Structure-Enabled Discovery of a Stapled Peptide Inhibitor to Target the Oncogenic Transcriptional Repressor TLE1

Article abstract of DOI:10.1002/chem.201700747

TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of transcription factors. Inhibition of this protein–protein interaction represents a putative cancer target, but no small-molecule inhibitors have been published for this challenging interface. Herein, the structure-enabled design and synthesis of a constrained peptide inhibitor of TLE1 is reported. The design features the introduction of a four-carbon-atom linker into the peptide epitope found in many TLE1 binding partners. A concise synthetic route to a proof-of-concept peptide, cycFWRPW, has been developed. Biophysical testing by isothermal titration calorimetry and thermal shift assays showed that, although the constrained peptide bound potently, it had an approximately five-fold higher K_d than that of the unconstrained peptide. The co-crystal structure suggested that the reduced affinity was likely to be due to a small shift of one side chain, relative to the otherwise well-conserved conformation of the acyclic peptide. This work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors.

Full text of DOI:10.1002/chem.201700747

A Journal of
Accepted Article
Title: Structure Enabled Discovery of a Stapled Peptide Inhibitor to
Target the Oncogenic Transcriptional Repressor TLE1
Authors: Sally McGrath, Marcello Tortorici, Ludovic Drouin, Savade
Solanki, Lewis Vidler, Isaac Westwood, Peter Gimeson, Rob
vanMontfort, and Swen Hoelder
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700747
Link to VoR: http://dx.doi.org/10.1002/chem.201700747
Supported by

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Structure Enabled Discovery of a Stapled Peptide Inhibitor to
Target the Oncogenic Transcriptional Repressor TLE1.
Sally McGrath,[a] Marcello Tortorici,[a] Ludovic Drouin,[a] Savade Solanki,[a] Lewis Vidler,[a] Isaac
Westwood,[a] Peter Gimeson,[b] Rob Van Montfort,[a] Swen Hoelder*[a]
Abstract: TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of
transcription factors. Inhibition of this protein-protein interaction represents a putative cancer target but no small molecule
inhibitors have been published for this challenging interface. In this manuscript, we report the structure enabled design and
synthesis of a constrained peptide inhibitor of TLE1. Our design featured introduction of a four carbon atom linker into the
peptide epitope found in many TLE1 binding partners. We developed a concise synthetic route to a proof of concept peptide
cycFWRPW. Biophysical testing by ITC and thermal shift assays showed that whilst the constrained peptide bound potently, it
had an approximately five fold higher Kd than the unconstrained peptide. Our co-crystal structure suggested that the reduced
affinity is likely due to a small shift of one side-chain compared to the otherwise well conserved conformation of the acyclic
peptide. Our work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors.
Introduction
cancers with elevated TLE1 activity.[3] However, to date no TLE
inhibitors have been described in the literature.
TLE proteins (Transducin-Like Enhancer proteins) are
transcriptional co-repressors that modulate key pathways for
developmental as well as oncogenic signalling such as the
Notch and Wnt pathway. The TLE proteins do not bind directly to
DNA to exert their repressive effect on gene transcription.
Instead they utilise their WDR domains to bind to DNA bound
transcription factors.[1] Given their role in pathways known to be
deregulated in many cancers, it is not surprising that members
of the TLE family, particularly TLE1 have been implicated in the
development and maintenance of malignancies. Elevated levels
of TLE1 have been observed in a growing list of tumours,
including cervical, lung and colon carcinomas and TLE1 has
been recognised as a putative oncogene.[2] Given that TLE1
does not bind to DNA directly and that its repressive and
potentially oncogenic role relies on the ability of the WDR
domain to bind to transcription factors, blocking of this
interaction has been suggested as a possible treatment for
The crystal structures of the WDR domain of TLE1 in complex
with peptides derived from two different transcription factor
binding partners have been solved, thus characterising the
binding interface in detail.[3] One of these peptides (SMWRPW)
shows relatively potent (Kd = 1 μM) binding to TLE1. As will be
discussed in more detail below, the bioactive conformation of
this peptide is characterised by a compactly folded core formed
by the central three amino acids. This compact core engages in
extensive interactions with the WDR1 domain and positions key
amino acid side-chains such that they can form additional polar
and non-polar interactions (vide infra).[3] Given that this peptide
binds with micromolar activity and that detailed knowledge of its
binding mode and bioactive conformation are available, it
represents an attractive starting point for the discovery of TLE
inhibitors. Here we report a peptidomimetic approach based on
the hypothesis that the compact conformation of this peptide can
be stabilised by a hydrocarbon linker.
