Conjugation of spermine enhances cellular uptake of the stapled peptide-based inhibitors of p53-Mdm2 interaction

Article abstract of DOI:10.1016/j.bmcl.2011.10.009

We report the first synthesis of the C-terminally spermine-conjugated stapled peptide-based inhibitors of the p53-Mdm2 interaction. Subsequent biological, biophysical and cellular uptake assays with the spermine-conjugated stapled peptides revealed that s

Full text of DOI:10.1016/j.bmcl.2011.10.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7412–7415
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Bioorganic & Medicinal Chemistry Letters
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Conjugation of spermine enhances cellular uptake of the stapled
peptide-based inhibitors of p53-Mdm2 interaction
Avinash Muppidi a, Xiaolong Li b, Jiandong Chen b, Qing Lin a,
⇑
a Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA
b Department of Molecular Oncology, H. Lee Moffitt Cancer Center, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the first synthesis of the C-terminally spermine-conjugated stapled peptide-based inhibitors of
the p53-Mdm2 interaction. Subsequent biological, biophysical and cellular uptake assays with the sperm-
ine-conjugated stapled peptides revealed that spermine conjugation minimally affects biological activity
while significantly increases peptide helicity and cellular uptake without apparent cytotoxicity.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 10 August 2011
Revised 30 September 2011
Accepted 4 October 2011
Available online 12 October 2011
Keywords:
Spermine
Peptides
Helical structure
Protein–protein interaction
Cellular uptake
Peptide helices are found frequently at the interfaces of
protein–protein interactions that mediate all facets of cellular pro-
cesses including cell cycle control and apoptosis.1 As a result, there
has been a growing interest recently aimed at translating these
helical peptides into biological therapies targeting the intracellular
protein–protein interactions.2 Since short peptides exhibit poor
pharmaceutical properties, that is, poor cell permeability and low
metabolic stability, numerous strategies have been developed to
increase their proteolytic stability as well as their cellular uptake.
Among them, selective cross-linking of peptide side-chains (also
referred to as peptide ‘stapling’) via selective chemical reactions,
for example, disulfide bond formation,3 olefin cross metathesis,4
lactam formation,5 and 1,3-dipolar cycloaddition,6 has emerged
to be one of the most promising approaches.7
of the effect of polyamine conjugation on peptide bioactivity, helic-
ity, and cell permeability.
Polyamines such as spermine are positively charged at physio-
logical pH, and have been used through conjugation as an intracel-
lular delivery aid for the lipophilic small-molecule drugs11 as well
as for the negatively charged siRNA.12 However, the effect of poly-
amine conjugation to synthetic peptides on cellular transport of
peptides has not been reported.13 According to helical dipole mod-
el,14 placement of the positive-charged capping groups at the
carboxyl-termini of short helical peptides leads to increased
helicity due to the favorable charge–dipole interactions.15 To
probe whether this model is operative in the case of short stapled
peptides, we conjugated spermine at the C-terminus of
a
dicysteine-containing peptide inhibitor of p53-Mdm2 interaction
(sequence = LTFCHYWAQLCS), which was used previously as a
model in our peptide stapling studies.8 Scheme 1 shows a repre-
sentative synthesis of the spermine-conjugated stapled peptide 4
in seven steps based on a solid phase procedure. Briefly, spermine
was first immobilized onto solid support by treating 2-chlorotrityl
chloride resin with excess amount of spermine in dichloromethane
for 30 min. After extensive washing, selective protection of the
secondary amines with Boc was achieved by first masking
the terminal primary amine with 2-acetyldimedone16 to form the
N-[1,(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) deriva-
tive followed by treatment with di-tert-butyl dicarbonate. The
Dde-protecting group was selectively removed by treating the
resin with 2% hydrazine in DMF for 15 min. With the spermine-
functionalized resin in hand, standard Fmoc-based solid phase
Recently, we reported the discovery of a distance-matching
cross-linker, 6,60-bisbromomethyl-[3,30]bipyridine (Bpy), that re-
acts with a pair of cysteines located at i,i+7 positions of a short heli-
cal peptide, resulting in
a significant enhancement in cell
permeability.8 While it is known that attachment of the positively
charged groups to peptides generally leads to enhanced cellular
uptake, most studies in the literature have focused on either direct
fusion of polyarginine-like sequences9 such as TAT sequence or
substitution of arginines at strategically selected noncontinuous
sites on the peptides.10 Herein, we report the first synthesis of
polyamine-conjugated stapled peptides and the characterization
⇑
Corresponding author.
E-mail address: qinglin@buffalo.edu (Q. Lin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.009

