Peptide-Directed Binding for the Discovery of Modulators of α-Helix-Mediated Protein–Protein Interactions: Proof-of-Concept Studies with the Apoptosis Regulator Mcl-1

Article abstract of DOI:10.1002/anie.201705008

Targeting PPIs with small molecules can be challenging owing to large, hydrophobic binding surfaces. Herein, we describe a strategy that exploits selective α-helical PPIs, transferring these characteristics to small molecules. The proof of concept is demonstrated with the apoptosis regulator Mcl-1, commonly exploited by cancers to avoid cell death. Peptide-directed binding uses few synthetic transformations, requires the production of a small number of compounds, and generates a high percentage of hits. In this example, about 50 % of the small molecules prepared showed an IC₅₀ value of less than 100 μm, and approximately 25 % had IC₅₀ values below 1 μm to Mcl-1. Compounds show selectivity for Mcl-1 over other anti-apoptotic proteins, possess cytotoxicity to cancer cell lines, and induce hallmarks of apoptosis. This approach represents a novel and economic process for the rapid discovery of new α-helical PPI modulators.

Full text of DOI:10.1002/anie.201705008

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Peptide directed binding; a novel approach for the discovery of
modulators of alpha-helix mediated protein-protein interactions
demonstrated with apoptosis regulating Mcl-1
Authors: Andrew Michael Beekman, Maria Anne O'Connell, and Lesley
Ann Howell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705008
Angew. Chem. 10.1002/ange.201705008
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10.1002/anie.201705008
Angewandte Chemie International Edition
Peptide directed binding; a novel approach for the discovery of modulators of alpha-
helix mediated protein-protein interactions demonstrated with apoptosis regulating
Mcl-1
Andrew Michael Beekman, Maria Anne O’Connell and Lesley Ann Howell*
Abstract: Targeting PPIs with small molecules can be challenging due 14-3-3/Tau PPI of Ottmann et al.[10] The work also exploits the
to large, hydrophobic binding surfaces. Here, we describe a strategy advantages of altering peptidic binders, demonstrated by
that exploits selective alpha-helical PPIs, transferring these Gellman[11]
,
Fairlie[12] and Wilson.[13] The technique
characteristics to small molecules. The proof-of-concept is demonstrated here improves these strategies, substituting up
exemplified with the apoptosis regulator Mcl-1, commonly exploited to ten amino acids with one small molecule fragment, and
by cancers to avoid cell death. Peptide directed binding uses few rapidly supplanting the entire peptide chain with a small
synthetic transformations, requires the production of a small number molecule modulator.
of compounds and generates a high percentage of hits. In this In this proof-of-concept study the alpha-helical PPI of Mcl-1 and
example ~50% of the small molecules prepared showed an IC50 less Noxa, members of the apoptosis regulating Bcl-2 family of
than 100 µM and ~25% had IC50 values less than 1 µM to Mcl-1. proteins, was employed as an example.[14] The anti-apoptotic
Compounds show selectivity for Mcl-1 over other anti-apoptotic proteins (e.g Bcl-2, Bcl-xL and Mcl-1) are often overexpressed in
proteins, possess cytotoxicity to cancer cell lines, and induce cancer contributing to the development of the tumour and
hallmarks of apoptosis. This approach represents a novel and resistance to current therapies.[15]
economic process for the rapid discovery of new alpha-helical PPI Noxa displays high selectivity towards Mcl-1.[16] The NoxaB
modulators.
(NoxaB-(75-93)-
C
75A
)
peptide
is 19 amino
acids
(
AAQLRRIGDKVNLRQKLLN) from the BH3 binding region of
Protein-protein interactions (PPIs) regulate many processes in
life, both in healthy and disease states[1] and almost two-thirds
of protein-protein interfaces have alpha-helical binding
motifs.[2] However, targeting PPIs can be difficult due to their
large hydrophobic binding surfaces.[3] There are currently three
commonly employed approaches to develop modulators of
PPIs;[4] fragment screening,[5] computational screening and drug
Noxa, and has been shown to bind tightly as an alpha-helix in
the binding groove of Mcl-1.[17] NoxaB was divided into amino
acids 75-84 (AAQLRRIGD) and 85-93 (KVNLRQKLLN), each
possessing two key binding residues (L78, I81, V85 and Q )
89[17]
and reactive terminals were attached (Scheme 1 A). These
peptides possessing reactive terminals displayed no appreciable
binding affinity for Mcl-1 (IC50 values >100 µM in a previously
reported competitive fluorescence anisotropy (FA) assay[16c]) in-
line with results reported by Colman et al.[17] These reactive
terminals were then utilised to perform copper catalyzed azide-
alkyne cycloaddition (CuACC) reactions attaching small
molecule fragments (R1 and R2).[18] If binding affinity is restored
for these small molecule-peptide hybrids (AAQLRRIGD-R1 and
R2-KVNLRQKLLN), then the attached small molecule fragment
may represent a good emulator for that particular section of
NoxaB.
