2-O-Alkylated para-benzamide α-helix mimetics: The role of scaffold curvature

Article abstract of DOI:10.1039/c2ob26262b

The design and synthesis of a new 2-O-alklyated benzamide α-helix mimetic is described. Comparison with regioisomeric 3-O-alkylated benzamides permits a preliminary evaluation of the role that mimetic curvature has in determining molecular recognition properties.

Full text of DOI:10.1039/c2ob26262b

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2-O-Alkylated para-benzamide α-helix mimetics: the role of scaffold
curvature†
Valeria Azzarito,a,b Panchami Prabhakaran,a,b Alice I. Bartlett,a,b Natasha S. Murphy,a,b Michaele J. Hardie,a
Colin A. Kilner,a Thomas A. Edwards,b,c Stuart L. Warrinera,b and Andrew J. Wilson*a,b
Received 16th May 2012, Accepted 3rd July 2012
DOI: 10.1039/c2ob26262b
we describe the design, characterisation and testing of a new
α-helix mimetic based on a 2-O-alkylated template and compare
it to the regioisomeric 3-O-alkylated benzamide analogue that
we30 and others studied previously.24,31,35
We performed molecular modelling on both scaffolds using
isopropyl moieties as O-alkyl substituents (see ESI† for full
details). Scaffolds were evaluated for α-helix mimicry by com-
parison with the p53 transactivation domain from the p53/hDM2
crystal structure (PDB ID: 1YCR)36 in which three side chains –
Phe19, Trp23 and Leu26 – are shown to play a key role at this
interface (Fig. 1a). This PPI is a good model system given that it
has served as a target for small molecule development.37,38 Of
the ensemble of structures within 1.5 kJ mol−1 of the lowest
The design and synthesis of a new 2-O-alklyated benzamide
α-helix mimetic is described. Comparison with regioisomeric
3-O-alkylated benzamides permits a preliminary evaluation
of the role that mimetic curvature has in determining
molecular recognition properties.
The identification of ligands capable of modulating protein–
protein interactions (PPIs) represents an area of significant
focus.1–3 PPIs fulfil myriad roles in biology and represent targets
for therapeutic intervention. However, identification of competi-
tive inhibitors is considered challenging; larger surfaces are
involved at protein–protein interfaces alongside less well defined
shapes and orientations of recognition handles4 when compared
with the conventional cavities that have been the traditional
focus of medicinal chemistry.5 One class of PPI that may be
amenable to generic approaches of inhibitor design is the α-helix
mediated PPI, in which a helical motif from one protein projects
side chains into a cleft in its partner protein.6 Chemoinformatic
analyses reveal that these interactions can involve a range of
different side chains but that interactions mediated by a single
face (e.g. i, i + 4 (or 3) and i + 7 (or 8) residues) are more preva-
lent.7 Several approaches have been described that exploit a
common scaffold with appropriately positioned side chains to act
as effective inhibitors of a range of target PPIs.6,8,9 Mimicking
an α-helix can be achieved with a constrained backbone
mimetic,10–14 helical foldamer15–18 or a helix mimetic whereby a
scaffold positions key functional motifs in an identical spatial
orientation to those presented by the original α-helix.19–34 Our
group previously reported on the use of aromatic oligobenza-
mides as μM inhibitors of the p53/hDM2 interaction.29,30 Herein
aSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2
9JT, United Kingdom. E-mail: A.J.Wilson@leeds.ac.uk;
Fax: +44 (0)113 3436565; Tel: +44 (0)113 3431409
bAstbury Centre for Structural Molecular Biology, University of Leeds,
Woodhouse Lane, Leeds LS2 9JT, United Kingdom
Fig. 1 Molecular modelling studies for helix mimetics 1 and 2. (a)
Structure of p53/hDM2 (PDB ID: 1YCR, Phe19 in red, Trp23 in green
and Leu26 in blue). (b) Model of 1a. (c) Overlay of 1a with p53 (paral-
lel, RMSD = 0.2170 (Å)). (d) Overlay of 1a with p53 (antiparallel,
RMSD = 0.2171). (e) Model of 2a. (f) Overlay of 2a with p53 (parallel,
RMSD = 0.4951). (g) Overlay of 2a with p53 (antiparallel, RMSD =
0.4953). (h) Overlay of 1a (blue) with 2a (red). Side chains in the
mimetics are coloured (N to C terminus) to match the coding for p53.
cInstitute of Molecular and Cellular Biology, University of Leeds,
Woodhouse Lane, Leeds LS2 9JT, United Kingdom
†Electronic supplementary information (ESI) available: Synthesis and
characterisation, details of modelling, procedures for protein expression,
purification and anisotropy. CCDC 870274 and 870275. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c2ob26262b
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energy conformation, several were observed to position side
chains on one face, suggesting conformations mimicking the
α-helix were accessible (Fig. 1b and e). Structures were aligned
with the p53 helix and the root mean square deviation (RMSD)
calculated on the basis of the agreement between the polypeptide
and the scaffold with oxygen representing the α-carbon. Both