Hydrocarbon stapled macrocyclic peptides are increasingly
being explored as drug candidates and chemical probes,
particularly for challenging targets such as protein-protein
interactions.[4] Introducing conformational constraint through
macrocyclisation has a number of benefits. It particularly
reduces the entropic penalty upon binding to the target and has
been shown to have the potential to improve cell penetration and
metabolic stability.[5]
[a]
Dr. S. McGrath, Dr. M. Tortorici, Dr. L. Drouin, Dr. S. Solanki, Dr. L.
Vidler, Dr. I. Westwood, Dr. R. Van Montfort, Dr. S. Hoelder
The Institute of Cancer Research
Division of Cancer Therapeutics Unit
Cancer Research UK Cancer Therapeutics Unit
15 Costwold Road, Sutton, Surrey, SM2 5NG, UK
Phone: +44 2087224353
E-mail: swen.hoelder@icr.ac.uk
P. Gimeson
Malvern Instruments Nordic AB
Vallongatan 1, Uppsala, 752 28, Sweden
Phone: +46 704411919
[b]
E-mail: peter.gimeson@malvern.com
Supporting information for this article is given via a link at the end of
the document.
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HN
HN
the structure of the constrained peptide bound to the WDR
domain of TLE1.
Trp5
OH
NH
NH
HN
Arg3
H2N
H2N
HN
OH
HN
O
N
HN
O
O
O
O
O
O
H2N
O
H2N
O
O
N
NH
NH
HN
HN
Design
Pro4
S
S
Met1
NH
The crystal structure of the SMWRPW peptide bound to the
WDR domain of TLE1 was obtained by soaking apo TLE1
crystals in a solution of the slightly extended SMWRPW
peptide.[3] The indole moiety of the N-terminal tryptophan (Trp5)
and the central proline (Pro3) of the bound peptide tightly pack
against each other forming the core of the binding epitope
(Figure 1). This core engages in extensive hydrophobic
interaction with the protein.
N
Trp2
1
2
The compact conformation positions sidechains and backbone
moieties of the peptide such that they are ideally placed to
engage in additional polar and hydrophobic interactions.[3] The
N-terminal serine residue of the SMWRPW peptide is not
resolved, suggesting that it is disordered and does not make any
specific interactions.
Figure 1: Upper panel. Chemical structures of the SMWRPW peptide 1 and
the constrained peptide 2 (the linker is drawn in red) Lower panel. Co-crystal
structure of SMWRPW bound to the WDR domain of TLE1 (pdb code 2CE9)
and superposition of the modelled binding pose of the constrained peptide with
the crystallised pose of the unconstrained peptide 1.
HN
OMe
O
S
O
Pro-allyl
building block
NHCbz
NH2
NH
O
H2N
O
6
O
HN
O
Arg(Pbf)
N
Arg
N
HN
O
Arg
N
HN
O
N
NHCbz
O
N
N
OH
O
OMe
O
O
NH
O
O
N
OH
N-allyl
building
block
Trp
2
4
5
Scheme 1. Retrosynthesis of constrained peptide 2.
7
Designing and synthesising constrained macrocyclic peptides
still remains a formidable challenge.[4] Nevertheless, successful
examples have been reported, particularly for constraining and
stabilising α-helices, β- sheets and β-turns.[4]
Our strategy to generate a constrained macrocyclic inhibitor is
illustrated in figure 1: we hypothesised that connecting the Cα-
atom of the proline residue and the N1 nitrogen of the N-terminal
tryptophan with a hydrocarbon linker will lock the peptide in the
bioactive conformation. We modelled various linker lengths in
MOE by introducing the linker in silico into the bound
conformation of the peptide (PDB code 2CE9) and energy
minimising the modified peptides in the TLE binding site. The
resulting poses were visually inspected for minimal movement of
the peptide sidechains and low energy conformations of the
linker. These experiments together with an analysis of synthetic
accessibility (see below) suggested an ideal length of 4 carbon
atoms and compound 2 (Figure 1) as a promising synthesis
target.
