A novel low molecular weight antagonist of vascular endothelial
growth factor receptor binding: VGA1155
Yasuji Ueda,1 Takehiro Yamagishi,1
Kazunori Samata,1 Hisao Ikeya,1
Noriko Hirayama,1 Hajime Takashima,1
Shiro Nakaike,1 Makoto Tanaka,1 and Ikuo Saiki2
1Taisho Pharmaceutical Co., Ltd., Saitama, Japan and 2Depart-
ment of Pathogenic Biochemistry, Institute of Natural Medicine,
Toyama Medical and Pharmaceutical University, Toyama, Japan
Abstract
Vascular endothelial growth factor (VEGF) plays a pivotal
role in the processes of angiogenesis, which is essential
for the growth of solid tumors and their metastasis.
Because VEGF is a critical factor in tumor survival,
inhibiting VEGF would provide significant benefits in
tumor therapy. To identify a compound that inhibits the
binding of VEGF to its receptor, we used a high
throughput screening method, finding that small molecu-
lar compounds inhibited VEGF binding. Among active
compounds, 5-[N-methyl-N-(4-octadecyloxyphenyl)ace-
tyl]amino-2-methylthiobenzoic acid (VGA1155) was se-
lected for its potent inhibition of binding. VGA1155
inhibited [125I] VEGF binding to two cell lines, NIH3T3-
fms-like tyrosine kinase-1 (VEGF receptor 1 transfected)
cells and NIH3T3-kinase insert domain containing recep-
tor/fetal liver kinase-1 (KDR/Flk-1; VEGF receptor 2
transfected), in a concentration-dependent manner.
VGA1155 did not inhibit the binding of several other
growth factors or cytokines to their receptors. Based on
the results of surface plasmon resonance analysis using
Biacore S51 system, it appears that this binding inhibitory
property may be based on the association of VGA1155
with VEGF receptor 2 (KDR/Flk-1). Further, the interfer-
ence in VEGF binding by VGA1155 in turn induces the
inhibition of VEGF-induced KDR/Flk-1 autophosphoryla-
tion. VGA1155 also reduced intradermal VEGF-induced
vascular permeability in guinea pigs. These findings
indicate that VGA1155 inhibits not only VEGF binding
to its receptors through association with KDR/Flk-1 but
also VEGF function in vivo. These VGA1155 activities
may provide a useful basis for the development of
antiangiogenic and antitumor agents. (Mol Cancer Ther.
2003;2:1105–1111)
Introduction
Angiogenesis, the formation of new blood vessels sprout-
ing from preexisting vessels, plays a crucial role in
physiological and pathological phenomena, including
embryonic development, wound healing, diabetic retinop-
athy, psoriasis, rheumatoid arthritis, and solid tumor
growth and metastasis (1, 2).
In the process of angiognesis, vascular endothelial growth
factor (VEGF) is essential for growth, mitogenesis, and tube
formation of endothelial cells (3–6). This importance of the
function of VEGF suggests that blocking this function may
provide a promising new therapeutic strategy for inhibiting
angiogenesis and tumor growths. Practically, several anti-
VEGF agents have already been reported. One, SU5416, the
first VEGF receptor tyrosine kinase inhibitor, has produced
certain clinical benefits in clinical trials involving patients
suffering from advanced malignancies (7).
VEGF binds to two tyrosine kinase receptors, fms-like
tyrosine kinase-1 (Flt-1) and kinase insert domain contain-
ing receptor/fetal liver kinase-1 (KDR/Flk-1), on the surface
of endothelial cells, thereby activating signal transduction
and regulating physiological and pathological angiogenesis
(8). We hypothesized that an agent that inhibits binding
between VEGF and its receptors may serve as a valuable
antiangiogenic and antitumor agent. Several VEGF binding
antagonists, including anti-VEGF antibody (9, 10), anti-
KDR/Flk-1 antibody (11), 2V-fluoropyrimidine RNA-based
aptamers (12), various peptides (13, 14), and porphyrin
analogues (15), have been reported. However, these
antagonists may demand high cost of synthesis because of
their large molecule weight. Furthermore, there is concern
that antibodies and peptides may have antigenicity and in
vivo instability. Therefore, we thought that it was worth
finding a low molecular weight antagonist.
To identify the small compounds that inhibit the binding
of VEGF to its receptor, we deployed a high throughput
screening method using the VEGF receptor (Flt-1) binding
scintillation proximity assay kit (Amersham, Biosciences,
Uppsala, Sweden) and our chemical compound library. We
found that novel benzoic acid derivatives inhibited VEGF
binding to the two receptors. Among VEGF binding
antagonist compounds, we selected 5-[N-methyl-N-(4-
octadecyloxyphenyl)acetyl]amino-2-methylthiobenzoic ac-
id (VGA1155) for its potent binding inhibitory action.
This report discusses the inhibitory action and mecha-
nism of VGA1155 and its effects on signal transduction and
the function of VEGF in vivo.
Materials and Methods
Materials
VGA1155 and 4-{N-[3-(4-octadecyloxyphenyl)propio-
nyl]}aminophthalic acid (VGA1154) (Fig. 1, A and B) were
synthesized in our laboratory. VGA1154 and VGA1155
Received 6/20/03; revised 9/2/03; accepted 9/3/03.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for Reprints: Yasuji Ueda, Medicinal Pharmacology Laboratory,
Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Kita-ku, Saitama
331-9530, Japan. Phone: 81-48-669-3028; Fax: 81-48-652-7254.
