Interfacing native and non-native peptides: using Affimers to recognise α-helix mimicking foldamers

Article abstract of DOI:10.1039/c6cc09395g

Selection methods are used to identify Affimers that recognise α-helix mimicking N-alkylated aromatic oligoamides thus demonstrating foldamer and natural α-amino acid codes are compatible.

Full text of DOI:10.1039/c6cc09395g

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Interfacing native and non-native peptides:
using Affimers to recognise a-helix mimicking
foldamers†
Irene Arrata,ab Anna Barnard,‡ab Darren C. Tomlinsonbc and Andrew J. Wilson*ab
Cite this:DOI: 10.1039/c6cc09395g
Received 24th November 2016,
Accepted 10th February 2017
DOI: 10.1039/c6cc09395g
rsc.li/chemcomm
Selection methods are used to identify Affimers that recognise
a-helix mimicking N-alkylated aromatic oligoamides thus demon-
strating foldamer and natural a-amino acid codes are compatible.
Proteins adopt specific three-dimensional compact conforma-
tions comprising helices, sheets, loops, turns and disordered
domains to orient distinct groups for function e.g. molecular
recognition and catalysis. Inspired by the diversity of natural
protein structure, foldamers are defined as sequences of non-
natural monomers designed to adopt well defined secondary
and tertiary or quaternary structures and ultimately 3D archi-
tectures with novel, enhanced or emergent function.1–3 This
goal aligns closely with efforts to build functional proteins
de novo in synthetic biology.4–10 Although a major challenge
in supramolecular chemical biology,11 considerable progress
has been made in the de novo or ‘‘bottom-up’’ design of tertiary
foldamers (Fig. 1a)12,13 whilst efforts to understand and control
their dynamic topology have broadened potential applications
(e.g. PPI inhibition, Fig. 1b).14–21 An alternative strategy for
the design of functional foldamers is to replace segments of
protein sequence with non-natural foldamer22–27 an approach
termed ‘‘protein-prosthesis’’28–30 and leading to ‘‘bionic proteins’’
(Fig. 1c).31 A third related approach would be to exploit the
potential of combinatorial biology to identify natural biomacro-
molecule sequences (comprised of amino-acid or nucleotide
building blocks) that recognise a single synthetic foldamer
(Fig. 1d).32 In identifying compatible natural and non-natural
components driven by complementary molecular recognition,
Fig. 1 Schematics for elaborating functional foldamer structures (a) bottom-up
foldamer construction (b) PPI inhibition using foldamers (c) protein-prosthesis
(d) combinatorial selection of peptides for foldamers (e) N-alkylated
aromatic oligoamide proteomimetic illustrating a-helix mimicry (f) structure
of an Affimer (PDB ID: 4N6U).
such an approach could be used to identify potential biological
targets of a given foldamer,32 a potentially more rapid route to
ligand discovery than the painstaking construction of libraries
using synthetic foldamer assembly strategies. Alternatively, the
biological selection approach may generate non-covalent
foldamer-biomacromolecule complexes that serve as starting
points for the construction of foldamer-biomacromolecule
hybrid tertiary structures (making an assumption that the
former differs from the later only in respect of chain entropy).
a School of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.
E-mail: a.j.wilson@leeds.ac.uk
b Astbury Centre for Structural Molecular Biology, University of Leeds,
Woodhouse Lane, Leeds, LS2 9JT, UK
c School of Molecular and Cellular Biology, Faculty of Biological Sciences,
University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6cc09395g
‡ Current address: Department of Chemistry and Institute of Chemical Biology,
Imperial College London, London, SW7 2AZ, UK.
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Our group recently developed a series of proteomimetic optimised phytocystatin protein with high stability comprising
aromatic oligoamide foldamers designed to mimic the a-helix four b-strands and an a-helix with randomisation at the loops
and discovered selective inhibitors of a-helix mediated protein– connecting each pair of b-strands and has been successfully used
protein interactions (PPIs).33,34 In the proteomimetic approach, in a number of discovery settings.38–41 Using this scaffold and
a scaffold replicates the spatial and angular projection of essential trimeric aromatic oligoamide foldamers, we identified selective
recognition groups that represent ‘hot-spot’ residues at the PPI peptide-foldamer interactions using phage display screening.
