Linear aliphatic dialkynes as alternative linkers for double-click stapling of p53-derived peptides

Article abstract of DOI:10.1002/cbic.201402374

We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double-click stapling of p53-based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53-activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.

Full text of DOI:10.1002/cbic.201402374

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DOI: 10.1002/cbic.201402374
Linear Aliphatic Dialkynes as Alternative Linkers for
Double-Click Stapling of p53-Derived Peptides
Yu Heng Lau,[a] Peterson de Andrade,[a] Grahame J. McKenzie,[b] Ashok R. Venkitaraman,[b]
and David R. Spring*[a]
We investigated linear aliphatic dialkynes as a new structural
class of i,i+7 linkers for the double-click stapling of p53-based
peptides. The optimal combination of azido amino acids and
dialkynyl linker length for MDM2 binding was determined. In
a direct comparison between aliphatic and aromatic staple
scaffolds, the aliphatic staples resulted in superior binding to
MDM2 in vitro and superior p53-activating capability in cells
when using a diazidopeptide derived from phage display. This
work demonstrates that the nature of the staple scaffold is an
important factor that can affect peptide bioactivity in cells.
from those generated by metathesis—an important consider-
ation as the staple linkage can interact directly with the target
protein.[9] To address the synthetic challenge of efficiently vary-
ing the staple component, we previously developed a diver-
gent double-click strategy for generating i,i+7 stapled pep-
tides,[10] after pioneering work by Torres et al. on an i,i+4 helix-
dimer system.[11] The double-click strategy is one of several
reported triazole-stapling techniques.[12] The most common
approach involves single-click i,i+4 stapling.[13] Kawamoto et al.
implemented two i,i+4 staples onto the same peptide by this
technique,[8f] and Ingale and Dawson developed peptides with
i,i+3 triazole-stapling.[14]
Protein–protein interactions (PPIs) are involved in most cellular
functions in living organisms, and aberrant PPIs are often
linked to the onset of human diseases such as cancer.[1] One of
the most important proteins in cancer biology is the tumour
suppressor p53, which can trigger cell cycle arrest and apopto-
sis in cells with DNA damage or oxidative stress.[2] In healthy
cells, the E3 ubiquitin ligase MDM2 suppresses the apoptotic
function of p53 by inducing proteosomal degradation.[3] How-
ever, some cancer cell lines overexpress MDM2, thus leading to
loss of p53 function and uncontrolled cell growth. Inhibiting
the p53/MDM2 interaction is therefore seen as a promising
therapeutic strategy in the treatment of such cancers.[4]
Starting from a single p53 diazidopeptide developed in our
previous study,[10] different dialkynyl staple linkers were intro-
duced under CuI catalysis to generate bis-triazole stapled pep-
tides bearing different functionalities on the linker (Figure 1).
The p53/MDM2 interaction is mediated by the binding of
a key a-helix of p53 to a corresponding hotspot at the N-ter-
minal domain of MDM2.[5] Several different strategies have
shown promise in the development of p53/MDM2 inhibitors,
including peptidomimetic and helix-stabilising approaches.[6]
One of the most extensively studied of these techniques is all-
hydrocarbon peptide stapling, which involves the introduction
of two olefinic amino acids into a peptide followed by ring-
closing metathesis to reinforce an a-helical conformation.[7]
There is also an increasing number of alternative methods
for creating constrained a-helical peptides by different macro-
cyclisation chemistries.[6a,8] These methods generate peptides
with staple linkages that differ structurally and functionally
Figure 1. Double-click peptide stapling. Diazidopeptide is combined with
a dialkynyl linker under CuI catalysis to give a bis-triazole stapled peptide.
Previously, we appended linkers with different staple functionalities (top).
We now vary the structure of the linker scaffold (middle), as well as the pep-
tide sequence (bottom).
The ability of these peptides to inhibit MDM2 in cells was im-
proved by simply changing the functionality appended to the
staple linkage.
Our previously reported dialkynyl linkers 1–5 are based on
a 1,3-dialkynylbenzene scaffold, with different functions at
position 5 to vary the properties of the stapled peptide
(Scheme 1A).[10] As these dialkynyl linkers contain the same
core aromatic staple structure, we were interested in finding
new dialkynyl scaffolds that would allow us to explore the
effect of structural changes in the linker. We therefore decided
to investigate linear aliphatic dialkynes as a structurally distinct
family of linkers for stapling (Scheme 1B) with the aim of com-
paring their effectiveness to that of our previous aromatic di-
alkynyl linkers.
