Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins

Article abstract of DOI:10.1021/jm060460o

A structure-based approach was employed to design a new class of small-molecule inhibitors of Bcl-2. The most potent compound 5 (TW-37) binds to Bcl-2 with a K_i value of 290 nM and also to Bcl-xL and Mcl-1 with high affinities. Compound 5 potently inhibits cell growth in PC-3 prostate cancer cells with an IC₅₀ value of 200 nM and effectively induces apoptosis in a dose-dependent manner.

Full text of DOI:10.1021/jm060460o

© Copyright 2006 by the American Chemical Society
Volume 49, Number 21
October 19, 2006
Letters
proteins such as Bad, Bid, and Bim.1-4 The experimental
structures of Bcl-2 and its closely related homologous protein
Bcl-xL show that the BH1a (Bcl-2 homology domain 1), BH2,
and BH3 domains in Bcl-2 and Bcl-xL form a well-defined
hydrophobic surface binding groove, known as the BH3 binding
groove, into which Bad, Bid, and Bim bind.5-7 This binding
groove in Bcl-2 and Bcl-xL proteins is essential for their anti-
apoptotic function.5-7 We and others have hypothesized that
small molecules binding in this BH3 binding groove in Bcl-2/
Bcl-xL may be capable of blocking the heterodimerization of
Bcl-2 and Bcl-xL with pro-apoptotic members in the Bcl-2
protein family. This in turn may inhibit the anti-apoptotic
function of Bcl-2 and Bcl-xL and induce apoptosis in cancer
cells in which Bcl-2/Bcl-xL is overexpressed. Design of non-
peptide small molecule inhibitors of Bcl-2 and Bcl-xL is
currently an exciting research area for the development of new
cancer agents.8-14
Structure-Based Design of Potent Small-Molecule
Inhibitors of Anti-Apoptotic Bcl-2 Proteins
Guoping Wang,† Zaneta Nikolovska-Coleska,†
Chao-Yie Yang,† Renxiao Wang,† Guozhi Tang,† Jie Guo,†
Sanjeev Shangary,† Su Qiu,† Wei Gao,† Dajun Yang,†
Jennifer Meagher,‡ Jeanne Stuckey,‡ Krzysztof Krajewski,§
Sheng Jiang,§ Peter P. Roller,§ Hatice Ozel Abaan,
York Tomita, and Shaomeng Wang*,†
ComprehensiVe Cancer Center and Departments of Internal
Medicine, Pharmacology and Medicinal Chemistry, and Life
Sciences Institute, UniVersity of Michigan, 1500 E. Medical Center
DriVe, Ann Arbor, Michigan 48109, Laboratory of Medicinal
Chemistry, National Cancer Institute-Frederick, National Institutes
of Health, Frederick, Maryland 21702, and Lombardi
ComprehensiVe Cancer Center, Georgetown UniVersity Medical
Center, Washington, D.C. 20057
ReceiVed April 18, 2006
Using structure-based database screening, we discovered that
(-)-gossypol15 (1), a natural product isolated from cotton seeds
and roots, binds to Bcl-2 and Bcl-xL with quite high affinities.
(-)-Gossypol has Ki values of 320 and 480 nM for Bcl-2 and
Bcl-xL proteins, respectively, in our competitive FP-based
binding assays (Table 1 and Supporting Information). The
binding of racemic gossypol to Bcl-xL has been confirmed
independently.12 Interestingly, our competitive binding data
show that (-)-gossypol also binds to Mcl-1 protein, another
anti-apoptotic Bcl-2 member, with a Ki value of 180 nM (Table
1). (-)-Gossypol is currently being clinically evaluated as an
anticancer drug and demonstrates acceptable and manageable
toxicity with evidence of single-agent antitumor activity in
patients with advanced malignancies.16 To date, (-)-gossypol
is the only orally available small-molecule inhibitor of Bcl-2,
Bcl-xL, and Mcl-1 that has advanced into clinical trials. Hence
it represents a promising lead compound for the development
of non-peptidic small-molecule inhibitors of anti-apoptotic Bcl-2
proteins as a new class of anticancer drugs. Herein, we wish to
report our structure-based design of a new class of small-
Abstract: A structure-based approach was employed to design a new
class of small-molecule inhibitors of Bcl-2. The most potent compound
5 (TW-37) binds to Bcl-2 with a Ki value of 290 nM and also to Bcl-
xL and Mcl-1 with high affinities. Compound 5 potently inhibits cell
growth in PC-3 prostate cancer cells with an IC50 value of 200 nM and
effectively induces apoptosis in a dose-dependent manner.
