[CANCER RESEARCH 60, 2973–2980, June 1, 2000]
Porphyrin Analogues as Novel Antagonists of Fibroblast Growth Factor and
Vascular Endothelial Growth Factor Receptor Binding That Inhibit
Endothelial Cell Proliferation, Tumor Progression, and Metastasis
David Aviezer,1 Sara Cotton, Magda David, Amit Segev, Nona Khaselev, Nitsa Galili, Zeev Gross, and Avner Yayon2
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100 [D. A., S. C., M. D., A. S., A. Y.], and Department of Chemistry, Technion-Israel Institute of
Technology, Haifa 32000 [N. K., N. G., Z. G.], Israel
ABSTRACT
Fibroblast growth factors (FGFs) and vascular endothelial growth
factor (VEGF) play a pivotal role in the multistep pathway of tumor
progression, metastasis, and angiogenesis. We have identified a porphyrin
analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetrap-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor
binding and activation. TMPP demonstrated potent inhibition of binding
of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at
submicromolar concentrations. TMPP inhibits binding of radiolabeled
FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding
of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an
in vitro angiogenic assay measuring the extent of endothelial cell growth,
tube formation, and sprouting, TMPP dramatically reduced the extent of
the FGF2-induced endothelial cell outgrowth and differentiation. In a
Lewis lung carcinoma model, mice receiving TMPP showed a marked
inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon
alternate daily injections of TMPP at 25 ␮g/g of body mass. Finally, novel
meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel
corrole-based derivative, with >50-fold increase in activity in vitro, had a
significantly improved efficacy in blocking tumor progression and metastasis in vivo.
INTRODUCTION
FGFs3 and VEGF act in concert to enhance tumor angiogenesis and
metastasis (1). In adults, FGF2 (basic FGF) is highly abundant in
tumors, whereas the oncogenes FGF-4 (hst/kfgf) and FGF-3 (int-2)
are found in tumors such as stomach cancer, Kaposi sarcoma, melanoma, and breast cancer (2). FGFR genes are also frequently amplified and overexpressed in a variety of cancers such as breast cancer,
gastric adenocarcinomas, melanoma, pancreatic cancers, and many
others (3– 6). Recently, activating mutations in FGFRs were also
found to be frequently expressed in both cervical and bladder carcinomas (7).
A most potent and selective angiogenic factor is VEGF. VEGF
is a multifunctional cytokine that plays a key role in physiological
and pathological angiogenesis in vivo by stimulating endothelial
cell proliferation and vessel hyperpermeability (8). VEGF binds to
Flt-1 and Flk-1/KDR cell membrane receptors that, similar to
Received 11/8/99; accepted 3/29/00.
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.
1
Present address: ProChon Biotech, P. O. Box 1482, Rehovot 76114, Israel.
2
To whom requests for reprints should be addressed.
3
The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor;
FRAP, FGFR1 alkaline phosphatase fusion protein; VEGF, vascular endothelial growth
factor; EGF, epidermal growth factor; TMPP, 5,10,15,20-tetrakis N-methyl-4-pyridylium)porphyrin tetraiodide; P1012, 5,10,15,20-tetrakis(N-methyl-3-pyridylium)-21H,23Hporphine tetra-p-tosylate; P1016, 5,10,15-tris (4-N-methylpyridylium)-20-(2,3,4,5,6-pentafluorophenyl) porphyrin triiodide; P1020, 5,10,15,20-tetra-[4-(N-methyl-2-pyridylium
iodide)-2,3,5,6-tetrafluorophenyl)] porphyrin; P1021, 5,10,15-tris[2,3,5,6-tetrafluoro-4(N-methyl-2-pyridylium] corrole triiodide; CHO, Chinese hamster ovary; NMR, nuclear
magnetic resonance; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet;
DMF, dimethylformamide; THF, tetrahydrofuran.
FGFRs, are members of the tyrosine kinase receptor superfamily
(9). The association between the growth factor ligands and their
respective receptors stimulates tyrosine kinase activity as one of
the initial biochemical events leading to DNA synthesis and cell
division. Therefore, compounds that inhibit ligand-mediated signal
transduction pathways may be useful for the treatment of cellular
proliferative disorders.
