Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

Article abstract of DOI:10.1002/anie.201916257

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein–protein interaction antagonism, while keeping control over pharmacological performance such as stability and mem

Full text of DOI:10.1002/anie.201916257

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Multicomponent Peptide Stapling as a Diversity-Driven Tool for
the Development of Inhibitors of Protein-Protein Interactions
Authors: Alexander Doemling, Manuel Ricardo, Ameena Ali, Jacek
Plewka, Ewa Surmiak, Beata Labuzek, Constantinos
Neochoritis, Jack Atmaj, Lukasz Skalniak, Ran Zhang, Tad
Holak, Matthew Groves, and Daniel Rivera
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916257
Angew. Chem. 10.1002/ange.201916257
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10.1002/anie.201916257
Angewandte Chemie International Edition
RESEARCH ARTICLE
Multicomponent Peptide Stapling as a Diversity-Driven Tool for
the Development of Inhibitors of Protein-Protein Interactions
Manuel G. Ricardo,[a],‡ Ameena M. Ali,[b],‡ Jacek Plewka,[c] Ewa Surmiak,[c] Beata Labuzek,[c]
Constantinos G. Neochoritis,[d],[b] Jack Atmaj,[b],[c] Lukasz Skalniak,[c] Ran Zhang,[b] Tad A. Holak,[c]
Matthew Groves,[b] Daniel G. Rivera,[a],* Alexander Dömling[b],*
‡These authors contributed equally.
Abstract: Stapled peptides are chemical entities in-between biologics
and small molecules, which have proven to be the solution to high
affinity protein-protein interaction antagonism while keeping control
over pharmacological performance such as stability and membrane
penetration. We demonstrate that the multicomponent reaction-based
stapling is an effective strategy for the development of α-helical
peptides with highly potent dual antagonistic action of MDM2 and
MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X
interactions was assessed by fluorescence polarization, microscale
thermophoresis and 2D NMR, while several cocrystal structures with
MDM2 were obtained. This MCR stapling protocol proved efficient and
versatile in terms of diversity generation at the staple, as evidenced
by the incorporation of both exo- and endo-cyclic hydrophobic
moieties at the side chain cross-linkers. The interaction of the Ugi-
staple fragments with the target protein was demonstrated by
crystallography, while the difference in ring sizes, flexibility and
number of amide bonds within the ring seem to be crucial for a potent
activity.
techniques have been developed over the last decade.[3,4,9,10] As
a result, peptide stapling is nowa mature technology[11–16] that has
been applied to target protein-protein interactions related to
infectious diseases, cancer, neurological, endocrine, metabolic
and cardiovascular disorders, and a first example is tested in
clinical trials.[1,2,8,10,17–19]
As depicted in scheme 1, besides RCM, one-component
stapling techniques (i.e. the peptide as the only component)
currently include the traditional construction of lactam and
disulfide bridges as well as others based on oxime and thioether
bond formation[3,20] or the Ugi cyclization between Lys and
Asp/Glu side chains.[21,22] On the other hand, there is an
increasing interest in two-component stapling approaches
(Scheme 1A), in which – eventually bioorthogonal – processes
such as the click CuI-catalyzed alkyne-azide cycloaddition[3,10,23]
and dithiol (Cys) bis-alkylation[24] introduce a stapling linker
capable to stabilize the α-helical structure, with the former one
also enabling the functionalization of the staple moiety. This
strategy could be potentially more efficient for the rapid
optimization of the linker, since parallel syntheses and biological
screening can be undertaken using a single peptide sequence
and varying the length, flexibility and hydrophobicity of the linker.
