α-Helix mimetics: Outwards and upwards

Article abstract of DOI:10.1016/j.bmcl.2013.12.003

α-Helices are common secondary structural elements forming key parts of the large, generally featureless interfacial regions of many therapeutically-relevant protein-protein interactions (PPIs). The rational design of helix mimetics is an appealing small-

Full text of DOI:10.1016/j.bmcl.2013.12.003

Bioorganic & Medicinal Chemistry Letters 24 (2014) 717–724
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BMCL Digest
a
-Helix mimetics: Outwards and upwards q
⇑
⇑
Madura K. P. Jayatunga, Sam Thompson , Andrew D. Hamilton
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
a
-Helices are common secondary structural elements forming key parts of the large, generally featureless
Received 12 August 2013
Revised 23 November 2013
Accepted 1 December 2013
Available online 8 December 2013
interfacial regions of many therapeutically-relevant protein–protein interactions (PPIs). The rational
design of helix mimetics is an appealing small-molecule strategy for the mediation of aberrant PPIs, how-
ever the first generation of scaffolds presented a relatively small number of residues on a single recogni-
tion surface. Increasingly, helices involved in PPIs are found to have more complex binding modes,
utilizing two or three recognition surfaces, or binding with extended points of contact. To address these
unmet needs the design and synthesis of new generations of multi-sided, extended, and supersecondary
structures are underway.
Keywords:
Protein–protein interaction
Rational design
Peptidomimetic
Proteomimetic
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Proteomics has emerged as an important tool in unraveling the
intricate, dynamic nature of many cellular processes.1 The preva-
lence of protein–protein interactions (PPIs) in signal transduction,
transcription, and apoptosis, make them attractive therapeutic
targets for numerous diseases (e.g., HIV, cancer, and neurodegener-
ative disorders).2–8 The development of chemical probes is impor-
tant to gain further insights into these critical biological systems
but despite their potential clinical importance, few small molecule
inhibitors of PPIs have been developed. Many established enzy-
matic targets have small, well-defined catalytic domains which
govern the majority of the enthalpic contribution to substrate
binding.9 Conversely, PPIs are mediated by the cumulative binding
energy of many amino acid residues, over an extended surface (as
large as 4500 Å2).10–12 Much of this surface is solvent exposed
when unbound, with binding domains arranged in a noncontigu-
ous manner, often necessitating the development of large molecu-
lar weight inhibitors.13 Strategies to disrupt PPIs frequently focus
on key ‘hot spot’ residues which contribute heavily to binding.14
protein-protein recognition.15,16 They are also found at more com-
plex recognition domains such as DNA binding motifs and mem-
brane-bound proteins.17,18
The increase in structural data over the last 20 years (Fig. 2) has
facilitated research into the mediation of therapeutically relevant
PPIs, with many groups targeting key helical elements. Whilst
the ratio of two- and three-sided helix-mediated PPIs relative to
single-sided analogues has not changed, there has been a growing
awareness of their presence at important interfaces.
Many groups have developed conformationally restricted
peptides employing covalent tethers including lactams,19,20 disul-
fide bridges,21 triazoles,22 and hydrocarbon linkers.23–26 Grubbs
showed that olefin metathesis can be used to promote helicity,27
with Verdine later showing that stapled peptides inhibit the
(a)
(b)
(c)
An
a-helix is the most common protein secondary structural
element, defined by a tight helical turn (3.4 residues per turn),
thereby creating three distinct binding surfaces, with the i, i + 4
and i + 7, residues aligned on the same face (Fig. 1). Of the PPIs
found in the Protein Data Bank (PDB), 62% have an
a-helix at the
interface, illustrating the importance of this structural element in
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding authors.
E-mail addresses: sam.thompson@chem.ox.ac.uk (S. Thompson), andrew.hamil-
Figure 1. Distribution of residues on a canonical
i + 7, i + 11 residues displayed on the same face (orange); (b) top-down view, (c)
helical wheel.
a
-helix; (a) side-view with i, i + 4,
a-
ton@chem.ox.ac.uk (A.D. Hamilton).