FGF2 and VEGF have synergistic activities on endothelial cell
activation and differentiation (10). Both FGF2 and VEGF require a
cooperative interaction with heparan sulfate proteoglycans to form
functional growth factor-receptor complexes that are essential for
high-affinity binding and activation of their cognate receptors (11–13)
and angiogenic activity (14). On the basis of the crucial role of growth
factor-heparin interaction, we designed a high throughput screening
system using FGF ligands immobilized on heparin and tagged soluble
receptors to identify molecules that can modulate heparin-FGFR
interactions. The screening process has identified several potent compounds, among which is TMPP, a member of the porphyrin molecule
family.
Porphyrins have been of interest to chemists and medical scientists for over a century. It has been known for many years that
porphyrins interact with neoplastic tumors (15), and the fact that
porphyrins demonstrate high affinity to tumorigenic cells in vitro
and solid tumors in vivo is well established (16, 17). Moreover,
porphyrin derivatives have been used for the treatment of tumors
and malignant tissues in combination with electromagnetic radiation or radioactive emissions. Because they strongly absorb light,
many porphyrins are still being used as photosensitizers in photodynamic therapy (17).
In this study, we demonstrate that TMPP (Mr 1363) is capable of
directly inhibiting FGF2 and VEGF receptor binding. TMPP inhibits
endothelial cell proliferation and differentiation in an in vitro angiogenesis model and dramatically reduces primary tumor growth and
metastatic spread of Lewis lung carcinoma tumors in mice. A series of
novel, rationally designed porphyrin analogues demonstrated significantly improved potency in inhibiting FGFR binding in vitro and
improved efficacy in blocking tumor progression and metastasis in
vivo.
MATERIALS AND METHODS
Materials
Human recombinant FGF2 and EGF were purchased from R&D systems.
Human recombinant VEGF was generated by H. Weich (GBF, Braunscwig,
Germany). Heparin-coated plates were prepared by Carmeda (Sweden). FRAP
was prepared as described (18). EGF receptor immunoglobulin fusion protein
was generated by Y. Yarden as described (19). CHO cells expressing FGFR1
were generated as described (11). DMEM and F12 growth mediums, bovine
calf serum, and glutamine were made by Biological Industries (Bet Haemek,
Israel).
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
Compound Synthesis
Preparation of TMPP
A mixture of 5.7 ml (60 mmol, 4 equivalents) of 4-pyridinecarboxaldehyde
and 4.15 ml (60 mol, 4 equivalents) pyrrole was dissolved in 300 ml of
propionic acid, and the mixture was heated to reflux for 2 h. After cooling to
room temperature, the solvent was evaporated to dryness, and the oily residue
was washed by hot water, neutralized by aqueous ammonia (25%), and washed
again with hot water. The purple solids obtained by this procedure were filtered
and dried. The dry solid material was treated with three portions of 50 ml of
dichloromethane, each followed by filtration. To the combined organic phases,
10 g of basic alumina (activity II) were added, and the solvent was evaporated
to dryness. Separation of 5,10,15,20-tetra(4-pyridyl)porphyrin was achieved
by column chromatography. Rf (2% ethanol in CH2Cl2) ⫽ 0.18; 1H NMR
(CDCl3, ␦): 9.04 (d, J ⫽ 5.5 Hz, 8H), 8.85 (s, 8H), 8.14 (d, J ⫽ 5.5 Hz, 8H),
⫺2.95 (s, 2H). Two hundred mg (0.3 mmol) of 5,10,15,20-tetra(4-pyridyl)porphyrin were dissolved in 30 ml of dry DMF, and 9 ml of CH3I were added in
one portion. The reaction was stirred at room temperature for 10 h, after which
the reaction mixture was evaporated to dryness by high vacuum at room
temperature. The resulting crystals are recrystallized from mixtures of methanol and EtOAc.
