Structure-based design, synthesis and anticancer effect of cyclic Smac–polyarginine peptides

Article abstract of DOI:10.1007/s00726-018-2637-0

The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mitochondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring field for cancer therapy. In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly showed that cyclic peptides P4, P5 and P7 gave rise to a significant level of cytotoxicity and apoptosis mediated cell death in multiple myeloma tumor cells (MM) comparing to linear peptide.

Full text of DOI:10.1007/s00726-018-2637-0

Amino Acids
https://doi.org/10.1007/s00726-018-2637-0
ORIGINAL ARTICLE
Structure‑based design, synthesis and anticancer efect of cyclic
Smac–polyarginine peptides
Melek Parlak Khalily1 · Selin Gerekçi2 · Ezgi A. Güleç2 · Can Özen2,3 · Salih Özçubukçu1,2
Received: 24 April 2018 / Accepted: 14 August 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mito-
chondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of
apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino
acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor
stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring feld for cancer therapy.
In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its
intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained
in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly
showed that cyclic peptides P4, P5 and P7 gave rise to a signifcant level of cytotoxicity and apoptosis mediated cell death
in multiple myeloma tumor cells (MM) comparing to linear peptide.
Keywords Smac/DIABLO · Constrained peptides · IAP · Proapoptotic peptides · CPPs · Multiple myeloma
Introduction
disorders such as Alzheimer (Green and Martin 1995;
Thompson 1995).
Apoptosis is a tightly controlled cell death mechanism which
regulates development and homeostasis of all multicellular
organisms (Elmore 2007). Inappropriate apoptosis is associ-
ated with wide variety of illnesses. Abnormal inhibition of
apoptosis is implicated in cancer and autoimmune diseases,
while excessive cell death contributes to neurodegenerative
The inhibitor of apoptosis proteins (IAPs) plays critical
role in regulation of programmed cell death in a variety of
organisms (Deveraux et al. 1997; Deveraux and Reed 1999).
Their main function is to suppress activity of caspases which
are ultimate actors of apoptosis (Shi 2002; Thornberry
1998). The apoptotic caspases are generally divided into
two classes: the initiator caspases, which include caspase-2,
-8, -9 and -10 and the efector caspases, which include cas-
pases-3, -6 and -7. Efector caspases are activated by ini-
tiator caspases. Once activated, the efector caspases are
responsible for the proteolytic cleavage of a broad spectrum
of cellular targets, which fnally leads to cell death. In mam-
mals, caspases-3, -7 and -9 are subject to inhibition by IAPs,
specifcally X-linked inhibitor of apoptosis protein (XIAP)
(Shi 2002).
Handling Editor: N. Sewald.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00726-018-2637-0) contains
supplementary material, which is available to authorized users.
* Salih Özçubukçu
osalih@metu.edu.tr
The IAPs suppress apoptosis by negatively regulat-
ing the caspases, whereas Smac/DIABLO has ability to
remove the IAP-mediated negative regulation of caspases.
Smac/DIABLO is a mitochondrial protein and released
from intermembrane space of mitochondria into the cyto-
plasm in response to apoptotic stimuli (Wang and Youle
2009). This protein contains a tetrapeptide at its N terminus
1
Department of Chemistry, Middle East Technical University
(METU), 06800 Ankara, Turkey
2
Biochemistry Graduate Program, METU, 06800 Ankara,
Turkey
3
Biotechnology Graduate Program, Central Laboratory,
Center of Excellence in Biomaterials and Tissue Engineering,
METU, 06800 Ankara, Turkey
Vol.:(0123456789)
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M. P. Khalily et al.
–Ala–Val–Pro–Ile– which specifcally binds to the con-
served surface groove on the BIR3 domain of XIAP and
disrupts its interaction with the caspases, prompting apopto-
sis (Wu et al. 2000; Liu et al. 2000). The structural analysis
of Smac binding to XIAP showed that that peptides or small
molecules modeled on this binding motif might serve as pro-
totypical drugs whose activity might simulate that of Smac/
DIABLO (Kipp et al. 2002). Smac peptides composed of the
frst four to eight amino-terminal residues were delivered
into cancer cells, and it is proved that these peptides enhance
apoptosis just like known chemotherapeutics do (Arnt et al.
2002; Fulda et al. 2002; Li 2004; Park et al. 2005; Yang
et al. 2003).
in proteolytic stability. The efects of cyclization of SmacN7-
Arg8 fused peptides on both cellular uptake and cytotoxicity
were examined.