A. Muppidi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7412–7415
7413
Table 1
H
N
Sequence and activity of peptide-based inhibitorsa
NH2
H2N
N
H
Sequenceb
Charge
IC50 (nM)
spermine
Peptide
1
2
3
4
5
6
7
8
LTFCHYWAQLCS
0
0
+2
+3
0
0
+2
+3
57
35
237 16
46
13
5
102
18
5
5
i) 2-chlorotrityl chloride resin/DCM
ii) 2-acetyldimedone/DMF
iii) Boc2O, DIEA
LTFC0HYWAQLC0Sc
LTFC0RYWARLC0S
LTFC0HYWAQLC0S-Spd
LTFcHYWAQLCSe
LTFc0HYWAQLC0Sf
LTFc0RYWARLC0S
LTFc0HYWAQLC0S-Sp
6
1
1
8
2
iv) 2% hydrazine/DMF
Boc
H
N
a
ELISA assays were repeated three times to obtain average IC50 values and
standard deviations.
N
H2N
N
Boc
Trt(Cl)
b
For nonspermine-containing peptides, the N-termini were acetylated and the C-
termini were amidated. For spermine-conjugated peptides, the N-termini were
v) SPPS
vi) TFA/EDT/TIPS/H2O
(95:2.5:1.5:1)
acetylated.
c
C0 denotes Bpy-linked cysteine.
Sp denotes spermine.
d
e
c denotes
D
-cysteine.
f
c0 denotes Bpy-linked
D
-cysteine.
HS
HS
H
N
NH2
Ac-L-T-F-C-H-Y-W-A-Q-L-C-S-NH
N
H
vii)
N
N
(Bpy)
Br
Br
ACN/NH4HCO3
N
N
S
S
H
NH2
N
Ac-L-T-F-C-H-Y-W-A-Q-L-C-S-NH
N
H
4
Scheme 1. Representative synthesis of a spermine-conjugated stapled peptide 4 on
solid support.
peptide synthesis was performed and the linear cysteine-contain-
ing peptide was released from the resin after treatment with a
TFA-containing cleavage cocktail. After HPLC purification, the fully
deprotected linear peptide was incubated with 1.5 equiv of Bpy in
a mixed acetonitrile/30 mM NH4HCO3 solution at pH 8.5. HPLC
analysis of the crude product indicated that the stapling reaction
of the spermine-conjugated peptide proceeded with greater than
95% conversion after 1.5 h (Fig. S1). Using this route, we also
prepared spermine-conjugated D,L-dicysteine-stapled peptide 8 be-
cause the
D,L-dicysteine-stapled peptides have shown greater
inhibitory activity towards Mdm2 in our previous work.8
To gauge how spermine conjugation affects the stapled peptide’s
biological activity, we determined the inhibitory activities of the
spermine-conjugated cross-linked peptides using ELISA assay and
compared them to other stapled peptides reported previously8
(Fig. S2 and Table 1). Interestingly, whereas substitution of two res-
idues at solvent-exposed side of the helix with arginines (His-5?Arg
and Gln9?Arg) led to addition of +2 charge accompanied by sub-
stantial drop (6ꢀ20-fold) in inhibitory activity8 (compare 3 to 2; 7
to 6), spermine conjugation resulted in addition of +3 charge and sig-
nificantly smaller reduction in inhibitory activity (compare 4 to 2; 8
to 6). Therefore, spermine conjugation appears to provide a less dis-
ruptive route in terms of the addition of net positive charge.
To examine how spermine conjugation affects cellular uptake,
we synthesized the N-terminal fluorescein-labeled, spermine con-
jugated, stapled peptides 4 and 8 (Fluo-4 and -8, Table S1) and
compared their uptake into HeLa cells as measured by fluorescence
activated cell sorting (FACS) to the previously reported fluorescein-
labeled stapled peptides8 (Fluo-1–3 and Fluo-5–7). As shown in
Figure 1. Flow cytometry analysis of HeLa cells after treatment with 10 lM
fluorescein-conjugated peptides, Fluo-1–8: (a) Representative histograms; (b) Bar
graph showing the normalized mean fluorescence. For simplicity, Fluo- prefixes
were omitted in the stapled peptide denotations.
Figure 1, peptide stapling significantly enhanced the cellular up-
take (compare Fluo-2–4 to -1; Fluo-6–8 to -5). Among the stapled
peptides, peptides carrying the positive charges (Fluo-3, -4, -7, and
-8) showed significantly higher uptake than the charge-neutral
peptides (Fluo-2 and -6); the spermine-conjugated stapled
peptides Fluo-4 and -8 exhibited the highest uptake, yielding 16-
and 22-fold increase, respectively, over their charge-neutral linear
counterparts (compare Fluo-4 to -1; Fluo-8 to -5), indicating that
spermine conjugation acts in concert with peptide stapling to