[a] Y. H. Lau,+ Dr. P. de Andrade,+ Prof. D. R. Spring
University Chemical Laboratory, University of Cambridge
Lensfield Road, Cambridge CB2 1EW (UK)
E-mail: spring@ch.cam.ac.uk
[b] Dr. G. J. McKenzie, Prof. A. R. Venkitaraman
MRC Cancer Unit, Hutchison/MRC Research Centre
Hills Road, Cambridge CB2 0XZ (UK)
[+] These authors contributed equally to this work.
Starting with our previously reported diazidopeptide SP0
(based on the wild-type p5317–29 sequence), we introduced ali-
phatic dialkynyl linkers 6–8 under our optimised solution-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402374.
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Table 1. Binding affinities of peptides to MDM2 as determined by com-
petitive fluorescence polarisation.
Peptide
Staple scaffold
MDM2 Kd [nm]
wt p5317–29
SP0
SP1
SP6
SP7
none
none
820ꢀ60
16ꢀ1
aromatic
aliphatic
aliphatic
aliphatic
none
aromatic
aliphatic
aliphatic
aliphatic
none
3.2ꢀ0.4
2.6ꢀ0.3
4.0ꢀ0.4
3.1ꢀ0.3
19ꢀ1
SP8
SP0a
SP1a
SP6a
SP7a
SP8a
PD0
PD1
PD5
PD6
10.5ꢀ0.8
5.1ꢀ0.6
5.2ꢀ0.6
8.2ꢀ0.8
36ꢀ3
aromatic
aromatic
aliphatic
14ꢀ1
67ꢀ4
7.5ꢀ0.7
displays fairly potent binding because the staple replaces the
helix-breaking Pro27 residue of the wild-type sequence. All the
resulting stapled peptides had higher affinity for MDM2 than
either the wild-type or the corresponding unstapled peptide.
Stapled peptide SP6 (binding affinity 2.63ꢀ0.30 nm) was the
most potent p53 peptide in our assay (including our previously
best peptide SP1, 3.21ꢀ0.38 nm). Comparing the different
azido amino acid side-chain lengths, we noted that SP6–8
(three methylene series) consistently showed more potent
binding than SP6a–8a (two methylene series). However, for
each series, varying the length of the dialkynyl staples 6–8 did
not greatly affect binding affinity. These comparisons indicate
that the three methylene side-chain positions the two triazoles
at a more favourable distance from the peptide backbone.
Despite the promising results in vitro, we did not expect
SP6–SP8 and SP6a–SP8a to be active in cells, as our previous
studies (and work by Verdine)[7d] suggested that peptides
based on the p53 wild-type sequence are inactive without an
increase in the overall net positive charge. When tested in
a cell-based gene reporter assay, there was indeed no activa-
tion of p53 with any of the six peptides at concentrations up
to 100 mm (Figure 3). We previously overcame this issue by
using the cationic-functionalised staple linker 5, so we decided
to try an alternative approach for achieving cellular activity by
using the new linear peptide sequence PD0, a diazido version
of the phage-display-derived sequence pDI first reported by
Chen and co-workers.[16] This approach was inspired by the
successful application of hydrocarbon stapling to this peptide
sequence by Sawyer and co-workers.[7f]
Scheme 1. A) Previously reported aromatic dialkynyl linkers 1–5. B) Aliphatic
dialkynyl linkers 6–8. C) Linear diazidopeptides SP0, SP0a and PD0.
phase double-click conditions (copper sulfate, sodium ascor-
bate and THPTA ligand in a 1:1 mixture of tert-butanol and
water) to generate the corresponding stapled peptides SP6–
SP8 (i.e., SP0 stapled with linkers 6–8). We also explored varia-
tions in the azido amino acid side-chain length by using the
diazidopeptide SP0a,[15] which contains Aha (two methylene
units on the side chain) in place of Orn(N3) (three methylene
units), thus generating the corresponding double-clicked sta-
pled peptides SP6a–SP8a (i.e., SP0a stapled with linkers 6–8).
We were delighted to find that the stapling reactions with ali-
phatic linkers 6–8 proceeded efficiently to give the desired sta-
pled peptides as the major product in all cases (Figure 2). Cir-
cular dichroism studies showed a high level of helicity for all
six stapled peptides (Figure S4 in the Supporting Information),
with linker 6 producing the most helical peptides SP6 and
SP6a (78 and 74%, respectively).
