Structure-based design, synthesis and biochemical testing of novel and potent Smac peptido-mimetics

Article abstract of DOI:10.1016/j.bmcl.2004.11.008

Structure-based design, chemical synthesis and biochemical testing of a series of novel Smac peptido-mimetics as inhibitors of XIAP protein are described. The most potent compound, 6j, has a binding affinity (K_i value) of 24 nM to XIAP BIR3 pro

Full text of DOI:10.1016/j.bmcl.2004.11.008

Bioorganic & Medicinal Chemistry Letters 15 (2005) 793–797
Structure-based design, synthesis and biochemical testing of
novel and potent Smac peptido-mimetics
Haiying Sun,a Zaneta Nikolovska-Coleska,a Jianyong Chen,a Chao-Yie Yang,a
York Tomita,b Hongguang Pan,b Yoshiko Yoshioka,b Krzysztof Krajewski,c
Peter P. Rollerc and Shaomeng Wanga,*
aUniversity of Michigan Comprehensive Cancer Center, Departments of Internal Medicine and Medicinal Chemistry,
University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0934, USA
bLombardi Cancer Center, Georgetown University Medical Center, W213 Research Building,
3970 Reservoir Road NW. Washington, DC 20057-1469, USA
cLaboratory of Medicinal Chemistry, National Cancer Institute, FCRDC, Bldg 376, Frederick, MD 21702-1201, USA
Received 23 June 2004; revised 29 October 2004; accepted 1 November 2004
Available online 25 November 2004
Abstract—Structure-based design, chemical synthesis and biochemical testing of a series of novel Smac peptido-mimetics as inhibi-
tors of XIAP protein are described. The most potent compound, 6j, has a binding affinity (Ki value) of 24nM to XIAP BIR3 protein
and is 24 times more potent than the native Smac AVPI peptide. Further optimization of these potent Smac mimetics may ultimately
lead to the development of a novel class of anticancer drugs for the treatment of human cancer by overcoming apoptosis-resistance
of cancer cells through targeting the inhibitor of apoptosis proteins.
Ó 2004 Elsevier Ltd. All rights reserved.
Apoptosis, or programmed cell death, is an essential cell
suicide process1 that is important for normal develop-
ment, host defense and suppression of oncogenesis.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
targets caspase-9, an initiator of apoptosis, while the lin-
ker region between BIR1 and BIR2 inhibits effectors
caspase-3 and -7.6–9
Smac/DIABLO (second mitochondria-derived activator
of caspase or direct IAP binding protein with low pI), a
protein released from mitochondria in response to apop-
totic 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 study9 and high-resolu-
tion experimental structures13,14 have convincingly dem-
onstrated 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.10 Consist-
ent with the structural data, Smac peptides as short as
4-residues, derived from Smac protein bind to the re-
combinant XIAP BIR3 domain protein with the same
affinities as the mature Smac protein.14,15 Several recent
studies have shown 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
Inhibitor of apoptosis proteins (IAPs) were recently
identified and characterized as an important class of
intrinsic cellular inhibitors of apoptosis, although their
functions may not be limited to the regulation of apop-
tosis.5,6 IAP proteins potently inhibit both intrinsic and
extrinsic apoptosis pathways.5,6 XIAP (X-linked IAP) is
the most potent inhibitor of apoptosis among all known
IAP proteins.5,6 XIAP contains three BIR (Baculovirus
IAP repeat) domains as well as a C-terminal RING fin-
ger domain.6 The third BIR domain (BIR3) selectively
Keywords: Smac; Peptido-mimetics; XIAP.
*
Corresponding author. Tel.: +1 734 615 0362; fax: +1 734 647
9647; e-mail: shaomeng@umich.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.008

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H. Sun et al. / Bioorg. Med. Chem. Lett. 15 (2005) 793–797
Of great significance, these cell-permeable Smac peptides
have little toxicity to normal cells in vitro and to normal
tissues in vivo.16–18 Collectively, these studies suggest
that potent and 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.