Multi-component reactions (MCRs) are excellent diversity-
generating tools and have recently emerged as powerful stapling
tools capable to lock specific peptide conformations and
simultaneously diversify the staple moiety by variation of endo-
and exo-cyclic moieties during the multicomponent formation of
the side chain cross-linker.[20,21,25,26]
Introduction
Peptide stapling is a technology in which the side chains of two
amino acid residues – separated along the sequence – are cross-
linked to render
a conformationally constrained α-helical
peptide.[1–3] This synthetic approach does not only aim at the
stabilization of such secondary structure and the increase of the
binding affinity for a specific target,[2,4,5] but it can also produce
proteolytically stable[6] and cell-permeable peptides.[1,7] An
effective strategy for α-helix stabilization comprises the tethering
of residues at either i, i+4 or i, i+7 positions in the peptide chain,
so that the covalent bridge expands one or two turns of the α-
helical peptide.[1–5] While the original term “stapled peptides” was
employed for hydrocarbon bridged -helical peptides synthesized
by ring-closing metathesis (RCM),[1,2,8] several stapling
However, so far applications describing the utilization of a
multicomponent stapling approach[9] for the development of
bioactive α-helical peptides are absent. Herein, we describe a
diversity-driven peptide stapling approach for the discovery of
potent, dual action p53-MDM2 and MDMX antagonists. The
binding inhibition of the transcription factor p53 to MDM2 and
MDMX is an efficient way for the p53 activation in tumor cells. [27–
29]
The structure of the complex exhibits helical conformation,[30]
whereas this domain in free N-terminal domain of p53 is natively
unfolded.[31,32] For that reason, peptide stapling is nowadays
recognized as an effective approach to produce α-helical peptides
mimicking the p53 transactivation domain.[23,33–35]
[a]
Dr M.G. Ricardo, Prof D.G. Rivera
Faculty of Chemistry, Center for Natural Product Research,
University of Havana, Cuba
E-mail: dgr@fq.uh.cu
By targeting the dual inhibition of p53-MDM2 and MDMX
interactions as validated targets, we aim to demonstrate the
potential of Ugi multicomponent macrocyclizations in the rapid
discovery[36] of peptide inhibitors of protein-protein interactions
(PPIs).
[b]
Dr A.M. Ali, Prof C.G. Neochoritis, MsC J. Atmaj, MsC R. Zhang, Prof
M. Groves, Prof A. Dömling
Department of Pharmacy, Drug Design group,
University of Groningen, The Netherlands
E-mail: a.s.s.domling@rug.nl
Dr E. Surmiak, Dr J. Plewka, Dr B. Labuzek, MsC J. Atmaj, Dr L.
Skalniak, Prof. T.A. Holak
Faculty of Chemistry, Jagiellonian University, Krakow, Poland
Prof C.G. Neochoritis
[c]
[d]
Department of Chemistry, University of Crete, Greece
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201916257
Angewandte Chemie International Edition
RESEARCH ARTICLE
Scheme 1. A) One and two-component approaches based on different peptide stapling technologies. B) Novel multicomponent stapling strategy towards -helical
peptides described in this work.
containing the preformed imine. Such pseudo-dilution conditions
have proven successful in multicomponent macrocyclizations
based on varied MCRs,[24,25] and it constitutes a very effective way
Results and Discussion
Scheme
1
depicts the two different multicomponent
to avoid large solvent volumes at the same time that – typically –
avoids formation of dimeric cyclic and acyclic byproducts.[37] The
same protocol was employed for the synthesis of SPs 7 and 8
from the linear one 2, also using methylamine as the simplest
amine component.
macrocyclization variants devised for fine-tuning the nature and
flexibility of the linker. Since the Ugi-4CR comprises the reaction
of a carboxylic acid, usually a primary amine, an oxo compound
and an isocyanide,[37,38] we focused on incorporating Asp or Glu
at i, i+7 residues of the p53 binding sequence, as these positions
avoid engaging amino acids that are crucial for MDM2/X binding,
i.e. around the triad of Phe19, Trp23 and Leu26. As shown in
scheme 1B, two different classes of bifunctional building blocks
were envisioned as counterparts of the peptide dicarboxylic acid,
i.e. diamines and diisocyanides, which upon double Ugi
multicomponent macrocyclization[39] would lead to α-helical
structures, ideally pointing the critical ‘hot-spot’ residues towards
the same face. The difference between the dicarboxylic
acid/diamine and the dicarboxylic acid/diisocyanide combinations
does not only lie at the dissimilar length of the Ugi-4CR-derived
moieties connecting the linker with the peptide chain, but also in
the presence of two exo-cyclic fragments in the former one.