Preparation of Compound P1012
The noncharged intermediate, 5,10,15,20-tetrakis (3-pyridyl)porphyrin
(chemical abstract registry no. 40882-83-5), was alkylated with methyl toluene
sulfonate. 1H NMR (CDCl3, ␦): 9.45 (s, 4H), 9.06 (d, J ⫽ 5.5 Hz, 4H), 8.85
(s, 8H), 8.52 (d, J ⫽ 7.8 Hz, 4H), 7.7 (dd, J ⫽ 7.8 Hz, 4H), ⫺2.86 (s, 2H).
Preparation of Compound P1016 and Related Porphyrins
100%); 1d, UV-vis (CH2Cl2) ␭max, nm: 414, 508, 582; 1H NMR (CDCl3, ␦): 9.06
(d, J ⫽ 5.8 Hz, 4H), 8.89 (d, J ⫽ 6.6 Hz, 4H), 8.84 (m, 4H), 8.15 (dd, J1 ⫽ 4.3
Hz, J2 ⫽ 1.5 Hz, 4H), ⫺2.90 (s, 2H); 19F NMR (CDCl3, ␦): ⫺137.1 (dd,
J1 ⫽ 23.4 Hz, J2 ⫽ 8.1 Hz, 4F), ⫺152.0 (t, J ⫽ 21.1 Hz, 2F), ⫺161.9 (td,
J1 ⫽ 22.8 Hz, J2 ⫽ 7.9 Hz, 4F); MS⫹ m/z: 797.4 (MH⫹, 100%), MS⫺ m/z: 794.9
([M ⫺ H]⫺, 100%); 1e, UV-vis (CH2Cl2) ␭max, nm: 416, 510, 586; 1H NMR
(CDCl3, ␦): 9.05 (d, J ⫽ 5.4 Hz, 6H), 8.90 (d, J ⫽ 4.8 Hz, 2H), 8.84 (m
(unresolved doublets), 6H), 8.14 (m (unresolved doublets), 6H), ⫺2.92 (s, 2H); 19F
NMR (CDCl3, ␦): ⫺137.3 (dd, J1 ⫽ 22.8 Hz, J2 ⫽ 7.9 Hz, 2F), ⫺152.1 (t,
J ⫽ 21.7 Hz, 1F), ⫺162.0 (J1 ⫽ 23.0 Hz, J2 ⫽ 7.7 Hz, 2F); MS⫹ m/z: 708.1
(MH⫹, 100%), MS⫺ m/z: 706.1 ([M ⫺ H]⫺, 100%); 1f, 1H NMR (CDCl3, ␦): 9.04
(d, J ⫽ 5.5 Hz, 8H), 8.85 (s, 8H), 8.14 (d, J ⫽ 5.5 Hz, 8H), ⫺2.95 (s, 2H).
(c) Alkylation Step. Seventy mg (0.1 mmol) of 5-(2,3,4,5,6-pentafluorophenyl)-10,15,20-tris(4-pyridyl) porphyrin (compound 1e) or any of the other
derivatives 1b–1f were stirred at room temperature with 3 ml (48 mmol) CH3I
in 10 ml of DMF for 12 h, after which the reaction mixture was evaporated to
dryness by high vacuum at room temperature. The resulting crystals were
recrystallized from mixtures of methanol and EtOAc, thus obtaining: 2d,
5,10-bis(2,3,4,5,6-pentafluorophenyl)-15,20-bis(N-methyl-4-pyridylium) porphyrin diiodide, UV-vis (H2O) ␭max, nm: 416, 514, 582; 2e (compound
P1016), UV-vis (H2O) ␭max, nm: 420, 516, 584; 1H NMR (DMSO-d6, ␦): 9.48
(d, J ⫽ 6.5 Hz, 6H), 9.44 (d, J ⫽ 5.4 Hz, 2H), 9.17 (m, 6H), 9.02 (d, J ⫽ 6.5
Hz, 4H), 8.99 (d, J ⫽ 6.5 Hz, 2H), 4.71 (s, 9 H), ⫺3.13 (s, 2H); 19F NMR
(DMSO-d6, ␦): ⫺139.3 (dd, J1 ⫽ 24.5 Hz, J2 ⫽ 5.8 Hz, 2H), ⫺153.4 (t,
J ⫽ 22.2 Hz, 1H), ⫺162.2 (J1 ⫽ 23.0 Hz, J2 ⫽ 5.1 Hz, 2H).