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.003

718
M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
pioneered the use of b-peptides to mimic
a-helical secondary
1-sided helix PPI 2-sided helix PPI 3-sided helix PPI
250
structure,47–49 with Schepartz later demonstrating inhibition of
the p53/MDM2 PPI.50 An example by Gellman showed an extended
helix which accurately mimicked the native sequence, and success-
fully inhibited the Bcl-2/Bim PPI.51
200
150
100
50
A fundamentally different strategy to mediate PPIs is to mimic
elements of protein secondary structure with entirely non-peptidic
constructs. The rational design of helix mimics aims to project
functional groups from a scaffold in order to reproduce the orien-
tation of the native side-chains, whilst avoiding the expression,
purification or chemical synthesis of peptidic elements. Such pep-
tidomimetics present an opportunity to design compounds which
have the same binding mode as the native protein, yet are meta-
bolically stable, can permeate membranes and are readily
absorbed.11 The structural diversity provided by chemical synthe-
sis allows a much greater array of groups, with varied steric and
electronic properties, to be projected than those provided by the
side chains of proteogenic amino acids. Majmudar et al. identified
phenol carboxylic acid natural products, sekikaic- and lobaric acid,
that inhibit the PPI between p300 and both MLL (Mixed-Lineage
Leukemia) and pKID (phosphorylated kinase-inducible domain of
CREB). These molecules bind at the same site as the helical region
of peptides, hinting that Nature has evolved its own helix
mimics.52
Early work focused on the mimicry of hydrophobic residues on
a single helical face, yet important targets often have amphiphilic
helices with hot-spot regions projected from multiple faces. Impor-
tant helical sequences with hot-spot residues in the i, i + 4 and i + 7
positions were frequently targeted since they are found on a com-
mon face (Fig. 1).14 Several classes of helix mimetic have been
developed, many inspired by the terphenyl scaffold introduced
by Orner et al. in 2001.53 The p53/MDM2 and Bcl-xl/Bak interac-
0
1990-1995
1996-2000
2001-2005
2006-2011
Figure 2. PDB structures with helices at the PPI interface over time. Subcategories
illustrate which proportion contained helices with one, two or three key binding
domains.15,16
Bcl-2/Bid interaction, resulting in decreased growth of human leu-
kemia xenografts in vivo.28 Strategies developed by Cabezas and
Arora involve replacement of the internal a-helix hydrogen bond-
ing network with a covalent linkage.29–32 A number of different
approaches have since been developed to introduce constraints
in an effort to promote helicity (Fig. 3).33,34 Arora et al. demon-
strated inhibition of the HIF-1a/p300 PPI with an olefin-stapled
hydrogen bond surrogate (HBS) where key residues are found at
the i, i + 2, i + 5, i + 6 and i + 10 positions.35 This strategy has the
benefit of retaining the i, i + 4 and i + 7 residues and avoiding the
use of synthetically challenging
a,a-di-substituted amino acids.
