Orthogonal functionalisation of α-helix mimetics

Article abstract of DOI:10.1039/c4ob00915k

α-Helix mediated protein-protein interactions are of major therapeutic importance. As such, the design of inhibitors of this class of interaction is of significant interest. We present methodology to modify N-alkylated aromatic oligoamide α-helix mimetics using 'click' chemistry. The effect is shown to modulate the binding properties of a series of selective p53/hDM2 inhibitors. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00915k

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Orthogonal functionalisation of α-helix mimetics†
Anna Barnard,a,b Kérya Long,a,b David J. Yeo,a,b Jennifer A. Miles,a,b
Valeria Azzarito,a,b George M. Burslem,a,b Panchami Prabhakaran,a,b
Thomas A. Edwardsb,c and Andrew J. Wilson*a,b
Cite this: Org. Biomol. Chem., 2014,
12, 6794
Received 4th May 2014,
Accepted 7th July 2014
DOI: 10.1039/c4ob00915k
www.rsc.org/obc
α-Helix mediated protein–protein interactions are of major thera-
peutic importance. As such, the design of inhibitors of this class of
interaction is of significant interest. We present methodology to
modify N-alkylated aromatic oligoamide α-helix mimetics using
‘click’ chemistry. The effect is shown to modulate the binding pro-
perties of a series of selective p53/hDM2 inhibitors.
Inhibition of protein–protein interactions (PPIs) represents a
considerable challenge in modern chemical biology.1,2 PPIs
mediate multiple critical biological processes and are pre-
valent in disease pathways.3 When compared to traditional
enzyme-substrate drug targets, PPIs are more complex due to
the wide range of topographies and larger surface areas
(>1000 Å2) present at the binding interfaces.4 However, it is
often possible to identify ‘hot-spot’ residues which account for
the majority of the binding energy.5 α-Helices are the most
abundant secondary structural motif and frequently mediate
PPIs.6 Several classes of inhibitors have thus been designed7
against this type of PPI, including β-peptides,8 mixed α/β-pep-
tides9,10 and constrained peptides.11–17 Proteomimetics mimic
the spatial arrangement of key binding residues on an α-helix
(Fig. 1a).18–30 Within this family, aromatic oligoamides have
emerged as a promising class of helix-mimetic.31–33 They have
been shown to be synthetically accessible34 including by solid-
phase synthesis35,36 and their side chains are projected in a
manner which effectively recapitulates the spatial orientation
of ‘hot-spot’ residues on a helical scaffold. To utilise helix
mimetics and tailor their properties for cellular studies (e.g.
cellular imaging, optimising cell uptake etc.) and/or appli-
cations in hit identification (e.g. immobilisation of libraries on
surfaces), it is necessary to functionalise the ligands in an
Fig. 1 Helix mimetics as inhibitors of PPIs: (a) schematic illustrating
proteomimetic concept, (b) p53/hDM2 interaction (PDB ID: 1YCR),
(c) N- and (d) O-alkylated aromatic oligoamides, (e) schematic illustrat-
ing concept for orthogonal functionalisation.
orthogonal manner and with minimal perturbation of the
helix mimicking epitope (Fig. 1e). Herein we describe a strat-
egy to achieve such a goal, through functionalisation of the
non-helix mimicking face of N-alkylated aromatic oligoamides
(Fig. 1c), by ‘click’ chemistry. Our results illustrate that, the
selectivity profile of the mimetic is retained as a consequence
of the modifications. The p53/hDM2 interaction represents a
PPI under intense current investigation37 (Fig. 1b); it exploits
residues Phe19, Trp23 and Leu26 on the p53 transactivation
domain bound in a helical conformation to hDM2 (PDB ID:
1YCR).38 It thus represents an ideal model target with which
to develop helix mimetics. In recent years, our group has tar-
geted the p53/hDM2 interaction using both N- and O-alkylated
aromatic oligoamide helix-mimetics (Fig. 1c and d).31–33 To
improve the solubility of the O-alkylated series an ethylene
glycol chain was introduced onto the non-binding face,39
which improved aqueous solubility without abrogating
aSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK
bAstbury Centre for Structural and Molecular Biology, University of Leeds,
Woodhouse Lane, Leeds, LS2 9JT, UK. E-mail: A.J.Wilson@leeds.ac.uk
cSchool of Molecular and Cellular Biology, University of Leeds, Woodhouse Lane,
Leeds, LS2 9JT, UK
†Electronic supplementary information (ESI) available: Additional binding data
and fluorescence characterisation. Experimental procedures and characteris-
ation of all new compounds. See DOI: 10.1039/c4ob00915k
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Scheme 1 Synthesis of alkyne-functionalised N- and O-alkylated Fmoc monomer, 10.
