Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein–Protein Interaction in Ovarian Cancer

Article abstract of DOI:10.1002/anie.201609427

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor

Full text of DOI:10.1002/anie.201609427

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Communications
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International Edition: DOI: 10.1002/anie.201609427
German Edition: DOI: 10.1002/ange.201609427
Peptide Therapeutics
Hot Paper
Development of Cell-Permeable, Non-Helical Constrained Peptides to
Target a Key Protein–Protein Interaction in Ovarian Cancer
Mareike M. Wiedmann, Yaw Sing Tan, Yuteng Wu, Shintaro Aibara, Wenshu Xu,
Hannah F. Sore, Chandra S. Verma, Laura Itzhaki, Murray Stewart, James D. Brenton, and
David R. Spring*
Dedicated to Professor Stuart L. Schreiber on the occasion of his 60th birthday
Abstract: There is a lack of current treatment options for
ovarian clear cell carcinoma (CCC) and the cancer is often
resistant to platinum-based chemotherapy. Hence there is an
urgent need for novel therapeutics. The transcription factor
hepatocyte nuclear factor 1b (HNF1b) is ubiquitously overex-
pressed in CCC and is seen as an attractive therapeutic target.
This was validated through shRNA-mediated knockdown of
the target protein, HNF1b, in five high- and low-HNF1b-
expressing CCC lines. To inhibit the protein function, cell-
permeable, non-helical constrained proteomimetics to target
the HNF1b–importin a protein–protein interaction were
designed, guided by X-ray crystallographic data and molecular
dynamics simulations. In this way, we developed the first
reported series of constrained peptide nuclear import inhib-
itors. Importantly, this general approach may be extended to
other transcription factors.
provided by Liu et al., who showed that downregulation of
HNF1b increased cisplatin- and paclitaxel-mediated cytotox-
icity.[4] HNF1b is expressed in the liver, digestive tract,
pancreas, and kidneys, where it plays a role in early differ-
entiation.[5] Human HNF1b is made up of three domains: the
dimerization domain; the transactivation domain, which is
involved in binding transcriptional coactivators; and the
DNA-binding domain (DBD). We have recently confirmed
the existence of a nuclear localization signal (NLS) within the
DBD of HNF1b,[6] which directs the nuclear import of the
protein.[7]
Many NLS sequences are recognized in the cytoplasm by
a heterodimeric transport carrier complex composed of
importin a and importin b.[8] Classical NLSs (cNLS) can
bind to importin a through either a major site, a minor site,
or both.[8,9] Monopartite cNLSs consist of a single cluster of
positively charged residues, primarily lysines or arginines, that
assume an ordered state once bound to importin a.[10]
Therapeutic targeting of the nuclear import of tran-
scription factors provides a strategy for inhibiting their
function, since activity depends on successful localization to
the nucleus for transcription to take place.[11] Lin et al.
developed a 41-residue synthetic peptide called cSN50 that
contains the NF-kB NLS and a cell-permeable motif.[12] The
peptide inhibits the nuclear translocation of NF-kB, attenu-
ates gene transcription in intact cells, and is not cytotoxic
within the concentration range of the experiments.[13] cSN50
also inhibits the nuclear import of the transcription factors
AP-1, NFAT, and STAT1.[13] However, it was readily digested
T
he prognosis for ovarian clear cell carcinoma (CCC)
patients with advanced-stage disease is poor owing to intrinsic
resistance to platinum-based chemotherapy and the lack of
targeted therapies available.[1] Overexpression of the hepato-
cyte nuclear factor 1b (HNF1b) transcription factor is the
most important clinical immunohistochemical marker for the
disease, since it is ubiquitously overexpressed in CCC.[2]
However, to date, drugs targeting HNF1b have not been
developed due to the high content of intrinsically disordered
regions in transcription factors.[3]
Evidence that targeting HNF1b is a viable and attractive
approach for developing a new targeted therapy was initially
[*] M. M. Wiedmann, Y. Wu, Dr. H. F. Sore, Prof. D. R. Spring
Department of Chemistry, University of Cambridge
Lensfield Road, Cambridge, CB2 1EW (UK)
E-mail: spring@ch.cam.ac.uk
Dr. W. Xu, Dr. L. Itzhaki
Department of Pharmacology
Tennis Court Road, Cambridge, CB2 1PD (UK)
Dr. C. S. Verma
M. M. Wiedmann, Dr. J. D. Brenton
Cancer Research UK Cambridge Institute, University of Cambridge
Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE (UK)
School of Biological Sciences, Nanyang Technological University
60 Nanyang Drive, Singapore 637551 (Singapore)
and
Department of Biological Sciences, National University of Singapore
14 Science Drive 4, Singapore 117543 (Singapore)
Dr. Y. S. Tan, Dr. C. S. Verma
Bioinformatics Institute, Agency for Science, Technology and
Research A*STAR
Supporting information (including all data supporting this study)
and the ORCID identification number(s) for the author(s) of this
article can be found under http://dx.doi.org/10.1002/anie.
