Helical peptides from VEGF and Vammin hotspots for modulating the VEGF-VEGFR interaction

Article abstract of DOI:10.1039/c3ob27312a

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.

Full text of DOI:10.1039/c3ob27312a

Organic &
Biomolecular Chemistry
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PAPER
Helical peptides from VEGF and Vammin hotspots for
modulating the VEGF–VEGFR interaction†
Cite this: Org. Biomol. Chem., 2013, 11,
1896
María Isabel García-Aranda,a Susana González-López,a Clara María Santiveri,b
Nathalie Gagey-Eilstein,c Marie Reille-Seroussi,c Mercedes Martín-Martínez,a
Nicolas Inguimbert,c,d Michel Vidal,c,e María Teresa García-López,a
María Angeles Jiménez,b Rosario González-Muñiza and María Jesús Pérez de Vega*a
The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of
linear and cyclic peptide analogues of the N-terminal α-helix fragments 13–25 of VEGF and 1–13 of
Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm
and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared
on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of
amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a
clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a
VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and
Tyr25), Val14 and Val20 seem to be relevant for affinity.
Received 28th November 2012,
Accepted 18th January 2013
DOI: 10.1039/c3ob27312a
www.rsc.org/obc
domains connected by a single transmembrane helix to the
intracellular tyrosine kinase domain. Most of the therapeutic
Introduction
Angiogenesis is crucial for normal generation of new blood strategies hitherto described are directed to the inhibition of
vessels, but it is also implicated in a number of pathological the tyrosine kinase activity of the VEGF receptors.12,13 There
conditions,1,2 such as cancer, rheumatoid arthritis, and diabetic are indeed several compounds with this profile that are in
retinopathy.3,4 One of the key factors promoting angiogenesis is advanced clinical trials for tumour angiogenesis treatment.
the Vascular Endothelial Growth Factor (VEGF) able to induce Four small molecule tyrosine kinase inhibitors have now been
multiple biological actions of endothelial cells, like proliferation approved by the FDA as anti-cancer agents – sunitinib, sorafe-
and migration, as well as vascular permeability enhancement.5
nib, pazopanib and axitinib. An alternative approach is to
VEGF, and particularly its predominant isoform VEGF165 disrupt VEGF–VEGFR interaction through neutralization of
(VEGF-A), exerts its pro-angiogenic action through its binding VEGF by monoclonal antibodies.14–16 Bevacizumab is a huma-
to specific receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR).6–9 nized recombinant monoclonal antibody approved in 2004 as
Recent studies have shown that both receptors are necessary first line therapy for metastatic colorectal cancer, and was the
for human tumor growth and metastasis formation.7 Whilst first antiangiogenic agent launched to the market for the treat-
VEGFR-2 appears to be directly involved in pathological angio- ment of tumor angiogenesis. Additionally, different com-
genesis, VEGFR-1 seems to work as a negative regulator of its pounds have been described as inhibitors of the VEGF–VEGFR
activity,7,10 although its implication in angiogenesis is clear.11 interaction through the binding to either VEGF17 or
VEGFR-1 and 2 receptors contain seven extracellular Ig-like VEGFRs18,19 or co-receptors.20,21 Most of these compounds
have emerged from high throughput screening of peptide and
diverse small-molecule libraries. VEGF and its receptors rep-
aInstituto de Química Médica, CSIC, C/Juan de la Cierva 3, 28006 Madrid, Spain.
resent one of the best validated signaling pathways in angio-
E-mail: pdevega@iqm.csic.es
genesis,22 however very little attention has been paid to the
bInstituto de Química-Física Rocasolano, CSIC, C/Serrano 119, 28006 Madrid, Spain
rational design of compounds to modulate the protein–protein
interactions involved in the molecular recognition between
VEGF and VEGFRs.23–29 In this respect, disruption of the
above-mentioned protein–protein interaction could be a valid
target for the search for inhibitors of angiogenesis. Muta-
genesis data indicated that the VEGF binding sites for
VEGFR-1 and VEGFR-2 receptors are very similar.30,31 Two
cUniversité Paris Descartes, UFR des Sciences Pharmaceutiques et Biologiques, UMR
CNRS 8638, 4 Avenue de l’Observatoire, Paris F-75270, France
dUniversité de Perpignan Via Domitia, Laboratoire de Chimie des Biomolécules et de
l’Environnement, EA 4215, 52 Avenue P. Alduy, Perpignan F-66860, France
eUF Pharmacinétique et Pharmacochimie, GH Cochin – Hôtel Dieu – Broca,
Hôpital Cochin, AP-HP, Paris, France
†Electronic supplementary information (ESI) available: Synthetic procedures,
compounds characterization, CD and NMR data. See DOI: 10.1039/c3ob27312a
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main hot-spots have been identified at the interaction inter- models for the search for new modulators of the VEGF–VEGFR
face between VEGF and these receptors. These are fragments interaction. For the design of VEGF13–25 analogues, key amino
of VEGF17–25, located at the N-terminal and VEGF81–91, located acids Phe17, Tyr21 and Tyr25 (corresponding to residues 5, 9
at loop 3.32–36 We have recently reported the preparation of and 13 in the designed peptides) were maintained while the
cyclic peptide analogues of the second of these two hot-spots, other residues were replaced. Alignment of the N-terminal
37,38
VEGF81–91
.