Bcl-2 is a critical arbiter of apoptosis and functions as a potent
anti-death molecule.1 Bcl-2 is overexpressed in many types of
human cancer,2-4 and such overexpression protects cancer cells
from a variety of apoptotic stimuli, including those associated
with cancer chemotherapeutic agents, and confers on cancer cells
resistance to current therapeutic agents.2-4 Bcl-2 is a promising
molecular target for the design of an entirely new class of
anticancer drugs aimed at overcoming resistance of cancer cells
to apoptosis.2-4
The anti-apoptotic function of Bcl-2 is attributed, at least in
part, to its ability to heterodimerize with pro-apoptotic Bcl-2
* To whom correspondence should be addressed. Phone: 734-615-0362.
Fax: 734-647-9674. E-mail: shaomeng@umich.edu.
† Comprehensive Cancer Center and Departments of Internal Medicine,
Pharmacology and Medicinal Chemistry, University of Michigan.
‡ Life Sciences Institute, University of Michigan.
a Abbreviations: BH1-3, Bcl-2 homology domain 1-3; FP, fluorescence
polarization; ELISA, enzyme-linked immunosorbent assay; HSQC, hetero-
nuclear single quantum coherence; TUNEL, terminal dUTP nick-end
labeling; WST, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium monosodium salt.
§ National Cancer Institute-Frederick.
Georgetown University Medical Center.
10.1021/jm060460o CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/12/2006

6140 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Letters
Table 1. Binding Affinities of Small-Molecule Inhibitors to Bcl-2,
Bcl-xL, and Mcl-l as Determined Using a
Fluorescence-Polarization-Based Binding Assay (FP) and
Enzyme-Linked Immunosorbent Assay (ELISA)a
Bcl-2
Bcl-xL
Mcl-l
FP
ELISA
IC50 ( SD
(µM)
FP
FP
Ki ( SD
Ki ( SD
Ki ( SD
compound
(µM)
(µM)
(µM)
(-)-gossypol
0.32 ( 0.02
24.1 ( 2.1
8.3 ( 1.1
0.93 ( 0.11
0.29 ( 0.06
25b
0.5 ( 0.1
53.1 ( 0.1
7.2 ( 1.6
1.6 ( 0.5
0.7 ( 0.3
0.48 ( 0.04
0.18 ( 0.01
2
3
4
5
6
1.11 ( 0.4
0.26 ( 0.01
Bid BH3
0.011 ( 0.001
0.007 ( 0.001 0.012 ( 0.001
peptidec
Bim BH3
peptided
0.011 ( 0.003
a Three to seven independent experiments were performed for each
compound. b Partial binding was observed at 100 µM, and the Ki value was
an estimate. c Residues 79-99 (also see Supporting Information). d Residues
81-106 (also see Supporting Information).
Figure 2. Predicted binding models of Bcl-2 in complex with (a) (-)-
gossypol, (b) Bim BH3 peptide, and designed compounds (c) 2 and
(d) 5. Bcl-2 is shown in surface representation where carbon, oxygen,
nitrogen, and sulfur atoms are colored in gray, red, blue, and orange,
respectively. The carbon and oxygen atoms in gossypol, 2, and 5 are
shown in yellow and red, respectively. The Bim BH3 peptide is shown
as a light-blue helix. Hydrogen bonds are depicted as dotted lines in
cyan.
We used a simple phenyl ring tethered to the polyphenol ring
via an amide linker to mimic the hydrophobic interaction
between this part of (-)-gossypol and Bcl-2. Our predicted
binding model (Figure 2) shows that 2 indeed mimics most,
but not all, of the crucial interactions observed between (-)-
gossypol and Bcl-2.