design,[6] and the exploration of peptides
and
peptidomimetics.[7] However, computational design and
fragment screening require large libraries of molecules and
extensive synthetic work, often resulting in non-selective
compounds.[3] Peptides are challenging drug leads because in
vivo their efficacy can be compromised due to a loss of
secondary structure, poor cellular uptake and susceptibility to
proteolysis.[8]
The work described here exploits the advantages of the above
approaches while limiting their weaknesses. This approach,
termed peptide directed binding, utilises the tight and selective
binding of alpha-helical peptides which govern PPIs as a
framework for the discovery of small molecules. Sections of the
natural peptide are employed to identify a small molecule
fragment which emulates the peptide.
Inspiration for this approach was taken from the REPLACE
strategy of McInnes et al.[9] and the chimeric inhibitors of the
[*] Dr A. M. Beekman, Prof. M. A. O’Connell, Dr L. A. Howell
School of Pharmacy, University of East Anglia, Norwich
Research Park, Norwich, Norfolk, NR4 7TJ, United Kingdom
Dr L. A. Howell
School of Biological and Chemical Sciences, Queen Mary
University of London, Mile End Road, E1 4NS, United
Kingdom
Email: L.Howell@qmul.ac.uk
Supporting Information Statement
Scheme 1. A: Conceptual diagram of peptide directed binding to target PPIs. B:
Extended NoxaB peptide fragment which demonstrated a restored affinity for
Mcl-1
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10.1002/anie.201705008
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As an initial control for this strategy, the two peptide fragments alkynes were exposed to the peptide on the resin in the
were clicked together to generate an extended NoxaB peptide presence of Cu(MeCN)4PF6 and DIPEA in DMF to achieve CuAAC.
which had an IC50 of 7.23 ± 0.88 µM, compared to 650 ± 130 nM The use of a N-coordinated Cu(I) source was found to be
for NoxaB, highlighting the suitability of the approach (Scheme advantageous, as other Cu(I) sources gave lower yields,
1, B).
presumably due to sequestration of the copper catalyst by
To economise peptide directed binding, covalent docking peptidic co-ordination. Cleavage from the resin and RP-HPLC
studies using the Schrodinger Suite were employed to assist in provided a library of thirty-five R2-KVNLRQKLLN hybrids. The
the identification of the small molecule fragments most likely to analogous methodology was applied to prepare twenty-five
mimic a section of the NoxaB peptide. The crystal structure AAQLRRIGD-R1 hybrids, via a propargylglycine-terminated
geometry of the NoxaB peptide with Mcl-1 (PDB Entry: 2NLA[17]
)
SPPS resin (Scheme 2).
was modified; virtually, amino acids 85-93 of NoxaB were The ability of these 60 hybrids to inhibit the interaction of Mcl-
removed and propargylglycine was attached to the C-terminus 1 and FITC-Noxa was examined in a FA assay, 23 compounds
of amino acids 75-84. Covalent docking studies were performed (30%) were identified as hits (defined as having an IC50 <100
on a catalogue of azides, modelling a Huisgen cycloaddition µM). Eight of the hits contained amino acids 75-84, and 13 hits
(detailed in SI). The results were scored and ranked,[19] and were derived from amino acids 85-93 (Table 1 and SI Table 1).
those highly ranked structures which were synthetically and The orthogonal nature of the reaction enabled the combination
economically viable were chosen for synthesis. Similarly, amino of the azide and alkyne small molecule fragments to generate a
acids 75-84 of the peptide were virtually removed and library of small molecule-triazoles which in theory have an
azidoacetic acid was attached to the N-terminus of amino acids increased likelihood of possessing characteristics that emulate
85-93.
the entire NoxaB peptide. The identified hybrid molecules
In this manner, we selected sixty hybrid compounds, to suggest 104 triazoles for preparation. We selected thirty-five for
synthesise and screen for the ability to disrupt the Mcl-1/Noxa synthesis and evaluated them in the FA assay (Scheme 2).
PPI. Using solid phase peptide synthesis (SPPS) amino acids 85- Nineteen (54%) of the triazole compounds showed an IC50 <100
93 (KVNLRQKLLN) of NoxaB were prepared and azidoacetic µM, and ten (27%) of the compounds displayed an IC50 <1 µM
acid was used to cap the peptide, (Scheme 2). Subsequently (Table 2 and SI Table 2).
Scheme 2. Synthesis of the small molecule-peptide hybrids via solid phase peptide synthesis, and subsequent small molecule peptide mimic synthesis. DIPEA,
diisopropylethylamine; DMF, dimethylformamide; TFA, trifluoroacetic acid; TIPS, triisopropylsilane
J. Name., 2013, 00, 1-3 | 2
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10.1002/anie.201705008
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Table 1. Binding inhibition IC50 of FITC-NoxaB to Mcl-1 of small molecule-peptide hybrid compounds.
