Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction

Article abstract of DOI:10.1021/jm901755g

We report the molecular design and synthesis of EG00229,2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRPl) and the structural characterization of NRPl-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRPl bl domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRPl and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

Full text of DOI:10.1021/jm901755g

J. Med. Chem. 2010, 53, 2215–2226 2215
DOI: 10.1021/jm901755g
Small Molecule Inhibitors of the Neuropilin-1 Vascular Endothelial Growth Factor A (VEGF-A)
Interaction†
Ashley Jarvis,‡ Charles K. Allerston,§ Haiyan Jia, ,# Birger Herzog, ,# Acely Garza-Garcia,^ Natalie Winfield,‡ Katie Ellard,‡
,#
Rehan Aqil,‡ Rosemary Lynch,‡ Chris Chapman,‡ Basil Hartzoulakis,‡ James Nally,‡ Mark Stewart,‡ Lili Cheng,
Malini Menon, ,# Michelle Tickner, ,# Snezana Djordjevic,¥ Paul C. Driscoll,^ Ian Zachary,# and David L. Selwood*,ꢀ
‡Domainex Ltd. (NCE Discovery), 324 Cambridge Science Park, Cambridge CB4 0WG, U.K., §Ark Therapeutics, Structural and Molecular
Biology, UCL, Gower Street, London WC1E 6BT, U.K., Ark Therapeutics Ltd., Rayne Building, 5 University Street, London WC1E 6JJ, U.K.,
^Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K., ¥Structural
and Molecular Biology, UCL, Gower Street, London WC1E 6BT, U.K., #Centre for Cardiovascular Biology and Medicine, BHF Laboratories at
UCL, 5 University Street, London WC1E 6JJ, U.K., and ꢀBiological and Medicinal Chemistry Group, Wolfson Institute for Biomedical Research,
UCL, Gower Street, London WC1E 6BT, U.K.
Received November 27, 2009
We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A
receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR
spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1
domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis
for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and
attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was
also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of
the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis
for design of specific small molecule inhibitors of ligand binding to NRP1.
Introduction
effects of the inhibitory anti-VEGF-A antibody, bevacizumab,
in mouse xenograft models.3 As an alternative to biological
therapeutics, small molecule inhibitors of NRP1 function would
be desirable, but development of protein-protein interaction
inhibitors is not a trivial task.4,5 We utilized the bicyclic peptide
1, corresponding to the C-terminal 28 amino acids of VEGF-
A165 (Figure 2) as a starting point for small molecule design.
From this peptide we developed EG00229, 2 (Figure 2), a small
molecule designed to interact with the VEGF-A165 binding
pocket of NRP1. Mutational analysis, NMR, and X-ray crys-
tallography establish that the interaction with NRP1 of peptide
ligands (and by inference VEGF-A) and the new small mole-
cules described herein is with the same binding site formed by
the loops at the end of the b1 domain.6 These molecules act as
inhibitors of VEGF-A function, reducing VEGF-A receptor
phosphorylation and endothelial cell migration. The in vitro
cytotoxic effect of paclitaxel and 5-fluorouracil was enhanced in
the presence of 2. Small molecule inhibitors of NRP1 have
considerable potential as novel anticancer therapeutics.
Neuropilin 1 (NRP1a)1 is a receptor for vascular endothelial
growth factor A165 (VEGF-A165) and the neuronal guidance
molecule semaphorin 3A (SEMA3A)2 with key roles in vascular
and neuronal development (Figure 1). In endothelial cells,
NRP1 enhances the biological signals of VEGF-A mediated
by binding to its receptor vascular endothelial growth factor 2
(VEGFR2). NRP1 has also been implicated in tumor growth
and angiogenesis; inhibition by a blocking antibody that pre-
vents VEGF-A binding to NRP1 enhanced the antitumor
†Atomic coordinates and structure factors for the reported crystal
structure have been deposited in the Protein Data Bank under accession
code 3I97.
*To whom correspondence should be addressed. Phone: þ44 207 679
6716. Fax: þ44 207 209 0470. E-mail: d.selwood@ucl.ac.uk.
a Abbreviations: G, glycine; P, proline; A, alanine; V, valine; L,
leucine; I, isoleucine; M, methionine; C, cysteine; F, phenylalanine; Y,
tyrosine; W, tryptophan; H, histidine; K, lysine; R, arginine; Q, gluta-
mine; N, asparagine; E, glutamic acid; D, aspartic acid; S, serine; T,
threonine; bt, biotinylated; Boc, tert-butyloxycarbonyl; BSA, bovine
serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; DMSO,
dimethyl sulfoxide; EBM, endothelial cell basal medium; ELISA, en-
zyme-linked immunosorbent assay; FBS, fetal bovine serum; FMOC, 9-
fluorenylmethyl)oxy]carbonyl; 5-FU, 5-fluorouracil; HEPES, N-2-hy-
droxyethylpiperazine-N0-2-ethanesulfonic acid; HSQC, heteronuclear
single quantum coherence; HUVECs, human umbilical vein endothelial
cells; NRP1, neuropilin 1; NRP2, neuropilin 2; Pbf, 2,2,4,6,7-penta-
methyldihydrobenzofuran-5-sulfonyl; PBS, phosphate buffered saline;
PyBrOP, bromo-tris-pyrrolidinophosphonium hexafluorophosphate;
PAE, porcine aortic endothelial; SDS, sodium dodecyl sulfate; SE-
MA3A, semaphorin 3A; TIPS, triisopropylsilane; TFA, trifluoroacetic
acid; Tris, tris(hydroxymethyl)aminomethane; VEGF, vascular en-
dothelial growth factor; VEGFR2, vascular endothelial growth factor
receptor 2.
Results and Discussion
Computational Prediction of the Binding Pocket on NRP1
and Mutational Analysis of VEGF-A Binding. The reported
crystal structures6,7 and our own computational analysis of the
NRP b1 domain using SYBYL SITEID identified the cleft
formed by the loops at one end of the β-barrel as a potential
binding site (Figure 3a). Residues clustered in this region8 were
conserved in mammalian NRP1 species and in human NRP2, a
closely related receptor for VEGF-A1 (Figure 3b), implying an
important functional role in VEGF-A binding. Mutational
r
2010 American Chemical Society
Published on Web 02/12/2010
pubs.acs.org/jmc

2216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Jarvis et al.