Preparation of Compound P1020
(a) Preparation of the Precursor, 5,10,15,20-Tetra[(4-(2-pyridyl)2,3,5,6-tetrafluorophenyl)]porphyrin. One ml of an 1.6 M n-BuLi solution
(1.6 mmol) was added to a stirred solution of 0.14 ml (1.5 mol) 2-bromopyridine in 8 ml of dry THF under an argon atmosphere at ⫺78°C at such a rate
that the temperature of the reaction mixture did not exceed ⫺70°C. After the
addition was complete, the reaction mixture was stirred for 1 h at ⫺78°C,
resulting in a clear yellow solution. Next, a solution of 0.1 g (0.1 mmol) of
5,10,15,20-tetra(2,3,4,5,6-pentafluorophenyl)porphyrin (1a) in 5 ml of dry
THF was added dropwise. The mixture was stirred for 3 h at ⫺78°C and then
hydrolyzed with saturated aqueous bicarbonate solution. The layers were
separated, the aqueous layer was washed with ether, and the combined ether
extracts were dried and evaporated to a solid residue. The product was purified
by column chromatography on silica gel (2:1 EtOAc:hexane) and recrystallized from EtOAc:ethanol to give 26 –30 mg (20 –25% yield) of the pure
product as violet solids. 1H NMR (CDCl3, ␦): 9.06 (s, 8H), 8.97 (d, J ⫽ 3.9 Hz,
4H), 8.03 (t, J ⫽ 7.7 Hz, 4H), 7.89 (d, J ⫽ 7.5 Hz, 4H), 7.54 (t, J ⫽ 6 Hz, 4H),
⫺2.82 (s, 2H). 19F NMR (CDCl3, ␦): ⫺137.57 (q, J ⫽ 24.8 Hz, 8F), 144.11 (q,
J ⫽ 24.6 Hz, 8F). MS⫹ m/z: 1211.4 (MH⫹, 100%), MS⫺ m/z: 1208.3
([M ⫺ H]⫺, 100%).
(b) Alkylation of the Precursor to Obtain Compound P1020. A mixture
of 40 mg (33 mmol) of 5,10,15,20-tetra(4-(2-pyridyl)-2,3,5,6-tetrafluorophenyl)porphyrin and 2.5 ml (40 mmol) of CH3I in 6 ml of freshly distilled DMF
was heated to 70°C for 5 h. After evaporation of the solvent, the product
recrystallized from methanol:ether to give 55 mg (95% yield) of the title
compound as violet solids. UV-vis (H2O) ␭max, nm (ex103): 410 (238), 508
(17.6), 574. 1H NMR (DMSO-d6, ␦): 9.70 (s, 4H), 9.59 (s, 4H), 9.52 (d,
J ⫽ 5.8 Hz, 4H), 9.04 (t, J ⫽ 7.4 Hz, 4H), 8.79 (d, J ⫽ 7.6 Hz, 4H), 8.54 (t,
J ⫽ 6.8 Hz, 4H), 4.72 (s, 12H), ⫺3.05 (s, 2H). 19F NMR (DMSO-d6, ␦):
⫺137.23 (m, 8F), 137.68 (m, 8F).
(a) Preparation of the Intermediate Compounds (Condensation Step).
A mixture of 4.3 ml (45 mmol, 3 equivalents) of 4-pyridinecarboxaldehyde,
2.06 ml (16.5 mmol, 1 equivalent) of pentafluorobenzaldehyde, and 4.15 ml
(60 mmol, 4 equivalents) of pyrrole was dissolved in 300 ml of acetic acid, and
the mixture was heated to reflux for 2 h. After cooling to room temperature, the
solvent was evaporated to dryness by vacuum, and the oily residue was washed
by hot water, neutralized by aqueous ammonia (25%), and washed again with
hot water. The purple solids obtained by this procedure were filtered and dried.
The dry solid material was treated with three portions of 50 ml of dichloromethane, each followed by filtration. To the combined organic phases, 10 g
of silica were added, and the solvent was evaporated to dryness.