201609427.
30 Biopolis Street, #07-01 Matrix, Singapore 138671 (Singapore)
Dr. S. Aibara
SciLifeLab, Tomtebodavꢀgen 23A, 171 65 Solna, Stockholm (Sweden)
Dr. S. Aibara, Dr. M. Stewart
MRC Laboratory of Molecular Biology, Francis Crick Avenue
Cambridge Biomedical Campus, Cambridge CB2 0QH (UK)
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during protease treatment with trypsin and pronase.[12] cSN50
is the first nuclear-import inhibitor that has shown importin a
isoform specificity, binding with nanomolar affinity to impor-
tin a5 and only weakly to the other importin a isoforms.[14] It
also represents the first example of targeting the nuclear
import of a transcription factor at the level of NLS
recognition.[13] To date, there has been no use reported of
the technique of stapling[15] to stabilize these intrinsically
disordered NLS peptides. The aim of this work was to develop
constrained peptide-based inhibitors that target the HNF1b–
importin a protein–protein interaction (PPI) and inhibit the
activity of HNF1b. The proposed nuclear import targeting
approach for the HNF1b–importin a PPI is summarized in
Figure 1. The constrained peptide competes with HNF1b
protein for importin a binding in the cytoplasm and is
imported into the nucleus.
PPIs are crucial for many biological processes in the living
cell and are responsible for the majority of cellular func-
tions.[17] Interestingly, it has been predicted that up to 49% of
transcription factor sequences are intrinsically disordered.[18]
Intrinsically disordered protein domains (IDD) do not
assume well-defined folded structures, but rapidly intercon-
vert between different conformations.[19] An example of IDDs
are targeting motifs such as NLSs.[7] Because IDDs have
unique binding properties, conventional drug-discovery strat-
egies are less applicable for finding inhibitors, and novel
strategies such as constrained-peptide-based approaches may
be required.[19,20] Peptide-based drugs are attractive alterna-
tives to small-molecule inhibitors owing to their high potency,
specificity, and therapeutic safety.[21] Compared to protein-
based drugs, they are less likely to initiate an immune
response and their synthesis is more economical and less time
consuming.[22] Synthetic macrocylization through linking of
the side chains of two non-proteogenic amino acid residues
allows peptides to be constrained in their bioactive confor-
mation, thereby resulting in less entropy lost upon binding.[23]
In addition, the rate of proteolytic degradation of constrained
peptides is often lower than that of their linear counter-
parts.[24] Current challenges in the field include the design of
cell-permeable constrained peptides.
Our goal was to stabilize the HNF1b NLS peptide, which
binds to importin a, in its binding conformation to give
a constrained peptide with increased permeability whilst
retaining potency. The crystal structure of the HNF1b NLS
peptide bound to mImportin a1 DIBB (PDB ID: 5K9S),
which has the autoinhibitory importin b binding (IBB)
domain deleted, was used to aid the design of the constrained
peptide.[6c]
We first performed cell proliferation experiments to
validate the potential of HNF1b as a therapeutic target for
the treatment of CCC. The effect on cell proliferation upon
HNF1b knockdown was studied in five high- and low-HNF1b-
expressing CCC cell lines and one high-grade serous ovarian
cancer (HGSOC) control cell line (PEO1), which does not
express HNF1b. All of the CCC lines apart from JHOC7,
OVISE, and PEO1 proliferated less upon small hairpin RNA
(shRNA)-mediated HNF1b knockdown (Figure 2). In the
Figure 2. Relative proliferation of PEO1, JHOC5, JHOC7, JHOC9,
OVISE, and SKOV3 CCC lines with n=4 after HNF1b shRNA knock-
down. The mean is shown, with error bars showing the SEM.
