Solid phase synthesis of Smac/DIABLO-derived peptides using a 'Safety-Catch' resin: Identification of potent XIAP BIR3 antagonists

Article abstract of DOI:10.1016/j.bmc.2013.06.055

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide 'Safety-Catch' resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500 nM) revealed N-methylalanyl- tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1 μM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.

Full text of DOI:10.1016/j.bmc.2013.06.055

Bioorganic & Medicinal Chemistry 21 (2013) 5004–5011
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Solid phase synthesis of Smac/DIABLO-derived peptides using a
‘Safety-Catch’ resin: Identification of potent XIAP BIR3 antagonists
⇑
Mohamed A. Elsawy, Lorraine Martin, Irina G. Tikhonova, Brian Walker
School of Pharmacy, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3
domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimet-
ics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer
cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a
small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using
docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized
employing Solid Phase Synthesis (SPS) on an alkane sulfonamide ‘Safety-Catch’ resin. This novel approach
afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of var-
ious C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and pur-
ity (>85%), upon nucleophilic release from the activated resin by treatment with various amine
nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the
recombinant BIR3-domain of human XIAP (500 nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-
(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of
Received 22 December 2012
Revised 20 June 2013
Accepted 24 June 2013
Available online 2 July 2013
Keywords:
SPPS
Safety-Catch resin
Peptidomimetics
Smac/DIABLO
XIAP BIR3
Cancer resistance
Apoptosis
the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1 lM concentration.
Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-
MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were
totally inactive in the same cell lines.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
demonstrated that only the N-terminal residues of Smac/DIABLO
are essential for its pro-apoptotic function.11–14 Moreover, other
The ability of cancer cells to suppress apoptosis induced by con-
ventional chemotherapeutic agents is achieved by the up-regula-
tion of various anti-apoptotic proteins. The Inhibitor of Apoptosis
Proteins (IAPs) constitute one such family of anti-apoptotic pro-
teins that are over-expressed in many tumours and which inhibit
caspase activation,1–9 making those tumours intensively impervi-
ous to traditional chemotherapeutics and radiotherapy. X-linked
IAP (XIAP) exhibits the most pronounced anti-apoptotic effect
among the IAP family.3–10 XIAP inhibits the apoptosome-mediated
activation of the initiator caspase, caspase-9, through binding to
the protease via its third Baculovirus IAP Repeat (BIR3) domain
and sequestering it in an inactive monomeric form.
Recently, a pro-apoptotic mitochondrial protein; Second mito-
chondria-derived activator of caspases (Smac), known also as Di-
rect IAP Binding protein with LOw pI (DIABLO), was discovered
to bind to and inhibit IAPs at the BIR3 domain, relieving capases
from their inhibition and thus permitting the re-activation of
the apoptosis machinery.5,6 Interestingly, many studies have
studies showed that the Smac/DIABLO-derived N-terminal short
peptides conjugated to cell penetrating carrier peptides are able
to overcome the XIAP-mediated apoptosis resistance, and re-sensi-
tized cancer cells, over-expressing XIAP, to various anticancer
drugs both in vitro and in vivo.15–17 Also, those peptides exhibited
a low toxicity profile in normal cells, which make them potentially
highly selective anticancer agents.
These attributes pinpoint the Smac/DIABLO-derived peptides as
potential anti-cancer therapeutic agents, though their entirely pep-
tidic nature may limit their therapeutic utilization. This is because
of the intrinsic and generally poor bioavailability of peptides owing
to their inability to penetrate cell membranes and instability to-
wards proteolytic cleavage. Consequently, many research groups
have focused on developing peptidomimetic18–21 and non-pep-
tidic22,23 Smac/DIABLO mimics, in attempts to generate XIAP
BIR3 tight-binders with improved bioavailability.
In this paper, we report on a new Solid Phase Synthesis (SPS)
strategy for the generation of novel Smac/DIABLO-derived peptides
using a ‘Safety-Catch’ resin. This strategy represents an original ap-
proach for the facile expedited synthesis of such peptides that were
previously only available through classical solution methods,
⇑
Corresponding author. Tel.: +44 2890972117; fax: +44 2890977794.
E-mail address: brian.walker@qub.ac.uk (B. Walker).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.055

M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
5005
which are more time consuming. The approach has been utilized
for the preparation of XIAP BIR3 antagonist sequences identified
using in silico structure-guided design. The peptides produced
have been shown to be potent XIAP BIR3 inhibitors in caspase-9
reactivation assays and demonstrated moderate cytotoxicity in cel-
lular context compared to the totally inactive Smac/DIABLO-de-
rived wild type sequences.
of peptides 10a and 11a–f from 4-Sulfamylbutyryl AM resin was
performed as described under Section 2.1.3. After a brief drying
in vacuum to remove all traces of solvents, the peptide products
were dissolved in 10% TFA aqueous solution and freeze dried. Pep-
tide purity was checked through RP-HPLC, using Waters HPLC sys-
tem fitted with Waters 1525 binary HPLC pump and Waters 2489
UV/visible detector (k216 nm) (Waters, Milford, Massachusetts,
USA) and employing a Phenomenex Jupiter C12 column (250
ꢁ 4.66 mm; particle size, 10
lm). The runs were carried out on
2. Materials and methods
2.1. Synthesis
an analytical scale with a flow rate of 1 ml/min. An elution gradient
was utilized to resolve the crude product components that went
from 90% solvent A (0.05% TFA in H2O)/10% Solvent B (0.05% TFA
in CH3OH) to 10% solvent A/90% solvent B in 60 min. Peptides with
crude purity less than 95% were purified with semi-preparative RP-
HPLC, using Phenomenex Jupiter C12 column (250 ꢁ 21.20 mm;
All amino acids were introduced as their Fmoc protected deriv-
atives. Fmoc-Ala-OH, Fmoc-Phe-OH and Fmoc-Pro-OH were pur-
chased from CEM (Buckingham, England, UK). 4-Sulfamylbutyryl
AM ‘Safety-Catch’ resin (substitution 0.8 mmol/g), Fmoc-Tic-OH,
Fmoc-Tyr-(tBu)-OH and Fmoc-Val-OH were obtained from Nova-
biochem (Darmstadt, Germany). Fmoc-N-Me-Ala-OH and Fmoc-
Tle-OH were supplied by BACHEM (Weil am Rhein, Germany).
