Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions

Article abstract of DOI:10.1002/anie.201410810

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modul

Full text of DOI:10.1002/anie.201410810

Angewandte
Chemie
DOI: 10.1002/anie.201410810
PPI Inhibitors
Hot Paper
Selective and Potent Proteomimetic Inhibitors of Intracellular Protein–
Protein Interactions**
Anna Barnard, Kꢀrya Long, Heather L. Martin, Jennifer A. Miles, Thomas A. Edwards,
Darren C. Tomlinson, Andrew Macdonald,* and Andrew J. Wilson*
Abstract: Inhibition of protein–protein interactions (PPIs)
represents a major challenge in chemical biology and drug
discovery. a-Helix mediated PPIs may be amenable to
modulation using generic chemotypes, termed “proteomimet-
ics”, which can be assembled in a modular manner to
reproduce the vectoral presentation of key side chains found
on a helical motif from one partner within the PPI. In this
work, it is demonstrated that by using a library of N-alkylated
aromatic oligoamide helix mimetics, potent helix mimetics
which reproduce their biophysical binding selectivity in
a cellular context can be identified.
(HTS),[6] fragment-based approaches,[7] and computer aided
ligand ID/optimization[8] have afforded small-molecule mod-
ulators of PPIs, generic approaches which target particular
classes of PPI are desirable. Helix-mediated PPIs[9] have
received considerable attention[10] as the secondary structure
motif represents a generic pharmacophore. Constrained
peptides[11,12] and ligands which mimic the helical topography
of the helix (e.g. a/b and b-peptides)[13–15] are proven
successful approaches and have entered clinical develop-
ment.[16] An alternative small-molecule approach has been
postulated whereby a generic scaffold is used to mimic the
spatial and angular projection of “hot-spot” side chains found
on the key helix mediating the PPI of interest.[17] Such ligands
have been termed proteomimetics,[18] a-helix mimetics,[19–22]
and topographical mimics.[23] Several studies on this general
class of ligand have illustrated that they can be used to
selectively recognize their target protein in biophysical
assays,[19,24,25] that they act in cells upon the pathway in
which the PPI is found,[23,26,27,52] and that they exhibit the
anticipated phenotypic effects in animals.[23] In this work we
performed biophysical and cellular experiments on a library
of N-alkylated aromatic oligoamide proteomimetics
(Figure 1). Our purpose was to study the correlation between
biophysical and cellular selectivity, and to highlight the
potential for off-target effects, which have not been described
for proteomimetics. Although strictly speaking our goal was
not to identify inhibitors of a specific PPI, we identified
potent inhibitors of p53/hDM2 and the B-cell lymphoma-2
(Bcl-2) family PPIs which induce apoptosis, and this may
P
rotein–protein interactions (PPIs) mediate all biological
processes and thus are actively involved in the development
and progression of disease.[1] Studies of the protein inter-
actome have estimated that there may be as many as 650000
pairwise interactions,[2] hence there is considerable therapeu-
tic potential in being able to modulate these interactions.
Despite this clear need, it has historically been considered
challenging to identify small molecules which selectively
recognize their protein targets based on the type of surface
involved in PPIs.[3–5] Although, high-throughput screening
[*] Dr. A. Barnard, Dr. K. Long,[+] Dr. J. A. Miles, Prof. A. J. Wilson
School of Chemistry, University of Leeds
Woodhouse Lane, Leeds LS2 9JT (UK)
E-mail: A.J.Wilson@leeds.ac.uk
Dr. A. Barnard, Dr. K. Long,[+] Dr. J. A. Miles, Dr. T. A. Edwards,
Dr. D. C. Tomlinson, Dr. A. Macdonald, Prof. A. J. Wilson
Astbury Centre For Structural Molecular Biology, University of Leeds
Woodhouse Lane, Leeds LS2 9JT (UK)
Dr. H. L. Martin[+]
Leeds Institute of Biomedical and Clinical Sciences, Wellcome Trust
Brenner Building, University of Leeds
St. James’s University Hospital, Leeds LS9 7TF (UK)
Dr. T. A. Edwards, Dr. D. C. Tomlinson, Dr. A. Macdonald
School of Molecular and Cellular Biology, University of Leeds
Woodhouse Lane, Leeds LS2 9JT (UK)
E-mail: A.Macdonald@leeds.ac.uk
[+] These authors contributed equally to this work.