However, in the case of TLE1, the bound SMWRPW peptide
adopts neither a typical α-helical nor a β-sheet conformation and
constraining the peptide thus required a different strategy. As
will be described in more detail below, we hypothesised that
connecting two amino acids that are a critical part of the binding
epitope, the side-chain of the first tryptophan and the proline,
through a hydrocarbon linker would stabilise the bioactive
conformation. Here we report the design and development of a
chemical route to this hydrocarbon linker-constrained, proof of
concept peptide. Furthermore we tested the binding affinity of
the constrained and corresponding acyclic peptides and solved
Retrosynthesis
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Our retrosynthetic analysis is depicted in Scheme 1. We
unprotected L-tryptophan using either a copper TMEDA catalyst
or sodium metal had been described.[6] We tested the copper
envisioned synthesising the constrained hydrocarbon stapled
Cbz
OH
Cbz
O
HN
NaH
AllBr
(1
C
NH2
O
eq.),
eq.),
HN
i)
(3
DMF, 0 o
DIPEA
HOBT
DMF,
N
eq.)
(1.5
eq.)
OH
ii) benzyl
(1
rt
chloroformate,
0 o
N
O
O
H
Na
2CO3
C
,
2O,
N
N
N
N
N
H
28%
7
Scheme 2. Synthesis of N-allyl-Trp 7.
peptide 2 from the macrocyclic intermediate 4 through addition
of the N-terminal methionine and the C-terminal tryptophan via
peptide coupling chemistry. Furthermore, we hypothesised that
intermediate 4 can be prepared from the acyclic tripeptide 5
through RCM, followed by concomitant saturation of the double
bond and removal of the Cbz protecting group under
hydrogenation conditions. To prepare the acyclic RCM precursor
5, two unnatural amino acids were required, substituted proline 6
and allyl substituted tryptophan 7.
mediated conditions but did not observe any conversion. More
recently a team from Sanofi published the synthesis of boc
protected 1-allyl-L-tryptophan but this work was not in the public
domain when we undertook our work.[7] We hypothesised that
selective allylation of unprotected tryptophan can be achieved
after deprotonating the carboxyl group and the NH indole with
two equivalents of a strong base such as NaH, since under
these conditions the deprotonated indole nitrogen represents the
strongest nucleophile. Pleasingly, reacting L-tryptophan with 2.5
equivalents of NaH and one equivalent of allyl bromide in DMF
gave the desired mono allylated product in 40% yield after HPLC
purification. To avoid HPLC purification of the polar, unprotected
amino acid we decided to attempt the allylation and subsequent
Cbz protection with benzyl chloroformate in a one pot procedure.
Gratifyingly, this procedure gave the desired, protected amino
This approach offered the advantage of conducting the critical
RCM in solution whilst all polar groups, particularly the basic
arginine side-chain were fully protected. Furthermore the cyclic
intermediate 4 offered the opportunity of late stage modification
of the C- and N-terminal amino acids.
Results and discussion
Fmoc
NH
Fmoc
NH
.
HN
HN
N
HCl
Pbf
N
N
Pbf
H
O
H
N
H
H
NH
HN
N
OH
O
H
Fmoc
+
N
O
N
Pbf
N
H
OMe
HCl-6
O
OMe
O
8
9
eq.)
O
Piperidine
(1
EtOH / EtONa
i)
ii)
Cbz
O
HN
rt,
100%
HCl
(MeOH)
Cbz
N
HN
Evaporation
N
O
O
N
N
N
NH
N
Arg(Pbf)
NH
+-H3N
Cl
N
HN
Arg(Pbf)
N
Pbf
N
H
N
H
N
O
O
O
rt
DMF, DIPEA,
OMe
OMe
11
O
O
5
70%
steps)
(two
10
Scheme 3. Optimised reaction conditions for the synthesis of arginine containing tripeptide 5.
The synthesis of the proline derivative 6 was described in the
literature and we followed the protocols with minor modifications.
We next turned our attention to the synthesis of Cbz protected 1-
allyl-L-tryptophan 7. We decided to use the Cbz protecting group
since it is stable to basic and acidic conditions and because we
anticipated that it can easily be removed during hydrogenation of
the double bond arising from the RCM thus making an additional
step unnecessary. At the start of this work direct allylation of
acid 7 in an acceptable yield of 28% over two steps.