XIAP through several hydrogen-bonding interactions
and van der Waals contacts. The experimental struc-
tures (Fig. 1) show that the backbone carbonyl group
of I40 of Smac does not have specific interactions with
the protein, and the hydrophobic side chain of I40 of
Smac inserts into a hydrophobic pocket. Hence, a simple
benzyl amine was used to replace the I40 residue (1 in
Table 1). Our computational modeling indicated that
this simple benzyl amino group mimics the I40 for
hydrophobic and hydrogen-bonding interactions with
the protein (Fig. 1). Compound 1, Smac AVPI peptide
and all other Smac peptido-mimetics reported in this
study (Table 1) were evaluated for their binding affinities
to XIAP BIR3 protein in a fluorescence-polarization-
based (FP-based) assay we have recently developed.19
In this competitive binding assay, these designed Smac
mimetics were measured for their affinities to displace
a fluorescently labelled, high-affinity, mutated Smac
Natural Smac peptides have several intrinsic limitations
(e.g., poor in vivo stability and poor bioavailability) as
pharmacological tools and as potentially useful thera-
peutic agents for the treatment of cancer. Furthermore,
the natural Smac AVPI peptide has only a moderate
binding affinity to XIAP BIR domain protein
(Ki = 0.58lM as determined in our binding assay).19 To-
wards developing potent Smac mimetics with improved
binding affinities, cellular permeability, in vivo stability
and pharmacokinetics, a number of laboratories, includ-
ing ours, are actively pursuing the design of Smac pep-
tido-mimetics15,21 and non-peptidic Smac mimetics.22
In this paper, we wish to report our structure-based de-
sign, synthesis and biochemical testing of a series of no-
vel and potent Smac peptido-mimetics.
Table 1. Chemical structures of Smac peptido-mimetics and their
binding affinities to XIAP BIR3 protein as determined using a
fluorescence-polarization-based binding assay19
O
High-resolution, experimental three-dimensional (3D)
structures of the BIR3 domain of XIAP in complex with
Smac protein and peptide have recently been determined
(Fig. 1). The N-terminal tetrapeptide of Smac (AVPI)
recognizes a surface groove on the BIR3 domain of
H N
2
N
N
H
R4
O
R1
R4
Compounds R1
Ki SD (lM)
0.29 0.07
13.40 1.6
2.45 0.7
1
CH3
-CO-NH-CH2
-CO-NH-CH2
6a
6b
6c
6d
CH3
CH3
CH3
CH3
-CO-NH-CH
2
4.41 1.5
-CO-NH-CH2
1.27 0.2
-CO-NH-CH
2
O
S
6e
6f
CH3
CH3
CH3
CH3
0.22 0.07
0.18 0.07
4.9 2.1
-CO-NH-CH
-CO-NH-CH
-CONH
2
2
6g
6h
0.15 0.09
-CO-NH-CH -CH
2
2
6i
CH3
0.028 0.020
-CO-NH
-CO-NH
6j
C2H5
CH3
0.024 0.020
1.2 0.4
10
-CH2-CH2-CH2
-CO-NH-(CH2)2
Figure 1. Superposition of modeled structure of compound 1 (yellow)
complexed with XIAP BIR3 and the X-ray structure of Smac protein
(green) complexed with XIAP BIR3. Compound 1 in complex with
XIAP BIR3 was modeled based upon the X-ray structure of XIAP
BIR3 in complex with Smac and minimized using the Sybyl program.23
For clarity, only the AVPI residues of Smac are shown. Carbon atoms
are colored in yellow, green and black in compound 1, Smac AVPI
peptide, and in the protein, respectively. Nitrogen and oxygen atoms in
these molecules are colored in blue and red, respectively. Hydrogen
bonds are depicted in blue dashed lines.
12a
12b
12c
12d
H
68
7
C2H5
i-C3H7
n-C3H7
0.081 0.06
4.15 1.2
-CO-NH-CH2
-CO-NH-CH2
54
7
-CO-NH-CH
2

H. Sun et al. / Bioorg. Med. Chem. Lett. 15 (2005) 793–797
795
peptide from recombinant XIAP BIR3 domain
protein.19
the binding affinity of the resulting compound (6i)
through: (a) controlling the second phenyl ring in an
optimal orientation for effective hydrophobic interac-
tions; (b) reducing the conformational flexibility of the
second phenyl ring as compared to compound 1. Com-
pound 6i was determined to have a Ki value of
0.028lM (28nM) and is 10 times more potent than 1,
representing a potent Smac peptido-mimetic.