Loss of binding was not due to impaired expression of NRP1
mutants, as Western blot analysis of transfected COS-7 cells
indicated similar levels of protein expression of all constructs
(Supporting Information Figure S1b). A triple mutant b1
protein (S346A, E348A, T349A) was previously shown to
prevent VEGF-A binding to rat NRP1.7
NMR Studies with the Peptide. 1, Tuftsin, and Ac-DKPRR-
OH Provide Additional Evidence To Identify the NRP1
Binding Pocket for the C-terminus of VEGF-A. The binding
of ligands was assessed by chemical shift perturbation map-
ping with 2D 15N,1H NMR of 15N-labeled NRP1 b1
(Figure 4b). In each case the residues with significant per-
turbations of the backbone NH cross-peak chemical shifts
were identified (compound Δδ > 0.15; see Experimental
Section). We employed tuftsin as a control ligand for these
studies because its binding site has been established by X-ray
crystallography.7 Titration with 1, tuftsin (H-TKPR-OH),
and the linear peptide Ac-DKPRR-OH (C-terminal five
residues of VEGF-A) yielded highly similar patterns of
chemical shift changes that mapped the VEGF-A interaction
site (Figure 4c,d and Supporting Information Figure S2).
The largest chemical shift changes were observed for the
backbone NH groups of W301 (adjacent to C-terminal end
of loop 1); T316, G318, Y322 (loop 5); A344, I345, K347,
T349, K351 (loop 3); and G414, M417 (loop 4) (Supporting
Information Figure S3). The binding site revealed by NMR
was identical to that predicted6 and previously shown for
tuftsin7 and very similar to a map of the key residues essential
for binding (Figure 4e). These data clearly indicate that 1 and
the peptide Ac-DKPRR-OH bind in a common mode to the
same site as tuftsin and VEGF-A, providing a strong ratio-
nale for modeling the interaction of small molecule inhibitors
with this binding pocket. These data imply that small
molecules blocking this site could interfere with the binding
(and function) of VEGF, and other molecules that bind this
site.
Figure 1. Model for binding of VEGF-A165 to NRP1. NRP1 has a
large extracellular (Ex) domain comprising tandem a1/a2, b1/2, and
a c domain, a single membrane-spanning domain, and a small
cytosolic domain (Cyt). The VEGF-A165 C-terminal domain en-
coded by exons 7 and 8 (yellow and blue oblongs, respectively) binds
to the extracellular NRP1 b1 domain. Concomitant binding of the
VEGF homology domain of VEGF-A165 (solid red ovals) to
VEGFR2 results in formation of a receptor complex of NRP1 with
VEGF-A165 and VEGFR2 and enhanced intracellular signaling,
essential for optimal migration and angiogenesis in development
and in tumors.
Synthetic Chemistry. Simple linear peptides were synthe-
sized by standard 9-fluorenylmethoxycarbonyl (FMOC)
solid phase methodology as previously reported.9 For the
small molecules a highly flexible mixed solution phase/solid
phase synthesis was devised that allowed the preparation of
multiple analogues in a timely fashion. The lysine analogue
and linking scaffold were assembled in solution phase and
then coupled in one unit to the arginine-O-Wang resin using
standard coupling conditions (Scheme 1). A representative
example is shown in Scheme 2, which illustrates the range of
chemical manipulations applied to different building blocks.
Thus, Suzuki coupling of 3-nitroiodobenzene 3 with 3-
methyphenylboronic acid 4 provided the biphenyl derivative
5 which was oxidized to the acid 6 using potassium perman-
ganate. Coupling to preformed Wang-Arg resin with the
reactive coupling agent bromo-tris-pyrrolidinophospho-
nium hexafluorophosphate (PyBrop) gave the nitro com-
pound 7 linked to solid phase. Tin(II) chloride reduction
followed by coupling with the Boc (tert-butyloxycarbonyl)
protected acid gave the intermediate 8. The target compound
9 was then produced by deprotection with trifluoroacetic
acid/triisopropylsilane (TFA/TIPS). For further testing lar-
ger amounts of compounds were required, and these were syn-
thesized by solution phase chemistry as shown in Scheme 3
for 2. Reaction of the thiadiazolesulfonyl chloride 10 with
methyl 3-aminothiophene-2-carboxylate 11 was achieved in
pyridine at room temperature to give the sulfonamide 12.
Hydrolysis to the acid 13 with lithium hydroxide and amide
Figure 2. Bicyclic peptide 1 (C-terminus of VEGF) and small
molecule neuropilin inhibitor 2.
analysis of VEGF-A binding to NRP1 was therefore performed
to confirm the identity of the binding pocket. Alanine substitu-
tion of amino acid Y297, W301, T316, D320, S346, T349,
Y353, or W411 resulted in complete loss of high affinity
biotinylated-VEGF-A binding to COS-7 cells transfected with
mutant NRP1 cDNA constructs (Figure 4a). In addition,
alanine substitution of K351 resulted in partial loss of
VEGF-A binding, while mutation of T337, P398, and S416
caused modest decreases in binding and mutation of E319 had
no effect (Figure 4a and Supporting Information Figure S1a).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2217
Figure 3. (a) VEGF/tuftsin binding site of NRP1 b1 domain (black arrow), with the protein surface and the loops L1 (green), L2 (yellow), L3
(cyan), L4 (pink), L5 (red), and L6 (black) shown. Model constructed from PDB code 2ORZ. (b) Protein sequence alignment of human, mouse,
and rat NRP1 (hNRP1, mNRP1, rNRP1) with human NRP2 (hNRP2). Highlighted residues were predicted to be in close contact with bound
ligand from the model in panel a.
coupling of 13 with the 2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl (Pbf) protected arginine methyl ester 14
gave 15. Finally deprotection with lithium hydroxide fol-
lowed by TFA and water gave 2.
heterocycles were less effective (data not shown). Reversal
of the sulfonamide linkage 31 or replacement of the thiophene
witha phenyl ring32removed activity, asdidremovalof the 4-
aminophenylsulfonamide group 33. The best scaffold exem-
plified by 30 was progressed with an extensive structure-
activity study; salient features are shown (Table 3). Thus,
H-bond acceptor groups in the 2 or 3 position of the aromatic
or aryl group were beneficial, 34 and 35, whereas methylation
of the sulfonamide NH removed activity, 36 and 40. Other 4-
Design of Small Molecules Mimicking the C-Terminal
Amino Acid Residues of 1. Our strategy for designing
small molecule inhibitors was initially to perform structure-
activity relationship studies of short peptides in assays of
VEGF-A binding to porcine aortic endothelial (PAE)
cells expressing NRP1 in the absence of other VEGF recep-
tors (PAE/NRP1) and thereby to identify the essential
properties of peptides necessary for inhibition of binding.
With this information, we then constructed a design template
for the peptide-NRP1 interaction (Scheme 1, box) and
undertook a systematic search for chemistry scaffolds that
would retain the high affinity of the peptides in binding
assays.
substitutions were tolerated, 37 and 38, while
a
2-nitro group, 39, showed the best activity of the simple
substituents. A fluorinated analogue, 41, was less active.