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A. Muppidi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7412–7415
showed additional 50% increase in percent helicity than the argi-
nine-substituted stapled peptides (compare 4 to 3; 8 to 7). This
enhancement in helicity is consistent with the helical dipole mod-
el15 in which the C-terminally conjugated, positive-charged sperm-
ine stabilizes helical dipole by forming additional hydrogen bonds
with C-terminal unpaired carbonyl groups. A correlation between
percent helicity and cell permeability appears to exist as peptides
with higher percent helicity invariably exhibited higher cellular
uptake.
To corroborate the FACS results on cellular uptake, a confocal
fluorescence microscopy study was performed. We found that
while the positive-charged stapled peptides showed stronger
intracellular fluorescence than the charge-neutral ones, Fluo-3
and -7 treated cells showed significant amount of cellular debris
which was absent in Fluo-4 and -8 treated cells (Fig. S3). This dif-
ference in apparent cytotoxicity was verified by ATP assay. Cells
treated with the spermine-conjugated stapled peptides (4 and 8)
were found to be completely viable whereas cells treated with
the arginine-substituted peptides 3 and 7 showed 50% and 30% de-
crease in viability, respectively, (Fig. 3). The cytotoxicity of 3 and 7
could be attributed to the nonspecific cell membrane insertion
activity of amphipathic arginine-containing peptide helices with
one side of the helix to be hydrophobic and the opposite side to
be cationic.17 In contrast, the devoid of cytotoxicity for the sperm-
ine-conjugated stapled peptides could be attributed to their unique
mode of helix stabilization. The possible mechanism for the uptake
of the spermine-conjugated stapled peptides was then probed with
a fluorescence-based subcellular localization study (Fig. 4). Fluo-4
showed a more diffusive intracellular distribution, with some over-
lap with Alexa Fluor 586 labeled transferrin, a fluorescent marker
for the recycling endosomes (panel d in Fig. 4), suggesting a possi-
ble endosome-mediated transport process.
Figure 2. CD spectra of the linear and Bpy-stapled peptides with or without
spermine conjugation and their calculated percent helicity values: The peptides
were dissolved in 20% ACN/H2O for a final concentration of 50 lM. The percent
helicity was calculated based on the [h]222 value.
In summary, we have demonstrated a small-molecule approach
in rendering stapled peptides cationic through the C-terminal
spermine conjugation. The attachment can be readily integrated
into the solid phase synthesis protocol and compatible with the
peptide stapling conditions. Compared to the arginine-substitution
approach, spermine conjugation minimally affected the biological
activity of the stapled peptides while significantly increased pep-
tide helicity. Together with peptide stapling, spermine conjugation
acted in concert to further enhance the cellular uptake of the bio-
active peptides without apparent cytotoxicity that is frequently
associated with arginine substitution. Inasmuch as the cationic
TAT peptide,9a spermine conjugation should provide a simple,
small-molecule strategy for intracellular delivery of bioactive pep-
tides and other biotherapeutics.
Figure 3. Spermine conjugation increases cellular uptake of the stapled peptides
without apparent cytotoxicity analyzed by ATP assay. The CellTiter-Glo reagent
(Promega) was used to measure HeLa cell viability after treatment with 10 lM of
the unstapled (1 and 5) or the stapled peptides (2–4 and 6–8).
further enhance the cellular uptake. The higher uptake observed
for the spermine conjugated stapled peptides Fluo-4 and -8 rela-
tive to the arginine-substituted, stapled peptides Fluo-3 and -7
could also be attributed to the localized positive charge at the C-
termini of Fluo-4 and -8 versus the dispersed positive charge in
Fluo-3 and -7.
To probe structural changes induced by spermine conjugation,
we determined the percent helicity of all stapled peptides with
far-UV CD measurement (Fig. 2). While cysteine cross-linking
invariably led to modest increase in percent helicity (compare 2–
4 to 1; 6–8 to 5), the spermine-conjugated stapled peptides
Acknowledgments
We gratefully acknowledge the Pardee Foundation (to Q.L.) and
the National Institutes of Health (CA 109636 and CA 118210 to J.C.)
for financial support.
Figure 4. Confocal fluorescence microscopy of live HeLa cells after treatment with 10 lM of fluorescein-conjugated 4 for 2 h followed by incubation with 10 lg/mL Alexa
Fluor 586 labeled transferrin for 30 min: (a) DIC channel; (b) FITC channel; (c) Fluor 586 channel; (c) merged images of channels a–c.

A. Muppidi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7412–7415
7415
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Supplementary data
Supplementary figures, experimental procedures, and charac-
terization data of peptides 4 and 8 associated with this article
can be found in the online version at doi:10.1016/j.bmcl.
2011.10.009.
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