Novel dimeric Smac analogs as prospective anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2014.02.024

A small library of monovalent Smac mimics with general structure NMeAla-Tle-(4R)-4-Benzyl-Pro-Xaa-cysteamide, was synthesized (Xaa = hydrophobic residue). The library was screened in vitro against human breast cancer cell lines MCF-7 and MDA-MB-231, and two most active compounds oligomerized via S-alkylation giving bivalent and trivalent derivatives. The most active bivalent analogue SMAC17-2X was tested in vivo and in physiological conditions (mouse model) it exerted a potent anticancer effect resulting in ～23.4 days of tumor growth delay at 7.5 mg/kg dose. Collectively, our findings suggest that bivalent Smac analogs obtained via S-alkylation protocol may be a suitable platform for the development of new anticancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2014.02.024

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1452–1457
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel dimeric Smac analogs as prospective anticancer agents
Ewa D. Micewicz a, Hai T. Luong b, , Chun-Ling Jung b, Alan J. Waring c,d, William H. McBride a,
Piotr Ruchala b,
⇑
a Department of Radiation Oncology, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
b Department of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
c Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90502, USA
d Department of Physiology and Biophysics, University of California Irvine, 1001 Health Sciences Road, Irvine, CA 92697, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of monovalent Smac mimics with general structure NMeAla-Tle-(4R)-4-Benzyl-Pro-
Xaa-cysteamide, was synthesized (Xaa = hydrophobic residue). The library was screened in vitro against
human breast cancer cell lines MCF-7 and MDA-MB-231, and two most active compounds oligomerized
via S-alkylation giving bivalent and trivalent derivatives. The most active bivalent analogue SMAC17-2X
was tested in vivo and in physiological conditions (mouse model) it exerted a potent anticancer effect
resulting in ꢀ23.4 days of tumor growth delay at 7.5 mg/kg dose. Collectively, our findings suggest that
bivalent Smac analogs obtained via S-alkylation protocol may be a suitable platform for the development
of new anticancer therapeutics.
Received 17 November 2013
Revised 5 February 2014
Accepted 7 February 2014
Available online 18 February 2014
Keywords:
Smac mimics
New anticancer agents
Peptides
Ó 2014 Elsevier Ltd. All rights reserved.
S-alkylation of peptides
Apoptosis
Apoptosis (programmed cell death, PCD) functions as an impor-
tant mechanism controlling homeostasis, normal development,
host defense, suppression of oncogenesis, and its dysfunctional
regulation is associated with a variety of human pathologies,
including cancer,1–5 inflammation6,7 and neurodegeneration.8,9
Inhibitors of Apoptosis Proteins (IAPs) are key regulators of
apoptosis.10–12 They contain one or more of Baculovirus IAP Repeat
(BIR) domains,12,13 which are approximately 70 amino acid long
structural motifs13,14 primarily responsible for the anti-apoptotic
activity of IAPs. Specifically, they bind and inhibit various caspases,
enzymes belonging to cysteine-aspartyl proteases family, which
are crucial for the apoptotic process.15 A total of eight mammalian
IAPs have been identified to date with the most potent caspase
inhibitor family member being XIAP (X-linked IAP),16,17 which
effectively inhibits three caspases: caspase-3, -7, and -9.18–21 An
apoptotic signaling is in turn regulated by the second mitochondria
derived activator of caspases (Smac), also called a direct IAP bind-
ing protein with low pI (DIABLO),22,23 which has been identified as
an endogenous proapoptotic antagonist of IAP proteins. After its
release from the mitochondria into cytosol, and subsequent pro-
cessing by proteases (removal of 55 N-terminal residues), a mature
form of Smac effectively antagonizes XIAP, cIAP1 and cIAP2
proteins22–26 promoting programmed cell death. Specifically,
N-terminal tetrapeptide AVPI (Ala-Val-Pro-Ile), so called binding
motif22,23 of mature Smac, is responsible for proapoptotic effects
of protein. In the case of XIAP, a homodimeric form of Smac is
capable of binding to both BIR2 and BIR3 domains of the protein
abrogating its inhibition of caspases-3, -7, and -9.25,27 In the case
of cIAP1 and cIAP2, only BIR3 domain is targeted by a single AVPI
binding motif.28
Targeting IAP proteins represents a promising therapeutic ap-
proach in cancer treatment14,29–31 and over the past 10 years a con-
siderable amount of research was done in this particular field32–35
including clinical trials.35,36 Recently bivalent Smac analogues con-
taining two AVPI mimics tethered with a linker and capable of bind-
ing to both BIR2 and BIR3 XIAP domains became the focus of
researchers due to their high potency.37–40 Available data41 suggest
that the overall hydrophobicity of the compounds may positively
influence biological activity, most likely promoting cell permeabil-
ity and increasing intracellular concentration of analogues resulting
in more potent therapeutic effects. Therefore in the case of dimeric
Smac mimics, we concluded that the overall therapeutic effect
generally is dependent on 3 factors: (1) binding potency of the
monomer(s), (2) length of the linker and (3) linker’s hydrophobic-
ity, with more hydrophobic compounds being generally more
active due to increased cell permeability. Moreover, we theorized
that cell permeability is a crucial limiting factor for Smacs’ activity.