Statistical significance was assessed with multiple t-tests and the
ˇ
Holm–Sꢁdꢂk method with a=5%. Optical densities (ODs) are given
relative to their respective non-target knockdown OD value and back-
ground OD was subtracted. Only shRNA knockdown clone 583 at 96 h
was considered here. * indicates P<0.02.
JHOC5, JHOC9, and SKOV3 cell lines, this reduction was
found to be statistically significant, with P < 0.02. These
results are in agreement with previous results by Tomassetti
et al. and Tsuchiya et al.,[2b,25] but we provide a more exten-
sive investigation, with five cell lines and five time points. This
work further validates HNF1b as a target for CCC.
Because the binding affinity of cargo proteins for their
carrier is an important factor for efficient nuclear import,[26] it
was imperative to quantify the dissociation constant (Kd) of
the HNF1bDBD with its nuclear import protein (importin a) by
isothermal titration calorimetry (ITC; see Figure S4 in the
Supporting Information for ITC curve and binding parame-
ters). The tighter binding (Kd = 625 nm) that was observed
with the HNF1bDBD protein compared to the much shorter
Figure 1. Proposed scheme for targeting the nuclear import of HNF1b
through the HNF1b–importin a PPI: 1) The IBB domain of importin a
binds to importin b to free up the NLS-binding sites on importin a.
2) HNF1b NLS recognition by a heterodimeric complex composed of
importin a and importin b, 3,4) To enable HNF1b to be imported in
the nucleus, the HNF1b NLS has to bind to the importin a–b
heterodimer. The constrained peptide competes for this binding,
thereby impairing the import of HNF1b. 5) Release of the constrained
peptide through RanGTP binding to importinb. Reproduced and
modified from Kobe et al.[16]
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HNF1b NLS peptide (Kd = 13.6 mm)[6c] can be rationalized by
position,[28] based on the structures of HNF1b NLS peptide in
additional contributions to binding arising from the entire
complex with mImportin a1 obtained from the MD simula-
tions. Residues Asn2, Asn8, Phe10, and Lys11 contributed
very little to the total binding free energy, thus suggesting that
they could be removed from the peptide with minimal
disruption to the overall binding (Figure 4A). Computational
HNF1bDBD protein.
Using a fluorescence polarization (FP) assay, the linear
HNF1b NLS peptide sequence (Pep0: TAMRA-5-Ahx-6-
TNKKMRRNRFK-NH2) was determined to bind to mIm-
portin a1 DIBB with a comparable Kd of 5.32 mm. This
represents a roughly 2.5-fold difference from that obtained
previously by ITC (Kd = 13.6 mm).[6c] The FP assay was used
for inhibitor testing.
To predict the most important binding interactions of the
HNF1b NLS peptide with the mImportin a1 protein, molec-
ular dynamics (MD) simulations for the complex were
performed. Two key discrepancies between the crystal
structure (PDB ID: 5K9S)[6c] and the MD simulations were
identified. In the crystal structure, the backbone carbonyl of
Thr1 from the peptide interacts with Arg238 of importin a
(Figure 3A), whereas in two out of three simulation runs, this
Figure 4. Energetic analysis of the MD simulations of the complex of
HNF1b NLS peptide with mImportin a1. A) Binding free energy
contributions of HNF1b NLS peptide residues. B) Computational
alanine scanning of HNF1b NLS peptide residues. Hot, warm, cool,
and cold spots are shown in red, orange, green, and blue, respectively.
Figure 3. Binding interactions of HNF1b NLS peptide (orange) with
mImportin a1 (gray) determined from X-ray crystallography and MD
simulations. The trajectory structures shown are final snapshots taken
from the end of the simulations. A) The backbone carbonyl oxygen of
Thr1 hydrogen bonds to the side chain of Arg238 in the obtained
crystal structure (PDB ID: 5K9S). B) The side chain of Thr1 hydrogen
bonds with the side chain of Asp270 in the MD simulations. C) Arg9
forms a salt bridge with Glu465 from a neighboring protein chain
(pink) in the crystal structure. D) Arg9 forms a salt bridge with Glu107
in the MD simulations.