We next prepared the tripeptide RCM precursor 5 by coupling
the allylated proline with protected arginine (Scheme 3) under
conditions precedented for this proline derivative. However, we
only isolated the cyclised side product 8. The formation of this
side product is likely due to steric hindrance of the amine
functionality. Gratifyingly, increasing the reaction temperature
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and the concentrations of the reactants to favour biomolecular
reaction led to the desired dipeptide in 48% yield.
Next we attempted removal of the Fmoc protecting group from
dipeptide 9 (Scheme 3). However, standard conditions using
piperidine as the base gave the undesired side product 11 as a
single diastereomer.
and hydrogen at atmospheric pressure left the starting material
intact. Elevated temperature, addition of acid or increase of
catalyst loading did not significantly improve turnover. We next
investigated other catalysts and found that Pearlman’s catalyst
both reduced the double bond and removed the Cbz protecting
group. Complete conversion required one equivalent of
NH2
Cbz
Cbz
HN
HN
O
10 mol% Grubbs II
O
O
40 mol%
0.01 M CH Cl reflux
1,4-benzoquinone,
,
HN
O
5
Arg(Pbf)
N
,
2
2
H2
HN
O
HN
Arg(Pbf)
N
Arg(Pbf)
N
N
N
N
Pearlman's Cat.
76%
trans:cis 9:1
83%,
O
OMe
O
OMe
OMe
5
12
4
O
O
Scheme 4: Ring Closing Metathesis of 5, removal of Cbz and double bond hydrogenation.
Repeating the reaction with 1 equivalent of piperidine and at a
lower temperature (0 °C) resulted in a mixture of the unprotected
dipeptide and the side product. Unfortunately, all attempts to
isolate the unprotected dipeptide and to remove the piperidine,
resulted in complete conversion to the diketopiperazine 11 side
product. To solve this conundrum we reasoned that protonation
after Fmoc deprotection will lower the nucleophilicity of the free
amino group sufficiently to prevent cyclisation thus allowing
isolation by evaporation of the solvent. Furthermore, we tested
alternative bases, particularly bases likely to not affect the
subsequent peptide coupling step. This approach indeed proved
successful and compound HCl-10 was obtained as single
stereoisomer through clean Fmoc deprotection in EtOH using
one equivalent of NaOEt as base. Subsequent protonation of
the amine and residual traces of NaOEt by addition of an HCl
solution in MeOH thwarted formation of the side product upon
solvent evaporation (Scheme 3). Coupling of the crude product
with the HATU derivative of the allyl substituted tryptophan 7
using DIPEA as a base yielded the metathesis precursor 5 in
70% overall yield.
Pd(OH)2/C and the addition of 2 equivalents of HCl, but resulted
in an isolated yield of 76% of the reduced and deprotected
intermediate.
With intermediate 4 in hand we next performed the coupling to
Boc-protected methionine. Whilst this coupling proceeded
readily, we reproducibly observed a +16 Da increase in
molecular weight after isolation and purification. We attributed
this increase to oxidation of the methionine to the corresponding
sulfoxide derivative 14 (Scheme 5). This oxidation is
precedented in the literature, however the degree and rapidness
of the reaction was surprising given that methionine is frequently
incorporated into peptides.
As we will discuss in the more detail below, this methionine
residue can be replaced in the acyclic peptide by phenylalanine
without loss of activity. We thus focused our attention on the
phenylalanine derivative. Coupling of 4 with Boc-protected
phenylalanine proceeded in 68% yield after purification (Scheme
5).
To complete the synthesis we hydrolysed the ester using LiOH
in methanol (86% yield) and added the final amino acid by
coupling this intermediate onto commercially available solid
support bound tryptophan (Scheme 5).
To our delight, the pivotal RCM proceeded readily using Grubbs’
second generation catalyst[8] in the presence of 1,4-
benzoquinone[9] yielding the desired product 12 in 83% yield as
a 9:1 mixture of trans and cis isomers (Scheme 4).
Cleavage of the solid support of 17 and concomitant removal of
the remaining two protecting groups provided the desired
macrocyclic peptide 18 in 10% yield over three steps (Scheme
5).
We next investigated concomitant reduction of the double bond
and removal of the Cbz group by hydrogenation (Scheme 4).