(2S,4R)-Fmoc-4-phenyl-pyrrolidine-2-carboxylic acid and (2S,4S)-
Fmoc-4-phenoxy-pyrrolidine-2-carboxylic acid were purchased
from PolyPeptide Group (Strasbourg, France). Benzotriazole-1-yl-
oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop),
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate (HBTU) and Rink amide MBHA resin (substitution
0.65 mmol/g, 100–200 mesh) were obtained from Iris Biotech
GMBH (Marktredwitz, Germany). Anhydrous magnesium sulfate,
benzhydrylamine, carbonyldiimidazole, dichloromethane (CH2Cl2),
diethylether, diisopropylethylamine (DIEA), HPLC grade methanol,
HPLC grade trifluoroacetic acid, HPLC grade water, iodoacetonitrile,
Kaiser Test Kit, 1-methyl-2-pyrrolidone (NMP), N,N-dimethylform-
amide (DMF), piperidine, (R)-(ꢀ)-1,2,3,4-tetrahydro-1-naphthyl-
amine, tetrahydrofuran (THF), trifluoroacetic acid (TFA) and
triisopropylsilane were all obtained from Sigma–Aldrich (Gilling-
ham, Dorset, England, UK). C-(1H-Indol-3-yl)-methylamine was
purchased from Rare Chemicals GmbH (Kiel, Germany). Doubly
particle size, 10 lm) and using the same above stated elution gra-
dient with a flow rate of 10 ml/min.
2.1.2. General procedure for 4-sulfamylbutyryl AM ‘Safety-
Catch’ resin loading
4-Sulfamylbutyryl AM Resin (625 mg, 0.5 mmol) suspended in
17 ml CHCl3, DIEA (476.6 ll, 2.5 mmol) and an Fmoc-amino acid
[(2S,4R)-Fmoc-4-phenyl-pyrrolidine-2-carboxylic acid (620 mg,
1.5 mmol); (2S,4S)-Fmoc-4-phenoxy-pyrrolidine-2-carboxylic acid
(643 mg, 1.5 mmol); Fmoc-Tic-OH (600 mg, 1.5 mmol)] were
added to a 100 ml round bottom flask. The reaction mixture was
stirred for 10 min, cooled to ꢀ20 °C and after 20 min PyBop
(780 mg, 1.5 mmol) was added as solid. The reaction mixture
was stirred for 8 h at ꢀ20 °C after which the resin was filtered
and washed with CHCl3 (3 ꢁ 5 ml). The resin was dried in a desic-
cator under vacuum. The resin loading was checked spectrophoto-
metrically by performing Fmoc-group removal on accurately
weighed samples (ꢂ4 mg) of resin derivatized by these three
Fmoc-derivatives, and measuring the formation of the dibenzoful-
vene-piperidine adduct (molar absorptivity
k290 nm), using a UV spectrophotometer (50 Scan UV–Visible Spec-
trophotometer, VARIAN, Australia).
e
= 5253 Mꢀ1 cmꢀ1 at
distilled, deionised water was used throughout (17–18 mX).
2.1.1. Protocol for the microwave assisted solid phase peptide
synthesis (MW-SPPS) for sequence assembly
2.1.3. Peptide cleavage from 4-sulfamylbutyryl AM ‘Safety-
Catch’ resin
Peptides were synthesized on a CEM Liberty™ automated
microwave peptide synthesizer (CEM Microwave Technology Ltd,
Buckingham, England, UK). In essence, each peptide was synthes-
ised, on a 0.1–0.5 mmol scale, using either Rink Amide MBHA resin
(substitution 0.65 mmol/g, 100–200 mesh) for peptides (18–20) or
After peptide assembly on the 4-sulfamylbutyryl AM Resin, the
resin was activated by iodoacetonitrile to produce a resin-bound
N,N-cyanomethylacylsulfonamide (8, 9a–b). In the activation pro-
cedure, the resin (0.5 mmol) was washed several times with
NMP, followed by the addition of 8 ml NMP and DIEA (480
2.5 mmol) to the swollen resin. Then, iodoacetonitrile (734
l
l
l,
l,
a
pre-loaded 4-Sulfamylbutyryl AM resin (substitution 0.6–
0.8 mmol/g, loaded as described under Section 2.1.2) for peptides
(10a) and (11a–f). Fmoc removal was performed using 2 repeat cy-
cles employing 20% (V/V) piperidine as solution in DMF. A first
deprotection cycle of 30 s at 50 W was employed, followed by a
second deprotection cycle of 3 min at 50 W. Both cycles were car-
ried out at 75 °C. Coupling steps were carried out by introducing
each Fmoc-amino acid (0.2 M solution in DMF) at a fivefold excess
over resin loading, together with HBTU activation reagent and DIEA
activation base used in the molar ratios HBTU/DIEA/AA (1/2/1).