binding affinity. The synthesis of this modified O-alkylated binding residues of the p53 helix. A solid-phase synthetic strat-
helix mimetic proved challenging and informed our strategy in egy was employed (Scheme 2).35 Trimer 1 (a regioisomer of our
the current work; we decided to combine features of both previously described most potent hit from this series),32 was
scaffolds; the N-alkylated scaffold to display the binding synthesised using unfunctionalised N-alkylated monomers
groups and the O-alkyl function to install a ‘click’ chemistry whereas trimers 2–5 utilised the alkyne monomer 10 as the
functional handle. The copper(I) catalysed cyclisation between central residue. The ‘click’ reaction could then be performed
an alkyne and an azide has received considerable attention in on resin. A variety of commercially available azides were
recent years.40 The modified Huisgen 1,3-cyclisation41 develo- chosen; a protected acid, methyl 2-azidoacetate, and an ethyl-
ped by the groups of Sharpless and Meldal42,43 is high yielding ene glycol, O-(2-azidoethyl)-O′-methyl-triethylene glycol in
and tolerant of diverse substrates and conditions.44
order to synthesise a trimer related to the ‘wet-edge’ foldamer
Introducing a residue amenable to ‘click’ chemistry necessi- described previously.39 Two novel azides were also synthesised
tated the design and synthesis of a novel monomer unit (Scheme S1†) to append the trimers with coumarin and fluor-
(Scheme 1). Methyl 3-hydroxy-4-nitrobenzoate, 6, was alkylated escein fluorophores. After a copper(I) catalysed ‘click’ reac-
with propargyl bromide installing the alkyne functionality. tion42,43 the trimers were cleaved from the resin with TFA and
Reduction with tin(II) chloride resulted in synthesis of the the desired proteomimetics were isolated in excellent yield and
desired amine without any effect on the alkyne and, after sapo- purity (see ESI† for characterisation).
nification of the ester this could be N-alkylated. Subsequent
Fmoc protection afforded monomer, 10.
In order to ascertain if the modifications were likely to have
any effect upon the ability of the oligomers to mimic the
The ‘click-able’ monomer could be readily incorporated p53 helix, molecular modeling was performed (see ESI† for
into a trimer structure with side chains designed to mimic the details). The unfunctionalised analogue was compared with
Scheme 2 Solid-phase synthesis of triazole-functionalised trimers 1–5.
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Table 1 IC50 values for trimers 1–4 obtained from fluorescence
anisotropy assays against the p53/hDM2, Mcl-1/NOXA-B and Bcl-xL/BAK
protein–protein interactions
Trimer
IC50 p53/hDM2
IC50 Mcl-1/NOXA-B
IC50 Bcl-xL/BAK
1
2
3
4
5
12.3 0.4 μM
40 5 μM
>70 μM
>70 μM
>70 μM
>70 μM
13 4 μMa
No inhibition
No inhibition
No inhibition
No inhibition
No binding
42 6 μM
20.3 0.3 μM
20 6 μMa
a Kd obtained through direct titration.
peptide is initially bound to hDM2 and displaced by increasing
concentrations of inhibitor leading to a measurable decrease
in anisotropy from which IC50 values can be determined,
Table 1.