Commonly used conditions such as 10% palladium on carbon
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Ph
O
NHBoc
O
NH2
HN
S
O
NH
O
BocHN
Boc-Met-OH,
HBTU, DIPEA,
O
HN
O
Arg(Pbf)
N
HN
O
Boc-Phe-OH, HBTU,
DIPEA, DMF,
Arg(Pbf)
N
rt
DMF,
HN
Arg(Pbf)
rt
N
N
N
68%
N
O
OMe
O
OMe
13
4
O
OMe
15
O
H
LiOH,
MeOH,
(86%)
2O,
C
i)
THF, 50 o
ii) NMP, DIPEA, HATU
O
NHBoc
O
H2N
iii)
HN
O
S
O
O
BocN
Ph
HN
O
O
Arg(Pbf)
N
Ph
O
N
TFA,
TIS,
NH
H
H2N
2O
O
NH
NH
BocHN
O
OMe
HN
O
14
NH2
10% steps)
(3
N
H
HN
Arg(Pbf)
N
N
N
O
N
O
O
NH
O
OH
NH
O
18
O
HN
O
17
NBoc
Scheme 5. Synthesis of the final constrained peptide 18.
Despite initial challenges, our synthetic approach enabled us to
access 14 mg of the desired, constrained peptide. Some of the
optimised steps, e.g. the one pot alkylation and protection of
tryptophan as well as the convenient and mild deprotection of
the Fmoc group in solution may be useful for the synthesis of
other constrained peptides.
All three peptides showed significant thermal shifts indicative of
binding to the protein. Interestingly, the MWRPW peptide, which
is derived from the sequence of TLE1 binding partners shows
Table 1. Thermal shifts at 100 and 200 uM peptide concentrations.
Peptide
Ligand
ΔTm
(100 μM)
6.3°C
ΔTm
(200 μM)
6.9°C
1 0
MWRPW
cycFWRPW (18)
8.4°C
9.4°C
7.5°C
5
F W R P W
FWRPW
10.1°C
c y c F W R P W
M W R P W
0
0
5 0
1 0 0
1 5 0
2 0 0
the smallest thermal increase. The mutant FWRPW peptide
causes a significantly larger thermal shift (9.4 °C versus 6.3 °C).
The cyclic peptide cycFWRPW (18) at 100 μM shows a thermal
shift comparable to the corresponding acyclic peptide (Table 1).
However, the thermal shift decreases when the concentration is
further increased from 100 to 200 μM. This decrease is likely
due to precipitation of the peptide at higher concentrations. Our
thermal shift data thus suggested that all three peptides bound
to TLE1.
[p ep tid e] µM
Figure 2. ΔTm plot of peptides-hTLE1 443-770 interaction in thermal
shift experiments. All the measurements were carried out in triplicate
and the points are reported as mean + SD. The ΔTm at top
concentrations is also reported in the table.
We next investigated the binding of this macrocyclic as well as
the acyclic MWRPW and FWRPW peptides to TLE1. We used
two orthogonal binding assays, the thermal shift assay[10] and
isothermal titration calorimetry (ITC) [11] to test binding of 18 and
the linear peptides to the TLE1 WD40 domain (TLE1 residues
443-770). The thermal shift data for the three peptides are
shown in figure 2 and table 1.
To confirm these findings and to explore the enthalpic and
entropic contributions to binding of the linear and constrained
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peptides we performed ITC experiments. Given conformational
restriction, one might expect the constrained peptide to show a
smaller entropic penalty upon binding. However, all three
peptides showed potent binding driven by strong enthalpy
contributions.
between the different peptides is in agreement with this
hypothesis.
In the following paragraph we will focus the discussion of the
ITC results on the second binding event (Kd2, ΔH2 and -TΔS2)
for three reasons. The second binding event covers binding to
the large majority of the protein (~80%), the Kds are in
agreement with published values and finally, due to the
experimental set up, the relative errors are smaller. However, we
include the data for the first binding event (Kd1, ΔH1 and –TΔS1)
and they broadly follow the same trend.
Interestingly, for each peptide we observed a biphasic curve.
This was initially more pronounced for FWRPW and cycFWRPW
(18) but also recognisable for the MWRPW peptide (see
Supporting Information, Figure S1). We repeated the MWRPW
titration at slightly higher protein and peptide concentrations to
achieve a higher enthalpy signal and therefore better resolution
of the titration event. Under these conditions we also observed a
clear biphasic curve (Figure 4). The biphasic curves are
indicative of two binding events and we calculated the
thermodynamic data for both (Table 2).