Molecular modeling and 1H NMR studies on regions of VEGF-A and Vammin permitted the identification of
these peptides indicated a tendency to be structured around the fragment 1–13 of Vammin as the sequence corresponding
the central β-turn of the VEGF81–91 β-hairpin and some of to the VEGF13–25 fragment. In this case, residues Phe5, His9,
them showed significant affinity for VEGFR-1, thus supporting and Ala13, located at the equivalent positions identified as key
the design of mimics of this fragment as a valid approach to in VEGF13–25, were kept intact and only the rest of the residues
disrupt the VEGF–VEGFR-1 interaction.
were modified. From the indicated fragments, virtual collec-
Concerning the less explored N-terminal fragment of tions of linear peptides were generated by replacing each of
VEGF17–25, D’Andrea and co-workers have reported a 15-mer the modifiable residues by the 20 proteinogenic amino acids.
analogue of VEGF14–28 (Ac-KLTWMELYQLAYKGI-NH2) that Then, we examined the helical character of each virtual
exhibits stable helical structure in solution, and pro-angio- peptide using an AGADIR10 algorithm.45
genic properties both in vitro and in vivo.39,40 More recently,
Residues that increased the helical character of the peptide
the same authors described the preparation of a new peptide were then combined to create a second-generation virtual
analogue (Ac-KLTWQELYQLKYKGI-NH2) that shows anti-angio- library. From this virtual collection, two peptides were chosen
genic properties.41 Both peptides bind to the membrane of to be prepared: one analogue of VEGF13–25, compound 2, and
endothelial cells, as observed in cellular assays, but no one of Vammin1–13, 6 (Fig. 1). According to AGADIR prediction,
binding assays with isolated receptors were reported. Within these analogues have higher tendency to adopt helical confor-
the VEGF-A fragment 17–25, a Phe17 residue has been ident- mations (≥60%), than the native sequences 1 and 5 (Fig. 1). An
ified as key for the interaction with VEGF receptors 1 and 2, important issue regarding purification as well as confor-
while Tyr21 and Tyr25 seem to be important for the stabiliz- mational and biological studies of peptides is their aqueous
ation of the α-helical structure.34 Moreover, this fragment has solubility. Taking into account that modifications to increase
been also located at the interaction interface of VEGF and the helicity are often associated with a decrease in solubility, four
Fab fragment of the monoclonal antibody bevacizumab,42 and new peptide analogues (3, 4, 7 and 8), which contain more
some other antibodies and peptides.15,36
polar residues (Ser), were selected. These peptides presumably
Vammin is a VEGF protein isolated from snake venom that would be slightly more soluble in aqueous media than the pre-
exhibits potent antiangiogenic activity both in vitro and in vivo, vious analogues, as estimated by their log S values (calculated
and proved to be specific for the VEGFR-2 receptor in a surface by the AlOGPS 2.1 program),46,47 while still maintaining a
plasmon resonance (SPR) binding assay.43 In addition to good predisposition to adopt helical conformations.
VEGF, Vammin could be also considered a source of peptide
In addition, compounds 10 and 12, two constrained cyclic
analogues for VEGF–VEGFR modulators. The crystal structure peptide analogues of VEGF13–25, were designed (Fig. 2). In this
of Vammin, reported by Morita and co-workers, revealed high case, the strategy to fix the α-helix involved crosslinking amino
structural similarities with VEGF-A,44 and these authors cen- acid side-chains at relative positions i and i + 4, corresponding
tered their structural studies particularly on the Vammin frag- either to residues 3 and 7 (A) or 7 and 11 (B) of the peptide,
ment 69–80, which corresponds to VEGF81–91
.
situated at the opposite face to that containing the residues
Based on all the above precedents, we describe herein our reported as important for the molecular recognition. The
efforts towards the de novo design of helical peptides able to cyclization was performed through the generation of
mimic VEGF13–25 and Vammin1–13 fragments, selected as the
minimum sequence to maintain the native helix confor-
mation. Our main goal is to interfere with the VEGF–VEGFR
interaction and try to identify key elements driving the mole-
cular recognition between these two proteins. The small set of
designed peptides adopted the desired α-helical structures,
similar to those of the parent fragments in the native proteins,
1
as confirmed by CD and H NMR studies. Moreover, most of
them are able to interact with the VEGFR-1 receptor.