Scheme 2 depicts the implementation of such combinations
with peptides 1 and 2, which were previously produced by solid
phase peptide synthesis (SPPS) and released from the resin (see
Supplementary Information, SI). The sequence that is reported in
Scheme 2 is not the original p53 protein sequence but a
previously reported and optimized sequence.[18] The design was
focused in including two Ala at the C-terminus and changing the
previously stapling residues by either two Asp or two Glu to
enable the double Ugi macrocyclization. In addition, any
remaining Glu was change to Gln to avoid the participation in the
Ugi reactions. Thus, in both cases, two Asp and Glu residues
were placed at positions 4 and 11 of the tetradecapeptide p53
sequence. Peptide 1, bearing two Asp residues, was initially
chosen for the double Ugi-MCR macrocyclization using 1.5 equiv.
each of m- and p-xylylene diisocyanides[40] and 4.0 equiv. of
methylamine and paraformaldehyde as monofunctional
components. Stapled peptides (SPs) 3 and 4 were produced in
good macrocyclization yield – considering their complexity and
the formation of 8 covalent bonds in one pot – by a protocol
consisting of the slow addition with syringe pumps of solution of
both the peptide and the diisocyanide each to a reaction mixture
On the other hand, the implementation of the dicarboxylic
acid/diamine combination required
a
more elaborated
experimental setup, as it had never been used before to
macrocyclize bifunctional peptides.[9] Since preformation of the
imine is known to improve Ugi macrocyclization yields,[37] we
turned to initially stir the diamine component with
paraformaldehyde to enable imine formation followed by slow
addition of the diimine and the peptide dicarboxylic acid solutions
to a stirring solution of cyclohexyl isocyanide. As before,
satisfactory macrocyclization yields were obtained for SPs 5 and
6 after 72 h of reaction. These latter SPs include a flexible,
aliphatic staple and m-xylylene linker, respectively, but both bear
exo-cyclic cyclohexyl carboxamide groups positioned at the
tertiary amides. The easy incorporation of such additional exo-
cyclic fragments is one of the key and distinctive features of the
Ugi multicomponent stapling, which might even become relevant
in case these hydrophobic moieties participate in the binding
process to the protein target. Scheme 2B shows the CD spectra
of
the
linear
and
stapled
peptides
in
40%
trifluoroethanol/phosphate buffer (PB), because the hydrophobic
sequence of such peptides makes them insoluble in pure water or
PB at 0.1 mM. Despite the use of TFE in the solvent mixture is
known to stimulate α-helical structures,[41] the trend of increased
helical propensity in the stapled peptides is clearly proven by CD.
The relative percent of α-helicity for each peptide was estimated
(as described in the SI) by the mean residue ellipticity at 222 nm,
a value that is commonly downshifted for short peptides to 215-
220 nm. Importantly, while the multicomponent macrocyclization
led to a marked increment in the α-helical character of the stapled
peptides derived from peptide 2 (having two Glu), this was not the
case for stapled peptides 3 and 4 derived from 1. At this stage, it
was intriguing to assess whether the higher α-helicity of peptides
5-8, as compared with 3 and 4, would correspond to a better
inhibition of the p53-MDM2/X interactions.
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10.1002/anie.201916257
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Scheme 2. A) Synthesis of stapled peptides by Ugi multicomponent macrocyclization using Asp and Glu-containing peptides. B) CD spectra of the stapled peptides
as compared with the linear precursors. In parenthesis isolated yields.
Three orthogonal assays based on independent physicochemical
principles, fluorescence polarization (FP), microscale
the determination of the binding affinity.[44,45] Assuming 1:1
interaction data, it was then fitted using the Kd model. All tested
compounds showed an affinity towards MDM2 with Kd of a low nM
range 3-13 nM with low to very low measurement errors and high
goodness of fit (Table 1). Verifying the results from the FP assay,
the stapled peptides 5, 7 and 8 showed the higher activity with a
Kd of 4.5 nM, 4.8 nM and 2.9 nM, respectively. The efficiency of
the stapling is now confirmed compared to the acyclic peptides
(Table 1), as the binding affinities for the best stapled peptides
are significantly better than for corresponding linear peptides. SPs
3 and 4 are at least twice as potent as parent peptide 1 for both
MDM2 and MDMX. Similarly, SP 7 was twice as active as parent
peptide 2 proving that the cyclization of the peptides does not only
improve their stability, but also is essential for their affinity.
thermophoresis (MST) and 2D HSQC NMR were employed to test
the in vitro activity of the SPs. FP allows for the determination of
inhibitory constant IC50, which are readily obtained, but hard to
compare, as well as the inhibitory affinities (Ki), which should be
comparable and consistent throughout used techniques. The Ki
determination was performed based on the mass balance
relationships as described.[42] Thus, we found that all the stapled
peptides exhibited very high activities for both MDM2 and MDMX
in the nM range (Table 1). Peptides 3, 4 and 6 gave Ki in a range
of 5-10 nM against MDM2 and 20-380 nM against MDMX.