The rank order based on the ITC Kds (Kd2) confirms the rank
order from the thermal shift assay described above. The acyclic
FWRPW shows the highest affinity with a Kd of 79 nM. It thus
shows almost 10-fold more potent binding than the peptide
representing the original MWRPW sequence from the TLE1
binding partners. The Kd2 for the cyclic peptide 18 is 522 nM and
The first phase of the curves corresponded to a molar ratio of
Figure 4. ITC measurement of peptides-TLE1 binding. Data fitting to a two-sites independent binding model, integrated heats are shown in the inset.
Histograms showing ΔG, ΔH, and –TΔS are presented in the bottom part of the figure. The thermodynamic values are also presented in Table 1. a.
MWRPW-TLE1 binding, n =1. Experiment performed with TLE1 40 μM and MWRPW peptide 420 μM. b. FWRPW-TLE1 binding, n =2. Experiment
performed with TLE1 30 μM, FWRPW peptide 240 μM and then repeated with TLE1 24 μM, FWRPW peptide 180 μM. Histograms represent averaged
values, error bars denote SD. c. cyc-FWRPW-TLE1 binding, n =2. Experiment performed with TLE1 30 μM, cyc-FWRPW 200 μM and then repeated with
TLE1 30 μM, cyc-FWRPW 180μM. Histograms represent averaged values, error bars denote SD.
Table 2. Kd and thermodynamic values determined in ITC for all the peptides-TLE1 binding experiments.
N1
Kd1 (nM)
3.5 ± 1.9
ΔH1 (kcal/mol)
-12.8 ± 0.2
-20.5 ± 3.7
-16.9 ± 0
-TΔS1 (kcal/mol)
1.3
N2
Kd2 (nM)
772 ± 7.1
ΔH2 (kcal/mol)
-11.1 ± 0.11
- 14.6 ± 0
-TΔS2 (kcal/mol)
2.72
MWRPW
FWRPW
0.24 ± 0.001
0.18 ± 0.002
0.18 ± 0.01
0.6 ± 0.004
0.7 ± 0.004
0.8 ± 0.01
8.6 ± 3.9
9.2 ± 3.8
6.4 ± 0.6
79.4 ± 24.6
522 ± 39.6
4.6 ± 0.2
cyc-FWRPW
24.7 ± 24.2
-11.8 ± 1.8
3.2 ± 1.7
approximately 0.2 (that is 20% of the protein is bound) and the
second an approximate molar ratio of 0.8 and the overall curve
thus reached saturation at a molar ratio close to one. This
suggested that only one binding site per molecule of protein is
occupied by the ligand. A possible explanation for the biphasic
curve is that in the binding experiment the protein exists in two
conformations that do not rapidly interconvert and show different
binding affinities for the peptides. The observation that the molar
ratios for the two parts of the biphasic curve correspond
thus less potent than the corresponding acyclic peptide
suggesting that introduction of the hydrocarbon linker leads to a
small loss of activity. Interestingly, binding of 18 is accompanied
by a reduced loss of entropy compared to the acyclic peptide
which is in agreement with the hypothesis that introduction of a
constraint reduces the entropic penalty (albeit that this reduced
entropic loss is overcompensated by a larger enthalpic loss
leading to a higher Kd compared to the acyclic FWRPW peptide).
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To be able to interpret these thermodynamic data in light of the
binding modes we set out to determine the crystal structure of
the cyclic peptide 18. Briefly, we grew apo crystals of the TLE1
WD40 domain using slightly modified previously published
conditions[3] and succeeded in solving the structure of the cyclic
peptide 18 bound to TLE1 to 2.18 Å resolution by soaking with a
2.5 mM solution of 18. The asymmetric unit contained two TLE1
monomers and electron density was evident in both binding
sites. However the quality of the electron density differed in the
two independent TLE1 monomers. Chain A showed strong
ligand density and allowed us to model the cyclic peptide 18 with
full occupancy. The ligand density in chain B was weaker and
refined at a lower occupancy (0.83). Therefore we will focus the
discussion on the peptide bound to chain A.
Conclusion
TLE1 is a transcriptional co-repressor that exerts its oncogenic
function through binding to transcription factors. Inhibition of this
protein-protein interaction represents a putative cancer target
but no small molecule inhibitors targeting this challenging
interface have been published to date. In this manuscript, we
report the design and synthesis of a constrained peptide inhibitor
of TLE1.
We developed a concise synthetic route to a constrained proof
of concept peptide cycFWRPW (18). Biophysical analysis by ITC
and thermal shift assays and X-ray crystallography confirmed
that the constrained peptide binds to the WD40 domain of TLE1.