Results
Design
Fragments of VEGF-A (13–25) and Vammin (1–13), with helical
structure within the respective native protein, were taken as Fig. 1 Sequences of VEGF13–25, Vammin1–13 and designed analogues 1–8.
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Conformational analysis by circular dichroism
The tendency of the designed peptides to adopt helical confor-
mations was first examined by circular dichroism (CD). CD
spectra of compounds 1–8, 15–18 in aqueous solution are
indicative of mainly disordered peptides. The helix popu-
lations estimated from the observed ellipticity ([θ]) at 222 nm
in water at 5 °C are less than 10% for all peptides, except for
peptides 4, 7, 16 and 18 with helix populations in the 10–25%
range. These results contrast with the helical percentages of
up to 30–65% predicted from the sequence by AGADIR. These
discrepancies arise, in part, from the low solubility shown
by most of the peptides, particularly, the most hydrophobic
(3 and 8), and from inaccurate estimations of peptide concen-
trations from UV absorption values, especially in the case of
the aromatic-lacking Vammin analogues, which translate into
inaccurate ellipticity values ([θ], see Methods). All peptides
become more helical in the presence of TFE, as shown by their
CD spectra that display the helix-characteristic double
minimum at 208 and 222 nm (Fig. 3 and S1–3†). This indi-
cated that the designed peptides have high tendency to be
helical in the appropriate environment.
Fig. 2 Cyclic constrained VEGF13–25 peptide analogues 10 and 12, and the corres-
ponding linear precursors 9, 11.
covalent amide bridges from conveniently situated Glu and Lys
residues, leading to lactam constrained cyclic peptides.
This approach has been widely reported as a successful
strategy for the stabilization of helical conformations.48–50 For
comparative purposes, it is also planned to study linear precur-
sors 9 and 11.
Synthesis
NMR conformational studies
The synthesis of the designed peptides (Fig. 1 and 2) was per-
formed in parallel, following solid-phase protocols, using a To get further details of the structures adopted by the VEGF
Rink amide MBHA polystyrene resin and an Fmoc/tBu strategy. and Vammin analogues, we performed an NMR structural
To prevent aggregation problems, and to favor intramolecular study of the peptides. Taking into account that CD indicated
cyclization when required, a low load resin (0.34 mmol g−1
)
very low helical percentages for all the peptides in aqueous solu-
was used. All compounds were isolated as C-terminal amides tion, together with their low solubility, we decided to study the
and acetylated at the N-terminal.
peptides in the presence of trifluoroethanol.
Cyclic compounds 16 and 18 were prepared by formation of
The VEGF and Vammin analogues were demonstrated to
a side-chain-to-side-chain glutamate–lysine amide linkage, adopt helical structures in the mixed TFE–H2O solvent by the
from linear precursors 15 and 17 respectively (Scheme 1). The set of NOEs, which included the non-sequential helix-charac-
glutamic acid residue was always placed towards the N-term- teristic dαN(i, i+3), dαN (i, i+4) and dαβ (i, i+3) NOEs (see NOE sum-
inal side of the peptide, because a Glu-X-X-X-Lys arrangement maries in Fig. S7–S9†). Some of these non-sequential NOE
promotes higher helicity than the opposite Lys-X-X-X-Glu cross-peaks are seen in the NOESY spectral region shown in
organization, as reported by Mills et al. for a series of poly- Fig. 4 for compound 2.
arginine α-helical peptidomimetics.49 Glu and Lys side-chains
Further evidence to support the helical structure of the pep-
were conveniently protected with allyl and Alloc groups, tides came from the 1Hα and 13Cα chemical shifts, which
respectively, suitable for orthogonal deprotection before on- depend on the ϕ and ψ dihedral angles.56 Thus, the profiles of
resin cyclization. After N-terminal acetylation, and Pd-catalyzed conformational shifts (Δδ = δobserved − δrandom coil, ppm; where
deallylation of the linear precursors 13 and 14, lactamization δrandom coil were taken from Wishart et al.57) presented for all
was performed with the assistance of microwave irradiation, the peptides are characteristic of helical structures, i.e., nega-
using PyAOP as a coupling agent.51–53 15% TFE was used as a tive values for the Hα protons (Fig. 5) and positive values for Cα
cosolvent of DCE, to stabilize the helix conformation and to (Fig. S4–6†). The absolute values for the central residues are
facilitate the cyclization. The reaction was accomplished in 1 h larger than those at the N- and C-termini, which indicates
for both derivatives, leading to the corresponding cyclic com- that, as commonly found in linear helical peptides (i.e. 6), the
pounds. After cleavage from the resin final cyclic peptides were helices are frayed at both ends. This fraying effect is more
isolated, lyophilized and purified by semipreparative HPLC in apparent at the N-terminus of compound 5, the native frag-
good yields. Linear peptide analogues were obtained after a ment of Vammin, which is explained by the presence of a Pro
sequence comprising Fmoc-deprotection, acetylation, removal residue at position 4 (Fig. 1). This Pro probably distorts the ϕ
of allyl groups and final cleavage from the resin. The expected and ψ dihedral angles characteristic of α-helices and, prevents
peptides 9–12 were not isolated as such, but as sulphones, the formation of the first H-bond of the helix, between this
compounds 15–18 (Scheme 1) due to overoxidation of the Met residue and Glu1.