alanine scanning was then used to determine suitable stapling
locations. Each peptide residue was mutated to alanine and
the difference in the binding free energy between the mutant
and wild-type complexes calculated (Figure 4B). The results
were in agreement with the binding free energy decomposi-
tion analysis (Figure 4A). Thr1, Lys4, Arg6, Arg7, and Arg9
were identified as the most important residues for binding,
whereas Asn2, Asn8, Phe10, and Lys11 made only negligible
contributions to the binding. Both energetic analyses indicate
that the constrained peptide inhibitors should be designed
1
interaction was lost and the side chain of Thr1 formed
a hydrogen bond with Asp270 instead (Figure 3B). Secondly,
Arg9 of the peptide forms a salt bridge with Glu465 from
a neighboring importin a chain in the crystal structure due to
crystal packing (Figure 3C), whereas it formed a salt bridge
with Glu107 of importin a1 in all the simulation runs (Fig-
ure 3D). These observations highlight both the influence of
the crystal environment in inducing nonphysiological contacts
and the importance of using MD simulations to eliminate the
effect of crystal packing in the study of protein dynamics in
solution.[27] Our results suggest that both Thr1 and Arg9
should be retained to maintain the binding potency of the
constrained peptides.
based on the following peptide sequence: TNKKMRRNR9.
Since the constraining linker should preferably be placed on
residues where the side chains have little or negative
contribution to the binding,[29] residues Asn2 and Asn8 were
chosen for replacement with a linker. The constraints were
introduced by using unnatural azido amino acids and
dialkynyl linkers through two-component double-click
chemistry (Figure 5).[30]
The unconstrained peptides Pep1 and Pep2, as well as four
constrained peptides, were synthesized. Their binding affin-
ities for mImportin a1 DIBB were evaluated in fluorescence
polarization assays (Figure 5C). The introduction of the
unnatural azido amino acids did not have an adverse effect
on the binding of the peptides. Compared to the linear wild-
type peptide Pep0, there was a 2.5-fold improvement in the
The contribution of each HNF1b NLS residue to the
binding was then assessed by binding free energy decom-
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Figure 6. A model of the constrained peptide Pep2A (orange) bound
to mImportin a1 DIBB (gray).
ing side-chain lengths in the synthesis of “double-click”
peptides.[31] Significantly, this is the first time that a con-
strained HNF1b NLS peptide has been found to bind more
tightly than its unconstrained peptide precursor.
Figure 5. A) Synthesized peptide sequences containing azido amino
acids and linkers A–C. B) General structure of the bis-triazole-con-
strained peptides with n=1,2 and m=1–3. C) Direct FP assay binding
affinities for (constrained) peptides in mm. The full synthesis of the
intermediates and constrained peptides can be found in the Support-
ing Information.
Live-cell fluorescence microscopy studies were under-
taken to assess the cell permeability of the synthesized
TAMRA-labelled linear and constrained peptides (Figure 7).
Pep0 showed limited cell permeability, while Pep1 and in
particular Pep2 showed good cell permeability. The corre-
sponding constrained peptides Pep1B and Pep2A retained
their cell permeability upon stabilization. This work repre-
sents the first example of constraining an NLS peptide to
target the nuclear import pathway, and it does not require the
attachment of a cell-permeable peptide sequence. All of the
constrained peptides were more cell-permeable than the
linear HNF1b NLS control peptide Pep0.
binding of Pep1, while Pep2 exhibited slightly decreased
binding potency. The constrained peptides Pep1A–Pep1C
followed a rough trend in which binding affinities increased
with increasing linker length (Figure 5). However, their
binding affinities were still about an order of magnitude
weaker than that of Pep0. The linkers were possibly too short
to constrain these peptides in the appropriate conformation
for binding, thereby resulting in higher Kd values. In contrast,
Pep2A (Kd = 4.54 mm, Figure 6), which is formed from
unnatural azido amino acids with longer side chains, was
observed to bind with slightly improved binding affinity
compared to Pep0. This highlights the importance of optimiz-
In conclusion, we have further validated HNF1b as
a therapeutic target in CCC by including more CCC lines
and time points during knockdown studies than previous
efforts.[2a,b] A set of constrained peptide inhibitors based on
Figure 7. Cell-permeability studies for the linear and constrained peptides using JHOC9 cells. Images were taken on a Leica tandem confocal
microscope using a 40X objective.
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the HNF1b NLS sequence was developed using rational drug
design to competitively target the HNF1b–importin a PPI,
and binding data were obtained using both ITC and FP assays.