⇑
Corresponding author. Tel.: +1 310 825 0133; fax: +1 310 206 8766.
E-mail address: pruchala@mednet.ucla.edu (P. Ruchala).
Present address: Gilead Sciences, Inc., 4049 Avenida de la Plata, Oceanside, CA
92056, USA.
http://dx.doi.org/10.1016/j.bmcl.2014.02.024
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

E. D. Micewicz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1452–1457
1453
Subsequently, we decided to synthesize a small library of Smac ana-
logues with varying hydrophobic residues (Xaa) in position 4, based
on the modified structure of the previously described potent
(Kd = 5 nM) monovalent compound, NMe-Ala-Tle-(4S)-4-phenoxy-
Pro-(R)-tetrahydronaphth-1-yl amide42 which was developed in
Abbott
Laboratories
(NMeAla-(N-Methyl)alanine,
Tle-tert-
Leucine,). In our case we decided to use the following sequence:
NMeAla-Tle-(4R)-4-Benzyl-Pro-Xaa–NHCH2CH2–SH, where Xaa
stands for various hydrophobic residues and C-terminal cysteamide
provides means for further multimerization based on thiol group
reactivity (Fig. 1). This Letter describes synthesis and biological
properties of these novel compounds.
All monovalent Smac analogs were synthesized43 as C-terminal
cysteamine-amides by the solid phase method using CEM Liberty
automatic microwave peptide synthesizer (CEM Corporation Inc.,
Matthews, NC), applying 9-fluorenylmethyl-oxycarbonyl (Fmoc)
chemistry44 and standard, commercially available amino acid
derivatives and reagents (EMD Biosciences, San Diego, CA and
Chem-Impex International, Inc., Wood Dale, IL). Peptides were
purified by preparative reverse-phase high performance liquid
chromatography (RP-HPLC) to >90% homogeneity and their purity
evaluated by matrix-assisted laser desorption ionization spectrom-
etry (MALDI-MS) as well as analytical RP-HPLC.45 Analytical data
for obtained peptides as well as an example of MS-spectra and
corresponding analytical RP-HPLC profile are presented in
Supplementary material.
Since we assumed that hydrophobicity-dependent cell perme-
ability is a crucial limiting factor for Smacs’ bioactivity we decided
to use exclusively cell-based assay for an initial evaluation of our
compounds, namely cells’ growth inhibition assay.46 In our view,
this simple method provides more reliable, data which take into
account many factors like the compound’s cell permeability, its
binding potency, stability in the cell’s microenvironment, etc.,
and in this particular case is better suited for such screening than
pure biophysical method(s) for example, measurement of binding
affinity to BIR2/BIR3 XIAP domains. For our in vitro studies we se-
lected Smac-susceptible human non-metastatic breast cancer
MCF-7 and metastatic MDA-MB-231 cell lines. An example of cell
growth curves is presented in Figure 2. Initial screening of the
monovalent Smac library (Table 1) against both human breast can-
cer cell lines suggested that for the best ‘dual’ activity against both
MCF-7 and MDA-MB-231 cell lines, position 4 (Xaa) should be
occupied either by Bip, 1Nal, 2Nal or Dpa, residues that possess
fairly similar hydrophobic side chains. Interestingly, position 4
seems to also ‘differentiate’ between both tested cancer lines with
some analogues being more potent against MCF-7 cells, that is,
SMAC11 (Tic) and SMAC17 (Bip), and some being more potent
against MDA-MB-231 cells, that is, SMAC6 (Chg) and SMAC14
(1Nal).
