Cell-penetrating peptide-conjugated XIAP-inhibitory cyclic
hexapeptides enter into Jurkat cells and inhibit cell
proliferation
Yusuke Sasaki
1
, Motoko Minamizawa
1
, Akihiro Ambo
1
, Shigeki Sugawara
2
, Yukiko Ogawa
2,
* and
Kazuo Nitta
2
1 Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Japan
2 Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan
Keywords
antiproliferative activity; apoptosis;
cell-penetrating peptide; Jurkat cell;
XIAP inhibitory cyclic hexapeptide
Correspondence
Y. Sasaki, Tohoku Pharmaceutical
University, 4-4-1, Komatsushima, Aoba-ku,
Sendai 981 8558, Japan
Fax: +81 22 275 2013
Tel: +81 22 727 0213
E-mail: ysasaki@tohoku-pharm.ac.jp
*Present address
Department of Microbiology, Nagasaki
International University, Sasebo, Japan
(Received 24 August 2008, revised 29
September 2008, accepted 6 October 2008)
doi:10.1111/j.1742-4658.2008.06730.x
X-Linked inhibitor of apoptosis protein (XIAP) is a member of the
inhibitor of apoptosis protein family that is overexpressed in human can-
cers. There is great interest in the development of XIAP inhibitors,
which are predicted to promote apoptosis in cancer cells and thus have
therapeutic potential. A cyclic hexapeptide (CH), CPFKQC, which is
one of the consensus motifs that can bind to the baculovirus IAP
repeat 2 domain of XIAP, has been identified using phage-displayed
combinatorial chemistry techniques [Tamm I, Trepel M, Cardo-Vila M,
Sun Y, Welsh K, Cabezas E, Swatterthwait A, Arap W, Reed JC &
Pasqualini R (2003) Peptides targeting caspase inhibitors. J Biol Chem
278,14401–14405]. In this study, we designed and synthesized covalently
linked conjugates of CHs, cyclo[Cys-Pro-Xaa-Lys-Gln-Glu(-CO-)-NH
2
]
(Xaa = various amino acids; cyclization via a peptide bond between the
N-terminal amino group of Cys1 and the side-chain carboxylic acid of
Glu6), and a cell-penetrating peptide (CPP), Ac-Cys-Trp-(Arg)
8
-Lys-
NH
2
.CH–CPP conjugates (CHCPPs) with aromatic and hydrophobic
Xaa residues, such as Phe (CHCPP 1), 2,6-dimethyl-phenylalanine
(CHCPP 2) and 3-(1-naphthyl)-alanine ⁄3-(2-naphthyl)-alanine (CHCPPs 3
and 4), potently inhibited the proliferation of Jurkat cells in a dose-
dependent manner, whereas analogues with nonaromatic or less hydro-
phobic amino acids at the Xaa residue were less potent or caused no
inhibition. A morphological study of nuclei after treatment with
CHCPPs 1–4revealed that nuclear fragmentation occurred, suggesting
that these conjugates induce apoptosis. A kinetic study of the uptake of
fluorescein-labelled CHCPP 2into the cells showed that the conjugates
were translocated within a few minutes. The cellular uptake of fluores-
cein isothiocyanate-labelled CHCPP 1and CPP was greatly reduced in
high-K
+
buffers, suggesting that CPP and its conjugate are translocated
by a mechanism associated with cell membrane potential. Competitive
binding studies performed using fluorescence correlation spectroscopy
demonstrated that CHCPP 1binds to the baculovirus IAP repeat 2
domain of XIAP via the CH (Xaa = Phe) moiety. CHCPPs 1and 2
Abbreviations
BIR, baculovirus IAP repeat; CH, cyclic hexapeptide; Cha, cyclohexylalanine; CHCPP, cyclic hexapeptide–CPP conjugate; CPP,
cell-penetrating (or permeable) peptide; Dmp, 2,6-dimethyl-phenylalanine; FCS, fluorescence correlation spectroscopy; FITC, fluorescein
isothiocyanate; IAP, inhibitor of apoptosis protein; LHCPP, linear hexapeptide–CPP conjugate; Nal(1), 3-(1-naphthyl)-alanine; Nal(2),
3-(2-naphthyl)-alanine; Npys, 3-nitro-2-pyridinesulfenyl; PI, propidium iodide; PS, phosphatidylserine; SAL-resin, 4-(2¢,4¢-dimethoxyphenyl-
hydroxymethyl)-phenoxy resin; XIAP, X-linked inhibitor of apoptosis protein.