Each coupling reaction was performed for 10 min, at 22 W, at
75 °C. Finally, cleavage of peptides (18–20) from Rink Amide MBHA
was performed manually, at room temperature, using TFA/H2O/tri-
isopropylsilane (95/2.5/2.5, v/v/v), for two 1 h cycles, with washing
with dichloromethane after every cycle. The collected cleavage
reaction mixtures and washes were evaporated under vacuum, at
30 °C, cooled, and the products precipitated by the addition of cold
diethylether. The precipitates were collected by centrifugation (3–
5 min at 2000–3000g) and the pellet washed thoroughly with
diethylether. This process was repeated 2–3 times, each time the
solid was collected by centrifugation. On the other hand, cleavage
10 mmol) was added to the reaction mixture, which was protected
from direct exposure to light. The reaction mixture was stirred for
24 h, filtered and washed with NMP (5 ꢁ 5 ml, >10 min/wash),
CH2Cl2 (3 ꢁ 5 ml). The resin was then suspended in 15 ml THF
and transferred to 100 ml round bottom flask, to which the corre-
sponding primary amine was added [benzhydrylamine (458 mg,
2.5 mmol); (R)-(ꢀ)-1,2,3,4-tetrahydro-1-naphthylamine (368 mg,
2.5 mmol); C-(1H-Indol-3-yl)-methylamine (364 mg, 2.5 mmol)].
The reaction mixture was stirred for 4 h, filtered and washed with
THF several times. The combined filtrate and washes were evapo-
rated under vacuum, at 30 °C, dissolved in water and freeze dried.
2.2. Structural modeling
Automated docking was conducted with GLIDE 5.6 of Schrödinger
suite 9.0 using the Extra Precision XP algorithm.24,25 The crystal
structure of the BIR3 domain of XIAP with the PDB code 1G3F,
was refined with the Protein Preparation module of Schrödinger
suite 9.0 and used for the docking studies. The binding site was
defined as the residues located within 6 Å from the ligand. To

5006
M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
compensate for the rigid representation of the receptor, the Van
der Waals radii of the atoms were scaled by factor 0.65 to generate
the receptor grid. All compounds were pre-processed with LigPrep
2.4 of Schrödinger at pH 7. Default settings were used for the dock-
ing protocol. The final ligand poses were selected based on the GLIDE
empirical docking scores (GlideScore) and the overall complemen-
tary to the binding cavity identified visually. The OPLS2005 force
field was used for all calculations.
2.3.2. Cell viability assays (MTT assays)
Cells were seeded onto sterile 96-well culture plates at densi-
ties of 1 ꢁ 104 cells/well for MDA-MB-231 cells and 2 ꢁ 104 cells/
well for MCF-7 cells. Cells were incubated, for 24 h, at 37 °C and
5% CO2, after which media was aspirated from cells and 100 ll
fresh media with/without treatments were added. Control wells
were treated with media containing the DMSO vehicle at concen-
trations equivalent to those used in the peptide treatments. Cells
were further incubated at 37 °C and 5% CO2 for 72 h, followed by
2.3. In vitro assays
the addition of MTT (10 ll of a 10 mg/ml solution) to each well.
Following 2 h incubation of the plate at 37 °C and 5% CO2 and
shielded from light with tin foil, the media was removed and the
produced formazan crystals were solubilised by the addition of
200 ll of DMSO. Colour intensity was measured quantitatively at
550 nm using an EL808 HiTeck™ Spectra Thermo plate reader (Hi-
Cytochrome
c
from bovine heart, 20-deoxyadenosine 50-
triphosphate disodium salt (dATP), dimethylsulphoxide (DMSO),
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
Human recombinant Dipeptidyl Peptidase IV (DPP-IV) enzyme
expressed in SF9 cells, microsomal Leucine Aminopeptidase (LAP)
enzyme type IV-S from porcine kidney microsomes, tris(hydroxy-
methyl)aminomethane (TrizmaÒ base), sodium chloride (NaCl)
and potassium chloride (KCl) were obtained from Sigma–Aldrich
Chemical Company (Gillingham, Dorset, England, UK). Caspase-9
Fluorimetric Assay Assay Kit (BioVision) was purchased from EnzoÒ
Life Sciences, Exeter, UK). The kit contained lysis buffer, assay buffer
and LEHD-AFC substrate. A Protease inhibitor cocktail was prepared
from a proteases inhibitor set obtained from Roche Applied Sciences
(West Sussex, England, UK). RecombinantHuman XIAP BIR3 Domain
(rhXIAP BIR3) was supplied by R&D systemsÒ (Abingdon,
Oxfordshire, England, UK). Doubly distilled, deionised water was
Teck Instruments, USA).
3. Results and discussion
3.1. Synthesis
A library of new Smac/DIABLO-derived peptidomimetics (Ta-
ble 1) was designed and selected for synthesis on the basis of the
in silico study performed as a part of the present investigation
(see Section 3.2), which was guided/informed by the well defined
structural information for the binding of Smac/DIABLO analogues
to XIAP BIR3.13,14
A novel SPS strategy for the efficient preparation of the target
library was adapted, based on the use of an alkane-sulfonamide
‘Safety-Catch’ resin, first introduced by Backes and Ellman.26 The
feature of resins with a safety-catch linker is that the peptide prod-
uct cannot be released by treatment with TFA and thus these resins
are suitable for both Fmoc- and Boc-SPPS. Rather, product cleavage
from such resins can be accomplished by a two-step process that
requires activation of the linker followed by reaction with a nucle-
ophile.26–28 This is most usually achieved by alkylation of the sul-
fonamide linker by an electrophilic halide (activation step),
followed by cleavage with a nucleophile, which in the present
study was an aryl–alkyl amine so as to produce the target C-termi-
nal capped peptide amides. This synthetic strategy was deemed
attractive for the synthesis of the target library, since it affords
high flexibility for the introduction of various C-terminal capping
groups by releasing the resin-tethered tripeptide sequences
[N-Me-Ala-Tle-X-Resin, where X is either Tic (6) or 4-substituted
Pro (7a,b)] with different amine nucleophiles (Scheme 1).