In another strategy, tethers have been used to staple long chains
and stabilize extended helical structures. An impressive example
is a 36 amino acid anti-viral sequence stabilized by two hydrocar-
bon bridges formed through ring-closing metathesis (RCM),
increasing potency of a peptide inhibitor, enfuvirtide, four-fold to
2.1 nM and at the same time improving its pharmacokinetics.36
Short peptide truncates are frequently unstructured, prompting
researchers to explore the helical propensities of more metaboli-
cally stable b-peptides.37,38 Variations of this strategy have been
employed to promote additional helicity, with many groups
exploiting conformationally constrained variants,39–42 and mixed
tions are two well-studied targets, with
a-helices projecting
crucial residues on a single face.54,55 Both interactions inhibit cel-
lular apoptosis: MDM2 binds and inactivates p53, the principal
architect of cell-death, thereby arresting apoptosis.56 Similarly,
Bak is a pro-apoptotic protein whose function is disrupted on bind-
ing of Bcl-xl (Fig. 4a).57 Both interactions are thought to be prom-
ising cancer targets since neoplastic cells often disable intracellular
apoptotic mechanisms. Yin et al. designed terphenyl helix mimet-
ics that inhibited the p53/HDM2 interaction in vitro with a Ki of
sequences of
a
/b-amino acids.43–46 Seebach and Gellman
182 nM where a native peptide truncate had a Ki of 3.51 l
M.54
(a)
(b)
Arora has highlighted the importance of binding to multiple
surfaces of a single helix in many interactions.15 The current
collection of single-sided helix mimetics are ill-equipped to simul-
taneously mimic hot-spot residues displayed on more than one
face, presenting a need for the development of more complex scaf-
folds (Fig. 4).58 Moreover, the restricted length of mimics of two
a
-helix turns is often insufficient to inhibit interactions of ex-
tended helices. The Bcl-2/Bid interaction is an example with key
residues orientated on two faces of the
-helix.59 Key hydrophobic
a
residues Ile82, Ile86 and Leu90 lie on the same face, with Asp95
projected on a different face to form important hydrogen bonds
to Bcl-2 arginine and asparagine residues (Fig. 4b). Enzymes E1
and E2, involved in the replication of papillomavirus, are another
(c)
(d)
important cancer target displaying a two-faced
a-helix binding
mode.60
Examples with hot-spot residues found on all three faces of an
-helix include the interaction of calmodulin with CaM kinase I.61
a
Peptide truncates show that a particularly dense array of residues
bound in the N-terminal domain of calmodulin is crucial, with the i
(Val), i + 1 (Arg), i + 2 (His), i + 3 (Met) and i + 4 (Arg) positions of
the peptide contributing strongly to binding (Fig. 4c).
Burgess has reasoned that since there is homology in the way
many secondary structural elements display residues, it is possible
to design ‘universal peptidomimetics’, able to reproduce the
Figure 3.
bonding, (b) stapled
a
-Helix tethering strategies; (a) canonical
a-helix with 13-membered H-
a-helix through residues on the i and i + 4 positions (22-ring
macrocycle), (c) Cabezas’ hydrazone HBS (13-ring macrocycle), (d) Arora’s olefin
HBS (13-ring macrocycle). HBS = hydrogen bond surrogate.

M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
719
(b)
(c)
(a)
Two recognition faces
One recognition face
Three recognition faces
Calmodulin/
CaMKI
Bcl-xL/Bak
Bid/Bcl-2
Figure 4. Examples of different a-helix binding modes; (a) Bcl-xL/Bak PPI (PDB: 1BXL), (b) Bcl-2/Bid PPI (PDB: 2VOI), (c) Calmodulin/CaMKI PPI (PDB: 1MXE).
inherent plasticity of these scaffolds suggests that this approach
would be particularly useful when targeting PPIs where the hot-
spot residues are known, but the bound conformation is not. The
strategy led to a peptidomimic of the i, i + 2, i + 4 side chains of a
peptide that inhibited dimerization of the HIV-1 protease with a
Ki of 0.38 l
M.67 Some of the more conformationally flexible Ham-
ilton scaffolds may allow mimicry of residues on multiple faces of a
helix in a similar manner to the Burgess peptidomimetics, however
preorganising molecules in bioactive conformations would seem to
be more entropically favorable for ligand–protein binding.
Scheme 1. Reagents and conditions: (i) (a) AlCl3, MeNO2, (b) Ri + 1COCl, reflux, (c)
H2, (50 psi), 10% Pd on C, CF3CO2H, EtOH (88%); (ii) (a) LiHMDS, THF, À78 °C, (b) XRi,
(c) NaOH, MeOH; (iii) (a) (COCI)2, CH2Cl2, (b) Ri À 1NH2, NEt3. Ri+1 = Ph, XRi = BnBr or
gramine, MeI, Ri À 1 = CH2C(CH3)3 or CH2CH2Ph.