Insertion of a benzothiadiazole heteroaryl produced com-
pound 2, with an IC50 of 8 μM in a cell based assay. The
sulfur atom had a positive effect on potency because the O
analogue 42 was less active. Other similarly active compounds
were found, such as 43 and 44. Most alterations to the
arginine, including the unnatural D-enantiomer 45 or 46
(Table 4) or substitution by lysine 47, resulted in a partial or
complete loss of activity (Table 4). The methyl ester 48 had
poor activity, while replacement of the alkyl side chain by
phenyl, 49, also reduced activity. Extension of the alkyl chain
to four carbons, 50, again reduced activity; however, intro-
duction of a carbonyl group into the side chain of the arginine
51 was partially tolerated. Methylation of the guanidine as in
52 or the nitroguanidine 53 had poor activity. Further manip-
ulations to the carboxylic acid group in the thiadiazole series,
such as reduction to the alcohol 54 or conversion to an amino
acid mimic 55 were deleterious. In contrast the hydroxamic
acid group in 56 was at least partially tolerated . Thus, 2 was
identified as a non-peptide antagonist of VEGF-A binding to
NRP1.
The C-terminal peptide Ac-RXDKPRR9 of VEGF-A re-
tains significant affinity for NRP1. Substitution of the non-
essential penultimate arginine to alanine 16 and the synthesis
of increasingly N terminal truncated peptides produced
H-KPAR-OH, 17, a minimal length significantly active pep-
tide (Table 1). Substitution with D-amino acids for the lysine
or alanine 18, 19 lowered inhibitory activity. The penultimate
position was tolerant of proline 20 and phenylalanine 21 in
addition to alanine. The terminal arginine was essential for
activity, as replacement with D-arginine 22, lysine 23, or
glutamine 24 removed activity. The essential C-terminal
arginine was retained with different chemical scaffolds being
used to connect it to the lysine (equivalent to K162 in VEGF-
A165) (Table 2). These scaffolds were selected on the basis of
an approximate modeling fit of the full small molecule
mimetic with the H-KPAR-OH peptide, and a representative
subset is shown. Biphenyl scaffolds 9, 25, furanylenephenyl
26, and cycloalkylaryl scaffolds 27, 28, 29 gave modest inhi-
bitions. However a thiophene-based scaffold30 had an IC50 of
13 μM for inhibition of VEGF-A165 binding to NRP1.
Follow-up studies showed that other five-membered ring
NMR and X-ray Structural Data on 2 Confirm Binding to
the Cleft Formed by the NRP1 Loops and Indicate a Key
Hydrogen Bond in the Structure Stabilizing the Bound Con-
formation. Definitive evidence for the binding site of 2 was
provided by monitoring the NRP1 b1 domain 2D 15N,1H-
heteronuclear single quantum coherence (HSQC) NMR

2218 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Jarvis et al.
Figure 4. (a) Mutational analysis of the NRP1 pocket. COS-7 cells were transfected with expression plasmids for wild-type (WT)
or mutant NRP1 as indicated. Binding assays using bt-VEGF-A165 were performed 48 h after transfection. Values presented are the
means ( SD obtained from three to six independent experiments each performed in duplicate. (b) An excerpted region of the 2D 15N,1H
HSQC NMR spectrum of human NRP1 b1 in the free state (black contours) and with saturating concentrations of tuftsin (blue) and
1 (green). The cross-peak assignments for the free-state NRP1 b1 are indicated in bold. Arrows depict significant ligand-dependent
chemical shift changes. (c) Solvent accessible surface representations of the structure NRP1 b1 with color highlighting showing
those residues whose backbone amide NH cross-peak exhibits a significant chemical shift perturbation (compound Δδ > 0.15; see
Experimental Section) upon addition of a saturating concentration of tuftsin. (d) As for (c) but with 1 as the ligand. The intensity of the
color is scaled to the residue-by-residue value of the compound Δδ. (e) Pymol diagram showing positions of mutated residues on
the NRP1 b1 protein surface. Mutations giving 100% inhibition of binding are shown as red, and moderate inhibition is shown
as salmon (K351).
spectrum in the presence of increasing concentrations of the
ligand. 15N,1H HSQC spectra are universally used to moni-
tor the local “chemical environment” of NH groups, and in a
ligand titration, the exchange regime exhibited by the per-
turbed cross-peaks can be related to the ligand binding
kinetics such that slow-intermediate exchange behavior is
typically observed for ligands with a slow off-rate consistent
with tight binding. Consistent with the measured IC50 for
NRP1 b1 binding, the NMR titration yielded intermediate-
to-slow exchange characteristics for the majority of per-
turbed resonances. Both the distribution of residues that
exhibited ligand-dependent chemical shift perturbations
(Supporting Information Figures S2 and S3) and the direc-
tional pattern of those chemical shift changes (Supporting
Information Figure S4) are highly similar to those obtained
with 1, Ac-DKPRR-OH, and tuftsin. These data point to the
conservation of interactions between the backbone and side
chain elements of the terminal arginyl moiety of 2 with the
binding pocket on NRP1 b1.
b1. The crystal structure, containing a dimer in the asym-
metric unit (interchain CR root-mean-square deviation of
˚
˚
<0.5 A), was determined at 2.9 A resolution, with electron
density readily observable for 2 in the binding pockets of
both noncrystallographic symmetry related molecules
(Figure 5a). The carboxylate group of 2 superimposes
almost perfectly with the carboxylate group of tuftsin in
the rat NRP1 b1b2 crystal structure (Supporting Informa-
tion Figure S5a).7 The guanidine groups of the two ligands
also overlay. The structure also reveals an intramolecular
shared hydrogen bond (Figure 5b) between the carbonyl
amide NH and the sulfonamide nitrogen, providing a
stable central conformation of the molecule. We postulate
that this bond is critical for biological activity. In chain A
the benzothiadiazole ring adopts a conformation on the
same side to the guanidine chain with respect to the central
planar portion of the molecule containing the thiophene
ring. In chain B the benzothiadiazole ring is on the opposite
side. In both arrangements the benzothiadiazole ring is
involved in stacking interactions with a side chain residue.
In chain A this interaction is provided by the M276B side
The precise binding mode and ligand conformation of 2
were established by X-ray crystallography of human NRP1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2219
Scheme 3. Solution Phase Methodologya
Scheme 1. Mixed Solid Phase-Solution Phase Synthetic Strategy
Scheme 2. Typical Analogue Synthesisa
a Reagents: (a) pyridine, 20 ꢀC; (b) LiOH H2O, 50 ꢀC, THF, MeOH,
3
H2O; (c) PyBrOP, DIPEA, DCM, 20 ꢀC; (d) TFA/H2O 9:1.