The functionalised helix mimetics 2 and 3 inhibit the
p53/hDM2 interaction but the introduction of an additional
functional group onto the non-binding face of the helix mimetic
leads to a decrease in inhibitory potency. Several hypotheses
for this change are immediately obvious (a) although the func-
tionalised helix mimetic retains the ability to mimic the
helical pharmacophore (Fig. 2), the addition of the triazole
ring may introduce a steric clash between the mimetic and
helix binding cleft on hDM2 (Fig. 4a) or (b) the introduction of
the triazole ring in some way changes the conformational pro-
perties of the helix mimetic. Computational docking experiments
Fig. 2 Overlay of (a) unfunctionalised trimer 1 (RMSD = 0.983 Å),
(b) acid functionalised trimers 2 (RMSD = 0.772 Å) and (c) ethylene glycol-
functionalised trimer 3 (RMSD = 0.753 Å) with p53. Overlaid residues are
shown in CPK format. p53 residues are in dark colours and helix mimetic
residues are in light colours (side and top views are given).
the scaffolds functionalised with a carboxylate and an ethylene
glycol (Fig. 2a–c). The entire set of structures within 1.5 kJ
mol−1 of the minimum energy conformation were superim-
posed onto the p53 helix with the α-CH2 on the trimer repre-
senting the α-carbon on the peptide. A root mean square
deviation (RMSD) was calculated based on this degree of
overlap. The combinations with the best overlap (lowest
RMSD) are shown in Fig. 2. The modeling data show that by
introducing functionality onto the non-binding face the side
chain overlap is not significantly affected and these structures
are still capable of effective mimicry of the p53 helix.
The ability of the trimers to inhibit the p53/hDM2 inter-
action was then investigated using a fluorescence anisotropy
competition assay (Fig. 3a). Briefly, a fluorescein-labelled p53
Fig. 4 Effects of modification on helix mimicry (a) schematic illustrating
potential steric clash of functional handle with helix binding cleft.
(b) Dihedral angle analysis showing relative energies at different degrees
of rotation about (c) the central N-aryl bond (d) steric clash in the func-
tionalised trimers, between carbonyl and side chain oxygen atoms
(shown in CPK format).
Fig. 3 Fluorescence anisotropy binding assays in phosphate buffer
pH 7.5, 200 mM NaCl, 0.02 mg ml−1 BSA. (a) p53/hDM2 competition
assay hDM2 is fixed as 154 nM and FITC-p53 is at a concentration of
54.5 nM. (b) Direct binding assay of trimer 5 binding to hDM2 or Mcl-1.
Compound 5 is fixed at 50 nM.
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were attempted in order to investigate the first hypothesis; tested in the fluorescence anisotropy competition assays as
these suggested that the additional functional group does described above (Fig. S7 and S8†). The FITC-labelled trimer 5,
not introduce a steric clash with the p53 binding cleft on however, showed good fluorescence intensity (Fig. S16†).
hDM2 (Fig. S1†). In order to test the second hypothesis, we Direct binding experiments were performed using both fluo-
performed dihedral analysis on the oligomers (Fig. 4b and c). rescence anisotropy (Fig. 3b) and microscale thermophoresis52
The analysis shows that in the case of functionalised trimers 2 (Fig. S12†). From the direct binding anisotropy experiment Kd
and 3 there are additional energetic barriers to rotation about was found to be 20 6 μM whereas using thermophoresis it
the central N-aryl bond. These higher energy conformations was found to be 11 2 μM. The same fluorescence anisotropy
result from a steric clash between the O-alkyl group and carbo- experiment was then carried out for Mcl-1 and Bcl-xL. The Kd
nyl group on the adjacent aromatic ring (Fig. 4d) and may for binding Mcl-1 was found to be 13
4 μM whereas no
diminish the ability of the helix mimetic to adopt a confor- binding was observed for Bcl-xL (Fig. S11†). The absence of
mation in which the α-helix mimicking side chains are aligned selectivity between hDM2 and Mcl-1 in this instance may arise
on one face. An analogous control analysis with all-methyl side as a consequence of non-specific interactions imparted by the
chains was also performed (Fig. S2†) and the same steric clash fluorescein group – we note that Kd is lower for 5 than the
was observed. This indicates that the observed effect is not due IC50’s observed for both 2 and 3 which would support this
to the identity of the individual binding groups and is a hypothesis.
