Oligobenzamide proteomimetic inhibitors of the p53-hDM2 protein-protein interaction

Article abstract of DOI:10.1039/b908207g

Oligobenzamide inhibitors of the p53-hDM2 protein-protein interaction are described. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b908207g

Chemical Communications
www.rsc.org/chemcomm
Number 34 | 14 September 2009 | Pages 5045–5184
ISSN 1359-7345
COMMUNICATION
Jeffrey P. Plante, Thomas Burnley, Barbora Malkova, Michael E. Webb,
Stuart L. Warriner, Thomas A. Edwards and Andrew J. Wilson
Oligobenzamide proteomimetic inhibitors of the p53-hDM2 protein–protein
interaction

COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Oligobenzamide proteomimetic inhibitors of the p53–hDM2
protein–protein interactionw
Jeffrey P. Plante,ab Thomas Burnley,b Barbora Malkova,b Michael E. Webb,ab
Stuart L. Warriner,ab Thomas A. Edwards*b and Andrew J. Wilson*ab
Received (in Cambridge, UK) 24th April 2009, Accepted 10th June 2009
First published as an Advance Article on the web 24th June 2009
DOI: 10.1039/b908207g
Oligobenzamide inhibitors of the p53–hDM2 protein–protein
Trp23 and Leu26 from p53 binding in a helical conformation
to a hydrophobic cleft on hDM2 (Fig. 1).34
interaction are described.
Our design was driven by two criteria: (i) the scaffold should
maintain sufficient flexibility so as to optimise its conforma-
tion for maximum binding affinity and (ii) mimetic synthesis
should be amenable to library generation. The scaffold should
also position side chains to mimic the key residues at i, i + 4
and i + 7 of the p53 helix (Fig. 2a). Shown in Fig. 2b is the
minimum energy conformation of one such trimeric benzamide
as identified by a Monte Carlo search in Macromodel using the
MMFFs force field (see ESIw for details). Pleasingly, the
O-alkyl substituents mimic the spatial orientation of the i,
i + 4 and i + 7 residues of the p53 a-helix reasonably well.
Shown in Fig. 2c is mimetic 1aec superimposed upon the p53
a-helix—the RMSD = (0.1245 A) indicates a good geo-
metrical match between the oxygen of the scaffold and the
a-carbons of the helix. For this amino-terminated tri-
benzamide, the O-alkyl substituents all reside on the same
face—in contrast to the results obtained when a nitro group is
at the N-terminus of the tribenzamide as described earlier.25
However, conformational analysis in solution reveals these
compounds also adopt an extended conformation with intra-
molecular hydrogen-bonding fixing rotation about the Ar–NH
bond and free rotation about the CO–Ar bond (see ESIw).
We prepared a series of compounds (Fig. 3) and initially
tested compound 1aec in a fluorescence anisotropy assay
described previously.35 The assay involves displacement of a
fluorescein labelled analogue of p53 : p5315-31Flu, from hDM2
with concomitant loss of anisotropy. In our hands, direct
titration of p5315-31Flu with hDM2 resulted in the expected
increase in fluorescence anisotropy and a dissociation constant
of 74 nM was determined based on a 1 : 1 isotherm (Fig. S2,
ESIw). Upon titration with mimetic 1aec, the expected
Although many cellular processes depend on enzymatic
reactions, protein–protein interactions (PPIs) mediate many
regulatory pathways—the explosion of interest in their study
mirrors a key role in diseased states.1 Small molecules that
selectively target PPIs are therefore urgently required.2,3 What
is not clear is how to achieve inhibition using a small molecule,
given that it must cover B800–1100 A of a protein surface and
complement the discontinuous projection of hydrophobic and
charged domains on a relatively shapeless surface.4 In the
proteomimetic5 approach a scaffold is used to project binding
functionality in an identical spatial orientation to mimic that
presented by a given secondary structure (commonly an
a-helix) involved in the interaction.6 In addition to Hamilton’s
terphenyl,5,7 a number of proteomimetic scaffolds have been
described.8–15 Similarly, foldamers,16 if suitably designed,
function as inhibitors of PPIs.17–19 Aromatic oligoamides are
particularly attractive as foldamers because they exhibit
predictable folding patterns controlled largely by the preferred
conformation of the Aryl–NHCO–Aryl bond and adjacent
ortho interactions;20,21 however, such foldamers have rarely
been shown to act as a-helix mimicking antagonists of
PPIs.10,22,23
During our studies on aromatic oligoamides,24,25 we
reported a modular synthesis of rod shaped aromatic oligo-
benzamides that incorporate different side chains via an
O-alkyl substituent.25 Elsewhere, trimers of this type have
been proposed to act as a-helix mimetics23,26,27 and shown
to act as inhibitors of PPIs.23 We now report that such
compounds act as low mM affinity inhibitors of the
p53–hDM2 protein–protein interaction. p53 is the major
tumour suppressor in humans and is regulated by binding
to hDM2.28 Over-expression of hDM2 prevents p53 from
exerting its pro-apoptotic activity and so this PPI is a major
target for cancer chemotherapy, with several examples of small
molecule inhibitors reported.29–33 The interaction between p53
and hDM2 involves three key hydrophobic residues Phe19,
a School of Chemistry, University of Leeds, Woodhouse Lane, Leeds,
UK LS29JT. E-mail: A.J.Wilson@leeds.ac.uk;
Fax: +44 (0)113 3431409; Tel: +44 (0)113 3436565
b Astbury Centre for Structural Molecular Biology, University of
Leeds, Woodhouse Lane, Leeds, UK LS29JT.
E-mail: T.A.Edwards@leeds.ac.uk
w Electronic supplementary information (ESI) available: Synthetic
procedures, protein expression and FP assays. See DOI: 10.1039/b908207g
Fig. 1 (a) Crystal structure of hDM2 in complex with a p53 peptide
(PDB ID: 1YCR) and (b) the p53 peptide showing side chains key for
binding.
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Chem. Commun., 2009, 5091–5093 | 5091