The design and synthesis of double-sided mimetics is inherently
more challenging than single-sided scaffolds due to the density of
functionalization. Howson imitated the i and i + 1 geometry of a
dipeptide fragment with a 1,6-disubstituted indane.68 This was
expanded to the racemic 1,1,6-trisubstituted indane 1 (Scheme 1),
the (S)-enantiomer of which maps closely onto the i À 1, i and i + 1
recognition properties of a range of motifs.62–65 An induced fit
mode of binding is invoked in which conformationally flexible
molecules are able to mimic residues on multiple faces.66 The
residues of an
a-helix. These short helix mimics showed low
Figure 5. Schematic representation of an
benzoylurea 7.
a-helix alongside double-sided mimics: indane 1, anthracene 2, pyrimidine 3, diphenylindane 4, oligobenzamides 5 and 6, and

720
M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
(b)
(c)
(a)
(a)
(c)
(d)
(b)
Figure 6. (a) Energy minimized conformation of a diphenylindane scaffold, side
view.74 (b) Top view: projection of side-chain groups looking down the helix
mimetic. (c) Reagents and conditions: 11 six steps from o-cresol. (i) (a) NBS, AcOH,
85 °C, (b) ArOTf, Pd(PPh3)4, Cs2CO3, H2O, DMF, 120 °C (77% over two steps); (ii) (a)
BBr3, CH2Cl2, (b) Tf2O, Et3N, CH2Cl2, (c) ArSnBu3, CuBr, LiCl, DMF, 160 °C (overall
58%).
Figure 7. Syntheses of double-sided scaffolds: (a) Hamilton’s oligobenzamide; (b)
Marimganti’s oligobenzamide. Energy minimized conformation of the oligobenza-
mide: (c) side view; (d) top view: projection of side-chain groups looking down the
helix mimetic.74
Table 1
cLogP values for double-sided scaffolds and the polarity of side chains that may be
mimicked
(a)
(b)
Scaffold
cLogP
Side-chain mimicry
Reference
1
2
3
4
5
6
7
2.12
3.67
2.03
8.35
1.10
3.13
4.04
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Polar and hydrophobic
Polar and hydrophobic
Polar and hydrophobic
68,69
71
72
73
78
83
91
cLogP values calculated with methyl substituents as side-chain mimics (Chem-
BioDraw 13.0, Cambridgesoft).
micromolar IC50’s against tachykinin receptors, with many show-
ing selective inhibition for NK1R and NK3R over NK2R, which
control dopamine release in different areas of the brain.69,70
Zhang et al. demonstrated that a naphthalene group could effec-
(c)
tively span the width of an a-helix, allowing residues on opposite
sides to be mimicked. An anthracene-based scaffold 2, substituted
at the 2- and 7-positions, mimicked residues in the i and i + 2 posi-
tions (Fig. 5).71 A 24
lM inhibitor of the Bim/Bcl-2 PPI mimicked
the Asp67 and Ile65 residues of the Bim helix (Fig. 4b).
Rodriguez et al. expanded the concept of double-sided mimicry
in developing an inhibitor of the p106 helical peptide truncate/nu-
clear hormone receptor (NR) interaction.72 X-ray crystallography
showed that protein fragments with an LXXLL motif in a two-turn
helical orientation bound in a hydrophobic groove in the NR. A
pyrimidine scaffold 3 (Fig 5) mimicking residues in the i, i + 3
Figure 8. (a) Energy minimized conformation of double-sided benzoylurea scaffold,
side view.74 (b) Top view: projection of side-chain groups looking down the helix
mimetic. (c) Synthesis of double-sided benzoylurea scaffold. The monomeric units
were easily assembled from commercially available starting materials.
and i + 4 positions had a Ki of 29 lM. Substituting pyrimidine for
trithiane or cyclohexyl ablated binding, while a triazine decreased
inhibition by an order of magnitude. Larger hydrophobic side-
chains resulted in increased activity, illustrating the importance
of subtle structural modification and the advantage of non-proteo-
genic approaches in which a large library of synthetic building
blocks may be used. The strategy was further expanded with the
design of the diphenylindane i, i + 3, i + 4 and i + 7 mimic 4
(Fig. 6).73,74 The synthetic route provides the final product as a
racemate but with sufficiently low energy aryl–aryl rotational
barriers for interconversion of the diastereomers at room temper-
ature, thus allowing an induced fit mode of binding for one of the
enantiomers. To increase the utility of this scaffold, asymmetric
approaches for the synthesis of single enantiomer indanes are
required.