FA IC50
(µM)
FA IC50
(µM)
Peptide
Small Molecule
Peptide
Small Molecule
AAQLRRIGD
KVNLRQKLLN
2
3
0.4 ± 0.3
9
0.1 ± 0.1
1.2 ± 9.5
0.7 ± 0.8
10
4
5
5.8 ± 4.6
<100*
11
12
3.5 ± 1.5
4.3 ± 0.9
6
<100*
13
8.2 ± 1.9
7
8
<100*
<100*
14
15
8.3 ± 3.6
<100*
*Hybrid compound demonstrated binding inhibition less than 100 µM and greater than 10 µM, accurate IC50 not determined. Fmoc, 9-fluorenylmethylcarbonyl. IC50 determined by
non-linear regression of at least three experiments. Errors are the transformed greater extreme of the standard error.
Table 2. Binding inhibition IC50 of FITC-NoxaB to Mcl-1 of small molecules and cell growth inhibition of representative compounds towards pancreatic cancer cells lines MiaPaCa-2,
BxPC-3, and AsPC-1.
MiaPaCa-2
(µM)
BxPC-3
(µM)
AsPC-1
(µM)
MiaPaCa-2
(µM)
BxPC-3
(µM)
AsPC-1
(µM)
FA IC50
(nM)
FA IC50
(nM)
Structure
Structure
1.85
2.81
33
± 8
1260
± 142
16
21
>100
>100
>100
>100
>100
>100
>100
± 3.17
± 0.40
102
± 14
1680
± 694
17
18
19
22
23
24
>100
>100
>100
>100
>100
15.19
± 0.75
29.82
± 4.15
19.52
± 1.35
25.21
± 2.66
10.54
± 1.53
186
± 20
1700
± 230
21.60
± 8.83
2.14
10.66
± 2.72
2.28
217
± 58
2210
± 877
>100
± 0.56
± 1.44
6.57
5.98
2.04
249
± 51
5500
± 3923
20
25
>100
>100
>100
± 3.92
± 1.14
± 0.62
Fmoc, 9-fluorenylmethylcarbony. IC50 determined by non-linear regression of at least three experiments. Errors are the transformed greater extreme of the standard error.
The nature of this approach allows for SAR to be drawn without Fmoc protecting group were considered, but as these
targeting specific modifications. The most potent compounds, compounds are primarily meant as chemical probes or potential
16 and 17 both possess a heptyl chain which provokes concerns leads further development may discover more effective
about non-specific hydrophobic events. However, not all alternatives. Indeed, the Fmoc group is somewhat reminiscent
compounds synthesised with the heptyl chain demonstrated of structural features present in Souers’ A-1210477[16d] and
activity (Table S2). Additionally, the Fmoc-propargylglycine Fesik’s 2-indole-acysulfonamides,[20] both highly potent and
moiety proved effective in several of the identified binders (18, selective Mcl-1 binders. Interestingly, the most potent small
20, 21, 23 and 25), but again was not a feature which was molecules were not the combination of the most potent small-
sufficient for binding on its own (Table S2). Concerns over the molecule peptide hybrids 2 and 9, as might be expected. The
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10.1002/anie.201705008
Angewandte Chemie International Edition
23]
combination of 2 and 9 demonstrated no appreciable ability to permeability.[16d,
Indeed, some of the more potent
inhibit the interaction of Mcl-1 and FITC-Noxa. Additionally, the compounds in regard to the FA assay showed no activity in the
small molecule fragment of 9 did not appear in any small cellular assays, such as compound 17 (IC50 = 102 ± 14 nM),
molecule which inhibited the Mcl-1/FITC-Noxa interaction. A suggesting an inability to cross the cell membrane. Additionally,
possible explanation for this phenomenon is that some small often subnanomolar binding affinities are required for small
molecule fragments, when bound to the peptide fragment, alter molecules to compete with high-affinity endogenous ligands.[24]
the helicity of the peptide, perhaps increasing the binding Compounds 18, 20, 21 and 23 were highlighted by this assay,
affinity of the peptide segment, or altering the binding site.[21] and selected for further examination.
Further studies on the power of small molecule peptide hybrids
as protein-protein interaction modulators are underway.