Methods
Materials
All chemical reagents and solvents were ACS grade or
higher and purchased from commercial sources other than
tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4). All
Fmoc-protected-l-amino acids, coupling reagents, Rink
amide MBHA resin (200–400 mesh, 0.52 meq/g loading
capacity) and 2-chlorotrityl chloride resin (100–200 mesh,
1.0–2.0 meq/g loading capacity) were obtained from Chem-
Impex (USA). Dichloromethane (DCM) distilled over cal-
cium hydride. Dimethylsulfoxide was pre-dried over calcium
sulfate for approximately 2 days, and then dried with vac-
uum distillation. MilliQ water was double deionized using
a Milli-Q® Advantage A10® Water Purifcation Systems
(18.2 MΩ cm at 25 °C, TOC: 7 ppb). 5 mM stock peptide
solutions in MilliQ were prepared for cell assays and stored
at −20 °C.
Some studies showed that Smac peptides are sensitive to
proteolytic degradation, and have a poor capacity to pen-
etrate cells (Yang et al. 2003). Therefore, many research
groups have focused on developing a number of Smac con-
jugates (Arnt et al. 2002; Fulda et al. 2002; Yang et al. 2003)
and mimetics (Li 2004; Park et al. 2005; Oost et al. 2004)
and to circumvent the limitations of Smac peptides. Estab-
lished transmembrane transport peptides such as Penetratin
(Arnt et al. 2002), HIV-1 Tat (Fulda et al. 2002), and pol-
yarginine (hexa- or octaarginine) (Yang et al. 2003) have
been fused to the Smac sequence to promote uptake of Smac
peptides into the cell. These studies suggest that Smac–CPP
conjugates may have promising therapeutic potential as a
new class of anti-cancer drugs.
General method for solid‑phase peptide synthesis
Development of cyclic Smac–CPP peptide structure may
both lead to improvements in internalization efciency and
stability. Structural variants of the linear peptide backbone
have always played an important role on specifc characteris-
tics of a peptide. One possible consequence of linear nature
is rapid degradation by proteases (Adessi and Soto 2002;
Burton et al. 1996; Gilon et al. 1991; McGeary and Fairlie
1998; Pauletti 1997). Furthermore, to provide a therapeutic
efect, a peptide must bind to its target. The fexible nature
of linear peptides can limit their afnity making it difcult
to bind to some challenging cellular targets, such as protein
surfaces involved in protein–protein interactions. In con-
trast, the reduced fexibility of cyclic peptides decreases the
entropic penalty associated with binding, making them much
more suitable for inhibiting some difcult targets (Liskamp
2014). Overall, constraining a peptide into a cyclic structure
may signifcantly increase its efcacy in diferent ways.
In the present study, a heptapeptide AVPIAQK (SmacN7)
that contain binding sequence (AVPI) was targeted. SmacN7
peptide was fused with a well-established cell-penetrating
peptide octaarginine (Arg8) to achieve cellular uptake.
Diferent variants SmacN7–Arg8 peptides (Fig. 1) were
designed, synthesized, and tested for U266 multiple mye-
loma cell line. To increase therapeutic efcacy of the bifunc-
tional sequence, the peptide conformation was locked via
cyclization, which leads to reduction of polarity and increase
All peptides were synthesized with CEM Discover Bio-Man-
ual Microwave Peptide Synthesizer using Fmoc-based solid-
phase peptide synthesis (SPPS) strategy either on a Rink
amide resin or a 2-chlorotrityl chloride resin at diferent
scales between 0.1 and 0.25 mmol. N-terminus of peptides
except P6 and P3 were extended with an aminohexanoic
acid (Ahx) spacer to attach the fuorescein moiety needed
to follow cellular uptake by fow cytometry. Attachment of
fuorescein for P6 and P3 was done from the lysine after
selectively removal of allyloxycarbonyl (Alloc) or methyl-
trityl (Mtt) protections on resin. All arginines were coupled
with potent coupling reagent HCTU (2-(6-chloro-1-H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafuoro-
phosphate). Other amino acids were coupled with HBTU
(N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl) uronium
hexafuorophosphate). Couplings were carried out with 3.0
equivalents of Fmoc-l-amino acids, 2.85 equiv. of coupling
reagent, and 6 equiv. of N,N-diisopropylethylamine (DIEA)
in dimethylformamide (DMF) with assistance of MW heat-
ing (20 W, 10–15 min). All deprotections were achieved with
5 mL solution of 20% piperidine in DMF with MW heating
(20 W, 3×2 min). Completion of each coupling and depro-
tection was monitored by Kaiser test (Kaiser et al. 1970)
except proline coupling.