Compound 2 was synthesized (Supporting Information) and
determined to bind to Bcl-2 with a Ki value of 24.1 µM in our
FP-based binding assay (Table 1). Consistent with our modeling
prediction (Figure 2), 2 has a significant binding affinity to Bcl-2
but is 80-times less potent than (-)-gossypol.
Analysis of the binding models for (-)-gossypol and 2
suggests that 2 can be further optimized to enhance its
interaction with Bcl-2. For example, the hydrophobic pocket
occupied by the isopropyl group in 2 and in (-)-gossypol can
accommodate a larger hydrophobic group. Modeling suggests
that a benzyl group may be used to replace the isopropyl group
to optimize the interaction at this site (Supporting Information),
and this led to the design of 3 (Figure 1 and Supporting
Information). Compound 3 was synthesized (Supporting Infor-
mation) and was determined to bind to Bcl-2 with a Ki value of
8.3 µM (Table 1). Hence, 3 is 5-times more potent than 2,
supporting our modeling prediction.
The binding model of the Bim BH3 peptide in the complex
with Bcl-2 suggested that the hydrophobic pocket occupied by
Leu94 in Bim is not utilized by 3 (Figure 2 and Supporting
Information), and it was predicted that capture of this hydro-
phobic interaction should yield new inhibitors with higher
affinities. This led to the design of 4 (Figure 1), which contains
a phenyl ring tethered through a sulfone linker to the para-
position of the phenyl ring in 3. Modeling studies showed that
this additional phenyl group in 4 inserts into the hydrophobic
pocket occupied by the Leu94 in the Bim BH3 peptide
(Supporting Information) and predicted that 4 should have a
higher binding affinity to Bcl-2 than 3. Compound 4 was
synthesized (Supporting Information) and found to bind to Bcl-2
with a Ki value of 0.93 µM (Table 1), an affinity 8-times higher
than that of 3.
Figure 1. Structure-based design of a new class of Bcl-2 inhibitors.
molecule inhibitors of Bcl-2, using (-)-gossypol as the initial
lead compound.
To understand the structural basis of (-)-gossypol binding
to Bcl-2, we have performed computational docking of (-)-
gossypol into the BH3 binding groove in Bcl-2 (Supporting
Information). We have also modeled the binding of a Bim BH3
peptide in a complex with Bcl-2 since this Bim BH3 peptide
binds to Bcl-2 with a high affinity (Table 1). Based upon the
predicted binding model (Figure 2), (-)-gossypol forms a
hydrogen bonding network with residues Arg146 and Asn143
in Bcl-2 through the aldehyde group and the adjacent hydroxyl
group on the right naphthalene ring. This mimics the hydrogen
bonding network formed by Asp99 and Asn102 in Bim and
Arg146 and Asn143 in Bcl-2 (Figure 2). The isopropyl group
on the same naphthalene ring inserts into a hydrophobic pocket
in Bcl-2, in part mimicking the Phe101 in the Bim peptide. The
left half of the (-)-gossypol molecule interacts primarily with
Bcl-2 through hydrophobic contacts, mimicking Ile97 in the Bim
peptide (Figure 2). This binding model suggests that the two
halves of (-)-gossypol interact differently with Bcl-2 and
provides a structural basis for the design of novel small-molecule
inhibitors of Bcl-2.
Based upon our predicted binding model for (-)-gossypol
(Figure 2), we have sought to design completely new structural
classes of compounds that mimic the interaction between (-)-
gossypol and Bcl-2. The binding model calls for one-half of
the (-)-gossypol molecule to form an extensive hydrogen
bonding network with Bcl-2. Modeling shows that a polyphenol
ring containing three hydroxyl groups can mimic the hydrogen
bonding network between (-)-gossypol and Bcl-2, and this led
to the design of our initial template compound 2 (Figure 1). In
2, an isopropyl group was installed on the polyphenol ring to
mimic the hydrophobic interaction formed between the isopropyl
group on the right naphthalene ring of (-)-gossypol and Bcl-2.