Figure 2. An example of cell viability curves obtained for MCF-7 and MDA-MB-231
human breast cancer cell lines treated with bivalent analogue SMAC17-2X.
Table 1
Smac induced cell growth inhibition of MCF-7 and MDA-MB-231 human breast
cancer cells
Peptide
R
EC50
(
lM) MDA-MB-231
EC50 (lM) MCF-7
SMAC1
SMAC2
SMAC3
SMAC4
SMAC5
SMAC6
SMAC7
SMAC8
Phg
17.2 6.4
23.8 4.5
14.3 3.2
47.1 15.9
68.3 4.0
9.8 3.1
91.8 16.3
34.3 6.7
27.0 3.2
29.7 3.3
13.3 1.3
10.1 2.6
21.7 1.3
4.5 1.1
2.8 0.1
7.2 1.5
9.4 0.5
9.4 0.6
10.6 0.9
2.4 0.3
NA
31.2 1.4
43.6 1.6
49.1 2.6
41.6 3.7
476.7 48.4
31.2 2.1
86.3 10.5
33.3 1.5
19.8 3.0
23.9 2.1
5.3 0.6
21.2 2.4
12.3 0.5
13.4 0.5
3.5 1.6
120.4 8.4
10.5 0.6
11.3 1.8
5.7 0.9
NMePhg
Amp
DISC
Idc
Chg
Amc
Phe
PheF5
bhPhe
Tic
SMAC9
SMAC10
SMAC11
SMAC12
SMAC13
SMAC14
SMAC14-2X
SMAC14-3X
SMAC15
SMAC16
SMAC17
SMAC17-2X
SMAC17-3X
SMAC18
SMAC19
Cha
bhNalGly
1Nal
1Nal
1Nal
2Nal
Dpa
Bip
Bip
Bip
Ant
1.7 0.4
NA
15.1 0.9
27.6 1.8
17.3 1.0
36.7 3.2
Trp
Abbreviations: Amc—trans-4-(aminomethyl)cyclohexane carboxylic acid, Amp—
4-(aminomethyl)phenylacetic acid, Ant—3-(9-anthryl)alanine, Bip—biphenyl-alanine,
Cha—cyclohexylalanine, Chg—cyclohexylglycine, bhPhe—b-homophenylalanine,
bhNalGly—(R,S)-3-amino-3-(1-naphthyl)propionic acid, DISC—(R,S)-1,3-dihydro-
2H-isoindole carboxylic acid, Dpa—diphenylalanine, Idc—(S)-indoline-2-carboxylic
acid, 1Nal—1-naphthylalanine, 2Nal—2-naphthylalanine, NMePhg—(N-methyl)-
phenylglycine, Phg—phenylglycine, PheF5—pentafluorophenylalanine, Tic—(3S)-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, NA—not active, 2X-dimer,
3X-trimer.
Based on these results, we selected two compounds, SMAC14
(1Nal) and SMAC17 (Bip), for subsequent multimerization47 based
on S-alkylation principles. The multimerization of peptides is fre-
quently used as means to increase an immunogenicity (MAP
peptides), prolong serum half-life/stability or a way to increase
affinity to the receptor by harnessing multivalency effects. As a
convenient multimerization scaffold(s) we decided to use commer-
cially available 1,4-bis(bromomethyl)benzene (dimerization) and
1,3,5-tris(bromomethyl)benzene (trimerization). Moreover, com-
pounds resulting from dimerization of our monovalent analogues
with the use of 1,4-bis(bromomethyl)benzene produce a bivalent
Smacs that possess roughly the same length as optimal linkers pre-
viously reported41 and have hydrophobic properties.
Figure 1. General structure of synthesized monovalent Smac compounds.
R-various hydrophobic substituents (for list see Table 1).

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E. D. Micewicz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1452–1457
For S-alkylation based multimerization, as a method of choice,
overall anticancer activity of Smac-peptides. However promising,
dimerization is also associated with notable problems: (1) synthe-
sis of dimers requires at least one additional synthetic step in the
late stage of synthesis that lowers overall yield of the final product,
(2) dimerization increases significantly molecular weight (at least
doubles) of the bioactive compound(s) effectively multiplying cost
of production, (3) in the case of our analogues, multimerization is
also associated with decrease in solubility that can influence deliv-
ery, distribution and pharmacokinetics of the drug. Obviously, such
factors are of crucial importance for the drug development and may
heavily influence the fate of dimeric Smac-derivatives.
we decided to use a modified protocol previously described by
Salvatore et al.48 that we adapted to peptides (Scheme 1). Our
protocol allows for synthesis in relatively high concentrations
of peptides (ꢀ5 mg/ml) resulting in solution(s) that may be directly
loaded on the HPLC columns simplifying post-synthetic procedures.