Compound 1 was determined to have a Ki value of
0.29lM in our FP-based assay, and is thus 2 times more
potent than Smac AVPI peptide (Ki = 0.58lM) under
the same assay conditions. Compound 1 was subse-
quently used as the template for further design. Based
upon 1, a series of new compounds with different hydro-
phobic groups (compounds 6a–h in Table 1) were de-
signed, synthesized and tested to further explore the
importance of the hydrophobic interactions between
the phenyl ring in 1 and XIAP.
Previous X-ray structure showed that the methyl group
in alanine in position 1 (A10) interacts with a small but
well-defined hydrophobic pocket in XIAP BIR3 (Fig.
1) and this hydrophobic pocket appears to be large en-
ough to accommodate a slightly larger hydrophobic
group than a methyl group. To probe the interactions
at this site, we have designed and synthesized several
simple Smac mimetics (compounds 12a–d in Table 1).
Replacement of the benzyl group in 1 with an isobutyl
group (6a) reduced the binding affinity by about 45
times, indicating that isopropyl is not large enough for
achieving optimal hydrophobic interactions at this site.
Consistent with this result, replacement of the isobutyl
group with a larger 20-ethylbutyl group (6b) improved
the binding affinity by 5 times over 6a, but 6b is still 8
times less potent than 1. Replacement with a cycloprop-
ylmethyl group (6c) resulted in a reduction of 15 times
over 1 but 6c is 3 times more potent than 6a, suggesting
that not only the size but also the shape of the hydro-
phobic groups are important for hydrophobic interac-
tions at this site. Replacement of the phenyl ring in 1
with a saturated cyclohexyl ring (6d) caused a moderate
reduction of 4 times, indicating that an aromatic ring
appears to be more effective for hydrophobic interac-
tions. Accordingly, we synthesized two compounds with
a five-membered aromatic ring (6e and 6f in Table 1).
Compounds 6e and 6f have Ki values of 0.22 and
0.18lM, respectively, and both compounds are as po-
tent as 1.
Replacement of the methyl group in compound 1 by a
hydrogen atom resulted in compound 12a. Compound
12a has a Ki value of 68lM and is more than 200 times
less potent than 1, highlighting the importance of the
hydrophobic interactions. Consistent with a previous
study14 on Smac peptides, replacement of the methyl
group in compound 1 by an ethyl group (12b) improves
the binding affinity by 3 times. Consistent with our
modeling analysis that this hydrophobic pocket is quite
small, compound 12c, in which an isopropyl was used to
replace the methyl group in compound 1, is 14 times less
potent than 1. Moreover, compound 12d, in which an n-
propyl group was used to replace the methyl group in
compound 1, is more than 180 times less potent than
1. These data indicate that this small hydrophobic pock-
et in XIAP is not large enough to accommodate an iso-
propyl or n-propyl group, but can accommodate a
methyl or an ethyl group. Indeed, replacement of the
methyl group with an ethyl group in 6i resulted in 6j,
which has a Ki value of 24nM. Hence, compound 6j is
12 times more potent than 1 and 24 times more potent
than the natural Smac AVPI peptide, representing a
highly potent new Smac mimetic.
For effective hydrophobic interactions, the length of the
linker between the proline residue and the phenyl ring in
1 is important. Compounds 6g and 6h were designed to
investigate the optimal length for the linker. While com-
pound 6g with one carbon-atom shorter linker than 1 is
16 times less potent than 1, compound 6h with a carbon-
atom longer linker than 1 is 2 times more potent than 1
with a Ki value of 0.15lM (Table 1), confirming the
importance of the length of the linker.
