inhibition by several research groups.3 The association
of Bcl-xL with BAK is an example of a protein-protein interaction
that has provided a structural basis for the design of inhibi-
tors.40–43 These efforts have led to a well-known set of Bcl-xL
inhibitors that define the molecular feature for inhibition of this
protein with BAK.38,44 The compound Bang52 does not share any
chemical features with these known inhibitors of Bcl-xL. There-
fore, it is likely that Bang52 exerts its effects by a novel pathway
Figure 8. Analogs of Bang52 that were tested to determine the molecular
requirements for elimination of Bcl-xL. None of these molecules had the activity
of Bang52 based on FACS analysis using PI staining.
although much less dramatically and in variable amounts. For in-
stance, PRIMA-1 was most effective at reducing Bcl-xL in the
WTK1 cells, while it was moderately effective in the TK6 cells
90.0
HeLa and HeLa-XL
80.0
48h Bang52
treatment
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
25 uM Bang52
50 uM Bang52
HeLa HeLa-XL
100 uM Bang52
Figure 10. Bcl-xL hyperexpression inhibits Bang52-mediated cell death. HeLa cells hyperexpressing Bcl-xL (HeLa-xL)39 and the parental cell line transfected with the empty
vector (HeLa) were treated for 48 hours with 0, 25, 50 or 100 M Bang52. The degree of apoptosis was determined from the sub-G0/G1 DNA content fraction in cell cycle
histograms analyzed by flow cytometry as described in Figure 3. Data are presented as the means s.e.m. of three independent experiments.
l

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M. Rossi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2429–2434
4. Selivanova, G.; Wiman, K. G. Oncogene 2007, 26, 2243.
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that does not involve direct targeting of the protein. Recently,
two other group have independently reported the isolation of
natural products that similarly eliminate Bcl-xL. Wang and co-
workers reported that a pyrrolizidine alkaloid (clivorine) derived
from plants induced apoptosis in hepatocytes by downregulation
of Bcl-xL.45 Similarly, Imoto and colleagues found that incednine,
a molecule isolated from the culture of a Streptomyces, induced
apoptosis in HEK293T cells also by downregulation of Bcl-xL.46
As with Bang52, the natural products also have no obvious sim-
ilarity with the known binders of Bcl-xL. At the present time, the
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With the development of chemical biology, small molecules
that selectively perturb cellular pathways can be valuable re-
search tools. The recent emergence of p53 as a target for small
molecules has fueled the possibility of designing molecules to in-
duce apoptosis in some of the most resistant cancers. However, a
number of the p53-targeting molecules possess very complex
mechanisms of action that are difficult to fully characterize. In
this regard, there is increasing evidence that PRIMA-1 targets
multiple entities within a cell15,47 and the results from our study
indicate that p53 is not required for PRIMA-1 to induce cell
death. Since cancer is a highly heterogeneous disease, we feel
that it is necessary to develop a chemical arsenal of molecules
that may induce apoptosis by a number of different cellular
pathways. While such molecules may not developed into drugs
to treat cancer, well-defined chemical probes for apoptosis may
provide valuable insight into the biological functions of tumor
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The authors gratefully acknowledge financial support from the
Intramural Research Program of NIH, CA6320 and NIH/NCI Cancer
Center Support Grant CA21765. We are also grateful for the sup-
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