(b) Chromatographic Separation. Separation and purification of the components obtained in step a was achieved by column chromatography, in which the
polarity of the eluents was gradually increased from dichloromethane to mixtures
of dichloromethane and 2–10% ethanol. The order of elution (the Rf values are for
silica with 2% ethanol in CH2Cl2) and the chemical yields were as follows:
5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (1a ⫽ P2, traces;
Rf ⫽ 0.95); 5,10,15-tris(2,3,4,5,6-pentafluorophenyl)-20-(4-pyridyl)porphyrin (1b,
1.1%; Rf ⫽ 0.66); 5,15-bis(2,3,4,5,6-pentafluorophenyl)-10,20-bis(4-pyridyl)porphyrin (1c; Rf ⫽ 0.60); 5,10-bis(2,3,4,5,6-pentafluorophenyl)-15,20-bis(4-pyridyl)
porphyrin (1d; Rf ⫽ 0.54); 5-(2,3,4,5,6-pentafluorophenyl)-10,15,20-tris(4-pyridyl)porphyrin (1e, 9.4%; Rf ⫽ 0.45); and 5,10,15,20-tetrakis(4-pyridyl)porphyrin
(1f, traces; Rf ⫽ 0.18). The combined yield of compounds 1c and 1d was 13.4%.
Their separation required an additional column in which the eluent was 2% ethanol
in dichloromethane. Spectroscopic characteristics of the compounds (1a and 1f are
known compounds): 1a, UV-vis (CH2Cl2) ␭max, nm: 412, 506, 586; 1H NMR
(CDCl3, ␦): 8.91 (s, 8H), ⫺2.93 (s, 2H); 19F NMR (CDCl3, ␦): ⫺136.9 (dd, Preparation of Corrole P1021
J1 ⫽ 22.8 Hz, J2 ⫽ 7.0 Hz, 8F), ⫺151.6 (t, J ⫽ 20.7 Hz, 4F), ⫺161.7 (dd,
J1 ⫽ 22.4 Hz, J2 ⫽ 5.8 Hz, 8F); 1b, UV-vis (CH2Cl2) ␭max, nm: 414, 506, 582;
The preparation of P1021 and its precursors are described by Gross et al.
1
H NMR (CDCl3, ␦): 9.06 (d, J ⫽ 4.3 Hz, 2H), 8.89 (s, 6H), 8.16 (d, J ⫽ 4.2 Hz, (20). In brief, this is the preparation of intermediate 5,10,15-tris(4-(2-pyridyl)2H), 8.15 (s, 2H), ⫺2.92 (s, 2H); 19F NMR (CDCl3, ␦): 137.0 (m, 6F), ⫺151.8 (m tetrafluorophenyl) corrole. 0.42 ml of a 1.6 M n-BuLi solution (0.7 mmol) was
(2 overlaying t), 3F), ⫺161.8 (m, 6F); MS⫹ m/z: 886.1 (MH⫹, 100%), MS⫺ m/z: added to a stirred solution of 0.054 ml (0.56 mmol) 2-bromopyridine in 6 ml
884.6 (M⫺, 40%), ([M ⫺ H]⫺, 60%); 1c, UV-vis (CH2Cl2) ␭max, nm: 412, 508, of dry THF under an argon atmosphere at ⫺78°C at such a rate that the
584; 1H NMR (CDCl3, ␦): 9.06 (d, J ⫽ 4.4 Hz, 4H), 8.89 (s, 4H), 8.85 (s, 4H), 8.15 temperature of the mixture did not exceed 70°C. After the addition was
(d, J1 ⫽ 4.5 Hz, 4H), ⫺2.94 (s, 2H); 19F NMR (CDCl3, ␦): ⫺137.2 (dd, J1 ⫽ 23.2 complete, the reaction mixture was stirred for 1 h at 78°C to give a clear yellow
Hz, J2 ⫽ 7.2 Hz, 4F), ⫺152.0 (t, J ⫽ 20.9 Hz, 2F), ⫺161.9 (J1 ⫽ 22.8 Hz, solution. Next, a solution of 0.03 g (0.038 mmol) of 5,10,15-tri(2,3,4,5,6J2 ⫽ 7.3 Hz, 4F); MS⫹ m/z: 797.4 (MH⫹, 100%), MS⫺ m/z: 794.9 ([M ⫺ H]⫺, pentafluorophenyl)corrole in 6 ml of dry THF was added dropwise. The
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
mixture was stirred for 1 h at ⫺78°C and then hydrolyzed with saturated
aqueous bicarbonate solution. The layers were separated, the aqueous layer
was washed with ether, and the combined ether extracts were dried and
evaporated to a solid residue. The product was purified by column chromatography on silica gel (1:1 EtOAc:hexane) and recrystallized from CH2Cl2:
hexane to give 13 mg (35% yield) of the pure product as violet crystals.