Table 1. Establishing Small Peptide Structure-Activity Relationshipsa
inhibition of 125I-VEGF binding to
PAE/NRP1 cells
compd
structure
at 100 μM
IC50 (μM)
16
17
18
19
20
21
22
23
24
Ac-RXbDKPAR-OH
H-KPAR-OH
H-kPAR-OH
H-KPaR-OH
H-KPPR-OH
H-KPFR-OH
H-KPAr-OH
71 ( 6.8%
83 ( 1.2%
51 ( 7.9%
36 ( 5.1%
96 ( 1.2%
63 ( 3.3%
22 ( 9.5%
22 ( 1.2%
19 ( 7.6%
18 ( 0.11
30 ( 0.04
14 ( 0.01
H-KPAK-OH
H-KPAQ-OH
a IC50 values were measured on all compounds showing inhibition of
>70%. PAE/NRP1 cells in 24-well plates were incubated with different
concentrations (0.1-100 μM) of compounds followed by addition of
0.1 nM 125I-VEGF-A165. Nonspecific binding was determined in the
presence of 100-fold excess unlabeled VEGF. Results were obtained
from three independent experiments, each performed using triplicate
determinations at each concentration of compound, and are presented
as the mean ( SEM. R2 values ranged from 0.9874 to 0.9996. b X
is 2-aminobutyric acid.
a Reagents: (a) Pd2(dba)3, Pd(PPh3)4, KF, dioxane, reflux 24 h;
(b) KMnO4, py, H2O, reflux 3 h; (c) HOBt, DCI, DMF, Wang-Arg-OH;
(d) SnCl2 H2O, DMF; (e) PyBrOP, RCOOH, 2,6-lutidine, CH2Cl2;
pocket binding the arginine seems to be an almost perfect
“arginine receptor”, and few changes to the L-arginine were
tolerated.
3
(f) TFA, TIPS, H2O.
chain of a symmetry related molecule (not shown). In chain
B the benzthiadiazole ring stacks against the N300B side
chain (Supporting Information Figure S5b). The part of the
Binding and Functional Biological Activity of 2 in Endothe-
lial and Lung Carcinoma Cells. Compound 2 selectively
inhibited radiolabeled 125I-VEGF-A binding to PAE/NRP1,

2220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 2. Search for Active Core Scaffoldsa
Jarvis et al.
a IC50 values were measured on all compounds showing inhibition of >70%. PAE/NRP1 cells in 24-well plates were incubated with different
concentrations (0.1-100 μM) of compounds followed by addition of 0.1 nM 125I-VEGF-A165. Nonspecific binding was determined in the presence of
100-fold excess unlabeled VEGF. Results were obtained from three independent experiments, each performed using triplicate determinations at each
concentration of compound, and are presented as the mean ( SEM. R2 values ranged from 0.9874 to 0.9996.
but not VEGFR2-expressing cells, with an IC50 of 8 μM
(Table 3) and inhibited bt-VEGF-A binding to purified NRP1
b1 domain in a cell-free assay with an IC50 of 3 μM (Figure 6a).
Compound 2 also inhibited VEGF-A binding to lung carci-
noma A549 and prostate carcinoma DU145 cells, which express
NRP1, but not VEGFR1 and VEGFR2, with similar potency
(Supporting Information Figure S6). Binding of VEGF-A to
human umbilical vein endothelial cells (HUVECs), which ex-
press VEGFR2, VEGFR1, and NRP1, was also inhibited by 2
with an IC50 of 23 μM, the reduced potency reflecting the
specificity of 2 for NRP1 and its inability to compete with
VEGF-A binding to other receptors (Figure S6). The potency of
compound 2 in inhibiting VEGF-A binding to NRP1 was thus
very similar to that of the bicyclic peptide compound 1, which
inhibits 125I-VEGF-A binding to PAE/NRP1 and tumor cells
with IC50 values of 2-3 μM and inhibits 125I-VEGF-A binding
to HUVECs with an IC50 of 20 μM.9
The effects of 2 on VEGF-A signaling and function were
examined in human endothelial cells. Compound 2 reduced
VEGF-A-induced VEGFR2 tyrosine phosphorylation in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2221
Table 3. Structure-Activity Relationships for Phenyl Sulfonamidesa
a IC50 values were measured on all compounds showing inhibition of >70%. PAE/NRP1 cells in 24-well plates were incubated with different
concentrations (0.1-100 μM) of compounds followed by addition of 0.1 nM 125I-VEGF-A165. Nonspecific binding was determined in the presence of
100-fold excess unlabeled VEGF. Results were obtained from three independent experiments, each performed using triplicate determinations at each
concentration of compound, and are presented as the mean ( SEM. R2 values ranged from 0.9874 to 0.9996.
HUVECs in a dose-dependent fashion, with a maximum
inhibition of 34% at 100 μM (Figure 6b and Supporting
Information Figure S7). Compound 2 also significantly
reduced VEGF-A induced migration of HUVECs (Figure 6c).
These results agree well with our previous findings showing
partial inhibition of VEGF-A-induced VEGFR2 tyrosine
phosphorylation by the bicyclic peptide NRP1 antagonist
1 and partial inhibition of both VEGFR2 activation
and VEGF-induced migration by an inhibitory NRP1 anti-
body.3,9 Overall, compound 2 caused a partial inhibition of
VEGF receptor activity and biological function, consistent
with the current model for the role of NRP1 in VEGF
function, in which NRP1 is required for optimal signaling
and certain biological functions downstream of VEGFR2,
particularly migration.1
(Supporting Information Figure S7). Pretreatment of A549
cells with 2 significantly enhanced the cytotoxic effects of two
widely used chemotherapeutic drugs, paclitaxel (Figure 6d)
and 5-fluorouracil (Supporting Information Figure S8) by
approximately 4- and 10-fold, respectively. NRP1 down-
regulation by siRNA has previously been shown to increase
the chemosensitivity of pancreatic cancer cells,10 while cyto-
toxic synergy has been noted for VEGF pathway inhibition
with a combination of the multikinase inhibitor sorafenib
and the proteasome inhibitor bortezomib.11
Conclusion
Compound 2 exhibited a biological profile consistent with
inhibition of VEGF-A binding to NRP1 demonstrating an
impairment, butnot completeinhibition, of cell migrationand
VEGFR2 phosphorylation. The viability of carcinoma cells
was also decreased by 2, and it enhanced the potency of
cytotoxics. NRP1 blockers have the potential to target both
angiogenesis and tumor growth and metastasis and may
The functional effect of 2 was also examined in A549 lung
carcinoma cells, which express NRP1 but no detectable
VEGFR2. Treatment of A549 cells with 2 caused a signifi-
cant reduction in cell viability over a 48 h incubation

2222 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 4. Structure-Activity Relationships for Arginine Mimeticsa
Jarvis et al.