After peptide elongation was complete, peptide was cleaved
from the resin and fully deprotected by treatment of the resin
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Structure-based design, synthesis and anticancer efect of cyclic Smac–polyarginine peptides
Fig. 1 Sequences of P1–7. Lactam bridges are shown as thin lines. The thick line joining the N and C termini of P6 indicates backbone cycliza-
tion. For better visualization, schematic representation of each peptide is shown from the right-hand side
with a mixture of trifuoroacetic acid (TFA)/triisopropylsilane
(TIS)/H2O (95: 2.5: 2.5 v/v/v) for 3–4 h at room temperature.
Free peptides were separated from the resin by fltration and
the resin was further washed with TFA. Collected fltrate was
precipitated in diethyl ether and centrifuged. The supernatant
was removed and diethyl ether was further added to repeat the
washing step up to three times. The precipitate was dried, lyo-
philized in water and purifed using preparative reverse phase
high performance chromatography (RP-HPLC). Purity was
evaluated by analytical RP-HPLC and identity confrmed by
mass spectrometry.
Reverse phase high performance liquid
chromatography (RP‑HPLC) analysis
Reverse phase preparative HPLC was performed on a
Dionex UltiMate 3000 HPLC system, employing a Thermo
Scientifc Hypersil Gold C18 column (250×10 mm, 5 μm)
at fow rate of 2 mL/min at 40 °C. Acetonitrile/water gradi-
ent containing 0.1% trifuoroacetic acid (5–100%, 1–70 min)
was utilized as eluent. All polyarginine containing peptides
were dissolved in 10% acetic acid containing MilliQ water.
The concentration of the peptides was adjusted as 20 mg/
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M. P. Khalily et al.
mL. 1.5 mL of this peptide solution was purifed for one
batch using 2 mL injection loop and purifcation repeated
consecutively. Purity of each fraction was assessed with ana-
lytical RP-HPLC, before combining pure fractions.
Analytical HPLC was performed on a Dionex UltiMate
3000 Hplc system equipped with a Thermo Scientific
AcclaimTM 120 C18 column (46×150 mm, 3 μm). Elution
of the peptides was achieved using an acetonitrile/water
gradient containing 0.1% trifluoroacetic acid (5–100%,
1–30 min, fow 0.4 mL/min). After purity of collected frac-
tions confrmed by analytical HPLC, lyophilization was
achieved using a Telstar Cryodos Freeze Dryer and peptides
were stored at −20 °C.
DIEA in DMF and, the left gentle stirring overnight. The
completion of the reaction was tested using Kaiser test.
Cell culture
U266 MM cells were cultured in RPMI-1640 growth
medium containing 10% FBS, 1% penicillin–streptomycin
and 1% non-essential amino acids at 37 °C in a 5% CO2
incubator.
Flow cytometry
Cellular uptake assessment, cell cycle analysis and apoptosis
assays were performed on Accuri C6 fow cytometer (BD
Biosciences). Gating was performed so that cell debris, cell
clumps and doublets were excluded. Ten thousand events
were recorded for each measurement.
Mass analysis of peptides
The concentration synthesized peptides was adjusted as
1 mg/mL with water and characterized by mass spectrom-
etry on an Agilent 6530 Q-TOF mass spectrometer equipped
with ESI (electrospray ionization) source.
Cellular uptake
Cells were treated with 10 µM of designed peptides for
15 min. Cellular uptake assessment was carried out using
fow cytometer.
Preparation of Tetrakis(triphenylphosphine)
palladium(0) Pd(PPh3)4
40 mL dry DMSO was deoxygenated by vigorously fush-
ing nitrogen gas for 30 min in a two-neck round-bottom
fask equipped with a magnetic stirring bar and a refux
condenser. PdCl2 (0.50 g, 2.8 mmol, 1.0 equiv.) and PPh3
(3.7 g, 14 mmol, 5 equiv.) were added under nitrogen atmos-
phere. The orange mixture is heated to ~150 °C by means
of an oil bath with stirring until complete solution occurs
(~ 1 h). The bath is then taken away, and the solution is
stirred for approximately 15 min, then hydrazine monohy-
drate (0.55 mL, 11 mmol, and 4 equiv.) was added dropwise
over approximately 1 min to form a yellow precipitate. A
vigorous reaction takes place with evolution of nitrogen.