MD simulations were performed to guide the development of
constrained peptides that have enhanced conformational
similarity to the bound HNF1b NLS peptide, thus further
reducing the entropic penalty for binding.[29] A constrained
peptide, Pep2A, which had a higher binding affinity than that
of the unconstrained HNF1b NLS peptide Pep0, and which
bound more tightly than its unconstrained precursor Pep2,
was identified. This confirmed that an entropically-driven
gain in binding affinity was achieved for Pep2A. All of the
constrained peptides, including Pep2A, were more cell-
permeable than Pep0. This work provides the first example
of using constrained peptides that mimic the ordered state of
NLSs to target the nuclear import of transcription factors.
Further studies are now underway to elucidate the structural
conformation of the constrained peptides upon binding to the
target protein. The surrounding residues of an NLS are often
important for binding specificity.[32] For example, the tran-
scription factor Stat1 has been found to be specifically
imported by importin a5 both in vitro and in vivo, and this
specificity appears to rely on contacts made with the C-
terminal acidic region of importin a5.[33] Further structural
information on the binding of HNF1b to importin a is
required for the future design of isoform-selective importin a
inhibitors. HNF1b overexpression in breast cancer[34] and
pancreatic clear cell carcinoma[35] also correlates with worse
survival rates, and the developed constrained peptide inhib-
itors may have a therapeutic effect on breast, pancreatic, and
ovarian clear cell carcinoma proliferation.[36] This method of
rational design of constrained-peptide drug candidates should
also be applicable to other IDDs.
[1] a) D. S. P. Tan, S. Kaye, J. Clin. Pathol. 2007, 60, 355 – 360;
b) D. S. Tan, R. E. Miller, S. B. Kaye, Br. J. Cancer 2013, 108,
1553 – 1559.
[2] a) H. Kajihara, Y. Yamada, S. Kanayama, N. Furukawa, T.
Noguchi, S. Haruta, S. Yoshida, T. Sado, H. Oi, H. Kobayashi,
Oncol. Rep. 2010, 23, 1193 – 1203; b) A. Tsuchiya, M. Sakamoto,
J. Yasuda, M. Chuma, T. Ohta, M. Ohki, T. Yasugi, Y. Taketani,
S. Hirohashi, Am. J. Pathol. 2003, 163, 2503 – 2512; c) N. Kato, S.
Sasou, T. Motoyama, Mod. Pathol. 2006, 19, 83 – 89; d) K.
Yamaguchi, M. Mandai, T. Oura, N. Matsumura, J. Hamanishi, T.
Baba, S. Matsui, S. K. Murphy, I. Konishi, Oncogene 2010, 29,
1741 – 1752.
[3] a) S. Sammak, G. Zinzalla, Prog. Biophys. Mol. Biol. 2015, 119,
41 – 46; b) R. van der Lee, M. Buljan, B. Lang, R. J. Weatheritt,
G. W. Daughdrill, A. K. Dunker, M. Fuxreiter, J. Gough, J.
Gsponer, D. T. Jones, P. M. Kim, R. W. Kriwacki, C. J. Oldfield,
R. V. Pappu, P. Tompa, V. N. Uversky, P. E. Wright, M. M. Babu,
Chem. Rev. 2014, 114, 6589 – 6631.
[4] P. Liu, A. Khurana, R. Rattan, X. He, S. Kalloger, S. Dowdy, B.
Gilks, V. Shridhar, Cancer Res. 2009, 69, 4843 – 4850.
[5] P. Lu, G. B. Rha, Y.-I. Chi, Biochemistry 2007, 46, 12071 – 12080.
[6] a) G. Wu, S. Bohn, G. U. Ryffel, Eur. J. Biochem. 2004, 271,
3715 – 3728; b) S. Bohn, H. Thomas, G. Turan, S. Ellard, C.
Bingham, A. T. Hattersley, G. U. Ryffel, J. Am. Soc. Nephrol.
2003, 14, 2033 – 2041; c) M. M. Wiedmann, S. Aibara, D. R.
Spring, M. Stewart, J. D. Brenton, J. Struct. Biol. 2016, 195, 273 –
281.
[7] D. Kalderon, W. D. Richardson, A. F. Markham, A. E. Smith,
Nature 1984, 311, 33 – 38.
[8] a) E. Conti, M. Uy, L. Leighton, G. N. Blobel, J. Kuriyan, Cell
1998, 94, 193 – 204; b) M. R. M. Fontes, T. Teh, B. Kobe, J. Mol.