The ‘Safety-Catch’ 4-sulfamylbutyryl AM resin was first loaded
with either Fmoc-Tic-OH (2) or 4-substituted Fmoc-Pro-OH (3a
or 3b). The loading was carried out, conventionally, for 8 h, at
ꢀ20 °C, in CHCl3 using PyBop as an activator and DIEA as an activa-
tor base. The % resin loading achieved was 86% (0.69 mmol/g) for
(2), 81% (0.65 mmol/g) for (3a) and 90% (0.72 mmol/g) for (3b),
as determined spectrophotometrically. After confirming successful
resin loading, the target sequences were then extended by incorpo-
ration of Fmoc-Tle-OH and Fmoc-N-Me-Ala-OH, through brief cy-
cles of MW-assisted SPS.
used for buffers preparation (17–18 m
X).
2.3.1. Caspase-9 activity fluorimetric assay
MDA-MB-231 cell lysates were prepared by solubilising cell
pellets (ꢂ5 ꢁ 107 cells) in 1 ml ice cold lysis buffer (50 mM KCl,
5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 0.2% CHAPS and 50 mM
HEPES; pH 7.4), supplemented with a protease inhibitor cocktail
(50
g/ml Pepstatin, 50
SC, 0.5 mg/ml EDTA-Na2 and 2
ice prior to their use (<30 min). 5
and dATP (5 mM) in assay buffer (10 mM HEPES, 0.5 mM EGTA,
5 mM DTT and 10% Glycerol; pH 7.4) were added to 25 l of cell ly-
sate contained in a 1.5 ml microcentrifuge tube. To this was added
l
g/ml Antipain. 2HCl, 40
g/ml Phosphoramidon, 1 mg/ml Pefabloc
g/ml Aprotinin), and stored on
l of Cytochrome c (1 mg/ml)
lg/ml Bestatin, 6 lg/ml Chymostatin,
7
l
l
l
l
l
1
ll rhXIAP BIR3 (500 nM final concentration) and 0.5
l
l peptide
M and
treatment in DMSO (final concentrations of 1
0.01
l
M, 0.1 l
lM), which were then incubated, at 37 °C, for 1 h. Controls
were set up in which recombinant XIAP BIR3 domain and/or pep-
tide treatments were replaced with the vehicles used to dissolve
each, as appropriate. Each treatment/control was then diluted with
65
microtiter plate, and 5
Ac-LEHD-AFC (in DMF) was then added to each well, to give a final
substrate concentration of 50 M. Caspase-9 activity in each sam-
ll of assay buffer, transferred to individual wells of a 96-well
l
l of a solution of the fluorogenic substrate
l
ple well was then monitored (kex 380–400; kem 470–500) by
following the increase in fluorescence due to the formation of
7-amino-4-trifluoromethylcoumarin from the hydrolysis of Ac-
LEHD-AFC, catalysed by the protease, using a FLUOstar OPTIMA
spectrofluorimeter (BMG LABTECH, Ortenberg, Germany). Cas-
pase-9 activity was monitored for 1 h, at 37 °C. Reactions were car-
ried out in duplicate. Finally, the % caspase-9 ‘relief’ was calculated
by relating the rate of hydrolysis of the fluorogenic substrate in the
As mentioned above, peptide release from the resin requires
pre-activation of the ‘Safety-Catch’ linker by reaction with various
electrophiles. We employed the frequently used iodoacetonitrile as
the activator electrophile. The activated peptide-safety-catch res-
ins (8, 9a–b) were then treated with a range of aryl–alkyl amines
to produce the library of C-terminal capped Smac/DIABLO-derived
peptidomimetics listed in Table 1. As can be appreciated from this
table, the purity and yields of the crude products (11a–d) are rel-
atively high (>85%, see Table 1 and Supplementary Figs. S1 and
S2). In comparison, the crude compounds (10a) and (11e,f) were
obtained in low yields and purity. We suspect that the reason for
presence of 1 lM peptide +500 nM rhXIAP BIR3 to the maximum
rate obtained in the absence of the recombinant protein, in lysates
treated with cytochrome C and dATP according to the following
formula:
% Caspase 9relief ¼ 100 ꢁ ½slop of treatment curve fit=slop of
maximum activation ðdATP þ cyt:cÞ curve fitꢃ

M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
5007
Table 1
Library of Smac/DIABLO-derived peptidomimetics synthesized in the present study
CH3
CH3
O
O
O
O
NH
CH3
O
NHR2
NH
CH3
NH
NH
NHR2
O
N
N
10a
11a-f
R1
Compound
no.