To conclusively confirm that compound 6j binds to the
binding groove in XIAP BIR3 where Smac binds, we
performed an analysis using nuclear magnetic resonance
(NMR) methods. The human XIAP BIR3 domain (res-
idues 241–356) fused to His-tag was expressed from
BL21(DE3) cells in M9 medium containing 15N ammo-
nium chloride to uniformly label protein with 15N, and
was purified. 15N heteronuclear single quantum coher-
ence spectroscopy (HSQC) NMR spectra were recorded
with samples containing 100lM of the 15N protein in
50mM Tris (pH7.2), 50lM Zn(Cl)2, 1mM DTT with
100lM of 6j or without it at 30°C. Overlaying of the
two 15N HSQC spectra of the BIR3 domain of human
XIAP with 6j (red) and without (black) showed that 6j
bound to the protein and caused induced chemical shifts
in several residues in XIAP BIR3 (Fig. 2). To identify,
which residues in XIAP BIR3 were affected by
compound 6j, 13C and 15N double labeled XIAP
BIR3 was prepared and 3D NMR triple reson-
ance experiments were performed to make backbone
atom resonance assignments. HNCA, HNCACB,
The amino group of I40 in AVPI and of the benzylamine
in compound 1 forms a hydrogen bond with the back-
bone carbonyl group of G306 of XIAP (Fig. 1). To
probe the importance of this hydrogen bond for bind-
ing, we synthesized 10 in which the amide group in 1
is replaced by two carbon atoms (–CH2–CH2–). Com-
pound 10 has a Ki value of 1.2lM, which is 4 times less
potent than 1, indicating that the amide group plays a
modest role in the binding between 1 and XIAP.
Although the backbone carbonyl group of I40 of Smac
points toward the solvent (Fig. 1), we hypothesized that
it may play a role in restricting the conformation of the
hydrophobic side chain of I40 for effective hydrophobic
interactions with XIAP. To test this idea, we designed
6i in which an additional phenyl ring was introduced.
We reasoned that this additional phenyl may enhance

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H. Sun et al. / Bioorg. Med. Chem. Lett. 15 (2005) 793–797
The synthesis of peptido-mimetics with modification at
the I4 residue is shown in Scheme 1. Briefly, condensa-
tion of commercially available amino acid derivatives
2 and 3 in the presence of EDC and HOBt pro-
duced compound 4. Removal of the Boc protective
group in compound 4 followed by condensation with
L-N-Boc-alanine or L-N-Boc-2-aminobutyric acid re-
sulted in amides 5a and 5b, respectively. Hydrolysis of
the ester group in 5a and 5b afforded two acids. Con-
densation of the two acids with different amines fur-
nished a series of amides. Removal of the Boc
protective groups in these amides afforded the desired
compounds 6a–j.
Figure 2. Overlaid 15N heteronuclear single quantum coherence
spectroscopy (HSQC) NMR spectra of XIAP BIR3 with (red) or
without (black) compound 6j.
The synthesis of compound 10 is provided in Scheme 2.
Briefly, oxidation of the hydroxyl group in commercially
available compound 7 generated an aldehyde. Olefina-
tion of this aldehyde with the ylide derived from phenyl-
triphenylphosphonium bromide by treatment with BuLi
furnished an alkene. Hydrogenation of this alkene cata-
lyzed by 10% Pd–C afforded compound 8. Removal of
the Boc protective group in 8 followed by condensation
with dipeptide derivative 9 yielded an amide. Removal
of the Boc protective group in this amide gave com-
pound 10.
HN(CO)CBCA, HNCO, TOCSY-HSQC, C(CO)NH
and the published results14,20 were used to nearly com-
plete the backbone assignments, except for the two flex-
ible loops (residues 276–280 and 308–314).20 Based upon
the nearly complete backbone assignments of XIAP
BIR3, we found that the residues G306, K297, L292,
K299 are affected by compound 6j (Fig. 2). Moreover,
these residues were also found to be affected by the Smac
AVPI peptide in our NMR analysis (data not shown).
Based upon the experimental complex structures of
Smac/XIAP BIR3 (Fig. 1), these residues in XIAP
BIR3 that are affected by compound 6j and the Smac
AVPI peptide are in direct contact with Smac protein.
Taken together, our NMR experimental results conclu-
sively confirm that compound 6j binds to the binding
groove in XIAP BIR3 where Smac protein binds. Our
experiments also showed that our potent mimetic 6j does
not unfold XIAP BIR3 protein.
The synthesis of compounds (12a–d) with modifications
at A1 of Smac is shown in Scheme 2. Hydrolysis of the
ester group in 4 followed by condensation with benzyl
amine afforded compound 11. Removal of the Boc pro-
tective group in 11 by treatment with HCl in 1,4-diox-
ane–methanol, followed by condensation of the
resulting salt with different L-N-Boc-amino acids, gave
a series of amides. Removal of the N-terminal Boc pro-
tective group and the C-terminal benzyl group provided
a series of the amide derivatives, 12a–d.