side-chain.54 NMR and mass spectra data of the isolated com-
Apart from the fraying effect, the magnitudes of the confor-
mational shifts differ among the different peptides (Fig. 5).
pounds agreed with the sulphone structure (ESI†).55
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Scheme 1 Synthesis of amide-bridged cyclic peptides 16 and 18. (a) 20% piperidine–DMF; (b) Ac2O, DIEA–DMF; (c) PhSiH3, Pd(PPh3)4, DCM; (d) TFA–EDT–H2O–TIS
(94 : 2.5 : 2.5 : 1); (e) PyAOP–HOAt, DIEA, DMF, 120°, MW.
The larger the magnitudes, the more populated are the
To visualize the helices adopted by the studied peptides, we
helices. Based on the averaged ΔδHα- and ΔδCα-values (Fig. 5), performed structure calculations (see Experimental methods).
the peptides can be qualitatively ranked according to their The resulting structures are well defined (the range of average
helical populations. Additionally, the percentages of helix RMSD for backbone atoms is 0.1–0.4 Å; see Table ST13 in ESI†)
adopted for each peptide can be quantitatively estimated from and, as deduced from analysis of chemical shifts (see above),
the averaged ΔδHα-values, as previously described58,59 show some fraying at the ends (Fig. S10–11†). The C-termini
(Table 1). Qualitatively, the ranking of the peptides in terms of are generally better defined in the VEGF-A than in the
theoretical (Fig. 1) and experimental helicity is coincident. In Vammin analogues. This is probably due to the interactions
general, the peptides with larger helix populations show a between the aromatic residues F5/Y9/Y13, all at the same
larger number of non-sequential NOEs.
helix face, being more stabilizing than the corresponding
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Fig. 3 CD spectra for Vammin1–13 analogues 5–8, in 30% TFE–H2O at pH 5.5
and 5 °C.
Fig. 4 Selected NOESY spectral region for peptide 2 in 30% TFE–H2O at pH 5.5
and 5 °C. The intra-residual Hα-NH cross-peaks are labeled and the non-sequen-
tial dαN(i, i+3) and dαN(i, i+4) boxed. The sequential dαN(i, i+1) are not labeled.
interactions (F5/H9/A13) in the Vammin analogues, as seen in
Fig. 6 for VEGF analogue 2 and Vammin analogue 8. Concern-
ing the N-termini, the helices of the VEGF-A analogues with a
better N-cap residue, such as the N-terminal Glu in peptides 1
and 2, are less frayed than other compounds, for example,
compounds 3 and 4 with two consecutive Ser residues at the
N-end (Table 1). Among the Vammin analogues, the most dis-
ordered at the N-terminus is peptide 5 with a Pro at position 4,
as deduced from conformational shifts (see above).
Concerning the bridged VEGF peptides 16 and 18 (Fig. 2),
the helices adopted by the two peptides with an i/i + 4 bridge
are more populated than their corresponding linear analogues
15 and 17, respectively, but, peptide 16 shows a lower helix
population than peptide 18.
observed
RC
Fig. 5 Histogram showing the ΔδHα values (ΔδHα = δ
− δ , ppm) as a
Hα
Hα
function of the sequence number for: (A) VEGF13–25 analogues 1–4, (B)
Vammin1–13 analogues 5–8, and (C) VEGF13–25 analogues 15–18.