R1
R2
Puritya
(%)
RT
Yield
TOF-MSb
Calculated Found
1H NMR (DMSO-d6, 400 MHz), d ppm
(min) (%)
10a
11a
11b
11c
11d
NAc
Indolyl-3-methyl
45
31
28
18
26
15
28
85
87
76
64
503.2918
570.3261
535.3284
555.3335
519.3335
503.2909 0.879 (s, 9H), 1.228 (d, 3H), 2.83 (d, 2H), 3.298–3.679 (m, 3H), 3.85
(m, 1H), 4.13 (m, 1H), 4.34–4.68 (m, 3H), 6.78–7.04 (m, 6H), 7.36–
7.44 (m, 2H)
570.3284 0.91 (s, 9H), 1.296 (d, 3H), 2.327 (m, 2H), 2.46 (s, 3H), 3.65 (d, 2H),
4.08 (m, 1H), 4.21 (m, 1H), 4.73 (m, 1H), 6.28 (s, 1H), 6.98–7.12 (d,
6H), 7.23–7.45 (m, 9H)
535.3298 0.876 (s, 9H), 1.278 (d, 3H), 1.243–1.349 (m, 4H), 2.39 (m, 2H),
2.52–2.75 (m, 2H), 3.34–3.82 (m, 3H), 4.12 (m, 1H), 4.34 (s, 1H),
4.62 (m, 1H), 7.16–7.18 (m, 3H), 7.25–7.48 (m, 6H)
555.3336 0.857 (s, 9H), 1.316 (d, 3H), 2.27 (m, 2H), 2.43 (s, 3H), 3.38 (m, 1H),
3.62 (d, 2H), 3.98 (m, 1H), 4.18 (m, 1H), 6.37 (s, 1H), 7.06–7.14 (d,
6H), 7.28–7.48 (m, 9H)
C6H5O (C6H5)2CH
99
98
95
85
C6H5
C6H5
C6H5
(R)-1-
Tetrahydronaphthyl
(C6H5)2CH
(R)-1-
Tetrahydronaphthyl
519.3337 0.86 (s, 9H), 1.28 (d, 3H), 1.58 (d, 2H), 1.77–1.94 (m, 2H), 2.20 (m,
2H), 2.65–2.73 (m, 2H), 3.29 (m, 1H), 3.58–3.78 (m, 3H), 4.16 (m,
1H), 6.92–7.09 (m, 3H), 7.29–7.43 (m, 6H)
11e
11f
C6H5O Indolyl-3-methyl
C6H5 Indolyl-3-methyl
35
32
27
28
18
13
551.3426
535.3437
551.3411 0.995 (s, 9H), 1.24 (d, 3H), 2.31 (m, 2H), 3.34 (m, 1H), 3.62–3.85 (m,
6H), 4.18–4.2 (m, 3H), 6.83–7.09 (m, 6H), 7.27–7.39 (m, 3H)
535.3456 0.91 (s, 9H), 1.25 (d, 3H), 2.1 (m, 2H), 3.28 (m, 1H), 3.46–3.62 (m,
6H), 4.11 (s, 1H), 4.14 (s, 1H), 6.88–6.94 (m, 4H), 7.27–7.39 (m, 5H)
a
b
c
% Purity of the crude product as estimated from its RP-HPLC chromatogram.
TOF-MS, Time of Flight-Mass Spectrometry.
NA, not applicable.
this is that the Indol-3-yl methylamine, which is the common
amine nucleophile used for release and C-terminal capping of the
three products, was sourced commercially and was found to be
of dubious quality, and is likely to be the reason behind the low
purity profile for those compounds.
substituent at the gamma (4) position of the proline residue. An-
other hydrophobic channel formed between the aliphatic hydro-
carbon side-chains of residues K297 and K299 (–[CH2]4–)
accommodates the Ile residue at the P4 position of Smac/DIABLO
(Fig. 1). This suggested to us that an aromatic ring could possibly
be slotted between these two residues so as to improve/enhance
the binding affinity of Smac/DIABLO-derived molecule to XIAP
BIR3.
3.2. In silico studies and in vitro assays
The structural basis for the interaction of the short N-terminal
segment of Smac/DIABLO with XIAP BIR3 domain is well
defined.13,14 The structure of the Smac/DIABLO-XIAP BIR3 complex
(PDB code: 1G3F) shows that the Smac/DIABLO N-terminal tetra-
peptide sequence AVPI possesses an extended conformation along
the third b-strand of the XIAP BIR3 domain and has identified spe-
cific inter-molecular interactions that appear to be crucial for com-
plex stability (Fig. 1). For example, possible electrostatic
interaction between the Smac/DIABLO peptide N-terminal amino
group and the proximal carboxylate of E314 in XIAP BIR3 seem
likely to occur. Also, the side-chain methyl group of Ala is accom-
modated into a hydrophobic pocket formed by the W310 residue of
the protein, in which it most likely interacts with the indole ring of
the tryptophan residue. As can also by appreciated from Figure 1,
the isopropyl side chain of the P2 Val residue of Smac is protruding
outside the XIAP BIR3 binding pocket and is solvent exposed,
which reflects its minor role in the interaction. At P3, the Smac/
DIABLO main chain amide backbone is slightly twisted, due to
the presence of a proline residue at this position. It can be appreci-
ated that the orientation of the proline residue could possibly fa-
vour hydrophobic interactions between the gamma (position-4)
and delta (position-5) methylene groups of the pyrrolidine ring
with the indole ring of W323 (Fig. 1). It can also be observed from
Figure 1 that there is a deep hydrophobic pocket formed by the res-
idues W323 and Y324 that can accommodate an extra aromatic
Based on this analysis of the structure of the complex, we con-
structed two libraries in silico. The first library comprised mole-
cules in which the nature of the R1 substituent at the 4-position
of the P3 proline residue was varied, along with the aromatic C-ter-
minal amide capping group (R2) to give the general structures
(11a–s), shown below (Table 2 and Supplementary Table S3),
which includes the peptide N-methylalanyl-tertiarybutylglycinyl-
4-(S)-phenoxyprolyl-N-(R)-1-tetrahydronaphthyl carboxamide (11g),
previously reported to be a potent antagonist of rhXIAP BIR3
(Table 2).18 In the secondlibrary, the P3 Pro residue was replaced
by tetrahydroisoquinoline carboxylate (Tic). Tic is also an amino
acid and can be considered as a highly constrained analogue of
4-phenyl-proline. Once again, the aromatic C-terminal capping
group (R2) of the Tic-containing peptide was varied to give the gen-
eral structures (10a–c) (Table 2 and Supplementary Table S3).