Fig. 2 (a) p53 helix depicting key side chains (in green); (b) minimised
structure of an aromatic oligoamide with R3 = Bn, R2 = Me-2-Napth
i
and R3 = Pr (carbon in grey, oxygen in red and nitrogen in light
Fig.
4 Representative FA competition titration data (40 mM
purple); (c) p53 a-helix superimposed onto minimised aromatic
oligoamide.
sodium–potassium phosphate buffer pH 7.5, 54 nM p5315-31Flu
,
42 nM hDM2).
decrease in anisotropy was observed and an IC50 of 1.0 mM
was determined (Fig. 4 and Table 1). Similarly, a decrease in
anisotropy was observed for the positive control; unlabelled
p53 peptide (p5315-31Flu) gave a IC50 of 1.2 mM. We then tested
a further series of mimetics 1 and 2 containing different side
chains to probe for side chain specificity. Compounds 2aa and
2ba are negative controls, which do not possess a sufficient
number of side chains to mimic two turns of an a-helix.
Compounds 1aaa, 1bca, 1acd, 1ace and 1adc contain a selec-
tion of side chains matched and mismatched to the sequence of
p53 (naphthyl acting as a mimetic of indole). The results of our
screening are summarised in Table 1 (see also Fig. S4, ESIw).
As predicted, compounds 2aa and 2ba were both inactive
under the conditions of our assay. The remaining compounds
were found to inhibit the interaction with low mM affinity:
compounds with more and larger aromatic groups tended to
be more potent although this effect was only subtle. We could
not perform a titration with a nitro terminated compound
because our synthetic approach results in hydrolysis of the
4-nitro benzamide terminus rather than the methyl ester.25
IC50 values from competition assays have been used to
extract a direct measure of binding affinity (Ki) for a given
target.36 Upon careful analysis of our data, we observed that
in the competition experiment, anisotropy values for ligands 1
and p5315-31 decreased below the original value observed at the
Table 1 Dissociation constants determined by FP displacement
assaya
Compound
IC50/mM
p5315-31Flu
p5315-31Flu
2aa
2ac
1aaa
1bca
1acd
1ace
1aec
0.074 (Æ0.004)b
1.2 (Æ0.04)
2700 (Æ1950)
50.0 (Æ20.0)
10.0 (Æ3.8)
4.7 (Æ0.53)
2.4 (Æ0.31)
1.6 (Æ0.25)
1.0 (Æ0.11)
5.1 (Æ0.44)
1acc
a
b
Conditions as indicated in Fig. 4. Kd.
start of the direct titration of hDM2 into p5315-31Flu. This
points to a more complex equilibrium that precludes determi-
nation of Ki. One possibility is that p5315-31Flu is present in an
aggregated/dimeric form before titration with hDM2 and then
becomes bound to the competitor upon displacement. Indeed,
direct titration of p5315-31Flu with both p5315-31 and our
compounds resulted in a decrease in anisotropy value com-
parable to that observed in the competition experiments where
hDM2 is also present. Data fitting to a 1 : 1 association model
provided Kd values in the mM range (see ESIw) for the
Fig. 3 Compounds tested.
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interaction between p5315-31Flu and p5315-31/ligand. We
therefore derived a model to extract a binding constant for
interaction of the competitors with hDM2 (see ESIw). Not
surprisingly, as the Kd values for p5315-31Flu binding to
competitors are in the mM range and the concentration of
competitor is far in excess of all other components in the
experiment (mM vs. nM), no meaningful data can be extracted
and it is not possible to confirm that the compounds bind to
hDM2. We therefore derivatised one of the more soluble
compounds (1acc) with a fluorescein label to give FITC-
Gly-1acc and performed a direct binding experiment with
hDM2—this afforded a dissociation constant of 760 (Æ140) nM
and confirmed strong interaction with the target protein.
In summary, we have described the design, synthesis and
binding studies of potent mM inhibitors of the p53–hDM2
protein–protein interaction and highlighted complications asso-
ciated with interpretation of data from competition experiments.
The generality of the syntheses reported by us and others25
represents a powerful starting point for generation of libraries
for screening against a plethora of a-helix mediated PPIs. Our
own investigations will focus on optimising the current
compounds for binding to hDM2 and targeting other PPIs.
This work was supported by EPSRC (EP/D077842/1) and
the Wellcome Trust (080709/Z/06/Z) through a PhD student-
ship (BM). We would also like to thank John Robinson
(University of Zurich) for providing the hDM2 plasmid.
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