scaffolds align with the p53 helix in parallel and anti-parallel
orientations to the direction of the polypeptide chain (Fig. 1c–d
and 1f–g and ESI, Fig. S27–S28†). Despite this, the backbone
curvature is visibly different for both scaffolds (Fig. 1h). We
attempted to quantify this effect by measuring differences in
several key angles (e.g. the angle and pseudo-dihedrals between
aromatic rings). However, no single measure accurately rep-
resents the curvature of the backbone and relates it to the presen-
tation of individual side chains in a meaningful manner as the
different hydrogen-bonded geometries in scaffolds 1 and 2
impose constraints on both the backbone curvature and the twist
around the Ar–amide axes.
We were intrigued to determine whether these subtle
variations in backbone architecture and side chain presentation
would manifest a difference in binding affinity to target
proteins. Following the modelling studies we synthesised a
series of mimetics (Fig. 2) using minor variations to the
method described previously (see ESI, Schemes S1 and S2†).39
Compounds 1a and 2a incorporated isopropyl side chains
for structural studies, whereas 1b and 2b–c incorporated
benzyl, 2-methylnapthyl and isopropyl side chains in both
orientations to provide side chains mimicking those present in
the p53 transactivation domain (Fig. 2). We were unable to
obtain the reversed sequence of 1b i.e. 1c because the precursors
were not sufficiently soluble to permit the last 2 steps of the
synthesis.
indicate 1a adopts an extended conformation in which rotation
around the Ar–CO axes is restricted by S(6) intramolecular
hydrogen bonding which contrasts with our studies on 2a in
which rotation around the Ar–NH axes is restricted by S(5) intra-
molecular hydrogen bonding.30,39 2D 1H–1H NOESY exper-
iments on 1a (see ESI, Fig. S7†) and 2a,30,39 provide
confirmation of this behaviour. Crystallographic analyses support
the solution observations and provide additional information.
The crystal structure of the nitro-ester analogue of trimer 2a (3a,
Fig. 3a) places two side chains on the same face with the side
chain at the N-terminus on the opposite face and confirms the
presence of S(5) intramolecular hydrogen bonding. We also
obtained a structure of a synthetic intermediate, dimer 4a
(Fig. 3b) on route to 1a. The structure places the isopropyl side
chains on opposing faces and confirms the presence of S(6)
intramolecular hydrogen bonding. In support of the modelling
studies, the curvature of 3a and 4a is visibly different (Fig. 3c
and ESI, Fig. S29† for a more detailed analysis). The different
curvature in 3a relative to 4a arises because the placement of the
O-alkyl group in the 2-position allows 6-membered hydrogen-
bonding with minimal distortion of the idealized backbone geo-
metry. In contrast, for 3a 5-membered hydrogen-bonding
“bends” the backbone and imposes different twisting around the
Ar–amide axes. Notably – these analyses suggest the nitro and
amino terminal groups have little influence on the oligomer
conformation.
We tested the compounds against the p53/hDM2 interaction
using fluorescence anisotropy (FA) displacement (Table 1 and
Fig. S30†). Low potency was observed for 1a possessing isopro-
pyl side chains, consistent with our earlier results for 2a,30
whereas for 1b possessing side chains that mimic those present
on the p53 helix, single digit μM IC50 values were obtained. All
derivatives with side chains matched to the p53 sequence gave
low IC50’s in the assay (see ESI† for full details).
Structural studies of the conformational properties of both 1a
and 2a including VT NMR, dilution and H/D exchange (see
ESI†) indicate strong S(6) for 1a and S(5) for 2a intramolecular
hydrogen bonding between the amide NH and the alkoxy
oxygen atom. In both cases, the NH at the N terminus exchanges
slower than the NH at the carboxy terminus of both 1a and 2a
whilst the amide protons of 1a (S(6) hydrogen bonding)
exchange an order of magnitude slower than 2a (S(5) hydrogen
bonding) implying stronger hydrogen-bonding for 1a in line
with our work on model compounds.40 Taken together these data
To evaluate the role of helix mimetic curvature on binding
affinity we compared the results for 2b against 1b. The results
Fig. 3 (a) Solid state structure of 3a. (b) Solid state structure of 4a.
(c) Superimposition of 3a and 4a (RMSD = 0.2436).
Table 1 IC50 values obtained from fitting the FA displacement assay
Compound
IC50
p5315–31
1a
1b
2a
2b
1.35 0.09 μM
35.2 7.5 μM
4.80 0.43 μM
25.5 6.3 μM
6.35 0.30 μM
4.15 0.20 μM
2c
Fig. 2 Structures of compounds 1a–b, 2a,30 2b and 2c.30
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Acknowledgements
This work was supported by the European Research Council
[ERC-StG-240324], NSM acknowledges the University of Leeds
for the award of a Mary & Alice Smith PhD Scholarship.
Crystallographic assistance of Dr Amber L. Thompson, Oxford
University is gratefully acknowledged.
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