[**] This work was supported by the European Research Council [ERC-
StG-240324], Yorkshire Cancer Research (L348), and The Wellcome
Trust (104920/Z/14/Z). The authors thank Arnout Kalverda and
Gary Thompson for assistance with NMR experiments.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410810.
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
Figure 1. N-alkylated helix mimetics. a) The p53 helix illustrating key
side chains. b) Structures of principle compounds discussed in this
work.
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Figure 2. HCS summary for mimetic library. a) Example images of cells treated with five compounds (20 mm) and controls of 0.2% DMSO and
Nutlin-3a (5 mm) scale=50 mm. DAPI staining is shown in the blue channel, caspase 3 is shown in red, LC3B antibody in yellow, F-actin stained
with AlexaFluor488 conjugated phalloidin shown in green. b) Heat map illustrating results of HCS. c) Summary of cellular toxicity of the mimetics
added to U2OS (black) and SJSA-1 (grey) cells at a concentration of 50 mm. d) Cells were treated with FITC-labelled trimers and imaged using
high-content imaging and co-stained with Toto-3 iodide as a cytoplasmic stain. Scale=50 mm.
represent a novel avenue for anticancer therapeutics devel-
opment.
were furnished with mostly hydrophobic aliphatic and
aromatic side chains to imitate Phe19, Trp23, and Leu26.[28]
The 73 trimeric oligobenzamides were obtained alongside
four dimers (trimers comprise three monomers linked by
amides with dimers comprising two monomers; see the
Supporting Information), which were designed to act as
negative controls incapable of effective mimicry of the full
p53 hot-spot region.
The cellular levels of the transcription factor p53 are
controlled by a negative feedback loop involving hDM2.[28] In
normal cells, binding of the helical p53 N-terminal trans-
activation domain to a cleft on hDM2 results in its poly-
ubiquitination and subsequent degradation.[29] In response to
cellular stress p53 is activated and initiates apoptosis to
eliminate the damaged cell. This target has seen the develop-
ment of several small-molecule inhibitors as potential anti-
cancer agents.[30] Similarly, the Bcl-2 family plays a central
role in the regulation of apoptosis through control of
mitochondrial outer membrane permeabilization.[31] Proteins
within this family include the anti-apoptotic members (Bcl-2,
Bcl-xL and Mcl-1), pro-apoptotic members (BAK, BAX), and
effector proteins (BID, BIM, PUMA and NOXA-B). The
anti-apoptotic proteins contain a hydrophobic groove into
which an a-helical BH3 domain of effector or pro-apoptotic
proteins can bind. Although the exact mechanism by which
these proteins coordinate to determine cell fate remains
unclear,[32] in certain cancers, anti-apoptotic members are
overexpressed and sequester the activity of the pro-apoptotic
proteins, thus preventing apoptosis from taking place.
Building on our prior work[24,33] on oligobenzamide
foldamers,[34,35] we synthesized a library of N-alkylated helix
mimetics using a microwave-assisted solid-phase synthesis
method which affords compounds in about 4 hours and in
greater than 90% purity suitable for screening (representa-
tive compounds shown in Figure 1; see Schemes S1 and S2
and Table S1 in the Supporting Information).[36,37] In this
instance, the library of 77 members was purified further by
HPLC where appropriate. We initially selected p53/hDM2 as
a model target. The library composition was tailored to reflect
the key binding residues on the p53 helix, therefore members
To test the behavior of aromatic oligoamides in cells,
a high-content imaging screen was developed (Figure 2a; see
Figure S2 in the Supporting Information for additional
images). Initially U2OS osteosarcoma cells were treated
with 20 and 10 mm helix mimetics in low-serum media. The
former concentration allowed the DMSO concentration to be
kept at less than 0.2% starting from 10 mm stocks whilst the
second concentration was used to confirm statistical signifi-
cance. Four endpoints were assessed 48 hours after addition
of the mimetic with hits defined as described in the Support-
ing Information. Firstly, cell number was measured by nuclear
counting. Secondly an antibody against caspase 3 (which is
common to both the extrinsic and intrinsic apoptotic path-
ways),[38] was used to identify cells which may be apoptotic.