FEBS Journal 275 (2008) 6011–6021 ª2008 The Authors Journal compilation ª2008 FEBS 6011
Apoptosis, or programmed cell death, is important for
the normal development and homeostasis of multicel-
lular organisms. Inappropriate control of apoptosis is
closely related to human diseases, including cancer [1].
Inhibitor of apoptosis proteins (IAPs) are a family of
apoptosis-suppressing proteins [2–4]. X-linked IAP
(XIAP) is the most potent inhibitor among IAPs and
is commonly overexpressed in human cancers [5–7].
XIAP potently inhibits caspases whose activities are
crucial for the apoptosis process; baculovirus IAP
repeat 1 and 2 (BIR1 and BIR2) domains, or the
linker region between them, in the XIAP molecule
bind and inhibit caspase-3 and caspase-7, whereas
BIR3 specifically binds to and inhibits caspase-9
[6–12]. There is considerable interest in the design and
development of small-molecule inhibitors of XIAP,
because such inhibitors may have therapeutic potential
as anticancer drugs, i.e. TWX006 [13], Compound 3
[14] and embelin (a natural product derived from Japa-
nese Ardisia herb) [15]. Furthermore, the N-terminal
short peptides of the second mitochondrial activator of
caspases ⁄direct IAP-binding protein with low pI
(Smac ⁄DIABLO), linked to a carrier peptide for intra-
cellular delivery, enhance the induction of apoptosis by
anticancer drugs in tumour cells [16–18].
Recently, Tamm et al. [19] used a phage-displayed
combinatorial chemistry technique to resolve a consen-
sus motif, C(D ⁄E⁄P)(W ⁄F⁄Y)-acid ⁄basic-XC, which
binds specifically to the BIR2 domain of XIAP. They
also demonstrated that the peptide sequence CEFESC
interacts with the caspase-binding-related site of XIAP,
and that the cyclic peptide CPFKQC fused to a
penetratin fragment induces programmed cell death
in leukaemia cells [19].
In this study, using a motif of CPFKQC as a tem-
plate structure, we designed and synthesized a series
of cyclic hexapeptides (CHs 1–8) based on the struc-
ture of cyclo[Cys-Pro-Xaa-Lys-Gln-Glu(-CO-)-NH
2
]
(Xaa = aromatic or aliphatic amino acid; cyclization
via a peptide bond formed by the N-terminal amino
group and Glu6 side-chain carboxylic acid) (Fig. 1).
To deliver the CHs into the cells, they were covalently
linked by a disulfide bond to a cell-penetrating peptide
(CPP), Ac-Cys-Trp-(Arg)
8
-Lys-NH
2
, which belongs to
the oligoarginine peptides in the CPP family of
peptides [20–22]. The N-terminal Cys residue was
added so that the resulting CPP can link various
cargos containing a thiol group and deliver them into
the cell. Eight CH–CPP conjugates (CHCPPs 1–8)
were investigated for their ability to incorporate into
Jurkat cells and induce apoptosis. The structure–activ-
ity correlation study focusing on the Xaa residue of
CHs revealed structural elements required for the inhi-
bition of cell proliferation and the induction of apop-
tosis in Jurkat cells. In addition, a competitive binding
study of a typical fluorescein isothiocyanate (FITC)-
labelled CHCPP 1to the BIR2 domain of XIAP
demonstrated the importance of the CH moiety for
binding to XIAP.
Results
Preparation of CHCPPs and CPP
All peptides were synthesized by solid phase peptide
synthesis using Fmoc chemistry, according to the
method described previously [23]. The key step for
the synthesis of CHs was the cyclization of linear
hexapeptide, forming an amide linkage between the
N
a
-amino group of Cys1 and the side-chain carboxylic
acid of Glu6, which proceeded smoothly with ben-
zotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexa-
fluorophosphate ⁄N,N-diisopropylethylamine reagents.