However, SPs 5, 7 and 8 showed higher activity against MDM2
with a Ki of 2.2 nM, 1.5 nM and 2.0 nM, respectively. Furthermore,
these peptides were also more active against MDMX with Ki of
11.8 nM, 7.1 nM and 12.7 nM, respectively, suggesting that they
could serve as an excellent example of dual inhibition.[43]
Microscale thermophoresis (MST) was utilized to cross-validate
the binding affinity of the tested peptides. This technique depends
on the detection of the Temperature Related Intensity Change
(TRIC), especially with the use of 2nd generation labeling dyes.
This temperature-induced change in fluorescence of a target
depends on the surrounding of the fluorophore and, therefore, is
related to the concentration of a non-fluorescent ligand allowing
For binding confirmation, we performed an H-15N HSQC of
1
15N labeled MDM2 and MDMX protein with increasing amount of
the representative peptide 8 as a third orthogonal assay to FP and
MST.[46–48] This method is based on monitoring of chemical shift
changes in protein amide backbone resonances upon protein
interaction with a small molecule and its advantage lays in the
complete unambiguity of the results as there are no auxiliary
molecules required, such as dyes (Figure 1). During the
experiment with both proteins, significant changes in spectra were
observed. Tight binding (slow chemical exchange) is present in
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10.1002/anie.201916257
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multiple residues, which is indicated by doubling of corresponding
cross-peaks (Figure 1). Slow chemical exchange is characteristic
for inhibitors with Kd lower than 1 μM. In such case, when the
compound is added to the protein with amount lower than the total
concentration of the protein, the peaks originating from free and
bound protein are seen separately on the spectra. Moreover, the
peaks assignment of residues in MDM2 and MDMX proteins
spectra is known, which allows to confirm the inhibitor
binding.[49,50] Especially important are the residues which lie near
the binding pocket of the surface of MDM2/X proteins, e.g. in the
case of MDM2, the Leu82, which is placed deep inside the Trp23-
p53 binding sub-pocket. Furthermore, multiple peaks that appear
near the binding area disappeared from the spectrum, indicating
strong binding (e.g. Gly58, Leu57, Met62 and Tyr60). For the
MDMX analysis, the situation is much more complex than for the
MDM2 protein. The main downfall in this case is the fact that
approximately 30% of the MDMX residues are not seen in 1H-15N
HSQC spectra when the protein is not bound to a ligand.[51] Upon
binding, additional residues appear on the spectra, as it is also
the case with our titration experiment of MDMX with the peptide
8. The performed titration experiment of 8 with the MDMX, clearly
shows strong binding between peptide and protein which is
indicated by the doubling of several cross-peaks, especially those
which are placed near to the binding pocket, such as Gly35.
Likewise, the other cross-peaks in near vicinity of binding pocket
which are changed in the spectra are Val26, Leu76 and Phe68.
Table 1. Biophysical binding data of Ugi-SPs to MDM2/X.
Peptide
Structure
Ki (nM, FP)
Kd (nM, MST)
14.8 ± 0.2
1
21 ± 6
(86 ± 17)a
3.6 ± 0.1
(16 ± 3)a
2
3
20 ± 13
13 ± 4
6.1 ± 0.1
(380 ± 169)a
9.5 ± 0.1
(44 ± 2)a
4
9 ± 1
2.2 ± 0.1
5
4.5 ± 0.9
(11.8 ± 0.2)a
5.0 ± 0.1
6
9 ± 2
(20.9 ± 0.4)a
1.5 ± 0.1
7
4.8 ± 0.9
(7.1 ± 0.1)a
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2.0 ± 0.1
8
2.9 ± 0.8
(12.7 ± 0.2)a
aData in parentheses refers to the MDMX protein
(according to WT-p53 sequence numbering) in the stapled
peptide helix and the carbonyl oxygen of Leu54 on MDM2
hydrophobic deep pocket. The staple is making multiple
hydrophobic and electrostatic contacts – partially mediated by
water – to the MDM2 rim of Leu54, Ile103, Lys51, Phe55, Gln59,
Gly58 and Met62 (Figure 2C-E). For example, the aromatic linker
makes a T-shaped -stacking interaction with Phe55 with a
closest distance of 4 Å (Figure 2E). The ability of the staple in the
stapled peptide to interact with the surface next to the major
binding cleft is interesting; the 'exploitation' of this surface by a
MDM2 inhibitor was first observed with
a
beta-hairpin
peptidomimetic of p53.[52] MDM2 retains its native fold observed
in other structures and undergoes only minor ligand-induced
changes upon binding. For example, the Met62 side chain folds
away from the p53 binding pocket to make room for the staple.