Binding of Soluble FGFR Alkaline Phosphatase Fusion Protein to
Immobilized FGF2
FGF2 (100 ng/ml) was incubated on 96-well plates to which heparin had
been covalently attached (Carmeda). Subsequently, 200 ␮l of FRAP condition
medium and TMPP were added and incubated together for 2 h. After three
cycles of washing with HNTG [20 mM HEPES (pH 7.5), 150 mM NaCl, 1%
Triton X-100, and 10% glycerol], alkaline phosphatase substrate (Sigma; 15
mM) was added, and catalyzation of the chromogenic product was measured by
spectrophotometry at 405 nm.
Binding of
125
I-Labeled FGF2 to Soluble FGFR
Conditioned medium from NIH 3T3 cells secreting FGFR1-FRAP was
incubated for 45 min at room temperature with rabbit antihuman placental AP
antibodies prebound to agarose-protein A beads (Pierce). The FRAP-coupled
beads were washed three times with 1 ml of HNTG and incubated with 2 ng/ml
of 125I-labeled FGF, 1 ␮g/ml heparin, and TMPP at different concentrations for
1 h at room temperature. High affinity-bound 125I-labeled FGF2 was determined by counting the tubes in a gamma counter.
Binding of
125
I-Labeled FGF2 to Cells
Confluent cultures of CHO cells expressing FGFR1 (11) in 24-well plates
(Falcon) were precooled and washed twice with cold DMEM supplemented
with 20 mM HEPES (pH 7.5) and 0.1% BSA (DMEM/BSA). They were then
incubated for 1.5 h at 4°C with 125I-labeled FGF2 (2 ng/ml) and increasing
concentrations of TMPP. The binding medium was discarded, and the cells
were washed once with ice-cold DMEM/BSA and twice with cold PBS (pH
7.5) containing 1.6 M NaCl. High-affinity, receptor-bound FGF2 was determined by extraction of the cells with 20 mM sodium acetate (pH 4.0) containing 2.0 M NaCl. Nonspecific binding was determined in the presence of a
100-fold excess of unlabeled FGF2.
Binding of
125
I-Labeled VEGF to Cells
Confluent cultures of bovine aortic endothelial cells in 24-well plates
(Falcon) were precooled and washed twice with cold DMEM supplemented
with 20 mM HEPES (pH 7.5) and 0.1% BSA (DMEM/BSA). The cells were
then incubated for 1.5 h at 4°C with 125I-labeled VEGF (2 ng/ml) and
increasing concentrations of TMPP. The binding medium was discarded, and
the cells were washed once with ice-cold DMEM/BSA and twice with cold
PBS (pH 7.5) containing 1.6 M NaCl. High-affinity, receptor-bound FGF2 was
determined by extraction of the cells with 20 mM sodium acetate (pH 4.0)
containing 2.0 M NaCl. Nonspecific binding was determined in the presence of
a 100-fold excess of unlabeled VEGF.