Table 1 Helix populations estimated for peptides 1–8, 15–18 in 30% TFE at pH 5.5 and 25 °C from ΔδHα-values as described in Methods
Peptide
1
2
3
4
5
6
7
8
15
16
17
18
Av. ΔδHα (ppm)
% Helix
−0.20
51
−0.29
74
−0.26
67
−0.27
69
−0.14
36
−0.28
72
−0.23
59
−0.24
62
−0.23
59
−0.30
77
−0.22
56
−0.48
100
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Fig. 6 NMR lowest target function structures of VEGF analogue 2 (left) and
Vammin analogue 8 (right): backbone atoms are shown as ribbons. Side chains
are colored in red for the negatively charged residues, in blue for the positively
charged residues, in cyan for the polar residues, in magenta for the hydrophobic
residues (Val/Leu), and in green for the residues reported as key for the inter-
action with VEGF receptors. Hydrogen atoms are not shown. All the residues are
labeled and the N- and C-ends are indicated. Side chains of Val8 and Ala8 are
not seen because they lie behind the ribbon.
Fig. 7 Dose–response curves for peptides 5, 15–18 on VEGF-1 D1–D3 displace-
ment assays.
shown by cyclic peptides 16 and 18 together with their linear
parent compounds 15 and 17, while AGADIR-based designed
peptides, 2–4, did not show significant binding. Concerning
Vammin analogues, 5–8, the parent Vammin 1–13 native
sequence, 5, together with analogues 6 and 8, showed signifi-
cant affinity for VEGFR-1, with values of the same order as
those found for the VEGF derivatives (Table 2).
Dose–response experiments to determine IC50 values for
most compounds that have shown significant affinity for the
VEGFR-1 receptor were performed. Cyclic constrained VEGF
analogues 16 and 18 and their parent linear precursors, 9 and
17, showed good binding affinities. Cyclic analogue 16 having
the conformational restriction closer to the N-terminus of the
sequence exhibited the best IC50 value found for this collection
of peptides (Table 2, Fig. 7).
Table 2 Inhibitory potency of peptides 1–8, 15–18 on VEGFR-1. Displacement
assays
%Inhibitiona IC50
ECD
Compound Sequence
b(μM)
1
2
3
4
5
6
7
8
Ac-EVVKFMDVYQRSY-NH2
20
3
9
—
—
—
Ac-EVQKFLEVYLRLY-NH2
Ac-SSQKFLEVYQRLYN-NH2
Ac-SSQKFLEVYLRLYN-NH2
Ac-EVRPFLEVHERSA-NH2
Ac-DVRRFLEVHLRLA-NH2
Ac-DLRAFLEQHLRSA-NH2
Ac-SVRRFLEAHLRLA-NH2
Ac-EVEKFM(O2)KVYQRSY-NH2
9
7
N/Ac
N/A
46
47
—
—
—
95 17
36
6
62 18
N/A
42
6
15
16
62 15
Ac-EV-c(NH-CO)3,7[EKFM(O2) K] 56 11
9
VYQRSY-NH2
17
18
Ac-EVVKFM(O2)EVYQKSY-NH2
Ac-EVVKFM(O2)-c(NH-CO)7,11
[EVYQK]SY-NH2
74
53 11
8
43
52
9
6
Discussion and conclusions
A small collection of linear and cyclic peptide analogues (1–8,
15–18) of the fragment 13–25 of VEGF, previously identified as
a hot-spot of the VEGF–VEGFR interaction, and of the corres-
ponding fragment of Vammin, 1–13, have been designed and
synthesized by solid-phase techniques. NMR conformational
studies (chemical shift deviation and non-sequential NOE)
showed that, in TFE–H2O solution, these peptides adopted the
desired native-like helical conformation. In general, and as
a Activity corresponds to the percentage of biotinylated VEGF165
displaced by a concentration of peptide of 100 μM on the whole
extracellular domain (ECD, D1–D7) of VEGFR-1. b Inhibitory
concentration able to displace 50% binding of biotinylated VEGF165
on D1–D3 VEGFR-1. c N/A = no affinity observed.
Binding affinity for VEGFR-1
Synthesized peptides were evaluated for their ability to dis- expected, the designed peptides were more structured than the
place biotinylated VEGF165, in a chemiluminescent assay parent compounds 1 and 5.
relying on competition between tested compounds and bioti-
When evaluated as inhibitors of the VEGF–VEGFR inter-
nylated VEGF165 for binding to the extracellular domain of action most peptides proved to bind to the VEGFR-1 receptor,
recombinant VEGFR-128,60 (D1–D7, Table 2). Some of the best the VEGF cyclic analogue 16 exhibiting the best IC50 value. A
compounds were selected for dose–response binding studies combined analysis of structural and biological results indi-
on the D1–D3 domain of the VEGFR-1 receptor, the specific cated that to interact with VEGFR-1, the peptides needed to
domains for VEGF binding.
have a certain tendency to be helical, but once a minimum
Some of the prepared peptides exhibited good inhibition helical population is reached the affinity of the peptides does
percentages. Thus, for VEGF analogues, the best results were not increase with increments in helicity. Detailed comparison
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among the sequences of the designed analogues and the can be attributed to the fact that their spatial conformation
native VEGF17–25 and Vammin1–13 native sequences indicates mimics better the helical conformation adopted by the parent
that the two N-terminal residues, Glu1 and Val2, of 1 and 5 native fragments in the protein, while in the VEGF81–91 ana-
could be directly participating in the VEGF–VEGFR interaction. logues only the β-turn of the native β-hairpin was mimicked.