The yellow solution is cooled to 0 °C with an ice bath and
solid was collected by fltration and washed successively
with 3 × 50 mL of ethanol and 3 × 50 mL of diethyl ether
(Coulson et al. 2007). The bright yellow solid was dried
by passing a slow stream of nitrogen through the funnel for
1 h, then with vacuum pump for overnight. The resulting
yellow crystalline product weights 3.2 g (96–98% yield).
The compound is sensitive to air, light and moisture. It was
stored cold under nitrogen.
Cell viability assays
Toxicity assays was done using the Cell Titer Blue Cell
Viability Assay (Promega). Cells were treated with 50 µM
of corresponding peptides for 48 h at a cell density of
100,000 cells/mL. For each sample, fve technical replicates
were used. At the end of the treatment duration, assay rea-
gent was added and plates were incubated for an additional
4 h.
Fluorescence was measured with excitation wavelength
at 555 nm and emission wavelength at 595 nm using
SpectraMax® Paradigm Multi-Mode Microplate Reader
®
(Molecular Devices).
Cell cycle analysis
Cells were treated with 25 µM peptides for 48 h and har-
vested. After cold PBS wash, samples were fxed with 70%
ethanol and kept on ice for 2 h. Following centrifugation
at 800 g for 5 min, cells were washed with PBS and then
incubated with 25 µg/mL propidium iodide (PI) and 3 mg/
mL RNAse, at 37 °C for 30 min. Stained cells were then
analyzed with fow cytometry.
Attachment of FITC
After obtaining free amine via either deprotection of Fmoc
group from N termini of peptide or orthogonal removal
of protecting groups (Mtt or Alloc) from lysine, resin was
treated with solution of 1.5 equiv. of FITC and 2 equiv. of
Apoptosis assays
Cells were treated with 25 µM P4, P5 and P7 peptides and
analyzed at 48 h with fow cytometry. To study the efect of
peptide treatment on mitochondrial membrane polarization
1 3

Structure-based design, synthesis and anticancer efect of cyclic Smac–polyarginine peptides
MitoScreen Flow Cytometry Mitochondrial Membrane
Potential Detection Kit (BD Biosciences) was used and
samples were prepared as described by the manufacturer.
Active caspase-3 was detected with PE Rabbit Anti-Active
Caspase-3 Kit (BD Biosciences). Annexin V PE-7AAD
Apoptosis Detection Kit from the same manufacturer was
used to detect early- and late-stage apoptosis.
Results and discussion
Macrocyclization methods
Macrocyclization strategy that we typically utilized is a side-
chain-to-side-chain lactamization (P4–P7), but head to tail
lactam formation was also used to obtain P6. Fluorescein
isothiocyanate (FITC) was coupled to these fusion peptides
at diferent positions (P2 and P3) to measure cellular uptake
by fow cytometry. Linear P1 was also synthesized to dem-
onstrate that SmacN7 alone cannot pass cellular membranes
and needs to be coupled with octaarginine (Fig. 1).
Statistical analysis
Statistical signifcance of results was determined using one-
way ANOVA with the Bonferroni post-test module or an
unpaired t test with two tails using GraphPad Prism soft-
ware. P value of less than 0.05 was accepted as signifcant.
To proceed side chain-to-side chain cyclization via lac-
tam formation on resin, amine and acid functionality of
lysine and glutamic acid must be free while other amino
acids are fully protected. For this purpose, we utilized two
diferent strategies for diferent peptides. Initially, we used
orthogonal Alloc/OAll deprotection strategy (Grieco et al.
2001). Alloc\OAll deprotection was done with freshly pre-
pared Pd(PPh3)4 (0.5 equiv.) as a catalyst and PhSiH3 (12
Fig. 2 Synthetic pathway of bicyclic P7 peptide. Both Alloc/OAll and Mtt/OPip strategies were used for side-chain-to-side-chain lactam forma-
tion
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M. P. Khalily et al.
Fig. 3 Synthetic pathway of bicyclic P6 peptide. Both side-chain-to-side-chain and head to tail cyclization were employed
Fig. 5 Assessment of U266 cell viability after 48 h treatment with
Fig. 4 Cellular uptake assessment of linear and cyclic Smac peptides.