M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
721
An example of a heteroatom-containing scaffold is the double-
sided oligobenzamide 5 based on an earlier single-sided variant
(Fig. 7a).78,79 The single-sided oligobenzamide has been employed
successfully to inhibit the Bak/Bcl-xL, p53/hMDM2 interactions,
and to inhibit islet amyloid peptide aggregation.80–82 The oligoben-
zamide scaffold allows for facile incorporation of a variety of differ-
ent side-chain mimics and with increased aqueous solubility. This
new mimic uses an amide linker to install the i + 2 and i + 6 resi-
dues, which also defines the geometry of the molecule through
the formation of two further hydrogen-bonded six-membered
rings. The densely functionalized monomer 13 (Fig. 7a) was syn-
thesized in three steps (50% overall yield) from meta-cresidine.
Sequential coupling of different monomeric units from a common
starting material adds to the synthetic flexibility.
The Ahn group developed a conformationally restricted bisben-
zamide scaffold 6 (Fig. 7b).83 Five-membered hydrogen bonds
between the backbone amide proton and ortho-oxy group places
the i + 2/i + 5 and i/i + 7 groups on opposite faces. A common pre-
cursor was used to synthesize both monomeric units 14 and 15.
Sequential alkylation of the hydroxyl groups allows for different
residue mimics to be installed in a linear manner, providing a rapid
and flexible synthesis. The Wilson group detailed the synthesis of
several benzamide helix mimics with N- and O-alkylation allowing
side-chain attachment.84,85 The synthesis of oligobenzamides was
further facilitated by loading of the monomer onto Wang resin
allowing for solid-phase assembly of large libraries.86 A single
sided benzamide from the same group showed an impressive 40
nM potency against the androgen receptor/PELP1 co-regulator
PPI, a pathway which is often upregulated in tumors.87 Cell-based
assays and in vivo mouse studies highlight the increased aqueous
solubility and cell permeability of these second generation
compounds.
(a)
(b)
Figure 9. Superimposition of lowest energy conformations of diphenylindane
(pink), benzamide (brown) and benzoylurea (cyan) scaffolds showing differing side
chain projections: (a) side view; (b) top view.74
Many of the early mimics mediate binding predominantly
through hydrophobic interactions and the exclusion of water—also
a major thermodynamic driving force in the association of pro-
teins.75,76 Interactions of this type are not necessarily non-specific:
for example a 1-naphthyl-substituted terphenyl displayed selec-
tive inhibition of the HDM2/p53 PPI over the Bcl-2/Bak PPI, while
a 2-naphthyl analogue reversed the selectivity.54 The hydrophobic-
ity of these molecules is an important factor in the good cell
permeability of this series,77 but limits aqueous solubility,58
providing an impetus for the development of new scaffolds. For
example scaffolds 1–3 have moderate clogP values (Table 1), how-
ever only hydrophobic groups can be appended, further increasing
their hydrophobicity. Later scaffolds, for example, 5–7 incorporate
heteroatoms that act to increase aqueous solubility and provide
additional synthetic versatility for the incorporation of a greater
range of side-chains.
The planarity of benzamides limits side-chain projection to
opposite faces, restricting the use of such scaffolds to PPIs with
(a)
(c)
(b)
Figure 10. (a) Extended benzoylurea 18 and enaminone 19 scaffolds, (b) nine turn a-helix from a membrane spanning opsin GPCR, (c) PDB: 3CAP. GPCR = G protein-coupled
receptor.

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M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
The benzoylurea scaffold can similarly mimic a more complex
presentation of residues, with a convergent synthesis allowing
incorporation of hydrophobic and hydrophilic groups.89,90 The
Hamilton group synthesized double-sided mimic 7 from a benzam-
ide 16 and an isocyanate 17 (Fig. 8c).91 The helical backbone is
reminiscent of earlier benzamide helices created by the group,82,91
but allows for a more comprehensive distribution of side chains
(Fig. 8b). Existing scaffolds reproduce groups on a number of differ-
ent faces of the helix (Fig. 9), suggesting that while a three-sided
helical peptidomimetic remains elusive, several frameworks exist
for targeting PPIs with complex binding modes.