Thus, VEGF analogues 3 and 4, in which Val2 has been This is also in accordance with the improved ability to displace
replaced by a Ser, and Vammin analogue 7, in which it has the VEGFR-1–VEGF interaction observed for the helical 16–27
been replaced by Leu, showed no affinity for the VEGFR-1 fragment of VEGF as compared to the VEGF61–68 sequence,
receptor. Although this last change is more conservative, the proving once more that the 16–27 helical fragment constitutes
concomitant replacement of Val8 by Gln in 7 could also con- a hot-spot in the VEGF–VEGFR-1 interaction.24
tribute to the lack of affinity. This Val residue is in the same
In summary, Vammin analogues 5, 6 and 8, and cyclic con-
face of the helix as key residues Phe17, Tyr21, suggesting the strained VEGF-A derivatives 16 and 18 as well as their precur-
importance of a hydrophobic region for the interaction. On sors 15 and 17 were able to interfere with the process of
the other hand, the Glu1 residue may play a stabilizing role on VEGF–VEGFR-1 recognition. Compound 16, one of the pep-
the helical structure, as it is able to form H-bonds between its tides with higher helical character, showed the best IC50 value
side chain and unpaired main-chain NH and CO groups of the in the binding assay. As a conclusion, it can be inferred that
first turn. Accordingly, it can be replaced by residues with VEGF13–25, corresponding to one of the identified hot-spots for
N-cap properties as Ser or Asp (compare compound 5 with 6 the VEGF–VEGFR interaction, and its homologue Vammin1–13
,
and 8).61 In the case of peptides 3 and 4, the extra C-terminal can be taken as templates for the design of peptide mimics
residue (Asn) might also be responsible for the drop in affinity. able to interfere with the interaction of VEGF with its recep-
As for the inhibition value found for analogue 2 was not very tors. The detailed analysis of the peptide sequences suggests a
reliable, taking into account the low water solubility and the role for Glu1, as a helix initiator, and for Val2 and Val8 resi-
high ability for aggregation shown by this peptide at the con- dues as relevant for the interaction with VEGFR-1, likely being
centration used in the binding assay.
important as a hydrophobic patch, together with Phe5, Tyr9,
Cyclic constrained VEGF analogues 16 and 18 and their Tyr13. In addition, the obtained results suggest that the speci-
parent linear precursors 15 and 17 showed good VEGFR-1 ficity of Vammin for the VEGFR-2 receptor does not rely on the
affinity values. Linear peptides 15 and 17 showed 3–4 fold N-terminal region of the molecule, although minor modifi-
increased affinity for VEGFR-1 over the native fragment 1, and cations in this fragment can lead to the loss of VEGFR-1
a slightly higher helicity. Compound 15 encompasses two affinity (i.e., compound 7). These results encourage us to
radical, non-conservative changes: Val3 has been replaced by prepare more VEGF and Vammin analogues to selectively inter-
Glu, and Asp7 by Lys, while in the case of peptide 17, the fere with the VEGF–VEGFR-1 or VEGF–VEGFR-2 interaction,
changes are much more conservative: Asp7 by Glu and Arg11 which can constitute interesting tools in pharmacology.
by Lys. However, all these modifications improve the affinity
for the receptor, suggesting that the original residues are not
very important for binding. Therefore, the improved affinities
of 15 and 17 could be ascribed to the increased helical charac-
ter, since the formation of ionic bridges between Glu and Lys
Experimental
Synthesis of linear and cyclic derivatives 1–8, 15–18. General
procedure
residues could contribute to the stabilization of their helical
conformation (Table 2, Fig. 7).62 Cyclic analogues 16 and 18 Resin bound peptides were synthesized by conventional Fmoc
also proved to have more helical character than the parent solid-phase chemistry on an MBHA-Rink amide resin.
compound 1, and more than linear precursors 15 and 17. As
Resins were swollen in DCM/DMF/DCM/DMF (4 × 0.5 min).