Asterisks (**and ***) denote statistical signifcance at p<0.01 and
p<0.001, respectively (n=2)
50 µM peptide solutions
2002). After completion of synthesis, Rink amide resin car-
rying Mtt/OPip protected peptide was swollen in dichlo-
romethane (DCM) prior to use and then treated with 1.8%
trifuoroacetic acid (TFA) (v/v) in DCM for 3 min at room
temperature, repeated 10 times, using 1 mL solution per
100 mg of resin. The peptide resin was then re-suspended
in DMF, followed by on-resin cyclization of the peptide via
the free carboxylic acid side-chain of glutamic acid and the
free amino group side-chain of lysine by addition of PyBroP
equiv.) as an allyl scavenger. Lactam formation between
free amine and acid functional groups were carried out
with 3.0 equiv. of phosphonium based coupling reagent,
bromo-tris(pyrrolidino)-phosphonium hexafluorophos-
phate (PyBroP) and 6.0 equiv of N,N-diisopropylethylamine
(DIEA) for 90 min to 2 h, at room temperature in DMF. The
other convenient and straightforward strategy that we used
is the removal of acid-labile Mtt/OPip groups (Li and Elbert
1 3

Structure-based design, synthesis and anticancer efect of cyclic Smac–polyarginine peptides
Fig. 6 Cell cycle distribution of U266 cells after P4, P5 and P7 treat-
ment. a Flow cytometry fuorescence intensity histograms of cells
stained with propidium iodide. Intensity ranges for the corresponding
cell cycle phases (G0/G1, S, and G2/M) are shown on the untreated
sample. b Bar plots of normalized count values of each phase for
untreated treat cells with 25 µM peptides. Asterisks (**and ****)
denote statistical signifcance at p<0.01 and p<0.001, respectively
(n=3)
(3.0 equiv.), DIEA (8 equiv.) for 1.5–2 h. Excess DIEA was
used to neutralize TFA which could be trapped into the resin
beads. Coupling time was extended until a negative Kaiser
test obtained. After each coupling, unreacted amine was
capped with acetic anhydride/pyridine/DMF (1:1:10, v/v/v)
solution (Fig. 2).
Cellular uptake of designed peptides
Cellular uptake studies showed that P1 has the lowest cel-
lular entry, as expected, due to lack of transmembrane trans-
port polyarginine peptide (Arnt et al. 2002; Fulda et al. 2002;
Yang et al. 2003). We found strong cellular uptake (>65%)
for most of the Smac conjugates, except of P6 (Fig. 4).
Position of FITC had an also efect on cellular uptake.
When FITC-labelled lysine was fanked by Smac and Arg8,
peptide (P3) resulted less internalization than other linear
variant (P2) which shows that slight alterations of structure
may result unpredictable outcomes. On the contrary to the
previous report (Lättig-Tünnemann et al. 2011) when octaar-
ginine part of the peptide was constrained in a cycle, while
keeping the Smac part linear (P4), expected enhancement
of uptake was not observed. Similarly, when Smac part was
cyclized, while octaarginine part was linear (P5), there was
no improvement either. We conferred that cyclic octaargi-
nine is not conformationally constrained enough to increase
cellular uptake and acts in a similar manner to its linear
counterpart. The recent study of Oba et al. (2017) supports
our argument. When two parts of the peptide cyclized as two
adjacent cycle (P6), cellular uptake went even worse. An
explanation for this can be that second backbone cyclization
As an alternative strategy, head-to tail cyclization was
also employed for the synthesis of P6. When the synthesis
is complete, the frst monocyclic peptide was constructed
on resin, then labeled with FITC after selective removal of
Alloc group from the lysine. Following the Fmoc removal
from the N-terminus of peptide (head), peptide was cleaved
from the resin by the treatment with acetic acid/trifuoro-
ethanol (TFE)/DCM = 1:1:6 (v/v/v) for 60 min at room
temperature. The solution was fltered and concentrated
in vacuo and precipitated with ether. The monocyclic pep-
tide that all side chains are still protected was subjected to
head-to-tail cyclization in solution for 3 days. The reaction
using PyBroP under the conditions high dilution in DCM
(< 1 mM) was aforded the desired fully-protected cyclic
peptide. Finally, deprotection of side chains was done with
TFA/TIS/H2O=90:5:5 (v/v/v) for 2.5 h, ended up with bicy-
clic P6 (Fig. 3).
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M. P. Khalily et al.