Analytical data for synthesized mono-, bi-, and trivalent analogues
are presented in Supplementary material. Obtained compounds (di-
mers and trimmers) were tested in vitro yielding the most potent
bivalent analog in this series, SMAC17-2X which shows EC50 values
of 1.7 0.4 and 2.4 0.3 lM for MCF-7 and MDA-MB-231, respec-
tively. Notably, trivalent compound SMAC14-3X shows signifi-
cantly lower activity than respective bivalent analog SMAC14-2X.
In the case of SMAC17-3X, no anticancer activity was observed.
According to published results37,40,41,49,50 and our in vitro data mul-
timerization, specifically dimerization, seems to be beneficial for
To test whether our approach may yield compounds with ther-
apeutic potential we performed animal studies using subcutaneous
engraftment mouse model and a human metastatic breast cancer
line, MDA-MB-23.51 Treatment of the experimental, cancer bearing
animals with bivalent compound SMAC17-2X resulted in potent
Scheme 1. General synthetic route for the synthesis of bivalent (SMAC17-2X) and trivalent (SMAC17-3X) analogs. Reagents and conditions: (a) 1,4-bis(bromomethyl)ben-
zene, Cs2CO3, TBAI, 50% DMSO in DMF (b) 1,3,5-Tris(bromomethyl) benzene, Cs2CO3, TBAI, 50% DMSO in DMF. Analogues SMAC14-2X and SMAC14-3X were synthesized in
similar manner.

E. D. Micewicz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1452–1457
1455
anticancer effects (Fig. 3). Animals treated with 10 doses of the
compound at the concentration of 2.5 mg/kg showed ꢀ10.2 days
delay in tumor growth and treatment of animals with 3 times
higher dose (7.5 mg/kg) resulted in ꢀ23.4 days of tumor growth
delay. Notably, no adverse effects were observed during animal
experiments.
To confirm that newly synthesized peptides indeed promote
apoptosis, we measured enzymatic activity of caspases-3/7 and -
9 in a metastatic breast cancer cell line, MDA-MB-231 that was
treated with selected SMAC peptides.52 Comparison of SMAC14
and SMAC17 with their respective dimers (SMAC14-2X and
SMAC17-2X) at 10 lM concentration resulted in significant in-
crease in caspases’ enzymatic activity (ꢀ2.1 Ä 12.8 fold increase,
Fig. 4). In both cases caspase-3/7 and caspase-9, dimers were more
active than respective monomers. In agreement with cell viability
data, SMAC17-2X showed the most potent effects causing ꢀ12.8
fold increase in caspase-3/7 activity and ꢀ6.1 fold increase in cas-
pase-9 activity, and observed effects are dose dependent (Fig. 5).
To examine whether oligomerization of monovalent SMAC17
peptide induces a higher order structural features in multivalent
counterparts we performed circular dichroism (CD) experiments53
which are summarized in Table 2. Data suggest that oligomeriza-
tion has no immediate effect on secondary structure of unbound
analogs in experimental conditions.
Figure 4. Increase in enzymatic activity of caspases-3/7 and -9 in MDA-MB-231
cells treated with SMAC peptides.
The binding affinity of SMAC17-2X analog to the rhXIAP and its
domains was assessed using surface plasmon resonance (SPR)
experiments.54 The peptide showed moderate affinity to the full-
length rhXIAP (Kd = 317 nM), a low affinity to its BIR3 domain
(Kd = 2.4 lM) and has no binding affinity to BIR2 domain (binding
undetectable in experimental conditions).