interaction (Fig. 1e).35,36 Given this known compatibility of our
In this preliminary study we used our previously described
proteomimetic scaffolds with protein structure, we were keen to N-alkylated aromatic oligoamide foldamer series.34,42,43 These
explore the extent to which biological selection methods might oligomers are amenable to solid-phase peptide synthesis44 and
generate peptide sequences for future exploitation in construction act as effective inhibitors of a-helix mediated PPIs.34 A series of
of foldamer-peptide hybrids. We used a non-antibody-based foldamers was prepared following established methods (see
scaffold termed an Affimer (Fig. 1f) in tandem with phage ESI,† Schemes S1 and S2).42,44 In total, six trimers (1–6) and
display screening.37 The Affimer scaffold37 is a consensus their biotinylated equivalents (Biotin-1–6) were synthesised
(Fig. 2 and ESI,† Scheme S2 for additional structures).
Biotinylated trimers Biotin-1–6 were immobilized alternatively
on: streptavidin-coated wells, neutravidin-coated wells, and
streptavidin-coated magnetic beads. Immobilized trimers were
incubated against an Affimer library (diversity of 3 Â 1010).37 The
phage library was pre-panned three times on each respective
surface before panning against the immobilized targets. Bound
phage were eluted and used to infect ER2738 E. coli cells in the
presence of M13K07 helper phage over four panning rounds (see
ESI,† Fig. S1). Following the final round of panning, 48 mono-
clonal Affimers for each trimer were analysed by ELISA; bound
phage were detected using Anti-Fd-Bacteriophage-horse radish
peroxidase (HRP) and SeramunBlaus fast TMB (Fig. 3 and ESI,†
Fig. S2). We assessed the extent of binding and selectivity for all
48 Affimers: for Biotin-1, there were 25 Affimers which bound, 12
of which were fully selective for Biotin-1; for Biotin-2, 41 Affimers,
30 of which were selective; 7 Affimers for Biotin-3, 2 of which
were selective; 19 Affimers for Biotin-4, none of which were
selective; 18 Affimers for Biotin-5, none of which were selective;
and, 25 Affimers for Biotin-6, 4 of which were selective (see
further discussion in ESI†). These preliminary data establish
that N-alkylated aromatic oligoamide foldamers can interact
with natural a-polypeptide/protein structures in an effective
and highly selective manner i.e. the natural and non-natural
foldamer ‘‘codes’’ are compatible.
Affimers showing differential values between test and negative
Fig. 2 Structures of N-alkylated trimeric aromatic oligoamide foldamers
control by ELISA were sequenced using a T7P primer – mixed
colonies were removed from the list. Each Affimer was named
1–2 and their biotin/fluorescein derivatives that represent the primary
focus of this study.
Fig. 3 ELISA showing the 48 monoclonal Affimers picked after four panning rounds of phage display against Biotin-1 (left) and Biotin-2 (right). Affimers
selected for analysis are indicated by a yellow arrow. The UV absorbance for each bar is indicative of the extent to which an Affimer binds to the target
and is colour coded according to the foldamer it binds.
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trend for the biotinylated trimer to more effectively compete for
the Affimer than the unlabelled trimer. For 1-AF8, 2-AF1, 2-AF23
and 2-AF25, the data suggest the biotin enhanced competition
for the Affimer. Data for 1-AF17 and 1-AF26 did not allow any
conclusion to be drawn although streptavidin also appeared to
compete for 1-AF26 indicating strong binding between the two.
This correlates with the initial screening results where 1-AF26
showed high affinity but no selectivity (Fig. 3). Further analyses
using 10 and 100-fold excess of competitor (see ESI,† Fig. S5),
showed increased competitor concentration enhanced competi-
tion for the Affimers.