a IC50 values were measured on all compounds showing inhibition of >70%. PAE/NRP1 cells in 24-well plates were incubated with different
concentrations (0.1-100 μM) of compounds followed by addition of 0.1 nM 125I-VEGF-A165. Nonspecific binding was determined in the presence of
100-fold excess unlabeled VEGF. Results were obtained from three independent experiments, each performed using triplicate determinations at each
concentration of compound, and are presented as the mean ( SEM. R2 values ranged from 0.9874 to 0.9996.
provide a valuable alternative targeted cancer therapy, parti-
cularly relevant in the light of concern over recent findings
showing that anti-VEGF agents may stimulate metastasis.12
Compound 2 and its analogues make excellent tools for
the study of NRP1 biology and provide a point of departure
for development of more potent anti-NRP1 drugs in the
future.
Analysis of compounds by reverse-phase LCMS was carried out
on an analytical C18 column (Phenomenex Gemini, 50 mm ꢀ
3.0 mm, 5 μm) and an AB gradient of 5-95% for B at a flow rate of
1 mL/min, where eluent A was 0.1% formic acid/water and eluent B
˚
was 0.1% formic acid/acetonitrile. Silica gel (60 A, 40-63 μm,
Fisher) was used for flash column chromatography. Routine
analytical thin layer chromatography was performed on precoated
plates (60F254, Machery-Nagel). All compounds tested were at least
95% pure by LCMS and NMR.
Experimental Section
Chemistry. Linear Peptide Synthesis. Linear peptides were
synthesized by Fmoc solid-phase synthesis using Wang or
2-chlorotrityl linkers in accordance with our previously re-
ported methods.9 The resins and the amino acid derivatives,
Fmoc-Abu-OH, Fmoc(Me)-Ala-OH, Fmoc-Ala-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Phe-OH, and Fmoc-Pro-OH were purchased from
Calbiochem Novabiochem (Nottingham, U.K.) and Bachem
AG (Bubendorf, Switzerland). All solvents used were of
HPLC-grade quality.
All commercially available solvents and reagents were used
without further treatment as received unless otherwise noted.
NMR spectra were measured with a Bruker DRX 400 or 600
MHz spectrometer; chemical shifts are expressed in ppm relative to
TMS as an internal standard and coupling constants (J) in Hz.
Mass spectra were obtained using a Waters ZQ2000 single quad-
rupole mass spectrometer with electrospray ionization (ESI). High
resolution mass spectra were acquired on a Waters LCT time-
of-flight mass spectrometer with electrospray ionization (ESI).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2223
pale-yellow precipitate was isolated by suction filtration and
was washed with water (3 ꢀ 10 mL) followed by isohexane (3 ꢀ
10 mL), then dried (in vacuo) to afford the desired product. Yield:
9.30 g, 96%. Pale-yellow solid. TLC Rf = 0.2 (ethyl acetate/
isohexane, 1:2). LCMS: tR = 3.09 min; purity >95%. MS: m/z
354.1 [M - 1]-.
3-(Benzo[1,2,5]thiadiazole-4-sulfonylamino)thiophene-2-car-
boxylic Acid (13). The ester 12 (9.28 g, 26.1 mmol, 1 equiv) was
stirred with lithium hydroxide monohydrate (5.48 g, 130.5
mmol, 5 equiv) in a tetrahydrofuran/methanol/water mixture
(5:3:2; 20 mL) at 50 ꢀC for 4 h. The reaction mixture was
monitored using TLC; when deemed complete, the solvents
were removed in vacuo. The product was then directly isolated
by precipitation using 2 M hydrochloric acid (100 mL) followed
by suction filtration. The precipitate was washed with water (3 ꢀ
50 mL) and then with isohexane (3 ꢀ 50 mL) and dried in vacuo
at 40 ꢀC for 14 h to afford the desired product. Yield: 8.75 g, 25.7
mmol, 98%. Beige solid. LCMS: tR = 2.74 min; purity >95%.
MS m/z 340 [M - 1]-. 1H NMR (400 MHz, DMSO-d6): δ 10.43
(1H, br, s), 8.44-8.38 (2H, m), 7.86 (1H, d, J = 8.9, 7.1 Hz), 7.75
(1H, d, J = 5.5 Hz), 7.25 (1H, d, J = 5.5 Hz).
(S)-2-{[3-(Benzo[1,2,5]thiadiazole-4-sulfonylamino)thiophene-
2-carbonyl]amino}-5-guanidino(Pbf)pentanoic Acid Methyl Es-
ter (15). The acid (13) (5.0 g, 14.7 mmol, 1 equiv) and bromo-tris-
pyrrolidinophosphonium hexafluorophosphate (PyBroP, 8.25
g, 17.7 mmol, 1.2 equiv) were suspended in dichloromethane (50
mL), under nitrogen (balloon), and stirred for 2 min at 20 ꢀC. N,
N-Diisopropylethylamine (23 mL, 132.3 mmol, 9 equiv) was
added in one portion and the orange solution stirred at room
temperature for 10 min. The protected amino acid (H-Arg(Pbf)-
OMe; 8.40 g, 17.7 mmol, 1.2 equiv) was added as a single
portion, and the reaction mixture was stirred at 20 ꢀC for 4 h.
The solvent was removed in vacuo and the residue taken up in
ethyl acetate (approximately 200 mL). The solution was washed
with 1 M hydrochloric acid (10 ꢀ 50 mL) and then evaporated
directly to give a viscous brown gum that was maintained under
vacuum for 17 h. The resulting brown glassy solid was carried
forward without further purification. Yield: 11.2 g, 14.7 mmol,
100%. Brown glassy solid. TLC Rf = 0.44 (methanol/ethyl
acetate (10:90)). LCMS tR = 1.81 min; purity >85%.
Figure 5. X-ray analysis of 2. (a) FOM weighted difference map of
2-bound NRP1 b1 crystal structure calculated from the model
refined without atomic coordinates for 2. The calculated electron
density (green mesh) clearly shows the presence of the ligand in two
different conformations in the binding sites A and B. Final stick
models for 2 in chain A (left) and chain B (right) are overlaid over
the density. Colors are green, red, blue, and yellow for carbon,
oxygen, nitrogen, and sulfur, respectively. (b) Detail of conforma-
tion of chain A bound 2. The position of the (putative) shared
hydrogen is indicated by a magenta sphere and arrow.