Direct comparison of our in vitro and in vivo results with
published data is somewhat difficult due to the differences in
experimental conditions.37,40,41,49,50 Nonetheless, reported results
for cell growth inhibition assay in MDA-MB-231 cell line show
activity in low nanomolar range37,41,49,50 (i.e. compound 24:IC50
= 1.2 0.3 nM,41 compound 13:IC50 = 3.4 0.6 nM,49 compound
16: IC50 = 0.9 0.2 nM50) versus low micromolar range in the case
of our compounds (Table 1). Notably, in the case of this particular
assay the time of cells’ incubation with Smac-compounds was in
our case 48 h versus 96 h for abovementioned analogs 13, 16 and
24. Interestingly, a comparison of in vivo data for our most active
bivalent analog, SMAC17-2X with previously reported compounds
is more favorable. For example, SM-16437 shows very similar tu-
mor growth profile in the shown experimental timeframe
(<50 days). Similar results were also reported for compound 2741
with slightly better bioactivity (27 was tested in lower dosage with
Figure 5. Increase in enzymatic activity of caspases-3/7 and -9 in MDA-MB-231
cells treated with various concentrations of SMAC17-2X.
Table 2
Proportions of different components of secondary structure for selected SMAC
peptides in TFE-buffer based on circular dichroic spectroscopic analysis
Peptide*
% Conformation
a-Helix
Turns
b-Sheet
Disordered
SMAC17
SMAC17-2X
SMAC17-3X
4.0
2.0
2.0
27.0
25.0
29.0
43.0
45.0
44.0
26.0
28.0
25.0
*
Peptides (100 lM) in TFE:10 mM HEPES buffer pH = 7.4, 4:6 (v/v) were analyzed
for secondary conformation based on secondary structural analysis using SELCON.
similar results in the reported experimental range <50 days). Nota-
bly, recently reported results for SM-120050 show markedly im-
proved activity comparing to all previously reported Smac-
analogs. This potent bivalent Smac mimetic appears to promote
complete and durable tumor regression in mouse model
(<75 days).
In conclusion, a new family of anticancer Smac peptides was
synthesized, characterized and screened for anticancer activity
against human breast cancer cell lines, MCF-7 (non-metastatic)
and MDA-MB-231 (metastatic). Selected analogues were oligomer-
ized resulting in bivalent and trivalent Smac compounds. The most
Figure 3. Anticancer effects of SMAC17-2X treatment in xenograft mouse model.

1456
E. D. Micewicz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1452–1457
39. Sun, H.; Nikolovska-Coleska, Z.; Lu, J.; Qiu, S.; Yang, C. Y.; Gao, W.; Meagher, J.;
Stuckey, J.; Wang, S. J. Med. Chem. 2006, 49, 7916.
40. Sun, H.; Nikolovska-Coleska, Z.; Lu, J.; Meagher, J. L.; Yang, C. Y.; Qiu, S.; Tomita,
Y.; Ueda, Y.; Jiang, S.; Krajewski, K.; Roller, P. P.; Stuckey, J. A.; Wang, S. J. Am.
Chem. Soc. 2007, 129, 15279.
active analogue (SMAC17-2X) was tested in vivo showing dose
dependent, potent anticancer activity and appears to be a suitable
template for the development of new anticancer Smac
therapeutics.
41. Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.; Nikolovska-Coleska, Z.; McEachern, D.; Yang,
C. Y.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. J. Med. Chem. 2011, 54, 3306.
42. Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A. S.; Betz, S. F.; Deckwerth, T.
L.; Ding, H.; Elmore, S. W.; Meadows, R. P.; Olejniczak, E. T.; Oleksijew, A.;
Oltersdorf, T.; Rosenberg, S. H.; Shoemaker, A. R.; Tomaselli, K. J.; Zou, H.; Fesik,
S. W. J. Med. Chem. 2004, 47, 4417.
43. All peptides were synthesized as C-terminal cysteamine-amides by the solid
phase method using CEM Liberty automatic microwave peptide synthesizer
(CEM Corporation Inc., Matthews, NC), applying 9-fluorenylmethyloxycarbonyl
(Fmoc) chemistry and commercially available amino acid derivatives and
reagents (EMD Biosciences, San Diego, CA and Chem-Impex International Inc,
Wood Dale, IL). Cysteamine 2-Chlorotrityl Resin (EMD Biosciences, San Diego,
CA) was used as a solid support. Peptides were cleaved from resin using
modified reagent K (TFA 94% (v/v); phenol, 2% (w/v); water, 2% (v/v); TIS, 1% (v/
v); EDT, 1% (v/v); 2 h) and precipitated by addition of ice-cold diethyl ether.