general feature of this scaffold. The dihedral analysis thus may
be considered to contradict the results obtained by molecular
modelling however it should be noted that a perfect alignment
of side-chains may not be a requirement for effective α-helix
Conclusions
mimicry31 and, that both modelling analyses are not In conclusion, we have described the development of versatile
sufficiently high resolution to make meaningful comparative methodology for orthogonal functionalisation of N-alkylated
interpretation of energy differences e.g. between high/low aromatic oligoamide helix mimetics with a wide range of func-
energy conformers/rotamers.
tional groups by exploiting alkyne–azide ‘click’ chemistry. The
The selectivity of the trimer family for the p53/hDM2 inter- modifications to the non-binding face of the helix mimetic
action was also investigated by screening the same compounds facilitate a range of additional experiments to be performed
against two additional α-helix mediated PPIs; Mcl-1/NOXA-B e.g. direct binding to target proteins to be assessed (via fluo-
and Bcl-xL/BAK, both of which are involved in the apoptosis rescence). Although functionalisation of the non-binding face
pathway.45,46 The IC50 values are presented in Table 1. The resulted in a loss of potency for trimers 2 and 3 in comparison
data shows some interesting trends in binding selectivity. The to unfunctionalised trimer 1, the compounds were still
unfunctionalised scaffold 1 shows some selectivity for hDM2 p53/hDM2 inhibitors and demonstrated selectivity towards the
over Mcl-1 and exhibits no inhibition of the Bcl-xL/BAK inter- p53/hDM2 interaction over Mcl-1/NOXA-B and Bcl-xL/BAK; thus
action (Fig. S7 and S8†). We were unable to fit the data to a the binding specificity can be reproduced exactly with only a
reliable IC50 value for the Mcl-1/NOXA-B competition exper- minor loss of inhibitory potency. Exploiting this methodology
iment as the lower limit for the anisotropy is not reached for to add groups that promote cell-uptake, facilitate detection in
these compounds. However, it is clear that the inhibition of cells and make favourable interactions with the solvent
Mcl-1/NOXA-B is weaker than that observed for the p53/hDM2 exposed protein surface to improve potency will be the focus of
interaction for all three compounds. The moderate inhibition future studies by our group.
of Mcl-1/NOXA-B is in some respects unsurprising given that
p53 itself has been shown to interact with Mcl-1.47 Further-
more, we note a recent publication from the Abbot group
where a fragment screen and subsequent crystal structure
Acknowledgements
studies illustrated that naphthyl groups are capable of This work was supported by the European Research Council
opening-up a pocket in the Mcl-1 helix binding groove.48 The [ERC-StG-240324] and Yorkshire Cancer Research (L348).
preference for Mcl-1 over Bcl-xL for these trimers is also note- Dr Alice Bartlett is thanked for protein preparation.
worthy given that several Bcl-2 family inhibitors target Bcl-xL
or Bcl-249,50 and that selective Mcl-1 inhibitors are rarer.51
Most significantly, when comparing 1 with 2 and 3 the result
illustrates that the selectivity profile of the helix mimetics can
Notes and references
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The fluorescently tagged trimers 4 and 5 were designed for
use in direct binding experiments. We observed that upon
‘click’ reaction, the fluorescence of the coumarin group in
trimer 4 was quenched (Fig. S14†). This trimer was therefore
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