Binding of
125
I-Labeled EGF to EGF Receptor
Confluent cultures of A431 cells in 24-well plates (Falcon) were precooled
and washed twice with cold DMEM supplemented with 20 mM HEPES (pH
7.5) and 0.1% BSA (DMEM/BSA). The cells were then incubated for 1.5 h at
4°C with 125I-labeled EGF (2 ng/ml) and increasing concentrations of TMPP
(19). Binding of radiolabeled EGF to soluble receptors was performed by
incubating conditioned medium from 293 cells secreting EGF receptor-Fc
immunoglobulin fusion protein for 45 min at room temperature with agaroseprotein A beads (Pierce). The EGF receptor-coupled beads were washed three
times with 1 ml of HNTG and incubated with 2 ng/ml of 125I-labeled EGF and
TMPP at for 1 h at room temperature as described by Tzahar et al. (19). High
affinity-bound, 125I-labeled EGF was determined by counting the tubes in a
gamma counter.
Rat Aorta in Vitro Angiogenesis Assay
pH and ionic strength of the collagen solution (22). Thoracic aortas were
obtained from 2-month-old Sprague Dawley rats. The fibroadipose tissue was
carefully removed under a dissecting microscope, and aortic rings were sectioned (1-mm long) and placed on top of a 0.2-ml collagen gel in 16-mm
culture wells. Collagen solution (0.4 ml) was carefully poured on top of the
ring. After the gel was formed, 0.4 ml of serum-free endothelial growth
medium was added and replaced every other day by fresh medium containing
FGF2 (2 ng/ml). TMPP was added to the growth medium twice a week.
Microvessel outgrowth was visualized by phase microscopy, and the number
of capillary vessels was counted throughout the course of the experiment. After
14 days, the cultures were fixed with 4% formaldehyde, embedded in paraffin,
and sectioned at 5 ␮m, and the extent of microvascular endothelial tube
outgrowth was measured under a light microscope.
Lewis Lung Carcinoma Tumor Assay
Murine Lewis lung carcinoma D122 cells (2 ⫻ 105 cells/50 ml PBS) were
injected into the foot pads of 10-week-old C57 black mice (23). Twenty-five
␮g/g body mass of porphyrins dissolved in PBS were injected i.p. twice a week
in the treated group, and tumor size was measured periodically to follow
primary tumor formation. To evaluate inhibition of lung metastasis by TMPP,
the primary tumors were allowed to develop over a period of 4 weeks to a
volume of ⬃8 mm3, after which the tumors were removed by amputation, and
metastases were allowed to develop for an additional 4 weeks, during which
the treated group received 25 ␮g/g body mass of porphyrin i.p. twice a week.
Subsequently, the mice were sacrificed and dissected, and the lungs were
removed and photographed. The extent of lung metastasis was measured by
weighing the lungs. C57/black mice were maintained on lab chow and tap
water and were housed with a 12-h day-night cycle.
RESULTS
TMPP Inhibits the Binding of FGF2 to the FGF Receptor. A
high throughput screening system composed of a heparin matrix,
FGF2, and a FGFR1 tagged by alkaline phosphatase (FRAP) was
designed using 96-well plates to which heparin had been covalently
attached. FGF2 binding to the immobilized heparin is then followed
by the addition of FRAP, and the compounds were to be screened for
their ability to modulate heparin-FGF, receptor-heparin, and receptorFGF interactions. The end point of the assay measures enzymatically
the formation of FGF-receptor complexes quantitated by the specifically associated alkaline phosphatase-catalyzed chromogenic product.
Thus, a lowered alkaline phosphatase activity would indicate inhibition at one or more of the three levels of interactions required for the
formation of the FGF-FGFR-heparin ternary complex. The screen of
the chemical synthetic library has identified several compounds for
their capacity to inhibit soluble FGFR binding. One of the most potent
ones was TMPP.