Ac-Cys-Trp-(Arg)
8
-Lys-NH
2
was designed and synthe-
sized as a CPP, in which Lys was incorporated as an
active centre for fluorescein labelling and Trp was used
to facilitate peptide monitoring during synthesis. The
reaction of [Cys(Npys)1]CH (Npys = 3-nitro-2-pyri-
dinesulfenyl) with CPP proceeded quantitatively to
yield CHCPPs. All peptides showed satisfactory ana-
lytical data by analytical HPLC, amino acid analysis
and MALDI-TOF-MS characterization.
Cytotoxicity of CHCPPs and CPP against
Jurkat cells
To examine the effects of the peptides on Jurkat cells
and their ability to translocate into the cells, a trypan
blue exclusion assay was performed against
CH-, CHCPP- and CPP-treated Jurkat cells. No
significant cytotoxic effects were observed with all
[Cys(Npys)1]CHs up to 20 lm, even after 24 h (data
not shown). However, significant cytotoxicity was
observed for the CPP conjugates that contained
showed the most potent inhibitory activity of the CHCPPs and embelin,
a nonpeptide inhibitor of XIAP, suggesting that they are good templates
for the design of a new class of anticancer drug.
CHCPPs as potent inhibitors of XIAP Y. Sasaki et al.
6012 FEBS Journal 275 (2008) 6011–6021 ª2008 The Authors Journal compilation ª2008 FEBS
aromatic and ⁄or hydrophobic amino acids at the Xaa3
residue [Phe (1), 2,6-dimethyl-phenylalanine (Dmp) (2),
3-(1-naphthyl)-alanine (Nal(1)) (3), 3-(2-naphthyl)-ala-
nine (Nal(2)) (4), Trp (5) and cyclohexylalanine (Cha)
(6)] at a concentration of 20 lm(Fig. 2). CHCPPs with
nonaromatic residues, such as Leu (7) and Ala (8), at
the Xaa3 residue were not cytotoxic (Fig. 2). CPP itself
also showed a weak cytotoxicity at a concentration of
20 lm. A linear [Phe3]hexapeptide–CPP conjugate (the
linear hexapeptide version of CHCPP 1, LHCPP) did
not show any cytotoxicity up to 20 lm. CHCPPs 1
and 2showed a dose-dependent cytotoxicity that was
the highest of all CHCPPs (Fig. 2).
Comparison of the antiproliferative activity of
CHCPPs 1 and 2 with that of embelin in Jurkat
cells
To evaluate the effectiveness of the CHCPPs as XIAP
inhibitors, the antiproliferative effects of CHCPP 1
and 2were compared with that of embelin, a nonpep-
tide XIAP inhibitor [15]. Jurkat cells were treated with
the peptides and embelin at different concentrations at
37 C for 24 h, and the cell viability was measured by
trypan blue dye exclusion assay (Fig. 3). Both peptides
exhibited a higher potency of inhibition of cell prolifer-
ation (IC
50
values of 9.8 lmfor CHCPP 1and
14.2 lmfor CHCPP 2) than that of embelin
(IC
50
= 70.1 lm).
Nuclear morphology after treatment with
CHCPPs
To investigate whether cell death occurred by apop-
tosis, CHCPP-treated cells were stained with Hoe-
chst33258 and observed by fluorescence microscopy.
As shown in Fig. 4, nuclear fragmentation was
observed in the cells treated with CHCPPs 1–5,
whereas no morphological changes were observed in
cells treated with CHCPPs 6,7and 8and CPP alone.
Flow cytometric analysis of annexin V binding
and propidium iodide (PI) incorporation
To further confirm apoptotic cell death, the ability to
bind to annexin V and to incorporate PI was investi-
gated by flow cytometric analysis of cells treated with
the highly cytotoxic CHCPPs 1and 2. As shown in
Fig. 5, treatment of the cells with CHCPPs 1and 2
at a concentration of 20 lmapparently increased the
Fig. 1. Chemical structure of CHCPPs (A) and Xaa residues in CHs (B).
Y. Sasaki et al. CHCPPs as potent inhibitors of XIAP
FEBS Journal 275 (2008) 6011–6021 ª2008 The Authors Journal compilation ª2008 FEBS 6013
annexin V-positive ⁄PI-positive population, which
reached 60–80% of the total population at 24 h. Con-
versely, treatment with the corresponding CH alone
resulted in no significant changes to the cell popula-
tion, even after 24 h of treatment. About one-half of
the double-positive population was observed in cells
treated with CPP alone.