E-mail: y.ueda@po.rd.taisho.co.jp
Molecular Cancer Therapeutics 1105

were dissolved in DMSO and diluted with the medium or
PBS before use. VEGF (165-amino acid type) was purchased
from Pepro Tech, EC, Ltd. (London, UK), KDR/Fc chimera
protein (carrier free) was from R&D Systems, Inc. (Minne-
apolis, MN), [125I] VEGF was from Amersham, trans-
forming growth factor (TGF)-h was from Genzyme/Techne
(Minneapolis, MN), and [125I] TGF-h was from NEN Life
Science Products, Inc. (Boston, MA).
Cells
NIH3T3-Flt-1 and NIH3T3-KDR/Flk-1 cells overexpress-
ing human Flt-1 and KDR/Flk-1, respectively, were
provided by Prof. Masabumi Shibuya (University of
Tokyo). These cell lines were established as described
previously (16) and maintained in DMEM containing 10%
calf serum and 200-Ag/ml G418 at 37jC in a humidified
atmosphere of 5% CO2/95% air. BALB/c-3T3 cells were
maintained in DMEM containing 10% calf serum under the
same conditions.
VEGF Receptor Binding Inhibition Assay
NIH3T3-Flt-1 and NIH3T3-KDR/Flk-1 cells were seeded
at 7
104 cells/well in 24-well collagen-coated plates 24 h
before experimental use. These cells were preincubated
with medium A (DMEM containing 10-mM HEPES, pH 7.2,
and 0.1% BSA) at 4jC for 30 min. The medium was then
replaced with 0.3-ml medium B (DMEM containing 10-mM
HEPES, pH 7.2, and 0.5% BSA) containing 25-pM [125I]
VEGF (37.5–62.5 nCi/ml; Amersham) and varying concen-
trations of VGA1155. The plates were incubated at 4jC for
90 min. All experiments were performed in triplicate wells.
After incubation, cells were washed thrice with ice-cold
medium A. After cells were solubilized with 0.5 ml of 0.5-N
NaOH at 37jC for 30 min, each solution in the wells was
transferred to test tubes. The wells were washed with
0.5-ml PBS, and the washing solution was combined with
the solution in the tubes. The radioactivity of each tube was
counted by a gamma counter. The percentage of radioac-
tivity of each group relative to the control group was
calculated, after which IC50 was calculated by nonlinear
regression analysis.
Nonspecific binding was determined by incubation in
the presence of 10-nM unlabelled VEGF (Pepro Tech, EC).
TGF-BReceptor Binding Inhibition Assay
BALB/c-3T3 cells were seeded at 5
104 cells/well in 24-
well collagen-coated plates at 48 h before experimental use.
These cells were preincubated for 30 min in a binding buffer
containing 50-mM HEPES (pH 7.5), 128-mM NaCl, 5-mM
KCl, 5-mM MgSO4, 1.2-mM CaCl2, and 0.2% BSA at 4jC. The
binding buffer was then refreshed and supplemented with
50-pM [125I] TGF-h (37.5–62.5 nCi/ml; Amersham) and
varying concentrations of VGA1155. The plates were
incubated at 4jC for 240 min. All experiments were
performed in duplicate wells. After incubation, cells
were washed four times with the ice-cold binding buffer.
After cells were solubilized with the cell solubilization
buffer containing 25-mM HEPES, 1% (v/v) Triton X-100,
10% (v/v) glycerol, and 1% BSA, each solution in the wells
was transferred to test tubes. The wells were washed with
0.5-ml PBS, and the washing solution was combined with
the solution in the tubes. The radioactivity of each tube was
counted by a gamma counter, and the percentage of
radioactivity of each group relative to the control group
was calculated, after which IC50 was calculated by nonlinear
regression analysis. Nonspecific binding was determined
by incubation in the presence of 10-nM unlabelled TGF-h
(Genzyme/Techne).
Receptor Binding Inhibition Assay in Cerep (Paris,
France)
Receptor binding assays of several ligands [(epidermal
growth factor (EGF), platelet-derived growth factor (PDGF),
platelet-activating factor (PAF)-interleukin (IL)-1h, IL-2, IL-
4, IL-6, IL-8, tumor necrosis factor (TNF)-a, macrophage
inflammatory protein (MIP)-1a, MIP-1h, and insulin] were
performed in Cerep. The membranes or cells included each
receptor incubated with radioisotope-labeled ligands (0.02–
1 nM) and VGA1155 (0.1–10 AM) for 0.5–4 h except insulin
(20 h) at 4jC or 22jC. The membranes or cells in suspension
were rapidly filtered under vacuum through glass fiber
filters (GF/B or GF/C, Whatman, Inc., Clifton, NJ, or
Packard, Meriden, CT). The filters were then washed
several times with an ice-cold buffer using a cell harvester
(Brandel, Gaithersburg, MD, or Packard). Cultured adher-
ent cells were rinsed with an ice-cold buffer and then lysed.
Bound radioactivity was measured with a scintillation
counter (LS 6000 or LS 1701, Beckman Instruments,
Fullerton, CA, or Topcount, Packard) using a liquid
scintillation cocktail (Formula 989 or Microscint 0, Packard).
Interaction Study by Surface Plasmon Resonance in
the Biacore S51System
Human VEGF (66.7 Ag/ml in 10-mM sodium acetate, pH
4.5) and human KDR/Fc chimera protein (5 Ag/ml in 10-mM
Figure 1. Chemical structure of VGA1155.A, VGA1155 (MW 583.87).
B, VGA1154, an analogue of VGA1155 without inhibition of VEGF-KDR/
Flk-1 binding (MW 581.79).
VEGF Receptor Binding Antagonist, VGA11551106