Reduced peptides were purified by preparative reverse-phase high
performance liquid chromatography (RP-HPLC) to >90% homogeneity and
their purity evaluated by matrix-assisted laser desorption ionization
spectrometry (MALDI-MS) as well as analytical RP-HPLC.
Acknowledgments
This project was partially supported by funds from the Adams
and Burnham endowments provided by the Dean’s Office of the
David Geffen School of Medicine at UCLA (P.R.).
Supplementary data
Supplementary data (analytical data for Smac analogs, repre-
sentative analytical RP-HPLC profile and corresponding MALDI-
MS spectra, and circular dichroism spectra) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.02.024.
44. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161.
45. Analytical RP-HPLC was performed on a Varian ProStar 210 HPLC system
equipped with ProStar 325 Dual Wavelength UV–Vis detector with the
wavelengths set at 220 and 280 nm (Varian Inc., Palo Alto, CA). Mobile
phases consisted of solvent A, 0.1% TFA in water, and solvent B, 0.1% TFA in
acetonitrile. Analyses of peptides were performed with an analytical C18 Vydac
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35. Wang, S. Curr. Top. Microbiol. Immunol. 2011, 348, 89.
36. Cai, Q.; Sun, H.; Peng, Y.; Lu, J.; Nikolovska-Coleska, Z.; McEachern, D.; Liu, L.;
Qiu, S.; Yang, C. Y.; Miller, R.; Yi, H.; Zhang, T.; Sun, D.; Kang, S.; Guo, M.;
Leopold, L.; Yang, D.; Wang, S. J. Med. Chem. 2011, 54, 2714.
37. Lu, J.; Bai, L.; Sun, H.; Nikolovska-Coleska, Z.; McEachern, D.; Qiu, S.; Miller, R.
S.; Yi, H.; Shangary, S.; Sun, Y.; Meagher, J. L.; Stuckey, J. A.; Wang, S. Cancer Res.
2008, 68, 9384.
218TP54 column, 4.6 Â 250 mm, 5
lm (Grace, Deerfield, IL) applying linear
gradient of solvent B from 0% to 100% over 100 min (flow rate: 1 ml/min).
Analytical data for synthesized peptides are presented in Supplementary
material.
46. Experiments were carried out using PrestoBlue™ Cell Viability Reagent
(Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. Briefly,
Smac-susceptible human non-metastatic breast cancer MCF-7 (or metastatic
MDA-MB-231) cells were plated in a 96-well plate at a density of 5 Â 103 cells/
well in a total volume of 50
ll of culture media and treated with various
concentrations of tested peptides (50
l
l of 0–200 M peptides in culture
l
media). The cells’ viability was assessed after 48 h by fluorescence
measurement (Ex/Em:560/590, incubation time 30 min) employing the
SpectraMAX M2 microplate reader (Molecular Devices, Sunnyvale, CA). All
experiments were carried out in triplicate.
47. Dimerization and trimerization of peptides was performed in 50% solution of
DMSO in DMF using modified S-alkylation protocol previously described by
Salvatore and co-workers48 that was adapted to peptides. Briefly, peptides
(3 equiv) were dissolved at final concentration 5 mg/ml. For dimerization 1,4-
bis(bromomethyl)benzene (1.5 equiv) and for trimerization 1,3,5-
tris(bromomethyl)benzene (1 equiv) were used with addition of anhydrous
cesium carbonate (Cs2CO3, 15 equiv) and tetrabutyl-ammonium iodide, (TBAI,
6 equiv). Solution was vigorously mixed on magnetic stirrer and progress of
reaction monitored by analytical RP-HPLC. Subsequently peptides were
purified and characterized as described in the peptide synthesis section.
48. Salvatore, R. N.; Smith, R. A.; Nischwitz, A. K.; Gavin, T. Tetrahedron Lett. 2005,
46, 8931.
49. Peng, Y.; Sun, H.; Lu, J.; Liu, L.; Cai, Q.; Shen, R.; Yang, C. Y.; Yi, H.; Wang, S. J.
Med. Chem. 2012, 55, 106.
50. Sheng, R.; Sun, H.; Liu, L.; Lu, J.; McEachern, D.; Wang, G.; Wen, J.; Min, P.; Du,
Z.; Lu, H.; Kang, S.; Guo, M.; Yang, D.; Wang, S. J. Med. Chem. 2013, 56, 3969.