The Tyr100 side chain is in the so-called “closed” conformation as
in most other MDM2 structures. Overall the observed binding
mode of the three cocrystallized peptides is similar to other
described MDM2/X-SPs. The buried surface area (BSA) of both
the peptide 8 and the p53 is given in figure 3 (see also SI).
Figure 1. 1H-15N HSQC titration experiment for the peptide 8 with 15 N labeled
proteins MDM2 (A) and MDMX (B). (A) MDM2 with the peptide 8 (PDB ID 6T2D),
(B) MDMX with the p53 (PDB ID 2MWY). Reference MDM2/X – red; MDM2/X:8
ratio 4:1 – blue; MDM2/X over titrated with 8 – green; Upper panel: residues
which undergo the largest changes have been plotted on the crystal structure
showing an interaction of the inhibitor with the p53-binding pocket. Cross-peaks
which undergo slow chemical exchanges are marked with orange; cross-peaks
which undergo other significant changes are marked in bright orange. Lower
panel: Enlarged fragment of 1H-15N spectrum showing cross peaks placed near
binding pocket (especially L82 for MDM2 and G35 for MDMX proteins) and
undergo slow chemical exchange (tight binding).
To elucidate the binding mode of the SPs to their receptor, we
crystallized three high affinity SPs with MDM2 (see SI). The three
SPs 3 (PDB ID 6T2F), 7 (PDB ID 6T2E) and 8 (PDB ID 6T2D)
were cocrystallized with MDM2 and the complex structures
diffracted at high resolution of 2.09, 2.24 and 1.80 Å, respectively.
The overall SP sequence resembles the p53 peptide and
especially the ‘hot-spot’ triad Phe19, Trp23 and Leu26 is identical
(Figure 2A). The bound peptide 8 forms a compact, short, two-
turn α-helix (Figure 2B). Therefore, the overall binding is similar
to the p53-MDM2 and also MDMX. When compared to the crystal
structure of p53 helix in complex with the N-terminal domain of
MDM2 (PDB ID 1YCR), all the peptides bind the MDM2 deep
grove in a similar way to the WT-p53 peptide, by mimicking the
conserved p53-derived MDM2 hot-spot interaction motif (Phe19,
Trp23 and Leu26) into an identical orientation (Figure 2). A
hydrogen bond was found between the indole NH of Trp23
Figure 2. Ugi stapled peptides cocrystallized in MDM2. A) Sequence of peptides.
B) Overall view of peptide 8 (stick presentation) bound to MDM2 (surface
presentation). The linker is shown in blue and the α-helical peptide in cyan. The
linker moiety interacting surface of MDM2 is shown in green. C-F) Close up view
of some linker MDM2 interactions; C) Water mediated hydrogen bonding
interaction between Gln59 and Glu-derived linker carbonyl. D) vdWaals
interaction of Met62 with the linker. E) Phe55 and the linker phenyl moiety in a
T-shaped conformation.
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10.1002/anie.201916257
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RESEARCH ARTICLE
of the Ugi-staple moiety in the binding process. It is worth
mentioning that our method provides a high diversification of the
staple. Therefore, it is not only the easy generation of staple
diversity which makes this multicomponent approach so
promising, but also the rapid access to very complex structures
created with formation of several covalent bonds in one synthetic
operation. On the other hand, orthogonal protection methods
could be needed if additional Asp or Glu residues used. As
previously recognized by Spring[23] and others,[24] this stapling
modality of using bifunctional peptides and linkers offers a better
possibility for fine-tuning the activity and also the pharmacological
properties.[16] Our work demonstrates that the implementation of
that concept in a multicomponent manner further increases the
diversity elements to be varied and the possibilities to succeed in
the discovery of PPI inhibitors. Because of the availability of
varied MCRs, we foresee that the multicomponent stapling
concept will become an enabling synthetic technology in the field
of peptide pharmaceuticals.
Figure 3. The buried surface area (BSA) of both the peptide 8 and the p53.