Biol. 2000, 297, 1183 – 1194.
[9] A. Lange, R. E. Mills, C. J. Lange, M. Stewart, S. E. Devine,
A. H. Corbett, J. Biol. Chem. 2007, 282, 5101 – 5105.
[10] a) G. M. Cooper, The Cell, A Molecular Approach, 2nd ed.,
Boston University, 2000; b) V. Neduva, R. B. Russell, FEBS Lett.
2005, 579, 3342 – 3345; c) M. Marfori, A. Mynott, J. J. Ellis, A. M.
Mehdi, N. F. Saunders, P. M. Curmi, J. K. Forwood, M. Boden, B.
Kobe, Biochim. Biophys. Acta Mol. Cell Res. 2011, 1813, 1562 –
1577.
[11] a) P. Ferrigno, P. A. Silver, Oncogene 1999, 18, 6129 – 6134; b) A.
Komeili, E. K. OꢀShea, Curr. Opin. Cell Biol. 2000, 12, 355 – 360.
[12] Y.-Z. Lin, S. Yao, R. A. Veach, T. R. Torgerson, J. Hawiger, J.
Biol. Chem. 1995, 270, 14255 – 14258.
[13] T. R. Torgerson, A. D. Colosia, J. P. Donahue, Y.-Z. Lin, J.
Hawiger, J. Immunol. 1998, 161, 6084 – 6092.
[14] J. Zienkiewicz, A. Armitage, J. Hawiger, J. Am. Heart Assoc.
2013, 2, e000386.
[15] Y. H. Lau, P. de Andrade, Y. Wu, D. R. Spring, Chem. Soc. Rev.
2015, 44, 91 – 102.
[16] B. Kobe, Nat. Struct. Mol. Biol. 1999, 6, 388 – 397.
[17] U. Stelzl, U. Worm, M. Lalowski, C. Haenig, F. H. Brembeck, H.
Goehler, M. Stroedicke, M. Zenkner, A. Schoenherr, S. Koep-
pen, J. Timm, S. Mintzlaff, C. Abraham, N. Bock, S. Kietzmann,
A. Goedde, E. Toksoz, A. Droege, S. Krobitsch, B. Korn, W.
Birchmeier, H. Lehrach, E. E. Wanker, Cell 2005, 122, 957 – 968.
[18] Y. Minezaki, K. Homma, A. R. Kinjo, K. Nishikawa, J. Mol.
Biol. 2006, 359, 1137 – 1149.
[19] S. J. Metallo, Curr. Opin. Chem. Biol. 2010, 14, 481 – 488.
[20] A. K. Dunker, V. N. Uversky, Curr. Opin. Pharmacol. 2010, 10,
782 – 788.
[21] S. Kosugi, M. Hasebe, T. Entani, S. Takayama, M. Tomita, H.
Yanagawa, Chem. Biol. 2008, 15, 940 – 949.
Acknowledgements
M.W. is funded by Cancer Research UK, Department of
Chemistry at the University of Cambridge, School of the
Physical Sciences and the Cambridge Cancer Centre. The
Spring lab acknowledges support from the European
Research Council under the European Unionꢀs Seventh
Framework Programme (FP7/2007-2013)/ERC grant agree-
ment no [279337/DOS]. D.R.S acknowledges support from
a Royal Society Wolfson Research Merit award. In addition,
the group research was supported by grants from the
Engineering and Physical Sciences Research Council, Bio-
technology and Biological Sciences Research Council, Med-
ical Research Council, Royal Society and Welcome Trust.
Funding in part was also provided by Medical Research
Council Grant U105178939 to M.S. We would like to thank
the Biorepository and Microscopy facilities at the Cancer
Research UK Cambridge Institute, University of Cambridge,
Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE,
UK for assistance.
Keywords: constrained peptides · drug discovery ·
nuclear import · peptide therapeutics · peptidomimetics
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[22] T. L. Nero, C. J. Morton, J. K. Holien, J. Wielens, M. W. Parker,
Nat. Rev. Cancer 2014, 14, 248 – 262.
Y. Wu, P. de Andrade, W. R. J. D. Galloway, D. R. Spring, Nat.
Protoc. 2015, 10, 585 – 594.
[23] J. S. McMurray, P. K. Mandal, W. S. Liao, J. Klostergaard, F. M.