The third endpoint assessed was autophagy, a self-digestion
process that cells in stressed conditions undergo as part of
their normal life cycle,[39] but which has been linked to
apoptosis.[40] Although autophagy has also been implicated in
cell survival, an apoptosis-inducing drug has been previously
shown to increase autophagosome numbers in osteosarcoma
cells,[41] whilst p53 has been shown to activate autophagy so as
to increase the efficiency of apoptosis.[42] To probe for cells
undergoing autophagy, cells were stained with the anti-LC3B
antibody, which is known to bind to autophagosomal mem-
branes.[43] Finally, the arrangement of actin filaments in cells
treated with the helix mimetics was investigated using
2
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a fluorescently labelled phalloidin.[44] To promote cell motility
and invasion, cancer cells alter the cytoskeleton and a number
of reports have indicated a relationship between this modi-
fication and the activity of p53.[44] To provide a qualitative
interpretation of the data, the data are shown in Figure 2b as
a heat map (a summary of all the data from the apoptosis
assay for the full library of compounds can be found in
Table S2 in the Supporting Information). A range of different
activities were found across the library with some mimetics
having no effect in any of the four categories, for example, 9,
17, 23, 32, and 62. Some compounds provided a hit with one or
two of the markers, for example 8, whilst a small number,
including the compounds 64 and 67, proved to be effective in
three or four areas.
An MTT cell toxicity assay using U2OS and SJSA-
1 osteosarcoma cells was also used to identify compounds
leading to an increase in cell death. The SJSA-1 cell line has
been frequently used in studies on p53/hDM2 inhibitors
because it overexpresses hDM2.[45] Cells were treated with
a compound and incubated for 18 hours, after which time cell
viability was measured using a Cell Titre 96ꢀ assay (Promega;
Figure 2c). There is generally good agreement between the
two cell lines for the entire library (see Figure S3 in the
Supporting Information). A number of compounds, including
23, 64, and 67, show a similar level of toxicity to that of Nutlin-
3. The MTT result for 23 is inconsistent with the HCS,
however MTT reports on mitochondrial activity (biomass)
rather than cell number. The reduced viability observed with
23 may therefore arise through its interaction with targets
other than hDM2. Several compounds with little response in
the HCS demonstrated little toxicity, for example, the
compounds 9, 17, 32, and 62. The dimers 74–77 had little
effect on cell population and fits well with the notion that
these compounds act as poor p53 helix mimetics.
To confirm that the observed effects were due to the
mimetics entering the cell, we labelled five mimetics with
a fluorescein tag using previously reported click chemistry
(see the Supporting Information for syntheses).[36] U2OS cells
were treated with FITC-labelled trimers (Figure 2d), and co-
stained with DAPI and Toto-3 iodide. The modified mimetics
co-localize with the cytoplasmic stain. An alternate method
using biotinylated trimers confirmed this result and is
described in the Supporting Information (Figure S4). Both
sets of compounds retained hDM2 affinity, although they
exhibited diminished activity in the HCS (see Figures S22 and
S23 and Table S2a–d in the Supporting Information).
To test for inhibition of the p53/hDM2 interaction,
selected helix mimetics were screened in a fluorescence
anisotropy (FA) competition assay. Six of the proteomimetics
[8, 23 (see Figure S5 in the Supporting Information), 31, 48,
64, and 67] displace p53 from hDM2 with low mm IC50 values
(Figure 3a). Despite containing a range of different side
chains, all six mimetics possess a large aromatic group on the
central residue, which is consistent with effective mimicry of
the central tryptophan residue on the p53 helix. Four
compounds (9, 17, 32, and 62) showed no inhibition of the
p53/hDM2 interaction up to a concentration of 100 mm
(Figure 3b). These compounds have either a small or polar
side chain in the central position which is likely to abrogate
Figure 3. Biophysical analyses of helix mimetics. Dose-response curves
for the inhibition of the p53/hDM2 interaction measured by fluores-
cence anisotropy for a) 8, 31, 48, 64, and 67 and b) 9, 17, 32, and 62.