N
a
COOH
BocHN
OMe
+
N
H
COOMe
O
NHBoc
O
4
3
2
O
Cl-
O
H3+N
N
b
BocHN
N
c
N
H
N
H
OMe
NHR2
R1
O
R1
O
6a-6j
O
O
5a R1 = Me, 86% over two steps
5b R2 = Et, 85% over two steps
Cl-
O
H3+N
N
e
Ph
d
N
N
N
CH2OH
Ph
H
Boc
Boc
O
7
8
10
O
BocHN
N
COOH
H
9
Scheme 1. Synthesis of peptido-mimetics with modifications at Ile residue. Reagents and conditions: (a) EDC, HOBt, N,N-diisopropylethylamine,
CH2Cl2, 92%; (b) i. 4N HCl in 1,4-dioxane, MeOH, ii. L-N-Boc-alanine or L-N-Boc-aminobutyric acid, EDC, HOBt, N,N-diisopropylethylamine,
CH2Cl2; (c) i. 2N LiOH, 1,4-dioxane, then 1N HCl, ii. amine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, iii. 4N HCl in 1,4-dioxane, MeOH,
structures were shown in Table 1, the yield over three steps is 48–72%; (d) i. Dess–Martin periodinane, CH2Cl2, ii. phenylethyltriphenylphosphonium
bromide, n-BuLi, ꢀ78°C, THF, iii. 10% Pd–C, H2, 51% over three steps; (e) i. 4N HCl in 1,4-dioxane, MeOH, ii. 9, EDC, HOBt, N,N-
diisopropylethylamine, CH2Cl2, 4N HCl in 1,4-dioxane, MeOH, 72% over three steps.

H. Sun et al. / Bioorg. Med. Chem. Lett. 15 (2005) 793–797
797
-
O
Cl
+
a
4
b
N
H
N
N
3
BocHN
N
H
NHBn
NHBn
O
R
1
O
O
O
12a-d
11
Scheme 2. Synthesis of peptido-mimetics with modifications at Ala residue. Reagents and conditions: (a) i. 2N LiOH, 1,4-dioxane, then 1N HCl, ii.
benzylamine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, 88% over two steps; (b) i. 4N HCl in 1,4-dioxane, MeOH, ii. L-N-Boc-amino acid,
EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, iii. 4N HCI in 1,4-dioxane, MeOH, structures are shown in Table 1, the yield over three steps is
68–74%.
9. Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.;
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In summary, our structure-based design, synthesis and
biochemical testing have yielded highly potent Smac
peptido-mimetics. Compounds 6i and 6j have Ki values
of 28 and 24nM, respectively, for their binding affinities
to the XIAP BIR3 protein, and are at least 20 times
more potent than the natural Smac AVPI peptide. Fur-
thermore, these compounds have reduced peptidic prop-
erties as compared to the natural Smac peptide.
Preliminary evaluations of compound 6j in human
breast cancer MDA-MB-231 cells and prostate PC-3
cancer cells with high levels of XIAP proteins show that
6j is effective in enhancing apoptosis induced by chemo-
therapeutic agents. Additional structural optimization
of these Smac peptido-mimetics and extensive biological
testing are underway and will be reported in due course.
It is predicted that highly potent and cell-permeable
Smac mimetics with in vivo stability may have the thera-
peutic potential to be developed as an entirely new class
of anticancer drugs for the treatment of human cancers
by overcoming apoptosis-resistance of cancer cells to
current therapeutic agents.
17. Arnt, C. R.; Chiorean, M. V.; Heldebrant, M. P.; Gores,
G. J.; Kaufmann, S. H. J. Biol. Chem. 2002, 277, 44236–
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Acknowledgements
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moto, H.; Yamori, T.; Oh-Hara, T.; Tsuruo, T. Cancer
Res. 2003, 63, 831–837.
19. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.;
Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N.
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We are grateful for the financial support from the Pros-
tate Cancer Research Foundation and from the
National Institutes of Health (1R01CA109025 to S.W.).
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