The binding of these six peptides to MDM2 was then tested
in a competitive fluorescence polarisation assay (Table 1). As
we have previously noted,[10] the unstapled peptide already
As SP6 gave the best results in our initial fluorescence polar-
isation assay, we chose linker 6 to staple onto PD0, thus giving
stapled peptide PD6. Although this stapling only resulted in
a modest improvement over PD0 in terms of peptide helicity
(by circular dichroism; Figure 4), importantly we observed a
fivefold increase in binding affinity after stapling (Table 1). In
the gene reporter assay, we were pleased to find that PD6 in-
duced dose-dependent activation of p53, whereas PD0 did not
(Figure 3). For comparison, we also synthesised PD1, which
bears the aromatic staple linker 1, and PD5, which has the cat-
Figure 2. Example HPLC chromatograph of crude reaction mixture of SP0a
stapled with 6 to form SP6a as the major product, as monitored by HPLC at
220 nm. See the Supporting Information for further examples.
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cationic tags for efficacy. We are now looking to systematically
explore more combinations of linker structure/functionality
and peptide sequence, in order to gain a better understanding
of what factors are important for peptide cellular activity in
general.
Experimental Section
Synthesis: Aromatic dialkynyl linkers 1 and 6 were synthesised as
previously described.[10] Aliphatic dialkynyl linkers were purchased
from Sigma–Aldrich.
Fmoc solid-phase peptide synthesis was carried out on a Liberty
Automated Microwave Peptide Synthesiser; CEM, Matthews, NC)
with Rink Amide MBHA resin LL (0.29–0.39 mmolgꢁ1 loading;
Merck Millipore). All peptide couplings were performed with Fmoc-
protected amino acids (5 equiv) in DMF, HATU or HBTU (5 equiv) in
DMF and DIPEA (10 equiv) in NMP (2m). Arginine was coupled by
using double couplings (15 min each) without microwave irradia-
tion; other amino acids were coupled by single couplings (25 W,
758C, 15 min). Fmoc deprotection was achieved with piperidine
(20% in DMF) at 758C (45 W, 3 min). N-terminal acetyl capping was
carried out manually by treating resin swelled in dichloromethane
with acetic anhydride (10 equiv) and N,N-diisopropylethylamine
(10 equiv) for 45 min. Cleavage from the resin was achieved with
triisopropylsilane (2.5%) and water (2.5%) in TFA for 2 h. The sol-
vent was removed under a stream of nitrogen, and the residue
was triturated with diethyl ether (3ꢁ5 mL) before HPLC purifica-
tion.
Figure 3. Activation of p53 by peptides in a cell-based gene reporter assay.
Fold-activation is relative to 1% DMSO.
HPLC analysis and purification: Analytical HPLC was run on a
model 1260 Infinity chromatographer (Agilent Technologies) with
a SUPELCOSIL ABZ+Plus column (150ꢁ4.6 mm, 3 mm) and linear
gradient elution (solvent A: TFA (0.05% v/v) in water; solvent B:
TFA (0.05% v/v) in acetonitrile); flow rate: 1 mLminꢁ1 over 15 min).
Semi-preparative HPLC was run on the Agilent 1260 Infinity with
a SUPELCOSIL ABZ+Plus column (250ꢁ21.2 mm, 5 mm) and linear
gradient elution (solvent A: TFA (0.1% v/v) in water; solvent B: TFA
(0.1% v/v) in acetonitrile); flow rate: 20 mLminꢁ1 over 20 min).
HPLC was monitored by UV absorbance at 220 and 254 nm.
Figure 4. Circular dichroism spectra of stapled peptides with different sta-
pling linkers based on a phage-derived diazidopeptide. a: PD0 (34%),
c: PD1 (32%), ····: PD5 (44%), a: PD6 (49%). Estimated helicity is based
on mean residue ellipticity at 222 nm.
ionic-functionalised aromatic linker 5. PD6 was superior to
both PD1 and PD6 in both the in vitro binding assay (Table 1)
and the in vivo gene reporter assay (Figure 3), thus demon-
strating the impact of different linker scaffolds on peptide ac-
tivity.
Double-click stapling:[10] A solution of diazido peptide (1 equiv;
1 mLmgꢁ1) and dialkynyl linker (1.1 equiv) in tert-butanol/water
(1:1) was degassed with nitrogen for 15 min, followed by the addi-
tion of copper(II) sulfate pentahydrate (1 equiv), tris(3-hydroxypro-
pyltriazolylmethyl)amine (1 equiv) and sodium ascorbate (3 equiv).
After stirring under nitrogen at room temperature for 16 h, the
reaction mixture was lyophilised and purified by HPLC to give the
final stapled peptide.
