Structure-based design, synthesis, and evaluation of conformationally constrained mimetics of the second mitochondria-derived activator of caspase that target the X-linked inhibitor of apoptosis protein/caspase-9 interaction site

Article abstract of DOI:10.1021/jm0499108

A successful structure-based design of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that target the XIAP/caspase-9 interaction site is described. The most potent Smac mimetic 12d has a K_i of 350

Full text of DOI:10.1021/jm0499108

J . Med. Chem. 2004, 47, 4147-4150
4147
Letters
the interaction with caspase-9 is the most critical for
its inhibition of apoptosis.8,9
Str u ctu r e-Ba sed Design , Syn th esis, a n d
Eva lu a tion of Con for m a tion a lly
Con str a in ed Mim etics of th e Secon d
Mitoch on d r ia -Der ived Activa tor of
Ca sp a se Th a t Ta r get th e X-Lin k ed
In h ibitor of Ap op tosis P r otein /Ca sp a se-9
In ter a ction Site
A recent crystallographic analysis10 showed that XIAP
BIR3 binds to caspase-9 and traps it in its inactive
monomeric form. Unlike most other protein-protein
interactions, the binding between XIAP and caspase-9
is mediated by a small but well-defined surface groove
in the XIAP BIR3 domain and four N-terminally ex-
posed amino acid residues (ATPF) in caspase-9.10 This
surface groove in the XIAP BIR3 domain represents an
attractive site for small-molecule inhibitors of XIAP and
other IAP proteins whose binding will block the inhibi-
tory activity of IAP proteins to caspase-9 and promote
apoptosis in cells.
Haiying Sun,† Zaneta Nikolovska-Coleska,†
Chao-Yie Yang,† Liang Xu,† York Tomita,#
Krzysztof Krajewski,‡ Peter P. Roller,‡ and
Shaomeng Wang*,†
Departments of Internal Medicine and Medicinal Chemistry
and Comprehensive Cancer Center, University of Michigan,
1500 E. Medical Center Drive,
Ann Arbor, Michigan 48109-0934, Lombardi Cancer Center,
Georgetown University Medical Center,
In fact, the second mitochondria-derived activator of
caspase or direct IAP binding protein with low pI (Smac/
DIABLO), a protein released from mitochondria in
response to apoptotic stimuli, directly interacts with the
BIR3 domain of XIAP, cIAP-1, and cIAP-2 and a single
BIR domain in ML-IAP and functions as a direct
endogenous inhibitor of IAP proteins.11,12 Biological
studies9 and high-resolution experimental structures13,14
demonstrated that Smac binds to the same surface
groove in the XIAP BIR3 domain where caspase-9 binds
via its N-terminally exposed four residues (AVPI) in a
manner similar to that in caspase-9/XIAP interaction.
Consistent with the structural data, Smac peptides as
short as four residues derived from Smac protein bind
to the recombinant XIAP BIR3 domain protein with the
same affinities as the mature Smac protein.14,15 Thus,
binding of Smac protein and peptides to XIAP effectively
removes the inhibition of XIAP to caspase-9. Several
recent studies showed that short Smac peptides fused
to a carrier peptide for intracellular delivery (cell-
permeable Smac peptides) overcome resistance of cancer
cells with high levels of XIAP protein to apoptosis and
enhance the activity of anticancer drugs in vitro and in
vivo.16-18 Of great significance, these cell-permeable
Smac peptides have little toxicity to normal cells in vitro
and to normal tissues in vivo.16-18 These studies suggest
that cell-permeable Smac mimetics may have great
therapeutic potential as a new class of anticancer drugs
for overcoming apoptosis resistance of cancer cells with
high levels of IAP proteins.
Smac peptides have several intrinsic limitations (e.g.,
poor in vivo stability and poor bioavailability) as phar-
macological tools and as potentially useful therapeutic
agents for the treatment of cancer. In this paper, we
present, to our knowledge, the first successful example
of structure-based design of a class of conformationally
constrained Smac mimetics.
The basic design idea is illustrated in Scheme 1 and
Figure 1. On the basis of the 3D experimental structures
of the XIAP BIR3 domain in complex with Smac protein
and peptide,13,14 the hydrophobic side chain of Ile4 in
the Smac AVPI (Ala1-Val2-Pro3-Ile4) binding motif
binds to a well-defined hydrophobic pocket in XIAP
Washington, D.C. 20007, and Laboratory of Medicinal
Chemistry, National Cancer Institute, FCRDC,
Building 376, Room 219, Frederick, Maryland 21702
Received February 1, 2004
Abstr a ct: A successful structure-based design of conforma-
tionally constrained second mitochondria-derived activator of
caspase (Smac) mimetics that target the XIAP/caspase-9
interaction site is described. The most potent Smac mimetic
12d has a Ki of 350 nM for binding to the XIAP BIR3 domain
protein. 12d is found to be effective in enhancing apoptosis
induced by cisplatin in PC-3 human prostate cancer cells.
Apoptosis, or programmed cell death, is an essential
cell suicide process1 that is important for normal
development, host defense, and suppression of oncogen-
esis.2 Inappropriate control of apoptosis plays a role in
many human diseases, including cancer, autoimmune
diseases, and neurodegenerative disorders.2-4 In recent
years, key apoptosis regulators have become attractive
molecular targets for designing new therapies to treat
a variety of human diseases and conditions.3,4
Inhibitor of apoptosis proteins (IAPs) were recently
discovered as important intrinsic cellular inhibitors of
apoptosis, although their functions may not be limited
to the regulation of apoptosis.5,6 XIAP (X-linked IAP) is
the most potent inhibitor of apoptosis among all the IAP
proteins.5,6 XIAP protein potently inhibits intrinsic and
extrinsic apoptosis pathways.5,6 XIAP contains three
BIR (birculovirus IAP repeat) domains and a C-terminal
ring finger domain.6 The third BIR domain (BIR3)
selectively targets caspase-9, an initiator caspase, while
the linker region between BIR1 and BIR2 inhibits
effectors caspase-3 and -7.6,7 Although XIAP prevents
the activation of all three caspases, it is apparent that
* To whom correspondence should be addressed. Phone: 734-615-
0362. Fax: 734-647-9647. E-mail: shaomeng@umich.edu.
† University of Michigan.
#
Georgetown University Medical Center.
‡ National Cancer Institute.
10.1021/jm0499108 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/16/2004