already mentioned, cyclic peptide 16 is the best compound All compounds were synthesized manually in parallel on resin,
within this collection in terms of affinity for the VEGFR-1. following the Fmoc/tBu strategy, using Fmoc-Glu(OtBu)-OH,
However, peptide 18, with higher helicity than 16, showed less Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Leu-
affinity for the receptor. The masking of the basic character of OH, Fmoc-Ile-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
the Arg11 residue, by forming the lactam cycle, could be His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-
responsible for the slight drop in affinity observed for 18 com- Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-
pared with its parent linear compound 17 that still has a basic OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH or Fmoc-Glu(All)-OH
Lys residue at the place of Arg11. The results found for com- as required.
pound 5, native sequence of Vammin, together with analogues
In each coupling step, the appropriate Fmoc amino acid
6 and 8, showing non-negligible IC50 values for binding with (1.5 equiv.) was treated with HOBt/DIC (1.5 equiv.) in anhy-
VEGFR-1, suggest that the specificity of Vammin for VEGFR-2 drous DMF or with HCTU/DIEA (2 equiv.). Couplings were
versus VEGFR-143 seems not to rely on this part of the Vammin allowed to proceed at room temperature overnight or for 1 h,
molecule (Table 2, Fig. 7).
respectively. The coupling efficiency was monitored by ninhy-
Compared with the previously prepared analogues of the drin or chloranil test, and when necessary repeated with a
VEGF81–91 fragment,37,38 it can be said that the affinities found fresh portion of Fmoc-amino acid and the indicated coupling
for the helical peptides described here are slightly better. This reagents. After each coupling step, the Fmoc group was
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removed by treatment with 20% piperidine in DMF spectrum and zero-filled to a 2K × 1K complex matrix prior to
(3 × 10 min). The resin was washed and drained with Fourier transformation. Baseline correction was applied in
DMF/DCM/DMF/DCM (4 × 0.5 min).
both dimensions. The 0 ppm 13C δ-value was obtained
For amide bridge formation, after removing Alloc and OAll indirectly by multiplying the spectrometer frequency that
protecting groups, the linear-resin-bound derivative (100 mg, corresponds to 0 ppm in the 1H spectrum, assigned to an
0.034 mmol) was treated with a solution of PyAOP (177 mg, internal DSS reference, by 0.25144953.65 Standard sequential
0.34 mmol), HOAt (46 mg, 0.34 mmol), DIEA (0.12 ml, assignment methods66,67 were applied to assign the 1H NMR
0.68 mmol) in anhydrous DMF (2 ml). The reaction was signals of the peptides. Then, the 13C resonances were straight-
allowed to proceed under microwave irradiation for 1 h until forwardly assigned based on the cross-correlations observed in
ninhydrin test was negative. The resin was washed and the HSQC spectra between the proton and the bound carbon
drained with DMF/DCM/DMF/DCM (4 × 0.5 min).
(1H and 13C chemical shifts for all the compounds are listed in
Before cleavage from the resin, all derivatives were acetyl- Tables ST1–ST12 as ESI†).
ated by treatment with a mixture of Ac2O–DIEA in DMF
NMR structure calculation
(4 × 10 min) and then washed with DMF and DCM. For cyclic
analogues, acetylation was performed before Alloc and OAll Distance constraints were derived from the 2D 150 ms mixing
deprotection prior to cyclization. Finally, the resin was treated time NOESY spectra. The NOE cross-peaks were integrated by
with the cleavage cocktail TFA–EDT–H2O–TIS (94 : 2.5 : 2.5 : 1) using the automatic integration subroutine of the SPARKY
for 5 h at room temperature. The filtrates were precipitated program68 and then calibrated and converted to upper-limit
from diethyl ether, centrifuged and lyophilised, and the result- distance constraints within the CYANA program. After cali-
ing mixture was purified by reverse phase semipreparative bration, we excluded from the input list of distance constraints
HPLC to obtain the peptides 1–8, 15–18 in high purities those for which contribution of random conformations can
(>98%, HPLC-MS data) and 1–9% overall yield (calculated on be large, i.e. intra-residual and sequential, except for the
the bases of resin substitution). See ESI† for details.
dNHi–NHi+1, which are characteristic of helices and absent or
very weak in random conformations. ϕ and ψ angle restraints
were obtained from 1Hα, 13Cα and 13Cβ chemical shifts using
Circular dichroism spectroscopy
CD spectra were recorded on
equipped with a Peltier temperature control unit, at 5 °C both which the derived angle restraints were ambiguous were
a
Jasco J-810 instrument the TALOS program.69 The ϕ angles for those residues for
in H2O and in 30% TFE–H2O solutions.