High-throughput docking of the 22 virtual ligands into the XIAP
BIR3 binding site was carried out using GLIDE (Schrödinger),
employing the GlideScore scoring function.24,25 The docking scores
for the best fitting ligands are shown in Table 2. On the other hand,
in silicocompounds (10b,c) and (11h–s) were not accommodated
in the XIAP BIR3 pocket (see Supplementary Table S3 for precise
structures), which is most likely due to their steric bulk. Conse-
quently, it was decided not to synthesize and test these examples.
The overlay of some of the analogues prepared in the
present study (shown in yellow) with the Smac/DIABLO-derived

5008
M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
O
NH
O
S
O
H2N
O
O
1
OH
OH
+
FmocN
Or
NFmoc
2
R1
3a-b
3a
3b
R1= C6H5O
= C6H5
(i) Resin/AA/DIEA/PyBop
(1/3/5/3) in CHCl3, -20 C, 8 hours
O
O
O
S
O
O
NH
O
O
Fmoc
N
NH
S
NH
NH
NFmoc
R1
O
4
5a-b
R1= C6H5O 5a
= C6H5 5b
(ii) 20% piperidine in DMF
(iii) HBTU / DIEA/ Fmoc-Tle-OH (1/2/1)
(ii) 20% piperidine in DMF
MW, 75C
22 Watts
(iv) HBTU / DIEA/ Fmoc-N-Me-Ala-OH (1/2/1)
(ii) 20% piperidine in DMF
NH CH3
CH3
O
O
NH
O
O
O
NH
O
O
S
O
O
NH
S
NH
O
R1
N
NH
N
O
7a-b
6
R1= C6H5O 7a
= C6H5 7b
NH
CH3
O
NH
CH3
(v) ICH2CN / DIEA (4/1), NMP,
in dark
(vi) NMP (5 x 5 ml, > 10 minutes/wash),
CH2Cl2 (3 x 5 ml)
NH CH3
CH3
O
O
S
O
O
O
NH
NH
O
O
O
S
O
N
R1
O
NH
N
O
N
C
N
N
9a-b
8
NH
C
N
R1= C6H5O 9a
= C6H5 9b
CH3
O
NH
CH3
(vii) R2NH2 (2 equivalent), THF, 4 hours
CH3
O
CH3
O
O
O
O
NH
CH3
NH
CH3
O
NHR2
NH
NH
NHR2
N
N
10a
11a-f
R1
Scheme 1. Scheme for the synthesis of Smac/DIABLO-derived peptidomimetics showing loading of the first amino acid onto and release of product by aminolysis from
4-sulfamylbutyryl AM ‘Safety-Catch’ resin.

M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
5009
grouping about the C–O ether bond, thus permitting interaction
with both W323 and Y324 residues. The replacement of the 4-
(R)-phenoxy with the more rigid 4-(S)-phenyl group at P3 does
not seem to make a big difference in the docking score of (11c)
compared to its phenoxy analogue (11a) (Table 2). However, there
is an appreciable drop in the docking score of the 4-(S)-phenyl
substituted analogues (11d) and (11f) when compared to their 4-
(R)-phenoxy congeners (11b) and (11e) (Table 2 and Fig. 2b). Final-
ly, no significant differences in the docking scores were observed
as a result of varying the C-terminal aromatic capping between tet-
rahydronaphthyl, indolyl and biphenyl groups with the exception
of the best fitting candidate (11b) (Table 2). On the other hand,
(10a) was accommodated particularly readily, with the Tic tetrahy-
droisoquinoline fused ring of this analogue overlapping with the 4-
(S)-phenoxy-substituted proline residue of the previously reported
analogue (11g). This predicted accommodation of (10a), preserves
the hydrophobic interaction with W323 (data not shown).
The top-ranked hits based on the GlideScore function and the
pocket fitting were conveniently synthesized as described in Sec-
tion 3.1 above and tested for their XIAP BIR3 inhibitory activity
through a caspase-9 activity reconstitution assay. In this assay,
the ability of the Smac/DIABLO-derived analogue to recover cas-
pase-9 activity in the presence of 500 nM rhXIAP BIR3 was deter-
mined by the % increase in caspase-9 activity that they promoted.
The XIAP BIR3 inhibition observed for the 4-(R)-phenoxy substi-
tuted compounds (11a), (11b) and (11e) of % caspase-9 relief
93.1 7.9, 87.6 6.2 and 64.9 9.3 is slightly-but not signifi-
cantly-higher than that caused by their 4-(S)-phenyl analogues
(11c), (11d) and (11f) of % caspase-9 relief 89.4 5.8, 83.7 7.1
and 61.6 8.6, respectively (Table 2 and Fig. 3). Also, the XIAP
BIR3 inhibition was slightly-but not significantly-improved when
the tetrahydronaphthyl amide C-terminal capping group of ana-
logues (11b) and (11d) of % caspase-9 relief 87.6 6.2 and
83.7 7.1 was replaced with the biphenyl amide group in ana-
Figure 1. Smac/DIABLO-derived N-terminal sequence AVPI (orange) in the XIAP
BIR3 binding pocket; generated from PDB: 1G3F.13 Only residues forming the direct
contacts with the peptide are visualized and the shape of the binding cavity are
shown with the surface-like representation coloured based on the location of the
negative and positive charge residues.
peptidomimetic (11g) (shown in green) within the XIAP BIR3 bind-
ing site are shown in Figure 2, where (11g) is to our knowledge the
tightest reported binder to XIAP BIR3 (Kd = 5 nM, Oost et al.18) It is
obvious from Figure 2a, that (11b) is one of the best overlapping
matching candidates with peptidomimetic (11g). The in silico
docking score of (11b) was ꢀ7.05 Kcal/mol compared to only
ꢀ3.81 Kcal/mol for its enantiomer (11g) (Table 2 and Fig. 2a).