Robertson, JAKSTAT 2012, 1, 263 – 347.
[31] Y. H. Lau, P. de Andrade, N. Skold, G. J. McKenzie, A. R.
Venkitaraman, C. Verma, D. P. Lane, D. R. Spring, Org. Biomol.
Chem. 2014, 12, 4074 – 4077.
[32] K. M. Wagstaff, D. A. Jans, Anal. Biochem. 2006, 348, 49 – 56.
[33] a) T. Sekimoto, N. Imamoto, K. Nakajima, T. Hirano, Y. Yoneda,
EMBO J. 1997, 16, 7067 – 7077; b) J. Nardozzi, N. Wenta, N.
Yasuhara, U. Vinkemeier, G. Cingolani, J. Mol. Biol. 2010, 402,
83 – 100.
[24] a) A. K. Yudin, Chem. Sci. 2015, 6, 30 – 49; b) G. L. Verdine, G. J.
Hilinski in Methods Enzymology, Vol. 503 (Eds.: K. D. Wittrup,
G. L. Verdine), Academic Press, New York, 2012, pp. 3 – 33.
[25] A. Tomassetti, G. De Santis, G. Castellano, S. Miotti, M. Mazzi,
D. Tomasoni, F. Van Roy, M. L. Carcangiu, S. Canevari, Neo-
plasia 2008, 10, 1481 – 1492.
[26] A. E. Hodel, M. T. Harreman, K. F. Pulliam, M. E. Harben, J. S.
Holmes, M. R. Hodel, K. M. Berland, A. H. Corbett, J. Biol.
Chem. 2006, 281, 23545 – 23556.
[27] T. Terada, A. Kidera, J. Phys. Chem. B 2012, 116, 6810 – 6818.
[28] H. Gohlke, C. Kiel, D. A. Case, J. Mol. Biol. 2003, 330, 891 – 913.
[29] Y. S. Tan, D. P. Lane, C. S. Verma, Drug Discovery Today 2016,
21, 1642 – 1653.
[34] A. Matsui, J. Fujimoto, K. Ishikawa, E. Ito, N. Goshima, S.
Watanabe, K. Semba, FEBS Lett. 2016, 590, 1211 – 1221.
[35] L. Kim, J. Liao, M. Zhang, M. Talamonti, D. Bentrem, S. Rao,
G. Y. Yang, Mod. Pathol. 2008, 21, 1075 – 1083.
[36] H. Katoh, H. Ojima, A. Kokubu, S. Saito, T. Kondo, T. Kosuge, F.
Hosoda, I. Imoto, J. Inazawa, S. Hirohashi, T. Shibata, Gastro-
enterology 2007, 133, 1475 – 1486.
[30] a) Y. H. Lau, P. de Andrade, G. J. McKenzie, A. R. Venkitara-
man, D. R. Spring, ChemBioChem 2014, 15, 2680 – 2683; b) Y. H.
Lau, P. de Andrade, S.-T. Quah, M. Rossmann, L. Laraia, N.
Skçld, T. J. Sum, P. J. E. Rowling, T. L. Joseph, C. Verma, M.
Hyvçnen, L. S. Itzhaki, A. R. Venkitaraman, C. J. Brown, D. P.
Lane, D. R. Spring, Chem. Sci. 2014, 5, 1804 – 1809; c) Y. H. Lau,
Manuscript received: September 26, 2016
Revised: October 18, 2016
Final Article published: && &&, &&&&
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Peptide Therapeutics
Import ban: The transcription factor
HNF1b is ubiquitously overexpressed in
ovarian clear cell carcinoma. Guided by X-
ray crystallographic data and molecular
dynamics simulations, cell-permeable,
non-helical constrained proteomimetics
(orange) were designed to target the
HNF1b–importin a protein–protein
interaction and thereby inhibit the nuclear
import of HNF1b. This general approach
may be extended to other transcription
factors.
M. M. Wiedmann, Y. S. Tan, Y. Wu,
S. Aibara, W. Xu, H. F. Sore, C. S. Verma,
L. Itzhaki, M. Stewart, J. D. Brenton,
D. R. Spring*
&&&& — &&&&
Development of Cell-Permeable, Non-
Helical Constrained Peptides to Target
a Key Protein–Protein Interaction in
Ovarian Cancer
Angew. Chem. Int. Ed. 2016, 55, 1 – 7
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!