We then performed full competition assays (Fig. 4b and
ESI,† Fig. S6). Data for 2-AF1 and 2-AF23 could not be inter-
preted, whilst data for 1-AF17 did not yield a full binding
curve. In accordance with single-concentration experiments,
streptavidin competed for 1-AF26 with similar IC50 values to 1
and Biotin-1. Data for 1-AF8 and 2-AF25 were more promising;
1-AF8 had weak affinity for 1 (IC50 4 100 mM), but using Biotin-1,
IC50 = 2.5 (Æ0.6) mM was obtained. This implies that trimer 1 on
its own is insufficient for recognition of 1-AF8 and that the
panning process selected for an Affimer that recognises the three
Fig. 4 (a) Direct titration (ELISA) for 1-AF8 (n = 3) and 2-AF25 (n = 2)
against the corresponding immobilized foldamer. For clarity, binding
curves for the remaining Affimers are shown in the ESI.† (b) Competition
assay (ELISA) using foldamers to displace 1-AF8 and 2-AF25 (n = 3) from
the corresponding immobilized foldamer. For clarity, binding curves for
the remaining foldamers are shown in the ESI.†
using the format X-AFY, where X is the number of the trimer helix mimicking side chains of the foldamer and the forth biotin
and Y the number of the Affimer out of 48 (see ESI,† Table S1 side-chain. In contrast, for 2-AF25 an IC50 = 56 (Æ2) mM
for further details). We focussed on Biotin-1 and Biotin-2 since was obtained upon competition with 2, whereas a comparable
they exhibited promising results in the ELISA assay (Fig. 3). IC50 = 28 (Æ10) mM was obtained for competition with Biotin-2,
Biotin-1 and Biotin-2 generated Affimers enriched predominantly suggesting the biotin has limited effect on binding and Affimer
in hydrophobic amino acids (see ESI† for further details). For each selection only for the three helix mimicking side-chains of 2.
trimer, two selective (1-AF8, 1-AF17, 2-AF23, 2-AF25) and one non-
To further confirm these data, an orthogonal fluorescence
selective Affimer (1-AF26 and 2-AF1) were chosen (Fig. 3, yellow anisotropy (FA) biophysical assay was used. Synthesis of fluorescein-
arrows) to be subcloned, expressed as his-tagged proteins and tagged analogues of 1 and 2 was performed using similar methods
finally purified on a Ni-NTA resin.
as for the biotinylated trimers (see ESI,† Scheme S1). Flu-1, Flu-2
To establish the affinity of interaction between foldamer and and Flu-2rev (an analogue of Flu-2 with the side-chain sequence
Affimer, we performed a direct ELISA based titration of the switched), were prepared (Fig. 2). In agreement with the data
Affimers against immobilised trimer (Fig. 4a and ESI,† Fig. S3); obtained using ELISA, Flu-2 was shown to bind to 2-AF25 with a
here, anti-6X his-tag HRP and SeramunBlaus fast TMB were Kd = 146 Æ 11 nM (Fig. 5a). Gratifyingly, binding was not observed
used for detection. Well-defined dose–response behaviour for Flu-2rev (Fig. 5b and ESI,† Fig. S7), which attests to the high
was observed for 1-AF8 and 2-AF25, which could be fit using selectivity of the Affimer for the exact sequence/order and composi-
a logistic model to obtain EC50 values of 4.8 (Æ0.6) and 0.98 tion of side chains. We also tested Flu-1 against 1-AF8 (Fig. 5b), but
(Æ0.08) mM respectively. The remaining Affimers gave noisy were unable to obtain a binding curve; this may arise due to the
data which made curve fitting challenging (see ESI† for data);
we attribute this to the hydrophobic nature of the side chains
and consequent amplification of hydrophobic Affimers.
To obtain evidence of a specific non-covalent interaction
between foldamer and Affimer, we performed competition
experiments. The extent to which immobilized trimers Biotin-1
and Biotin-2 recruited their complementary Affimers in the
presence of competing quantities of unlabelled unfunctionalized
1 or 2 was assessed at a single concentration. Based on the
results of the direct ELISA, we chose to work at the following
concentrations: 10 mM for 1-AF8 and 1-AF17, 1 mM for 1-AF26,
20 mM for 2-AF1, 2 mM for 2-AF23 and 5 mM for 2-AF25. We
also attempted to compete for Affimer binding with Biotin-1 or
Biotin-2, streptavidin or a complex of Biotin-1 and streptavidin
or Biotin-2 and streptavidin. The normalised average for each of
Fig. 5 Direct Fluorescence Anisotropy (FA) titration curve of (a) 2-AF25
the four conditions (n = 3 for each) is given in the ESI† (Fig. S4).
against Flu-2, and (b) 1-AF8 against Flu-1 (green), 2-AF25 against Flu-2rev
(blue) and on a special non-stick plate (orange).
Under single concentration conditions, we observed a general
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This work was supported by the European Research Council
[ERC-StG-240324], the Leverhulme Trust [RPG-2013-065] and
the EPSRC [EP/K039202/1]. We thank Dr Christian Tiede, Miss
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E Webb for useful discussions.
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