Analysis of the binding model of 4 to Bcl-2 (Supporting
Information) showed that the hydrophobic interaction between
4 and Bcl-2 as mediated by the two terminal phenyl groups

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6141
can be further optimized at these two sites. Guided by molecular
modeling, compound 5 was designed, in which an isopropyl
and a tert-butyl were installed on the ortho-positions of these
two terminal phenyl rings. Modeling predicted that 5 would bind
more potently than 4 (Figure 2 and Supporting Information).
Compound 5 was synthesized in eight steps (Supporting
Information) and determined to bind to Bcl-2 with a Ki value
of 290 nM. Therefore, 5 is 3-times more potent than 4 and 80-
times more potent than the initial compound 2.
Our modeling results suggest that the hydroxyl groups in 5
play an important role in the binding to Bcl-2 through the
formation of two hydrogen bonds (Figure 2). To investigate the
importance of these hydroxyl groups, we designed and synthe-
sized 6 (Figure 1 and Supporting Information), in which all three
hydroxyl groups in 5 are replaced by methoxyls. Our FP-based
binding assay determined that 6 has an estimated Ki value of
25 µM and is thus nearly 100-times less potent than 5,
confirming the importance of these hydroxyl groups for the
binding of 5 to Bcl-2.
Figure 3. Superposition of two 15N-HSQC spectra of Bcl-2 in free
form (black) and with compound 5 (red). Peaks circled in black showed
induced shifts when the Bid BH3 peptide was added to BCl-2.
Since (-)-gossypol binds to Bcl-xL and Mcl-1 proteins, we
have further evaluated 5 for its binding affinities to these two
Bcl-2 members using FP-based assays developed in our labora-
tory for these two proteins (Supporting Information). It was
found that 5 binds to Bcl-xL and Mcl-1 with Ki values of 1100
nM and 260 nM, respectively (Table 1).
FP-based assays can be influenced by the autofluorescence
of the tested inhibitors. We have developed an enzyme-linked
immunosorbent assay (ELISA) for Bcl-2 (Supporting Informa-
tion) and determined the binding of our designed inhibitors to
Bcl-2. As can be seen from Table 1, the relative binding
affinities for these inhibitors in our ELISA assay are highly
consistent with those obtained from the FP-based assay, and
compound 5 binds to Bcl-2 with an IC50 value of 0.7 µM in
our ELISA assay.
Figure 4. Inhibition of cell growth in human prostate cancer PC-3
cells as determined using a WST-based assay.
binding area than that covered by compound 5 in the BH3
binding groove on Bcl-2.
Compound 5 was designed to bind to the BH3 binding groove
in Bcl-2 protein, competing with BH3 peptides derived from
Bid, Bim, and Bad proteins. To further probe the mode of action
of 5, we have performed heteronuclear single quantum coher-
ence (HSQC) NMR spectroscopy using uniformly 15N-labeled
Bcl-2 protein. 15N HSQC spectra are very sensitive to structural
changes in the protein, and subtle changes such as an interaction
with a small molecule inhibitor will induce shifts in peak
positions in the spectra. Hence, the HSQC NMR spectroscopy
provides a convenient way to conclusively confirm the binding
of a small molecule inhibitor to Bcl-2 and to identify where
the inhibitor binds on the protein.
Using 15N labeled Bcl-2 (isoform 2),5 15N HSQC NMR
spectra of Bcl-2 were recorded with or without compound 5.
The NMR spectra of Bcl-2 alone showed well-dispersed peaks,
indicative of a folded and stable protein. The comparison of
the two HSQC spectra showed induced shifts in several peak
positions, indicating that compound 5 interacts with several
residues on Bcl-2 (Figure 3). Since the backbone chemical shift
assignments of Bcl-2 are yet to be completed, we compared
the induced shift patterns on Bcl-2 for compound 5 and a Bid
BH3 peptide. This Bid BH3 peptide (Table 1) is known to bind
to the BH3 binding groove on Bcl-2 and has a Ki value of 11
nM to Bcl-2 in our FP-based competitive binding assay (Table
1). The residues in Bcl-2 affected by 5 overlap nicely with those
by the Bid BH3 peptide (Figure 3), strongly suggesting that 5
and the Bid BH3 peptide bind to the same site on Bcl-2. The
number of residues on Bcl-2 affected by the Bid BH3 peptide
was greater than the number affected by compound 5, due
presumably to the fact that the Bid peptide covers a larger
A major advantage for non-peptide small-molecule inhibitors
of Bcl-2 over BH3 peptides is that they may have much superior
cell permeability and thus much better cellular activity. We have
evaluated our designed small-molecule inhibitors for their ability
to inhibit cell growth in PC-3 human prostate cancer cells with
a high level of Bcl-2 (Figure 4). Compound 5 inhibits cell
growth in PC-3 cancer cells with an IC50 value of 200 nM.