The improved in vitro binding affinity of PD6 compared to
PD1 and PD5 might arise from improved binding contacts of
the staple with MDM2, facilitated by the improved flexibility of
the aliphatic scaffold. We are currently working to obtain struc-
tural data to determine whether this is indeed the case. In the
cellular studies, it appeared that an additional cationic charge
is not always beneficial for peptide activity, given that PD6
was superior to PD5. In light of our studies with stapling SP0
and PD0, it appears that different rules apply to different pep-
tide sequences when optimising cellular activity. Furthermore,
the direct comparison between aromatic PD1 and aliphatic
PD6 (differing only by the staple structure) demonstrates how
the staple scaffold can affect the overall bioactive properties of
a staple peptide.
Competitive fluorescence polarisation: Fluorescence polarisation
and binding affinity calculations were carried out as previously de-
scribed,[10] with a PHERAstar Plus plate reader (BMG Labtech, Orten-
berg, Germany) and Prism software (GraphPad, La Jolla, CA). Kd
values were calculated using a non-linear least-squares analysis fit-
ting to the equations previously described for binding with recep-
tor depletion,[7e] and are reported with standard errors. Experi-
ments were conducted twice independently, each in triplicate.
Accurate peptide concentrations were determined by amino acid
analysis at the Peptide Nucleic Acid Chemistry Facility (Department
of Biochemistry, University of Cambridge). For binding curves see
the Supporting Information.
In summary, we have established dialkynes 6–8 as a linkers
for i,i+7 double-click stapling, with their aliphatic scaffolds
complementing our previously developed aromatic series of
linkers. After optimisation of the combination of linker and
peptide sequence, we found that dialkyne 6 had MDM2 inhibi-
tory activity in a cell-based assay, without relying on additional
p53 reporter assay: Assay was carried out as previously descri-
bed.[7e] T22 cells stably transfected with a p53-responsive b-galac-
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tosidase reporter were obtained from Prof David Lane.[7e] Cells
were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with
foetal bovine serum (FBS, 10%), penicillin (100 UmLꢁ1) and strepto-
mycin (100 mgmLꢁ1). Cells were seeded (8000 cells per 100 mL well)
for 24 h in a 96-well black-walled clear flat-bottom polystyrene
plate (#655090; Greiner Bio One). The cells were then treated with
peptide in triplicate for 18 h in DMEM with FBS (10%). b-galactosi-
dase activity was quantified with a FluoReporter LacZ/Galactosi-
dase Quantitation Kit (Life Technologies). Fluorescence was quanti-
fied on an Infinite 200 Pro plate reader (Tecan, Mꢂnnedorf, Swizer-
land).
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Circular dichroism: Circular dichroism spectra were obtained on
a Chirascan CD spectrometer (Applied Photophysics Ltd., Leather-
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,
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bandwidth 1.0 nm, response time 0.5 s). Each spectrum is the aver-
age of three scans. Peptides were dissolved in water/acetonitrile
(85:15). Helicity was calculated as previously reported[17] by com-
paring the mean residue ellipticity at 222 nm (MRE222) to the theo-
retical maximum as calculated by using the formula Eq. (1); n=
number of amino acid residues] described by Forood et al.:[18]
MRE222 ¼ ꢁ40 000 ꢂ ð1ꢁ2:5=nÞ
ð1Þ
Spectra were obtained at several concentrations (5–50 mm): no sig-
nificant change in the shape of the spectrum was observed at dif-
ferent dilutions. Accurate peptide concentrations were determined
by amino acid analysis at the Peptide Nucleic Acid Chemistry Facili-
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Acknowledgements
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This work was supported by the European Union, Engineering
and Physical Sciences Research Council, Biotechnology and Bio-
logical Sciences Research Council, Medical Research Council and
Wellcome Trust. Y.H.L. acknowledges a scholarship from the Cam-
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the T22 cell line for the gene reporter assay, and Dr. Pamela Row-
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Keywords: anticancer agents · click chemistry · macrocycles ·
peptides · stapling
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Received: July 11, 2014
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New scaffolds for stapling: A compari-
son of linear aliphatic dialkynes with
aromatic dialkynes for the double-click
stapling of p53 peptides demonstrates
that the linker scaffold, along with the
peptide sequence and staple functional-
ity, can influence the biological activity
of stapled peptides in a cellular context.
Y. H. Lau, P. de Andrade, G. J. McKenzie,
A. R. Venkitaraman, D. R. Spring*
&& – &&
Linear Aliphatic Dialkynes as
Alternative Linkers for Double-Click
Stapling of p53-Derived Peptides
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 4
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