51. All animal experiments were approved by the UCLA Animal Care and Use
Committee (ARC#1999-173-23) and conformed to local and national guidelines.
Presented in vivo results were calculated from at least
2 independent
experiments. Each group consisted 8 experimental animals. For Subcutaneous
engraftment model experiments BALB/SCID gnotobiotic mice (8 weeks old,
females) were obtained from the UCLA AALAC-accredited Department of
Radiation Oncology Facility and subcutaneously injected with 2.0 Â 106 cells
of human metastatic breast cancer line (MDA-MB-231, leg). After 3 weeks,
palpable tumors of approximately 5 mm diameter appeared and treatment was
initiated. In general, each animal received intraperitoneally a total of 10 doses of
SMAC17-2X at 2.5 or 7.5 mg/kg on days 1–5 and 8–12 with 8 mice per group. The
peptide was formulated in 2% Cremophor EL (Sigma–Aldrich, St Louis, MO) in
phosphate-buffered saline (PBS, vol/vol). Control animals were injected with
vehicle alone. Tumor size was assessed every two days and animals sacrificed as
necessary according to the UCLA Animal Care guidelines.
52. Enzymatic activity of caspases-3/7 and -9 was measured using commercially
available Caspase-GloÒ 3/7 and Caspase-GloÒ
9 assays (Promega Corp.,
Madison, WI) utilizing manufacturers protocols. Briefly, MDA-MB-231 cells
were plated in a white-walled 96-well plate at a density of 5 Â 103 cells/well in
a
total volume of 50 ll of culture media and treated with various
concentrations of tested peptides (50
ll of 0–100 lM peptides in culture
media) for 24 h. Subsequently, 100
l
l of appropriate Caspase-GloÒ reagent was
added to each well and cells incubated for additional 60 min. Luminescence
values were determined employing the SpectraMAX M2 microplate reader
(Molecular Devices, Sunnyvale, CA). All experiments were carried out in
triplicate.
38. Nikolovska-Coleska, Z.; Meagher, J. L.; Jiang, S.; Yang, C. Y.; Qiu, S.; Roller, P. P.;
Stuckey, J. A.; Wang, S. Biochemistry 2008, 47, 9811.

E. D. Micewicz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1452–1457
1457
53. Circular dichroism (CD) spectra (185–260 nm) of SMAC peptides were
measured in solution of trifluorethanol-buffer (TFE/HEPES 10 mM pH 7.4,
4:6, v:v) using a JASCO 715 spectropolarimeter (Jasco Inc., Easton, MD). The
instrument was routinely calibrated for wavelength and optical rotation using
10-camphorsulphonic acid and samples were scanned using 0.01 cm
pathlength cells at a rate of 20 nm per minute, a sample interval of 0.2 nm,
and a temperature of 25 °C. Sample concentration was determined by FTIR
quantitation. Sample spectra were baseline corrected by subtracting spectra of
rhXIAP BIR2 domain and rhXIAP BIR3 domain (R&D Systems Inc, Minneapolis,
MN) were immobilized on CM5 sensor chip using the amine coupling
method. The chip was activated by mixing 400 mM N-ethyl-N-(3-
dimethylaminopropyl)-carbodiimide hydrochloride and 100 mM N-
hydroxysuccinimide. Residual reactive groups on the chip surface were
blocked using 1.0 M ethanolamine/HCl (pH = 8.5). The flow cell-1 chip, which
served as a control, lacked immobilized protein but was treated with N-ethyl-
a
N-(3-dimethylaminopropyl)carbodiimide
hydrochloride,
N-hydroxy-
peptide-free solution and expressed as the Mean Residue Ellipticity [È]MRE
.
succinimide, and ethanolamine/HCl. Experiments were performed in HBS-EP
running buffer (pH = 7.4) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,
and 0.005% polysorbate 20. Binding signals were corrected for nonspecific
binding by subtracting the flow cell-1 signal. To regenerate chip surfaces,
bound ligands were removed with 10 mM HCl. Data were analyzed with
BIAevaluation 4.1 software (Biacore, Piscataway, NJ).
Quantitative estimates of the secondary structural contributions were made
with SELCON using the spectral basis set for proteins implemented in the Olis
Global Works™ software package (Olis Inc., Bogart, GA).
54. Binding studies were performed by surface plasmon resonance (SPR) on a
Biacore 3000 system (Biacore AB, Piscataway, NJ). The full-length rhXIAP,