restricted to the −180° to 0° range. A total of 50 structures
were calculated using the standard annealing strategy of the
CYANA program.70 The 20 structures with the lowest target
2D NMR spectroscopy
Samples were prepared by dissolving the lyophilized peptide function values were selected and energy-minimised within
(∼1 mg) in 0.4 ml of H2O–D2O 9 : 1 v/v, adjusting the pH to 5.5 the CYANA program. The structures were examined using the
by adding minimal amounts of NaOD or DCl, and adding the program MOLMOL.71
amount of deuterated TFE (0.17 ml) necessary to give a TFE/
water ratio of 30 : 70 in volume. Resulting peptide concen-
Chemiluminescent competition assays
trations were about 1 mM. pH was measured with a glass The surface of a white high-binding 96-well microplate was
microelectrode and not corrected for isotope effects. The temp- coated with 100 μl of phosphate-buffered saline solution (PBS,
erature of the NMR probe was calibrated using a methanol pH 7.4) containing either VEGFR-1 D1–D3/Fc Chimera (20 ng
sample. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) per well) or VEGFR-1 D1–D3/Fc chimera (15 ng per well) and
was used as an internal reference. NMR spectra were acquired incubated at 4 °C overnight. After three washes with 250 μl of
at 25 °C in a Bruker AV-600 spectrometer operating at a proton PBS 0.1%, (v/v) Tween 20 (buffer A), the plate was blocked by
frequency of 600.13 MHz and equipped with a cryoprobe. As 200 μl of PBS with 3% (w/v) of BSA and stirred at 37 °C for 2 h.
previously reported,63 phase-sensitive two-dimensional corre- The plate was washed three times with buffer A. Then, 100 μl
lated spectroscopy (COSY), total correlated spectroscopy of a solution of btVEGF165 at 131 pM (5 ng mL−1) and the
(TOCSY), nuclear Overhauser enhancement spectroscopy tested compounds at various concentrations diluted in PBS
(NOESY) spectra and 1H–13C heteronuclear single quantum containing 1% DMSO were added to each well. After 3 h stir-
coherence spectra (HSQC) at natural 13C abundance were ring at 37 °C, the plate was washed four times with buffer A
recorded by standard techniques using presaturation of the and 100 μl of streptavidin–horseradish peroxidase diluted at
water signal and the time-proportional phase incrementation 1 : 8000 in PBS containing 0.1% (v/v) Tween 20 and 0.3% (w/v)
mode. NOESY mixing times were 150 ms and TOCSY spectra BSA were added per well. After 1 h of incubation at 37 °C in
were acquired using 60 ms DIPSI2 with a z filter spin-lock the dark and stirring, the plate was washed five times with
sequence. Acquisition data matrices were defined by 2018 × 250 μl of buffer A and 100 μl of the chemiluminescent sub-
512 points in t2 and t1, respectively. Data were processed using strate were added. The remaining bt-VEGF165 was detected by
the standard TOPSPIN program.64 The 2D data matrix was chemiluminescence, which was quantified. The percentages of
multiplied by either a square-sine-bell or a sine-bell window btVEGF165 displacement were calculated by the following
function with the corresponding shift optimized for every formula: percentage of displacement = 100 × [1 − (S − NS)/
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Paper
Organic & Biomolecular Chemistry
(MS − NS)], where S is the signal measured, NS is the non- 18 D. Aviezer, S. Cotton, M. David, A. Segev, N. Khaselev,
specific binding signal and MS is the maximum binding
N. Galili, Z. Gross and A. Yayon, Cancer Res., 2000, 60,
signal observed with btVEGF165 without compounds tested.
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Acknowledgements
This work was supported by the Spanish Ministry of Science
and Innovation (SAF 2006-01205, SAF 2009-09323, CTQ2008- 21 A. Starzec, R. Vassy, A. Martin, M. Lecouvey,
0080/BQU and CTQ2011-22514), the CSIC (PIF 2005-80F0160
and PIE 2006-80I066) and the ANR (ANR-2012-BLAN-1533,
M. Di Benedetto, M. Crépin and G. Y. Perret, Life Sci., 2006,
79, 2370–2381.
project Salsa). M. I. G.-A. thanks the CSIC for a predoctoral 22 N. Ferrara, H. P. Gerber and J. LeCouter, Nat. Med., 2003, 9,
fellowship (JAE-Predoc, from “Junta para la Ampliación de 669–676.
Estudios”, co-financed by FSE), and the SEQT (Spanish Society 23 L. Zilberberg, S. Shinkaruk, O. Lequin, B. Rousseau,
of Therapeutic Chemistry) for the Ramón Madroñero award
(XIV edition, 2009).
M. Hagedorn, F. Costa, D. Caronzolo, M. Balke, X. Canron,
O. Convert, G. Laïn, K. Gionnet, M. Gonçalves, M. Bayle,
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C. Garbay, M. Vidal and N. Inguimbert, J. Med. Chem.,
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