(S)-2-{[3-(Benzo[1,2,5]thiadiazole-4-sulfonylamino)thiophene-
2-carbonyl]amino}-5-guanidino(Pbf)pentanoic Acid. The crude
ester 15 (11.2 g, 14.7 mmol) and lithium hydroxide hydrate (3.08
g, 73.5 mmol, 5 equiv) were stirred in tetrahydrofuran/water (2:1,
300 mL) for 30 min at 20 ꢀC. After this time, LCMS indicated
complete reaction, The organic solvent was removed in vacuo and
hydrochloric acid (1 M, aqueous solution, 20 mL) added to
precipitate the product. The resulting solid was collected by
filtration and washed with 1 M hydrochloric acid (100 mL), water
(2 ꢀ 100 mL), and isohexane (3 ꢀ 50 mL) and then carried forward
directly. Yield: quantitative recovery assumed. Yellow solid. Rf =
Molecular Modeling. Molecules were analyzed using SYBYL
7.0 (Tripos Inc., St. Louis, MO). The Biopolymer tools in SYBYL
were used to build some of the peptides, to check structures for
errors, to correct atom types, and to add/remove hydrogens as
necessary. Structures were minimized using the Tripos force field
with a steepest descent gradient of 100 iterations followed by a
conjugate gradient of 0.01 kcal/mol or a maximum of 10000K
1
˚
iterations as termination criteria. During minimizations, a 12 A
(ethyl acetate) baseline. LCMS tR = 3.17 min. H NMR (400
nonbonded cutoff was applied and when solvent was not present
and ionic strength was set to 4.00. The collected structural data
were analyzed with the graphic tools of SYBYL, version 7.0. All
calculations were performed on the dual Hewlett-Packard work-
station XW6000, two 2.8 GHz CPUs running REDHAT Enter-
prise Linux WS4. The possible binding sites of NRP1 b1 domain
were searched using the SYBYL SITEID module (SYBYL, ver-
sion 7.0; Tripos Inc., St. Louis, MO).
Large Scale Synthesis of 2. 3-(Benzo[1,2,5]thiadiazole-4-sul-
fonylamino)thiophene-2-carboxylic Acid Methyl Ester (12). To a
stirred solution of the arylsulfonyl chloride (10) (7.68 g, 32.7
mmol, 1.2 equiv) in pyridine (30 mL) under nitrogen (balloon)
at 45 ꢀC was added dropwise over 1 h a solution of methyl
3-aminothiophene-2-carboxylate (11) (4.28 g, 27.3 mmol, 1 equiv)
in pyridine (10 mL). The resulting suspension was stirred for
20 h at 20 ꢀC. The reaction was monitored using TLC and was
deemed complete after approximately 2 h. Water (25 mL) was
added and the resulting mixture stirred for 15 min. The resulting
MHz, DMSO-d6): δ 11.36 (1H, br s), 8.42-8.34 (3H, m), 7.84 (1H,
dd, J = 8.8 and 7.0 Hz), 7.66 (1H, d, J = 5.3 Hz), 7.19 (1H, d, J =
5.3 Hz), 4.23-4.20 (1H, m), 3.06-3.01 (2H, m), 2.92 (2H, s), 2.46
(3H, s), 2.41 (3H, s), 1.95 (3H, s), 1.82-1.73 (1H, m), 1.69-1.60
(1H, m), 1.40-1.35 (8H, m).
(S)-2-(3-(Benzo[c][1,2,5]thiadiazole-4-sulfonamido)thiophene-
2-carboxamido)-5-guanidinopentanoic Acid (2). The acid (11.0 g,
14.7 mmol, 1 equiv) was stirred with trifluoroacetic acid/water
(9:1, 150 mL) for 65 h at 25 ꢀC. After this time the acidic solution
was added to an ice/water mixture, causing the immediate
precipitation of a pale-green solid that was collected by filtration
and washed copiously with ether before being suspended in
further ether and subjected to ultrasonic irradiation for ∼5 min.
The resulting fine powder was collected and dried under vacuum
at ∼40 ꢀC. Yield: 5.16 g, 8.44 mmol, 57%. Pale-green solid.
LCMS tR = 4.77 min; purity >95%. MS m/z 498 [M þ 1]þ. 1H
NMR (400 MHz, (CD3)2SO/D2O): δ 8.26-8.23 (2H, m), 7.78
(1H, dd, J = 8.6 and 7.0 Hz), 7.45 (1H, d, J = 5.3 Hz), 7.17 (1H,

2224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Jarvis et al.
Figure 6. Binding and functional activity of 2. (a) 2 displaces biotinylated VEGF from NRP1 b1 in a cell-free assay. Plates precoated with
purified recombinant NRP1 b1 domain were incubated for 2 h at room temperature with 0.25 nM bt-VEGF-A165 in the presence of the
indicated concentrations of 2, and specific VEGF binding was determined. Values presented are the means ( SEM obtained from three
independent experiments each performed in triplicate. R2 = 0.9965. (b) 2 inhibits VEGF-stimulated VEGFR2 phosphorylation. Confluent
HUVECs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent (DMSO) and were then
treated with or without 25 ng/mL VEGF-A165 for 5 min at 37 ꢀC. Total VEGFR2 and VEGFR2 phosphorylated at Tyr1175 were then
determined in treated cell lysates using a specific ELISA. Values presented are the means ( SEM obtained from three independent experiments
each performed in duplicate: (/) p < 0.05 versus no 2; (//) p < 0.01 versus no 2; (///) p < 0.001 versus no 2. (c) 2 decreases chemotactic
responses to VEGF. HUVECs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent
(DMSO) and placed into top inserts. The chemotaxis of cells toward either VEGF at 25 ng/mL or medium control (C) in bottom wells was
determined after 4 h of incubation. Values presented are the means ( SEM obtained from three independent experiments each performed in
duplicate: (/) p < 0.05 for 100 μM versus 0 μM 2 pretreated cells toward VEGF. (d) Sensitization of carcinoma cells to a chemotherapeutic
agent by 2. A549 cells were incubated in serum-free medium containing paclitaxel at the indicated concentrations in the absence or presence of
100 μM 2. Cell viability was measured after 48 h of treatment. Values presented are the means ( SEM obtained from three independent
experiments each performed in triplicate: (//) p < 0.01 for the chemotherapeutic drug alone versus drug plus 2.
d, J = 5.3 Hz), 4.26 (1H, dd, J = 9.6 and 4.5 Hz), 3.16-
3.13 (2H, m), 1.88-1.81 (1H, m), 1.77-1.70 (1H, m), 1.60-1.54
(2H, m). 13C NMR (400 MHz, (CD3)2SO): δ 173.0, 163.2, 156.8,
154.7, 148.1, 131.9, 130.1, 128.7, 127.0, 119.8, 113.7, 52.0, 40.3,
27.3, 25.4. LCMS: tR = 4.48 min; purity >96%. MS m/z 498
[M þ 1]þ. HRMS (m/z): [M]þ calcd for C17H20N7O5S3,
498.0688; found, 498.0697.