To examine if these compounds are impacting on the intrinsic
apoptosis pathway, as would be expected if they are binding to
It has been shown that NoxaB is a selective Mcl-1 binder.[17] The Mcl-1 in cells,[25] assays were performed which demonstrate
deployment of the NoxaB peptide as a scaffold for discovering induction of the apoptosis pathway. Compounds 18, 20, 21 and
new compounds was envisaged to also impart this selectivity 23 demonstrated an increase in caspase-3 activation in BxPC-3
into the new small molecule mimics. To examine this, we cells 4 hours after treatment, indicated by the cleavage of
employed two in vitro fluorescence anisotropy assays with Bcl- DEVD-pNa and subsequent increase in optical density at 405
2 and Bcl-xL, using a FITC tagged Bid peptide as our fluorescent nm.[26] 18 (25 µM, 0.053 ± 0.02), 20 (5 µM, 0.036 ± 0.009), 21 (5
marker.[22] Navitoclax (ABT-263) was employed as a positive µM, 0.046 ± 0.004) and 23 (25 µM, 0.139 ± 0.06), all displayed
control, and as an additional indication that the FA assay was an increase in optical density at 405 nm when compared to a
performing adequately.[16b] Excitingly, compounds 16-25 and an vehicle control (DMSO, 0.1%, 0.003 ± 0.005) in this assay (Figure
additional nine compounds (see SI) displayed no appreciable 2, A). Additionally, compounds 18, 20, 21 and 23 were shown to
binding to Bcl-xL or Bcl-2 in our FA assays (Figure 1, see SI), induce the externalization of phosphatidylserine on the cell
demonstrating a minimum 20 fold selectivity for Mcl-1. To surface,[26] as demonstrated by the binding of annexin-V-FLUOS
confirm this result was not an artefact, all compounds were re- to BxPC-3 cells resulting in green fluorescence (Figure 2, B).
examined in the Mcl-1, Bcl-2 and Bcl-xL assays twice more, with
the controls of NoxaB peptide and Navitoclax performing as
expected. These results also provide some relief about the
potential of non-specific hydrophobic events caused by the
heptyl chain or Fmoc group.
Figure 1. Representative titration of compound 18 (left) and 21 (right) on 5 nM
FITC-NoxaB peptide in the presence of 10 nM Mcl-1 protein (red) and 5 nM FITC-
Bid peptide in the presence of 30 nM Bcl-2 (yellow) and Bcl-xL (green),
demonstrating no appreciable binding to Bcl-2 and Bcl-xL.
Figure 2. Representative small molecules induce hallmarks of apoptosis.
A.Increase of caspase-3 activation in BxPC-3 cells 4 hours after treatment.
Compounds 16-25 were examined to determine if they
displayed activity towards pancreatic cancer cells which are
known to overexpress members of the Bcl-2 family, including
B.Externalization of phosphatidylserine, a hallmark of apoptosis, was evaluated by
exposing BxPC-3 cells to compounds 18 (25 µM), 20 (5 µM), 21 (5µM) and 23 (25
µM) for 4 hours followed by annexin-V-FLUOS. Images were taken at 10x
magnification with a GFP filter (top) and white light (bottom).
Mcl-1. An MTS assay was employed to examine the ability of the
Utilising the selective NoxaB peptide as a framework, a library
of novel Mcl-1 binders which demonstrate selectivity for Mcl-1,
has been prepared. Peptide directed binding provided a high
percentage of compounds with increased potency when
compared to traditional methods of high throughput screening,
by the application of a natural peptide framework and simple
synthetic manipulations. Recent literature demonstrates that
fragment based methods require the screening of
approximately 15,000 fragments by NMR and extensive
subsequent synthetic manipulations to generate a selective
potent Mcl-1 binder.[23] Additionally, high throughput screening
has been shown to have a hit rate of 0.2% in recent studies
targeting the Bcl-2 family and other prominent cancer PPIs.[27]
compounds to inhibit cell growth and affect metabolism, a
potential indicator of cell death. Several compounds, notably
18, 20, 21, 23 and 24, displayed activity towards the pancreatic
cancer cell lines BxPC-3, known to over express Mcl-1, and
MiaPaCa-2, which over-expresses both Mcl-1 and Bcl-2.
Interestingly, compound 19 only had activity against BxPC-3
cells. The pancreatic cancer cell line AsPC-1, which does not
over-express Mcl-1 was also evaluated (Table 2). Two
compounds, 18 and 21, were ineffective against AsPC-1 cells at
the concentrations evaluated in our assay, which may suggest
they are acting through the inhibition of Mcl-1. The difference
in magnitude of activity in cells compared to the in vitro FA assay
is a commonly observed phenomenon, largely due to cell
J. Name., 2013, 00, 1-3 | 4
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10.1002/anie.201705008
Angewandte Chemie International Edition
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Acknowledgements
This work was funded by the EPSRC (EP/M006379/1) to LAH.
Mcl-1 expression plasmid was generously provided by Prof. R.
Wang. We acknowledge the EPSRC UK National Mass
Spectrometry Facility at Swansea University. We would like to
thank M. Searcey, J. Angulo and P. McCormick for their helpful
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