1
c) H-15N HSQC spectra of 15N-labelled hDM2. Spectra was recorded
in the absence (in black) and the presence (in red) of 64 (crosspeaks
that move or change in volume are mapped onto the surface of hDM2
and shown in blue).
inhibition. The dimers were generally poor inhibitors of the
p53/hDM2 interaction (see Figure S6 in the Supporting
Information). The compound 64 was tested for its ability to
bind to 15N-labelled hDM2 (Figure 3c). 1H-15N HSQC spectra
were recorded in the absence and presence of compound (see
Figures S7 and S8 in the Supporting Information). Upon
addition of 64, crosspeaks in the 1H-15N-HSQC spectrum
shifted and decreased in volume, thus indicating a direct
interaction with hDM2. The chemical shifts were mapped
onto the structure of hDM2 using a published NMR assign-
ment.[46] The compound 64 binds to hDM2, shown by
significant movement of the crosspeaks for Phe55 and
His73, at either side of the p53 binding site. These shifts are
similar in nature to those observed upon titration of the p53
peptide against 15N-labelled hDM2,[33] thus further suggesting
that the compounds act as effective p53 mimetics. Similar
results were obtained for 67 (see Figures S9–S11 and Sup-
porting Information).
Collectively, the HCS, MTT, and FA experiments identi-
fied library members most likely to act as effective mimics of
p53 function. Five promising trimers were taken forward for
further investigation along with four negative controls
identified above. These five potent compounds were sub-
jected to the HCS in U2OS and SJSA-1 cells at a range of
different concentrations to determine dose-response curves
for the mimetics and establish an active concentration
(Figure 4a and see Figure S12a in the Supporting Informa-
tion). The mimetics 64 and 67 proved to be the most effective,
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Figure 4. Cellular response to mimetics. a) Dose-response curves
based on analysis of cell number by nuclear staining from U2OS cells
treated with different concentrations of mimetics. b) U2OS and SJSA-
1 cell lines were incubated with mimetics (50 mm) or Nutlin-3 (10 mm)
for 4 h and lysates analyzed by western blotting for p21 and GAPDH.
c) U2OS and SJSA-1 cells were treated with biotinylated mimetics
(10 mm) for 4 h and cell lysates were subjected to Streptavidin pull-
down followed by analysis by western blotting for hDM2. d) Same as
for (a) except for the use of Saos-2 cells.
Figure 5. Bcl-2 family binding properties of mimetics. Dose-response
curves of the inhibition of the a) Mcl-1/NOXA-B and b) Bcl-xL/BAK
interactions measured with fluorescence anisotropy. c) 1H-15N HSQC
spectra of 15N-labelled Mcl-1. Spectra recorded in the absence (in
black) and presence (in red) of 64. Crosspeaks that move or change in
volume are mapped onto the surface of Mcl-1 and shown in blue.
d) U2OS and Saos-2 cells were treated with of biotinylated mimetics
(10 mm) for 4 h and cell lysates were subjected to Streptavidin pull-
down followed by analysis by western blotting for Mcl-1 or Bcl-xL
(GAPDH or actin used as loading controls).
presenting IC50 values of around 4 mm in both cell lines
(Nutlin-3 gives a value of 1.4 mm; see Figure S12b, the data of
which compares well with the literature[47] and a recently
described compound which has entered clinical develop-
ment).[47] We then sought to directly determine whether the
helix mimetics were able to act on the p53/hDM2 interaction
in cells. Western-blot analysis was carried out on both U2OS
and SJSA-1 cells. In both cases the activity of p53 as
a transcription factor was demonstrated by increased expres-
sion of its downstream target p21 (Figure 4b; see Figure S13
in the Supporting Information). Trimers shown to inhibit the
interaction in the FA assay and promote cell death through an
apoptotic mechanism induce p21 expression, whereas dimers
were ineffective (see Figure S14 in the Supporting Informa-
tion). Biotinylated trimers were then used in a streptavidin
pull-down experiment. In both U2OS and SJSA-1 cells, the
mimetics with good p53/hDM2 potency pull-down hDM2
(Figure 4c; full uncropped gel images see Figure S15 in the
Supporting Information). To assess the selectivity of the helix
mimetics, we performed an additional screen in Saos-2 cells
which are p53-null; compounds selective for p53/hDM2
should be inactive in this cell line. The mimetics were found
to have activity in the Saos-2 cell line (Figure 4d) comparable
to that observed in the U2OS and SJSA-1 cell lines. This
activity indicated that these compounds may act on additional
targets in the apoptosis pathway.