Structural Biology Methods. Protein NMR Methods. NMR
spectroscopy was performed at either 500 or 600 MHz 1H
frequency at 25 ꢀC. 15N or 13C,15N-isotope labeled NRP1 b1
samples were prepared in 50 mM phosphate buffered saline (100
mM sodium chloride), 1.0 mM dithiothreitol, 1.0 mM ethyle-
nediaminetetraacetic acid, pH 6.0. Backbone resonance assign-
ments were obtained by analysis of 3D HNCA, HN(CO)CA,
HNCACB, HNCO, HN(CA)CO spectra. Ligand titrations
were monitored by 2D 15N,1H HSQC spectroscopy of 0.3 mM
NRP1 b1 to which aliquots of buffered ligand (200 mM) were
added until saturation was achieved;13 the maximal protein
concentration dilution was <10%. Control experiments were
recorded to assess the sensitivity of the NRP1 b1 cross-peak
positions to small changes in protein concentration and sample
pH. NMR spectra were processed with nmrPipe14 and analyzed
in CCPN analysis.15 Cross-peak chemical shift perturbations
were declared significant when the compound change in 1H and
15N resonance position Δδ = {[(ΔδN/6.5)2 þ ΔδH2]}1/2 > 0.15
where ΔδN and ΔδH are measured in ppm. Essentially complete
backbone NMR assignments were obtained by standard triple
resonance NMR spectroscopy of a uniformly double 13C,15N-
isotope labeled sample of NRP1 b1. The pattern of secondary
13C chemical shifts obtained is consistent with the known
globular structure of NRP1 b1 from X-ray diffraction studies,
indicating that the overall structure in solution is unperturbed
(data not shown).
Protein Generation, Purification, and Crystallization with 2.
Recombinant human NRP1-b1 domain was expressed in E. coli
strain Rosetta (DE3) (Novagen) as a fusion protein containing a
tobacco etch virus protease cleavable N-terminal hexahistidine tag.
The protein was purified by combination of immobilized metal
affinity chromatography, size exclusion chromatography (Super-
dex75), and ion exchange chromatography on a SP FF Sepharose
column. Purified NRP1-b1 protein in 20 mM Tris-chloride (pH
7.9) and 50 mM sodium chloride buffer was incubated with a 10-
fold molar excess of 2 for 1 h at 4 ꢀC. The protein was concentrated
to 10 mg/mL and crystallized in a 1:1 volume mixture with 100 mM

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2225
Table 5. Data Collection and Refinement Statistics (Molecular Re-
placement)
carcinoma lung A549 and prostate DU145 cells were provided
by Quintiles Limited (Edinburgh, U.K.) and grown in RPMI
1640 medium (Invitrogen, Paisley, U.K.) containing 10% and L-
glutamine. HUVECs or DU145 cells were seeded at the density
of 2 ꢀ 104 cells per well of 96-well plates in 0.1 mL growth
medium and transfected with adenovirus vectors containing the
full-length open-reading frame of human NRP1. The adeno-
virus NRP1-transfected cells grew for 2 days prior to a binding
assay. COS7 cells were obtained from ECACC and maintained
in DMEM supplemented with 10% FBS (Invitrogen). COS7
cells were grown in 24-well plates to 80% confluency and
transfected with NRP1 plasmid constructs using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s instructions.
Immunoblotting. Cells were extracted by lysis buffer (64 mM
Tris-HCl, pH 6.8, 0.2 mM Na3VO4, 2% sodium dodecyl sulfate
(SDS), 10% glycerol, protease inhibitors for serine, cysteine,
and metalloproteases). The whole cell lysate samples were
separated by SDS-PAGE and transferred to Immobilon mem-
branes (Millipore). The membranes were immunoblotted with
specific primary antibodies. Immunoreactive bands were visua-
lized by chemiluminescence using horseradish peroxidase-con-
jugated secondary antibodies and enhanced chemiluminescence
reagent (Amersham Biosciences, Bucks, U.K.).
parameter
space group
cell dimension
P21
˚
a, b, c (A)
41.0, 89.6, 41.6
90, 97.7, 90
R, β, γ (deg)
30.3-2.9 (3.0-2.9)a
0.079 (0.191)a
8.5 (4.0)a
˚
resolution (A)
Rsym or Rmerge
I/σ(I)
completeness (%)
redundancy
88.0 (85.4)a
2.4 (2.3)a
Refinement
˚
resolution (A)
30.3-2.9
5852
21.6/28.2
no. reflections
R
work/Rfree
no. atoms
protein
2455
70
ligand/ion
water
rms deviation
8
˚
bond length (A)
bond angle (deg)
0.012
1.42
Cell-Based Ligand Binding. Radiolabeled binding displace-
ment experiments were performed in cells grown to confluence
in 24-well plates and using the indicated concentrations of
compounds followed by addition of 0.1 nM 125I-VEGF-A165
(1200-1800 Ci/mmol, GE Healthcare, U.K.) as described pre-
viously (Jia et al., 2006).9 Nonspecific binding was determined in
the presence of 100-fold excess unlabeled VEGF.
a Highest-resolution shell is shown in parentheses.
MES (pH 6.0), 10% PEG 8K, and 200 mM zinc acetate at 20 ꢀC
employing hanging drop vapor-diffusion method. Crystals ap-
peared within 3 days, with thick needle morphology and a yellow
hue, from compound 2. Crystals belonged to space group P21 with
˚
˚
˚
cell dimensions of a = 40.96 A, b = 89.60 A, c = 41.60 A, R =
90.00ꢀ, β = 97.72ꢀ, γ = 90.00ꢀ and contained two NRP1-b1 mole-
cules in the asymmetric unit.