We have implemented diversity-driven stapling approach for
constraining short peptides into a p53-type bioactive conformation
at the same time modulating the helical character, flexibility and
hydrophobicity of the staple moiety. By employing two different
combinations of the Ugi MCR-macrocyclization, we were able to
vary not only the macrocycle ring size but also the number of
amide bonds resulting at either endo- or exo-cyclic positions of
the ring. Despite all compounds showed a high in vitro activity,
such small differences arising from the variation of the
macrocyclization strategy turned out to be crucial for a very potent
inhibition effect. First of all, the peptides 1 and 2 have appeared
to be less active than the macrocyclized ones as measured by
both FP and MST (Table 1). Furthermore, using the same p53-
Acknowledgements
We are grateful to the Leibniz Institute of Plant Biochemistry, Halle
(Saale), for assistance in the MS and CD characterization of the
staple peptides. This research has also been supported to (AD)
by the National Institute of Health (NIH) (2R01GM097082-05), the
European Lead Factory (IMI) under grant agreement number
115489, the Qatar National Research Foundation (NPRP6-065-
3-012). Moreover, funding was received through ITN “Accelerated
Early stage drug dIScovery” (AEGIS, grant agreement No
675555) and COFUND ALERT (grant agreement No 665250),
based
dicarboxylic
acid
peptide
1,
the
diisocyanide/amine/aldehyde combination rendered SPs 3 and 4
which have the lowest helical character (Scheme 2) and proved
to be less potent than the others (Table 1). Utilizing the acyclic
peptide 2, yielded the SPs 5-8 with the highest helical character
(Scheme 2). It seems that the α-helical character is crucial for the
affinity, especially if we compared the SPs 3, 4 and 7, 8.
Comparison of SPs 5 and 6 is also intriguing, as both of them
Hartstichting
(ESCAPE-HF,
2018B012)
and
KWF
Kankerbestrijding grant (grant agreement No 10504). In addition,
this research has been supported by grants UMO-
derive from linear peptide
2
and from the same
diamine/isocyanide/aldehyde combination, but the change in the
flexibility of the linker from a flexible aliphatic one in 5 to the more
rigid m-xylylene in 6 seems to be determinant for decreasing the
activity from 2.3 nM to 5.0 nM, respectively (Table 1). SP 7 has
lower Ki and Kd than the corresponding peptide 6. This is
interesting, considering that both SPs 6 and 7 show a similar
helical character and include the same m-xylylene linker. A key
difference lies at the ring size and the presence of additional exo-
cyclic cyclohexyl moieties in SP 6, which – in this case – seems
to be quite detrimental for the binding to MDM2. Moreover, the
same amine/diisosyanide/aldehyde combination gave peptides 7
and 8 which are the most potent of the series as measured by all
methods. The ring size, the α-helical character, ring flexibility and
substitution pattern on the ring (especially the exocyclic moieties)
seem to influence the potency.
2012/06/A/ST5/00224
(to
T.A.H.)
and
UMO-
2016/21/D/NZ7/00596 (to L.S.) from the National Science Centre,
Poland.
Keywords: stapled peptides • Ugi reaction • p53-MDM2/X •
cancer • HSQC NMR
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Conclusion
To the best of our knowledge, the dual activity reported for SPs 5,
7 and 8 is amongst the highest described for p53-MDM2 and
MDMX inhibitors.[43,53,54] A deeper insight was achieved with the
analysis of the cocrystal structures, which demonstrates the
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[7]
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Angewandte Chemie International Edition
RESEARCH ARTICLE
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This article is protected by copyright. All rights reserved.

10.1002/anie.201916257
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
RESEARCH ARTICLE
An effective strategy of the
development of α-helical
peptides via Ugi stapling is
described. Highly potent
dual antagonistic action of
MDM2 and MDMX binding
p53 is shown based on
three biorthogonal assays,
e.g. fluorescence
Manuel G. Ricardo, Ameena
M. Ali, Jacek Plewka, Ewa
Surmiak, Beata Labuzek,
Constantinos G. Neochoritis,
Jack Atmaj, Lukasz Skalniak,
Ran Zhang, Tad A. Holak,
Matthew Groves, Daniel G.
Rivera,* Alexander Dömling*
Page No. – Page No.
polarization, microscale
thermophoresis and 2D
NMR. In addition, several
cocrystal structures with
MDM2 were obtained.
Multicomponent Peptide
Stapling as a Diversity-
Driven Tool for the
Development of Inhibitors
This article is protected by copyright. All rights reserved.