Significantly, the IC50 values of compounds 2-6 in inhibition
of cell growth in PC-3 cells correlate reasonably well with their
binding affinities to Bcl-2.
Since Bcl-2, Bcl-xL and Mcl-1 proteins function as critical
apoptosis regulators and potent anti-apoptotic molecules,1-4 it
is predicted that concurrent inhibition of these Bcl-2 members
by 5 may effectively induce apoptosis in cancer cells. To test
this prediction, we have evaluated 5 for its ability to induce
apoptosis in PC-3 cells using the TUNEL assay (Figure 5).
Compound 5 effectively and dose-dependently induces apoptosis
in PC-3 cells. At 1.25 and 5 µM, 11.2% and 89.5% of cells
underwent apoptosis when cells were treated for 4 days, while
2.6% of cells underwent apoptosis in an untreated control.
In summary, our structure-based design has led to the
discovery of a potent small-molecule inhibitor of compound 5.
Although (-)-gossypol was used as the starting point for our
design, compound 5 belongs to a totally new chemical class
different from that of (-)-gossypol. Compound 5 binds to Bcl-2
and Bcl-xL with Ki values of 290 and 1110 nM, respectively.
Interestingly, unlike N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-
piperazin-1-yl]-benzoyl}-4-((R)-3-(dimethylamino)-1-phenylsul-
fanylmethyl-propylamino)-3-nitro-benzenesulfonamide (ABT-
737),13 a previously reported potent small-molecule inhibitor

6142 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Letters
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Figure 5. Apoptosis induction by compound 5 and its inactive analogue
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of Bcl-2 and Bcl-xL, 5 also binds potently to Mcl-1, a Bcl-2
homologous protein, with a Ki value of 260 nM. Thus, 5 and
(-)-gossypol represent a new type of small-molecule inhibitors
that concurrently target Bcl-2, Bcl-xL, and Mcl-1 proteins. Such
inhibitors may be more effective inducers of apoptosis in cancer
cells, especially those with high levels of Mcl-1, than compounds
that target only Bcl-2 and Bcl-xL. Taken together, our data
indicate that 5 is a potent, cell-permeable small-molecule
inhibitor that targets multiple anti-apoptotic Bcl-2 members and
is therefore a promising lead compound for further optimization
and development as a novel therapy for the treatment of human
cancer.
Acknowledgment. We are grateful for the financial support
from the National Cancer Institute, National Institutes of Health
(Grant U19CA113317), the Department of Defense Breast
Cancer Program (Grant BC0009140), the Department of Defense
Prostate Cancer Program (Grant PC040537), the Prostate Cancer
Foundation, the Breast Cancer Research Foundation, the Susan
G. Komen Foundation, Ascenta Therapeutics, Inc., and the
Intramural Research Program of the National Institutes of Health
from the National Cancer Institute, Center for Cancer Research.
Supporting Information Available: An experimental section
including information on the synthesis and chemical data for
compounds 2-6, molecular modeling methods and results for 2-6,
the experimental procedures for the fluorescence polarization-based
binding assays for Bcl-2, Bcl-xL, and Mcl-1 and the enzyme-linked
immunosorbent assay for Bcl-2, and details on the cellular growth
inhibition and apopposis assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
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proteins, US patent application series no. 20030008924, May 30,
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of Bcl-2, in patients with advanced malignancies. Presented at the
2005 AACR-NCI-EORTC International Conference on Molecular
Targets and Cancer Therapeutics: Discovery, Biology, and Clinical
Applications, November 14-18, 2005, Philadelphia, Pennsylvania,
Abstract C89.
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JM060460O