Incorporation of FITC-labelled peptides into
Jurkat cells
To obtain information on the kinetics of incorporation
of CHCPPs into cells, a fluorescein group was intro-
duced into the side-chain amino group of Lys11 of the
CPP molecules using FITC. The rates of cellular
uptake of FITC-CHCPP 2and FITC-CPP were mea-
sured at different time periods (Fig. 6). Both peptides
were incorporated rapidly and the maximum uptake
was reached at 5–10 min. The cellular uptake rate of
FITC-CHCPP 2was higher than that of FITC-CPP
itself.
Fig. 3. Inhibition of cell proliferation by treatment with CHCPPs 1
and 2and embelin in Jurkat cells. Jurkat cells were treated with
CHCPP 1(d), CHCPP 2(r) and embelin ( )at37C for 24 h.
Varying concentrations from 0.5 to 34 lMfor 1and 2and from
1.25 to 80 lMfor embelin were used. Cell viability was measured
by the trypan blue dye exclusion assay. Each value represents the
mean ± SE of at least two or three independent experiments in
duplicate.
Fig. 2. Effects of peptide concentration on Jurkat cell viability. Jurkat cells were treated with or without CHs and CHCPPs (2.5, 5, 10 and
20 lM)at37C for 24 h. Cell viability was measured by trypan blue dye exclusion assay. Each column represents the mean ± SE of at least
two or three experiments. Cont, peptide-untreated Jurkat cells; CPP, CPP alone; LHCPP, linear hexapeptide version of CHCPP 1.
CHCPPs as potent inhibitors of XIAP Y. Sasaki et al.
6014 FEBS Journal 275 (2008) 6011–6021 ª2008 The Authors Journal compilation ª2008 FEBS
Effect of potassium ion concentration on the
cellular uptake of FITC-CHCPP 1 and FITC-CPP
into Jurkat cells
The membrane potential is essential for the uptake of
guanidine-rich transporters into cells, and the uptake
can be inhibited by an isotonic buffer with high potas-
sium ion concentrations similar to intracellular concen-
trations [24,25]. The effect of a high potassium ion
concentration on the cellular uptake of FITC-labelled
CHCPP 1and CPP was examined. As shown in
Fig. 7, the cellular uptake of both peptides was greatly
inhibited by higher K
+
concentrations; the uptake was
almost completely inhibited at K
+
concentrations
> 100 mm.
Binding and competition assays between FITC-
labelled CHCPP 1 and the BIR2 domain of XIAP
Using the automated fluorescence correlation spectros-
copy (FCS) system [26], we investigated whether
CHCPP 1binds to the recombinant human BIR2
domain of XIAP. The diffusion time of unbound
FITC-CHCPP 1was 319.5 ± 66.5 ls (Fig. 8A). When
the BIR2 domain protein was added, the diffusion
time was prolonged (1209.1 ± 244.9 ls). The pro-
longed diffusion time was reduced by two unlabelled
competitors (0.2 lm): CHCPP 1(43% reduction) and
Npys-free CH 1(Xaa = Phe) (58% reduction). In
addition, to confirm complex formation, competition
assays were performed with unlabelled CHCPP 1and
Npys-free CH 1. As shown in Fig. 8B, a parallel trend
was observed for data on the rate of complex forma-
tion in the absence (26.7%) and presence of CHCPP 1
(9.2%) and Npys-free CH 1(10.7%). These results
suggest that CHCPP 1binds to the BIR2 domain of
XIAP via its CH moiety (CH 1).
Discussion
The original CH (cyclo[CPFKQC]) reported by Tamm
et al. [19] has a cyclic ring formed by a disulfide bond
between two Cys side-chain groups. The CHs synthe-
sized in the present study are different from the origi-
nal in their cyclization linkage, which is cyclized by a
peptide bond between the N
a
-amino group of Cys1
Fig. 4. Nuclear morphology of CHCPP-treated Jurkat cells. Jurkat cells were treated with CHCPPs 1–6 (20 lM)at37C for 24 h, and then
stained with Hoechst33258. Nuclear morphology was observed by fluorescence microscopy. The arrow indicates apoptotic cells.
Y. Sasaki et al. CHCPPs as potent inhibitors of XIAP
FEBS Journal 275 (2008) 6011–6021 ª2008 The Authors Journal compilation ª2008 FEBS 6015