Furthermore, the observation that the constrained peptide shows
binding thermodynamics that are entropically favoured
compared to the acyclic FWRPW peptide is in agreement with
the hypothesis that rigidifying the peptide lowered the entropic
penalty upon binding to the target. However, the constrained
peptide also showed an approximately 6-fold lower affinity as
compared to the acyclic peptide. The crystal structure of the
constrained peptide bound to TLE1 suggests that the linker
causes some strain in the molecule that may, at least partially,
explain the lower affinity. These observations underscore the
known challenge of designing constrained peptides. Our
constrained peptide replicated the bioactive conformation very
well with an RMSD of 0.55 Å and yet a slight deviation caused
a sufficient penalty to compensate the gain achieved by
introducing the constraint.
Figure 5 depicts the constrained peptide bound to TLE1 and an
overlay with the published structure of our acyclic design
template, SMWRPW (pdb code 2CE9).
Figure 5. Left. Co-crystal structure of the constrained peptide 18 bound to the
WDR domain of TLE1. Right. Superpositioning of the acyclic (yellow) and
cyclic (green) peptides 1 and 18.
Overall, the binding mode of the constrained peptide is almost
identical to that of the published acyclic peptide bound structure.
The overall root-mean-square deviation (RMSD) between the
two structures is 0.55 Å. The N-terminal phenylalanine side-
chain of the constrained peptide occupies a similar position as
the methionine side-chain with the aromatic side chain efficiently
packing against the hydrophobic part of Glu 550, potentially
explaining the slightly higher affinity of the acyclic FWRPW
compared to the MWRPW peptide. The most significant
difference between the cyclic peptide conformation compared to
the bound SMRWPW conformation is the linker, which appears
to cause a slight change in position of the N-terminal tryptophan
and could go some way in explaining the lower affinity of 18 as
compared with the linear FWRPW peptide. This slight movement
may create an unfavourable, modestly repulsive interaction that
outweighs the gain achieved through constraining the peptide.
The observation that our cyclic peptide shows a higher Kd
despite replicating the bioactive conformation very accurately
underscores the challenge of designing constrained peptides.
Minor differences that are outside the predictive power of current
structure based design tools, even if high resolution crystal
structures are available, can have a significant effect on
bioactivity.
Experimental Section
Supporting Information
Experimental and characterization details for all new
compounds, computational data, ITC data, assays data,
crystallographic data, and NMR spectra are provided free of
charge at link.
Accession codes
Atomic coordinates and structure factors for the crystal structure
of TLE1 with contrained peptide 18 can be accessed using PDB
code: 5MWJ
AUTHOR INFORMATION
Corresponding Author
swen.hoelder@icr.ac.uk
Notes
The authors declare no competing financial interest.
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10.1002/chem.201700747
Chemistry - A European Journal
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ACKNOWLEDGMENTS
S. W. Seo, H. Lee, H.-I. Lee and H.-S. Kim, J. Orthop. Res. 2011, 29, 1131-
1136; e) X. Yao, S. K. Ireland, T. Pham, B. Temple, R. Chen, M. H. G. Raj and
H. Biliran, Biochem. Biophys. Res. Commun. 2014, 455, 277-284.
Lewis Vidler was funded by Cancer Research UK [grant number
C309/A11369], Sally McGrath was funded by Wellcome Trust
[grant number 090171/Z/09/Z]. We acknowledge NHS funding to
the NIHR Biomedical Research Centre and funding from Cancer
Research UK [grant number C309/A11566]. We are grateful to
Laurence Pearl, University of Sussex, Brighton, UK for his gift of
the construct of the TLE1 WD40 domain. We thank Dr Nora
Cronin and the staff of DIAMOND Light Source for their support
during data collection.
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This article is protected by copyright. All rights reserved.

10.1002/chem.201700747
Chemistry - A European Journal
FULL PAPER
FULL PAPER
Sally McGrath,[a] Marcello Tortorici,[a]
Ludovic Drouin,[a] Savade Solanki,[a]
Lewis Vidler,[a] Isaac Westwood,[a] Peter
Gimeson,[b] Rob Van Montfort,[a] Swen
Hoelder*[a]
Text for Table of Contents
N
NH
HN
N
O
O
NH2
O
O
O
NH
HO
NH
NH2
HN
NH
Page No. – Page No.
Title
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