4148 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Letters
Ta ble 1. Experimentally Determined Binding Affinities by
FP-Based Assay
Sch em e 1. Design for Deriving Conformationally
Constrained Smac Mimeticsa
configuration
compd
of C6
R1
R2
Ki ( SD,a µM
AVPI
1
2
0.58 ( 0.15
0.29 ( 0.06
4.47 ( 0.65
>100
R
S
Me
Me
Et
Bn
Bn
Bn
3
10a
10b
10c
12a
12b
12c
12d
R
R
R
R
R
R
R
1.41 ( 0.16
>100
n-Pr Bn
i-Pr
Me
Me
Me
Et
Bn
Ph
43.11 ( 1.51
>100
22.4 ( 1.87
2-phenylethyl
diphenylmethyl 2.33 ( 0.68
diphenylmethyl 0.35 ( 0.01
a
a
Starting from the AVPI peptide, simple Smac mimetic 1 was
The Ki values and standard deviation (SD) were determined
designed and synthesized. Cyclization of side chain of valine and
proline rings of 1 led to conformationally constrained Smac
mimetics 2 and 3.
by three to five independent experiments in triplicate.
Sch em e 2. Synthesis of Conformationally Constrained
Smac Mimeticsa
a
Reagents and conditions: (a) (i) 2 N, LiOH, then 1 N HCl; (ii)
benzylamine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2,
room temp; (iii) 1 N HCl in MeOH; (iv) N-(tert-butoxycarbonyl)-
L-alanine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, room
temp; (b) 1 N HCl in MeOH.
F igu r e 1. Superposition of modeled structure of 2 in complex
with XIAP BIR3 and the X-ray structure of Smac AVPI in
complex with XIAP BIR3. Carbon atoms are shown in yellow
and green for 2 and Smac peptide, respectively. Oxygen and
nitrogen atoms are shown in red and blue, respectively.
Hydrogen bonds are shown in light-blue dashed lines.
Ki greater than 100 µM, confirming our modeling results
and design strategy.
The chemical synthesis of these two compounds is
detailed in Scheme 2. Briefly, the key intermediates 5
and 6 were prepared according to the literature method.19
Hydrolysis of the methyl ester groups in 5 and 6
followed by condensation with benzylamine gave two
amides. Removal of the Boc protective groups in these
two amides followed by condensation with N-tert-bu-
toxycarbonyl-L-alanine yielded intermediates 7 and 8.
Removal of the Boc protective groups in these two
compounds afforded 2 and 3.
2 was used as the new template for further design
and optimization. On the basis of our modeled complex
structure for 2 (Figure 1), the methyl group in Ala1 and
the benzyl ring bind to small and large hydrophobic
pockets, respectively. The hydrophobic interactions at
these two sites have been shown to be important for the
interactions between Smac peptides and XIAP BIR3.13-15
We have therefore designed and synthesized new ana-
logues of 2 with modifications made at these two sites.
The synthesis of these new analogues was performed
similarly to that for 2 (Scheme 3), and their experimen-
tal binding affinities by the FP assay are provided in
Table 1.
BIR3 and the carbonyl group of Ile4 does not appear to
have specific interactions with the protein. Accordingly
we designed 1 (Scheme 1) in which the Ile4 residue was
replaced by a benzylamine. In our fluorescence polariza-
tion based (FP) binding assay (see Supporting Informa-
tion for assay details), 1 is as potent as the Smac AVPI
peptide (Table 1), confirming that the Ile residue can
be replaced by a suitable hydrophobic group. Cyclization
of the side chain of Val2 and Pro3 ring in 1 leads to
two bicyclic conformationally constrained Smac mimet-
ics (2 and 3 in Scheme 1). Detailed modeling studies
showed that although 2 has some deviations in com-
parison to the Smac AVPI peptide (e.g., the conforma-
tion of the five-membered ring), it quite closely mimics
the interactions of Smac/XIAP BIR3 (Figure 1). In
contrast, the other stereoisomer 3 is unable to mimic
the interaction between Smac/XIAP in both hydrogen
bonding and hydrophobic interaction (Supporting In-
formation). Thus, it was predicted that 2 would show
good binding affinity to XIAP BIR3, while 3 would be
much less potent than 2. Indeed, an FP-based binding
assay determined that while 2 has a Ki of 4.47 µM for
the recombinant XIAP BIR3 protein in displacing a
fluorescently labeled Smac peptide (Table 1), 3 has a
Our modeling analysis showed that the small hydro-
phobic pocket occupied by the methyl group in 2 can
accommodate a slightly larger hydrophobic group. Con-