Expression of p53 in Saos-2 cells has been shown to result
in potent induction of apoptosis, independent of its function
as a transcription factor,[48,49] whilst several studies have
shown p53 is capable of binding to Bcl-2 family members.[50]
The subfamily of trimers was therefore tested in FA
competition assays against Bcl-2 family interactions: Mcl-1/
NOXA-B and Bcl-xL/BAK (Figure 5a,b). The compounds 64
and 67 showed micromolar inhibition of the Mcl-1/NOXA-B
interaction, whereas 8, 31, and 48 were weak inhibitors.
Significantly, none of the mimetics were able to disrupt the
Bcl-xL/BAK interaction despite the similarity in the BH3
binding clefts of Mcl-1 and Bcl-xL. The compound 64 (Fig-
1
ure 5c) was tested for binding to 15N-labelled Mcl-1. H-15N
4
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[3] T. Berg, Angew. Chem. Int. Ed. 2003, 42, 2462; Angew. Chem.
HSQC spectra were recorded in the absence and presence of
compound (see Figure S16 and S17 in the Supporting
Information). Upon addition of 64, crosspeaks in the 1H-
15N-HSQC spectrum both shifted and decreased in volume,
thus indicating direct interaction between the compound and
Mcl-1. These chemical shifts were mapped onto the structure
of Mcl-1 using a recently published NMR assignment.[51] The
analysis suggests 64 binds in the BH3 binding cleft of Mcl-1, as
shown by significant movement of the crosspeaks for Phe270
and Arg263 along peptide binding site. Similar results were
obtained for 67 (see Figure S18–20 in the Supporting Infor-
mation). Finally we probed the ability of the trimers to bind
Mcl-1 and Bcl-xL in cells using the biotintylated trimers and
streptavidin pull-down on cell lysate from U2OS and Saos-2
cell lines. In all cases biotinylated trimers were capable of
pulling down Mcl-1 from cell lysate (the pull-down bands for
biotin/64 and biotin/67 were both more intense than the other
compounds reflecting their greater potency). We observe no
evidence of Bcl-xL pull-down, thus indicating that the
selectivity observed in the biophysical assay is replicated in
the cellular environment. Full, un-cropped gel images are
available in the Supporting Information (Figure S21)
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In conclusion, we have described the design, synthesis, and
testing of a library of N-alkylated helix mimetics. Whilst some
correlation between biophysical and cellular potency is
evident, the results reveal the interplay between the two is
complex, and emphasize the need to perform multiple
analyses on libraries of helix mimetics in order to a) identify
(and ultimately select for) desirable structure function
behavior and b) to understand strengths and areas for further
development (e.g. off target effects) of a-helix mimetics as
PPI inhibitors. For instance, 31 is a reasonable inhibitor of the
p53/hDM2 interaction in the FA screen, but does not score
highly for induction of apoptosis or cell viability. Similarly, 23
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and selective manner on Mcl-1/NOXA-B over Bcl-xL/BH3 in
both biophysical assays and a cellular context—a selectivity
profile which is challenging to achieve. Thus the results
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binding selectivity in cells, whilst dual inhibition of hDM2
and Mcl-1 may also represent a novel approach for elabo-
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Received: November 6, 2014
Revised: December 15, 2014
Published online: && &&, &&&&
Keywords: apoptosis · foldamers · helical structures ·
.
peptidomimetics · protein–protein interactions
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
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Angewandte
Chemie
Communications
PPI Inhibitors
A. Barnard, K. Long, H. L. Martin,
J. A. Miles, T. A. Edwards,
D. C. Tomlinson, A. Macdonald,*
A. J. Wilson*
&&&& — &&&&
Selective and Potent Proteomimetic
Inhibitors of Intracellular Protein–Protein
Interactions
Picky mimics: Inhibition of protein–pro-
tein interactions represents a major
challenge in chemical biology and drug
discovery. Using a library of N-alkylated
aromatic oligoamides it is demonstrated
that helix mimetics, which reproduce
their biophysical binding selectivity in
a cellular context, can be identified.
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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These are not the final page numbers!