Figure 11. Common super secondary structural motifs containing
(cylinders), b-strands (arrows) and loop sequences (red).
a-helices
Extended helices are found in important therapeutic targets;
for example transmembrane GPCR (G protein-coupled receptor)
helices which span cell membranes and assemble due to both
hydrophobic and hydrophilic side-chain interactions.92 Mem-
hot-spot residues at 180° to one another (Fig. 7c and d). The Spivey
group overcame this limitation by designing a scaffold with an
azabicyclo[2.2.2]octane moiety coupled to an aryl fragment,
mimicking the i, i + 1, i + 2, i + 4 and i + 5 residues.88
brane-spanning amphiphilic
a-helices form artificial ion-chan-
nels, mimicking the acetylcholine receptor.93 Synthetic variants
may be able to replicate this behavior or inhibit endogenous
Figure 12. (a) HTH (red) of Pax class homeodomain dimer bound to DNA (green, PDB: 1FJL). (b) Structure and energy minimized conformation of bis-pentabenzamide HTH
mimic 20.105

M. K. P. Jayatunga et al. / Bioorg. Med. Chem. Lett. 24 (2014) 717–724
723
ion channels. The Hamilton group elongated the single-sided
benzoylurea scaffold to an oligomeric species mimicking a 37 Å
-helix 18 (Fig. 10). The easily assembled benzoylurea ring surro-
gate enabled facile extension of the scaffold resulting in a mimic
that exhibited a preference for the extended conformation.89
similar result was attained by the same group with an enaminone
scaffold 19 which was extended to a ten turn
-helix mimic.94 It
was also possible to incorporate polar side-chains, thus broaden-
ing the potential for therapeutic applications. Other extended
scaffolds have been designed by the Rebek, Ahn and Wilson
groups, with the latter exploiting solid phase synthesis to facili-
tate oligomerisation.95–98
Acknowledgments
a
We thank Cancer Research UK (M.K.P.J.) and the University of
Oxford (S.T.) for funding, and Drs. Hayden Peacock and Hannah
Lingard (Oxford) for helpful discussions.
A
a
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cate elements of supersecondary structure.17 Commonly occur-
ring combinations of secondary structural elements constitute
a
-hairpins, a-corners and b-a-b motifs (Fig. 11). Helical hairpins
are important structures aiding protein insertion and transport
across cell membranes.99 Given the majority of folded protein is
buried in the hydrophobic core, loop sequences are found at
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that can be tuned for inter-helix angle and length (Fig. 12b).
Linked helix mimetics may find a role in inhibiting important pro-
tein–DNA interactions, such as the helix-turn-helix (Fig. 12a) or
basic-helix-loop-helix, which regulate gene transcription. The
DNA binding domain of the proto-oncogene c-Myc is an example
of a HTH tri-helical protein.106 The helical turn forms a hydropho-
bic cavity, while simultaneously projecting polar residues to-
wards the negatively charged phosphate DNA backbone. These
classes of HTH mimic allow controlled projection of side chains
in three-dimensional space and thus the reproduction of rela-
tively large protein surfaces. Surface mimicry has been success-
fully attempted with both cyclic peptides and backbone grafting
onto protein scaffolds.107,108 Synthetic scaffolds have also been
employed by Ghosh and Hamilton in immobilizing peptide loops
on G-quadruplexes and thus imitating multi-loop protein sufac-
es.109 The same group previously designed a large protein surface
mimic by coupling cyclic peptides on to a calixarene scaffold,
which showed strong binding to cytochrome c.110 Surface recog-
nition has been demonstrated with porphyrin species,111 copper-
and ruthenium-complexes,112,113 illustrating the diversity of
scaffolds available.
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program. We thank a reviewer for pointing out that the parameters used may
lead to the inclusion of a small number of 310 and
p helices.
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