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4149
Sch em e 3. Synthesis of New Analogues of 2a
F igu r e 2. Potentiating cisplatin (CDDP) induced apoptosis
in human prostate PC-3 cancer cells by Smac mimetic 12d
and in comparison with an inactive Smac mimetic 3, a Smac
peptide without a carrier peptide (Smac8), and a Smac peptide
tethered to a carrier peptide (Smac8-C).
a
Reagents and conditions: (a) (i) 2 N LiOH, then 1 N HCl; (ii)
benzylamine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2,
room temp, 92%; (b) (i) 1 N HCl in MeOH; (ii) N-(tert-butoxycar-
bonyl)-L-amino acid, EDC, HOBt, N,N-diisopropylethylamine,
CH2Cl2, room temp; (iii) 1 N HCl in MeOH; (c) (i) 1 N HCl in
MeOH; (ii) N-(tert-butoxycarbonyl)-L-alanine or N-(tert-butoxycar-
bonyl-L-2-aminobutyric acid, EDC, HOBt, N,N-diisopropylethyl-
amine, CH2Cl2, room temp; (d) (i) 2 N LiOH, then 1 N HCl; (ii)
amine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, room
temp; (iii) 1 N HCl in MeOH.
the binding of 12d to XIAP BIR3, we performed a
nuclear magnetic resonance analysis using the hetero-
nuclear single quantum coherence (HSQC) method. The
HSQC spectrum was recorded for a sample containing
the 15N-labeled human XIAP BIR3 protein with and
without 12d . Analysis of the HSQC spectra showed that
12d caused induced chemical shifts in several residues
in the XIAP BIR3, virtually identical to those caused
by the Smac AVPI peptide (Supporting Information),
indicating that 12d binds to the same surface groove
where Smac and caspase-9 bind.
sistent with the modeling analysis, replacement of the
methyl group with an ethyl group improves the binding
activity by 3-fold (10a vs 2), but replacement of this
methyl group by an n-Pr or i-Pr (10b and 10c) signifi-
cantly decreases the binding. The binding data thus
confirmed that the hydrophobic pocket occupied by the
methyl group can accommodate only a slightly larger
group (e.g., an ethyl group) but not an n-Pr or i-Pr
group.
The benzyl group in 2 mimics the hydrophobic side
chain of Ile4 in the Smac peptide. Replacement of the
benzyl group with a phenyl (12a ) or with a 2-phenyl-
ethyl (12b) reduces the binding affinity by more than
20- and 5-fold, respectively, demonstrating the impor-
tance of the linker length for optimal hydrophobic
interaction at this site.
Smac peptides tethered to a carrier peptide for
intracellular delivery have been shown to potentiate
chemodrug-induced apoptosis in cancer cells with high
levels of XIAP proteins.16-18 Accordingly, 12d was
evaluated for its ability to potentiate cisplatin-induced
apoptosis in the PC-3 human prostate cells with high
levels of XIAP protein (Supporting Information) and
compared to Smac peptide without (Smac8) or with
(Smac8-C) a carrier peptide derived from the Drosophila
antennapedia penetratin sequence.17 Consistent with
previous studies,16-18 Smac8, Smac8-C, and 12d do not
induce apoptosis on their own, while the cytotoxic drug
cisplatin (25 µM) significantly induces apoptosis in PC-3
human prostate cancer cells. The combination of cis-
platin with 100 µM Smac peptide without a carrier
peptide (Smac8 in Figure 2) only slightly increases the
cisplatin-induced apoptosis (from 9% to 13%) presum-
ably because of the poor cell permeability of this Smac
peptide without a carrier molecule. In contrast, combi-
nation of cisplatin with 100 µM Smac peptide with a
carrier peptide (Smac8-C in Figure 2) significantly
increases the apoptosis (from 9% to 23%). While the
combination of cisplatin with 10 µM 12d only slightly
increases cisplatin-induced apoptosis, 25 and 50 µM 12d
significantly increase the cisplatin-induced apoptosis.
In fact, 50 µM 12d is as effective as 100 µM Smac8-C
in potentiating the cisplatin-induced apoptosis (Figure
2). 3 (50 µM), which has a Ki greater than 100 µM, is
ineffective in potentiating the cisplatin-induced apop-
tosis. Taken together, our cellular studies suggest that
a potent Smac mimetic (12d ) is as effective as the
natural Smac peptide tethered to a carrier peptide
(Samc8-C) in increasing apoptosis induced by cisplatin,
while an inactive Smac mimetic (3) and a Smac peptide
without a carrier (Smac8) are ineffective.
In the experimental 3D complex structures,13,14 al-
though the carbonyl group of Ile4 in the Smac AVPI
peptide has no specific interaction with the XIAP
protein, we hypothesized that it plays a role in enhanc-
ing the binding affinity by controlling the orientation
and/or reducing the conformational flexibility of the
hydrophobic side chain of the Ile4 residue. To test this
idea, we designed and synthesized 12c, in which a
second phenyl group was introduced to the R carbon
atom of the benzyl group. Compound 12c has a Ki of
2.33 µM, twice as potent as 2. In our modeled structure
of 12c in complex with XIAP BIR3, this second phenyl
group does not appear to have a direct interaction with
the protein, suggesting that the slight improvement in
binding affinity of 12c over 2 is likely due to the
conformational restriction of the phenyl ring inserting
into the hydrophobic pocket (Figure 1).
Finally, 12d was designed by incorporating the re-
sults obtained for 10a and 12c and was determined to
have a Ki of 0.35 µM in our FP-based assay. Hence, 12d
is as potent as the Smac AVPI peptide and represents
a fairly potent Smac mimetic.
The FP-based binding assay showed that the Smac
mimetic 12d potently displaces the binding of a fluo-
rescently labeled Smac peptide from XIAP BIR, but the
assay does not provide direct information on which
residues in XIAP 12d interacts with. To further probe
In summary, we described a successful structure-
based design of a class of conformationally constrained