Fig. 7 Induction of apoptosis upon treatment with 25 µM peptides. a
Mitochondrial membrane depolarization state (MMD state). Bar plots
of normalized mitochondrial membrane polarization state values. b
Caspase-3 activity change. c Cell membrane asymmetry change. Dot
plots of Annexin V-PE vs 7-AAD signals gated as live, early apop-
totic and late apoptotic events, and cell population bar graphs. Aster-
isks (**and ***, ****) denote statistical signifcance at p<0.01 and
p<0.001, respectively (n=3)
causes distortion of the geometry which blocks cell-per-
meable function of octaarginine. To assess our claim, we
synthesized another bicyclic peptide (P7) with double side-
chain-to-side-chain cyclization with additional –GlyGly– as
a spacer. It is observed that cellular uptake of bicyclic P7
is higher than the other compact bicyclic peptide (P6). This
result showed that too much constrained in the cyclic struc-
ture of peptide would have adverse efect to its function.
Cytotoxicity of Smac derivatives
Cytotoxicity experiments were also conducted to show the
therapeutic potential of designed peptides. Toxicity of pep-
tides is largely proportional to their uptake efciency and
stability in cellular environment. The results displayed in
Fig. 5 showed that monocyclic P4 and P5 (50 µM) inhib-
ited cell growth at in 48 h. However, there was no further
1 3

Structure-based design, synthesis and anticancer efect of cyclic Smac–polyarginine peptides
improvement when both therapeutic (SmacN7) and cell-pen-
etrating (Arg8) parts were constrained in a cyclic structure
as in the bicyclic P7 peptide. Linear P2 and P3 peptides
did not show toxicity in micromolar concentration range,
even though their cellular uptake was very high. This result
could be due to their rapid proteolytic digestion in the cell
since linear peptides are less stable then their cyclic analogs
(Adessi and Soto 2002; Burton et al. 1996; Gilon et al. 1991;
McGeary and Fairlie 1998; Pauletti 1997). P6 showed the
lowest toxicity among all designed cyclic peptides due to
lower cellular uptake and reduction of Smac activity caused
by structural distortion. Taken together, these results suggest
that toxicity of the cyclic peptides is correlated with their
stability. In the follow-up experiments, cytotoxicity mech-
anism of potent cyclic peptides (P4, P5, P7) was further
investigated with cell cycle and apoptosis assays.
Conclusion
In this study, we performed the design, synthesis, and char-
acterization of conformationally constrained cell-permeable
and easily accessible Smac derivatives. Importantly, among
the designed peptides, monocyclic P4, P5 and bicyclic P7
have exhibited great cytotoxicity by increasing apoptosis and
suppressing proliferation in U266 MM cells. Although the
exact mechanism of cell death and cellular uptake remains
to be investigated, our study contributes useful knowledge
to the development of novel cyclic peptides which are good
candidates as therapeutics.
Acknowledgements Financial support provided by METU-BAP,
OYP program and TÜBİTAK BİDEB 2211/A scholarship is grate-
fully acknowledged.
Compliance with ethical standards
Cell cycle and apoptosis assays
Conflict of interest The authors declare that they do not have any com-
mercial or associative interest that represents a confict of interest in
connection with the work submitted.
Since typical cytotoxicity assays cannot diferentiate anti-
proliferative (growth inhibition) and toxic (cell death)
effects, we employed standard cell cycle and apoptosis
assays to better understand the mechanism behind the
observed cytotoxicity of P4, P5 and P7 derivatives. Smac
has been previously shown to have cell cycle arrest and
apoptotic effect on leukemic cells (Jia et al. 2003). As
expected, myeloma cell population treated with P4, P5 and
P7 signifcantly increased at G0/G1 with a linked reduction
at G2/M phases of the cell cycle (Fig. 6b).
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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Apoptotic efect of the constructs was also tested by
three independent assays measuring the mitochondrial
membrane depolarization state, caspase-3 activity and
change in the cell membrane asymmetry (Fig. 7). Flow
cytometry results indicate that mitochondria in U266 cells
treated with P4, P5 and P7 signifcantly shift to depolar-
ized state which is considered as one of the primary indi-
cators of apoptosis (Fig. 7a). Caspase-3 is another essen-
tial apoptotic marker which was detected in a follow-up
study using a fuorescently labeled antibody against the
active form of the enzyme. As shown in fow cytometry
bar plots (Fig. 7b), caspase-3 levels signifcantly increase
in myeloma cells treated with SMAC derivatives. Finally,
an Annexin V-based cell assay also supported previous
results by indicating a signifcant decrease in live cell pop-
ulations while both early and late apoptotic populations
increased in peptide applied samples (Fig. 7c). In sum-
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