For biotinylated (bt)-VEGF-A165 binding, confluent adenovirus
NRP1-transfected HUVECs or DU145 cells in 96-well plates were
washed twice with phosphate-buffered saline (PBS). The various
concentrations of compounds diluted in binding medium
(Dulbecco’s modified Eagle’s medium, 25 mM N-2-hydroxyethyl-
piperazine-N0-2-ethanesulfonic acid (HEPES), pH 7.3, containing
0.1% bovine serum albumin (BSA)) were added, followed by
addition of 2 nM bt-VEGF-A165. After 2 h of incubation at room
temperature, the medium was aspirated and washed three times
with PBS. The bound bt-VEGF-A165 to NRP1 was detected by
streptavidin-horseradish peroxidase conjugates and the enzyme
substrate and measured using a Tecan Genios plate reader at 450
nm absorbance with a reference wavelength at 595 nm. Nonspecific
binding was determined in the presence of 100-fold excess unlabeled
VEGF-A165. Half maximal inhibitory concentrations (IC50) for
selected compounds were obtained from competition curves ob-
tained from 125I-VEGF-A165 and bt-VEGF-A165 binding assays in
which different concentrations (0.1-100 μM) of compound were
tested, using Prism, version 4.0, software and the equation
˚
X-ray Crystallography. A data set at 2.9 A resolution was
obtained at the London School of Pharmacy by using a R-
AXISIV image plate detector system and Osmic mirrors. Data
were merged and indexed using the program d*TREK16 in the
CrystalClear17 software suite. Molecular replacement was per-
formed using PHASER18 with apo NRP1-b1 domain (PDB
code 1KEX). Iterative rounds of building and refinement were
carried out in COOT19 and REFMAC5.20 TLS groups were
generated using the TLSMD Web server.21 The dictionary file
for 2 was generated using the PRODRG Web server.22 The
structure was refined to final R/Rfree values of 21.6/28.2% with
reasonable geometry (Table 5).
Plasmids, Adenoviruses, and Mutagenesis. The full-length
open reading frame of human NRP1 (Origene) was cloned into
the pENTR directional TOPO vector. Subsequently plasmid
expression constructs (pcDNA3.2/V5-DEST) and adenoviral
vectors encoding NRP1 constructs (pAd/CMV/V5-DEST) were
generated by LR recombination using the Gateway system
(Invitrogen). Site-directed mutagenesis was performed with the
QuikChange XL site-directed mutagenesis kit (Stratagene). All
constructs were verified by DNA sequencing.
Reagents. Recombinant human VEGF (VEGF-A165) was
obtained from R & D Systems (Abingdon, U.K.). Collagen type
I, Dulbecco’s modified Eagle’s medium (DMEM)/25 mM
HEPES, 5-fluorouracil, and paclitaxel were purchased from
Sigma-Aldrich (Dorset, U.K.). All other reagents used were of
the purest grade available.
Cell Culture and Transfection. Human umbilical vein en-
dothelial cells (HUVECs) were purchased from TCS CellWorks
(Buckingham, U.K.) and cultured in endothelial cell basal
medium (EBM) supplemented with 10% fetal bovine serum
(FBS), 10 ng/mL human epidermal growth factor, 12 μg/mL
bovine brain extract, 50 μg/mL gentamicin sulfate, and 50 ng/
mL amphotericin-8. Porcine aortic endothelial cells expressing
NRP1 (PAE/NRP1) were provided by Dr. Shay Soker. The cells
were grown in Ham’s F12 medium containing 10% FBS and
25 μg/mL hygromycin B. Human tumor cell lines including
ðTop -BottomÞ
Y ¼ Bottom þ
50 -xÞHillslope
1 þ 10ðlogIC
where Top and Bottom are the responses, respectively, at the top
and bottom of the curve, Y is the inhibitory response at a given
compound concentration X, and the Hillslope describes the steep-
ness of the curve. R2 values measuring goodness of fit of the
competitioncurveswerealsodeterminedusingthePrism software.
Cell-Free bt-VEGF-A165 Binding. The 96-well plates were pre-
coated with NRP1-b1 protein at 3 μg/mL overnight at 4 ꢀC.
On the following day, the plates were treated with blocking
buffer (PBS containing 1% BSA) and washed three times with
wash buffer (PBS containing 0.1% Tween-20). The various
concentrations of compounds diluted in PBS containing 1%
DMSO were added, followed by addition of 0.25 nM bt-VEGF-
A
165. After 2 h of incubation at room temperature, the plates
were washed three times with wash buffer. The bt-VEGF-A165
bound to NRP1-b1 was detected by streptavidin-horseradish
peroxidase conjugates and the enzyme substrate and measured
using a Tecan Genios plate reader at 450 nm absorbance with a

2226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Jarvis et al.
reference wavelength at 595 nm. Nonspecific binding was
determined in the absence of NRP1-b1 coated wells of the plates.
Measurement of VEGFR2 Phosphorylation. Following treat-
ment of HUVECs, phosphorylation at Tyr1175 of VEGFR2
and total VEGFR2 in cell lysates were detected by PathScan
phopho-VEGFR2 and total VEGFR2 enzyme linked immuno-
sorbant assay (ELISA) kits (Cell Signalling) according to the
manufacturer’s instructions.
Cell Migration. Cell migration was measured in chemotaxis
24-transwell plates with collagen I-coated inserts incorporating
polyethylene terephthalate track-etched membranes with 8 μm
pores (Becton Dickinson Biosciences). VEGF in EBM/0.1% BSA
was placed in the bottom wells of the plates. Cells were trypsinized,
washed, and resuspended in EBM/0.1% BSA to give a final cell
concentration of 3 ꢀ 105/mL. Then 1.5 ꢀ 105 cells with or without 2
treatment as indicated were loaded into each top inserts. The
chemotaxis transwell plates were incubated at 37 ꢀC for 4 h. After
the incubation, nonmigrated cells on the top side of the transwell
membranes were removed and migrated cells on the under side of
the transwell membranes were stained with the REASTAIN
Quick-Diff kit (Reagena, Ltd., Toivala, Finland). The stained cells
from each well were counted in four fields at ꢀ100 magnification
using an eyepiece indexed graticule (100 grids).
Cell Viability. Cell viability was determined by measurement
of conversion of the tetrazolium salt XTT to form formazan dye.
Carcinoma cells were seeded at a density of 4 ꢀ 103 cells per well
of 96-well plates in 100 μL of serum-free medium containing
various concentrations of 5-FU or paclitaxel as indicated in the
absence or presence of 2 at 100 μM. After 44 h of incubation,
XTT labeling reagent mixture (Roche Diagnostics, East Sussex,
U.K.) was added to the cultures and they were incubated for a
further 4 h. The formazan product was then measured at 490 nm
with a reference wavelength at 595 nm.
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Acknowledgment. Research inthe PCD group is supported
by the MRC (file reference U117574559). Research in the IZ
group is supported by the British Heart Foundation (grant
RG/06/003). We thank Claudio Dagostin (compound
synthesis), Emma Dugdale (analysis and purification), Stew-
art Kirton (computational chemistry), and Kerry Jenkins and
Trevor Perrior (crystal structure interpretation of 2). We also
thank Gary Parkinson (London School of Pharmacy) for
assistance with data collection, Claire Newton for project
management, and John Martin for his unwavering support.
Supporting Information Available: Full experimental details
of the synthesis of the compounds mentioned in the text and
additional figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
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