4150 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Letters
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obtained from this study is as potent as the natural
Smac peptide binding to the XIAP BIR3 protein. Fur-
thermore, 12d contains only one unnatural amino acid
and no natural amino acid and thus has much reduced
peptide characteristics in its chemical structure. As
expected from its much reduced peptide nature, 12d is
as effective as the Smac peptide tethered to a carrier
peptide17 in enhancing apoptosis induced by cisplatin
in PC-3 prostate cancer cells, while the natural Smac
peptide without a carrier peptide is ineffective. Thus,
12d may be used as a powerful chemical and pharma-
cological tool to elucidate the roles of Smac and IAP
proteins in regulation of apoptosis in cells. Further
optimization of 12d may ultimately lead to the develop-
ment of an entirely new class of anticancer drugs that
specifically target IAP proteins and overcome apoptosis
resistance of cancer cells to current chemotherapeutic
agents.
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Ack n ow led gm en t. We are grateful for the partial
financial support from the Prostate Cancer Research
Foundation.
(15) Kipp, R. A.; Case, M. A.; Wist, A. D.; Cresson, C. M.; Carrell,
M.; Griner, E.; Wiita, A.; Albiniak, P. A.; Chai, J .; Shi, Y.;
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Su p p or tin g In for m a tion Ava ila ble: Information on the
synthesis of compounds in Table 1, the detailed conditions and
experimental procedure for FP based binding assay, the NMR
HSQC experiments with and without 12d , and detailed
cellular assays as indicated in Figure 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
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