The differences in docking score could be attributable to the ‘inver-
sion’ of the configuration of the 4-phenoxy group from S in (11g) to
R in (11b). This inversion would serve to position/place the phenyl
ring deeper into the hydrophobic pocket formed by residues W323
logues (11a) and (11c) of
% caspase-9 relief 93.1 7.9 and
89.4 5.8, respectively (Table 2). On the other hand, the C-terminal
capping with an indanylmethyl amide group caused ꢂ1.4-fold sig-
nificant reduction in the XIAP BIR3 inhibitory activity of (11e) and
(11f) of % caspase-9 relief 64.9 9.3 and 61.6 8.6, respectively,
when compared to their biphenyl amide analogues (11a) and
(11c) of % caspase-9 relief 93.1 7.9 and 89.4 5.8, respectively,
and Y324, which could lead to additional p–p interactions between
the 4-(R)-phenoxy phenyl ring of the peptide with the aromatic
side-chains of both residues (Fig. 2a). This would be possible be-
cause of the rotation of the phenyl ring of the 4-(R)-phenoxy
Table 2
Smac-derived peptidomimetic libraries docking scores and bioassays data
CH3
CH3
O
O
O
NH
CH3
O
NHR2
NH
CH3
NH
NH
NHR2
O
O
N
N
11a-g
10a
R1
Compound. no.
R1
R2
Glide docking score (Kcal/mol)
% Caspase-9 reliefa
% Viabilityb
MDA-MB-231
MCF-7
10a
11a
11b
11c
11d
11e
11f
Not applicable
(R)-C6H5O
(R)-C6H5O
(S)-C6H5
(S)-C6H5
(R)-C6H5O
(S)-C6H5
Indolyl-3-methyl
(C6H5)2CH
(R)-1-Tetrahydronaphthyl
(C6H5)2CH
(R)-1-Tetrahydronaphthyl
Indolyl-3-methyl
Indolyl-3-methyl
ꢀ4.598
ꢀ5.045
ꢀ7.05
66.3 8.4
93.1 7.9
87.6 6.2
89.4 5.8
83.7 7.1
64.9 9.3
61.6 8.6
NA
NDc
ND
50.3 7.9
58.1 7.2
50.1 6.5
54.6 11.9
ND
58.6 9.7
70.8 11.2
62.3 7.9
79.4 12.3
ND
ꢀ5.409
ꢀ4.68
ꢀ6.447
ꢀ4.215
ꢀ3.81
ND
NA
ND
NA
11gd
(S)-C6H5O
(R)-1-Tetrahydronaphthyl
a
b
c
Treatments were used at 1
lM concentration.
% Viability at 100 M concentration.
l
NA, data not available; ND, not determined.
d
Kd = 5 nM, as reported by Oost et al.18

5010
M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
Figure 2. Binding poses of analogues 11b (A) and 11c (B) (both shown in yellow) in XIAP BIR3, overlapped with the peptidomimetic 11g (shown in green).
Figure 3. Caspase-9 activation data for (11a) and (11f) as examples of peptidomimetics in comparison to the WT-Hid (18).
and their tetrahydronaphthyl amide analogues (11b) and (11d) of
% caspase-9 relief 87.6 6.2 and 83.7 7.1, respectively (Table 2);
even though the in silico docking predicted the complete overlap
of the indanyl ring with the tetrahydronaphthyl system. Further-
more, ligand (10a) demonstrated a 66.3 8.4% caspase-9 activity
recovery, which is equipotent to its 4-(R)-phenoxy proline ana-
logue (11e) of 64.9 9.3% caspase-9 activity recovery and
slightly-but not significantly-stronger than its 4-(S)-phenyl proline
analogue (11f) of 61.6 8.6% caspase-9 activity recovery (Table 2).
The rigidity of the tetrahydroisoquinoline ring fixed its phenyl ring
part in an optimal position for the hydrophobic interaction with
W323, although it does not project as deeply into the pocket as
the 4-phenoxy substituted proline containing analogue (11e) (data
not shown).
To validate the caspase-9 activity recovery assay, a series of
Hid-derived N-terminal pentapeptides (18–20) of known binding
affinity to XIAP BIR318 (Supplementary Table S4) were tested using
the same assay, where Hid is the Drosophila functional analogue of
human Smac/DIABLO.29 It was gratifying to find that, the % cas-
pase-9 relief recorded for those peptides matched their hierarchal
binding affinity order (Supplementary Table S4 and Fig. S5). Conse-
quently, the % caspase-9 activity recovery induced by our ligands
can be used as a basis to compare their relative XIAP BIR3 binding
affinities and also those of the Hid peptides. On this basis, the
Smac/DIABLO-derived peptidomimetics (11a–d) prepared in this
study and possessing % caspase-9 activity recovery range of ꢂ93–
83% are ꢂ1.8-fold significantly stronger XIAP BIR3 inhibitors than
the Hid peptides (18–20) of % caspase-9 activity recovery range
Consistent with the caspase-9 activity recovery assay, the % via-
bility (MTT) assays showed moderate cytotoxicity of Smac/DIA-
BLO-derived peptidomimetics (11a–d) at 100 lM concentration
in the breast cancer MDA-MB-231 and MCF-7 cell lines, with a
range of ꢂ79–50% cell viability compared to untreated control (Ta-
ble 2). In contrast, N-terminal Hid-derived peptides (18–20) exhib-
ited no cytotoxicity against the same cell lines, when used at the
same concentrations (data not shown).
Overall, the Smac/DIABLO-derived peptidomimetics (11a–d)
showed significant improvement in the release/relief of caspase-
9 inhibition caused by XIAP BIR3, compared to the N-terminal
Hid-derived peptides (18–20). In addition, they also exhibited
greater cytotoxicity against tumour cell lines, as assessed by MTT
assays, in comparison to the Hid-derived peptides. This improve-
ment is attributable to the SAR-based optimization and in silico
aided design methods used to decide on the combinations of C-ter-
minal capping groups and P3 residues of the sequences. These opti-
mized alterations conferred better binding of (11a–d) to XIAP BIR3,
and probably also improved their cell penetrability and resistance
towards proteolytic cleavage, in comparison to Hid-derived pep-
tides (18–20).
4. Conclusion
XIAP BIR3 inhibition by Smac/DIABLO-derived peptides can re-
sensitize cancer cells to apoptosis. Accordingly, Smac/DIABLO-de-
rived peptidomimetics were designed to improve both the XIAP
BIR3 inhibition capacity and pro-apoptotic activity against intact
cells. Based on the well-established SAR for Smac/DIABLO, 22
ꢂ43–55%, all tested at 1
lM concentration.

M. A. Elsawy et al. / Bioorg. Med. Chem. 21 (2013) 5004–5011
5011
virtual ligands were designed, which were docked to XIAP BIR3
Acknowledgments
binding pocket. The ligands that exhibited best-fit and appropriate
docking scores were synthesized by a novel SPPS strategy employ-
ing an Alkane sulfonamide ‘Safety-Catch’ resin. This approach
afforded the rapid synthesis of the target peptidomimetic library
and each of the member compounds was obtained in high % yield
and purity. The use of the ‘Safety-Catch’ resin greatly facilitated the
synthesis of the Smac/DIABLO-derived peptidomimetic library and
has proved a novel alternative approach for the expedited synthe-
sis of this class of compound that were previously only available
through the much tedious classical solution methods.
This work was supported by the Dr. John King Foundation,
through the provision of a Ph.D. studentship to Mohamed Elsawy.
Many thanks for Dr. Rebecca Craig, School of Pharmacy, Queen’s
University of Belfast, for performing the 1H NMR spectrometry
analysis and to Christopher Hutchison, School of Pharmacy,
Queen’s University of Belfast, for the scientific discussion.
Supplementary data
These were then tested for their XIAP BIR3 inhibitory activity
through a caspase-9 reactivation assay in presence of the apopto-
some elements (Apaf-1, cytochrome c and dATP) with/without
XIAP BIR3 domain. In general, candidates with 4-(R)-phenoxy sub-
stitution of the P3 pyrrolidine ring of the proline residue showed
higher caspase-9 reactivation than the 4-(S)-phenyl analogues.
This could be attributed to the deeper projection of phenoxy group
into the hydrophobic pocket formed by residues W323 and Y324
(of XIAP BIR3) when compared to the corresponding phenyl ana-
logues. This could reduce the inhibitory action of XIAP BIR3 and
thus improve the caspase-9 reactivation induced by the 4-(R)-
phenoxy analogues in comparison to the 4-(S)-phenyl analogues.
Additionally, the peptidomimetic (10a) with Tic at P3 showed very
similar caspase-9 reactivation to the 4-(R)-phenoxy analogues and
in silico ligand superimposition demonstrated that both analogue
types bound in a very similar manner. A study of the effectiveness
of using differing aromatic ring C-terminal amide capping groups
was also undertaken, since previous work has demonstrated that
such groups are accommodated snugly into the hydrophobic chan-
nel formed by the K297 and K299 side chains of XIAP BIR3. As a
general trend, it was found that the biphenyl amide capped com-
pounds (11a) and (11c) exhibited higher caspase-9 reactivation
than their tetrahydronaphthyl amide analogues (11b) and (11d),
and both were stronger caspase-9 activators than their indolyl-3-
methyl amide congeners (11e) and (11f). The biphenyl substituent
is constrained and holds the phenyl rings in optimal positions for
the hydrophobic interaction with K297 and K299 side chains.
Collectively, the Smac/DIABLO peptidomimetic (11a) with P1 N-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.055.
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methyl-L-alanine, P3 4-(R)-phenoxy proline and biphenyl amide
capping at P4 exhibited the most potent caspase-9 activity restora-
tion, with 93% caspase-9 relief being achieved at a concentration of
1
l
M.
It was anticipated that these collective molecular properties
would imbue these peptidomimetics with cell permeability and
resistance to hydrolysis by proteases and so they were examined
for their ability to cause loss of cell viability of a number of breast
cancer cell lines. Compounds (11a–d) did indeed demonstrate
moderate cytotoxicity against the breast cancer cell lines MDA-
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MB-231 and MCF-7, when used at a concentration of 100 lM.
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that Smac/DIABLO-derived peptidomimetics act as apoptosis re-
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per se.
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