TMPP demonstrated potent inhibition at submicromolar concentrations, with a distinct dose-dependent inhibition pattern (Fig. 1A). To
evaluate the capacity of TMPP to inhibit FGF2 receptor binding, we
measured the binding of radiolabeled FGF2 to FGFR1 in two independent experimental systems. In the first experiment, we used a
cell-free system, measuring the binding of radiolabeled FGF2 to a
dimeric soluble FGFR1 fused to alkaline phosphatase (18). The soluble FRAP was immobilized using an anti-alkaline phosphatase antibody prebound to agarose-protein A beads. As a second experimental model, we used heparan sulfate-deficient CHO cells, genetically
engineered to express FGFR1 (11). TMPP inhibited binding of 125Ilabeled FGF2 to the FGFR in both experimental systems. Fig. 1B
illustrates that TMPP is capable of profoundly inhibiting FGF2FGFR1 binding in the soluble receptor assay with an IC50 of ⬃1 ␮M
(Fig. 1B). In the cellular receptor system, TMPP was capable of
inhibiting FGF2 binding with an IC50 of ⬃2.5 ␮M (Fig. 1C). The
slightly higher concentrations of TMPP required for inhibition of
cellular FGF2 binding in the cellular assay result most likely from the
Type I collagen was prepared from the tail tendons of adult Sprague Dawley
rats (21). The collagen matrix gel was obtained by simultaneously raising the
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
2, there is complete inhibition of the formation of a typical FGF2receptor complex at TMPP concentrations as low as 5 ␮M, in agreement with the direct binding data (Fig. 1C).
TMPP Inhibits the Binding of VEGF to the VEGF Receptor.
Because FGF and VEGF share several similar characteristics and may
play synergistic role in tumor angiogenesis (2, 8, 10), we examined
the capacity of TMPP to inhibit VEGF binding to its receptor. Fig. 3A
demonstrates that TMPP efficiently inhibits VEGF binding to human
umbilical vein endothelial cells that express the Flk-1/KDR VEGF
receptor and with high potency. TMPP also inhibits VEGF binding to
bovine aortic endothelial cells expressing VEGF receptors (data not
shown) in a manner similar to the Flk-1/KDR-transfected cells.
TMPP Does Not Inhibit the Binding of EGF to the EGF Receptor. When TMPP was tested for its capacity to inhibit the binding
of radiolabeled EGF to the EGF receptor on A431 cells, no inhibition
of binding was noted, even at mM concentrations of TMPP (data not
shown). To unequivocally determine the specificity of this effect,
chemical cross-linking of 125I-labeled FGF2 to FGFR1-expressing
cells or 125I-labeled EGF to EGF receptor (19) was carried out in the
absence or presence of 10 ␮g/ml TMPP. As shown in Fig. 3B, TMPP
as expected inhibits FGF2 receptor binding and the formation of a
FGF-receptor complex but had no effect on the binding of 125I-labeled
EGF to the EGF receptor, in agreement with the results of the binding
experiment on A431 cells.
Inhibition of in Vitro Angiogenesis by TMPP. To establish the
biological effect of TMPP, we examined the compound for its effects
on an in vitro angiogenic assay using rat aorta sections embedded in
a collagen type I gel (21, 22, 24). The assay measures the extent of
endothelial cell growth and microvascular tubules sprouting from the
vessel tissue embedded in the gel. Basal tubule formation can be
detected, even when no additional factors were added. The addition of
Fig. 1. Inhibition of FGF2 binding to the FGFR by TMPP. A, binding of soluble FRAP to
FGF2 immobilized on heparin-coated 96-well plates. FGF was prebound to heparin-coated
plates; conditioned medium from NIH 3T3 cells expressing the FGFR-1 AP fusion protein was
added to the wells and incubated in the presence of increasing concentrations of TMPP. The
alkaline phosphatase enzymatically catalyzed formation of a chromogenic product was measured (405 nm) and represents the amount of FRAP present on the plates as a heparin-FGFFRAP ternary complex. B, effect of TMPP on binding of radiolabeled FGF2 to soluble
FGFR1. Radiolabeled FGF2 (2 ng/ml) was incubated (90 min at 4°C) with immobilized
FGFR1. Incubations were performed in the presence of 100 ng/ml heparin and increasing
concentrations of TMPP. Nonspecific binding was determined in the presence of 100-fold
excess of unlabeled FGF2 and did not exceed 10% of the total binding. Results represent the
means in one of at least two independent experiments. C, effect of TMPP on the binding of
125
I-labeled FGF2 to CHO cells transfected with FGFR1. HS-deficient CHO mutant cells
expressing FGFR1 were incubated for 90 min at 4°C in the presence of 1 ␮g/ml of heparin,
125
I-labeled FGF2 (2 ng/ml), and increasing concentrations of TMPP. The binding medium
was discarded, and the cells were washed with ice-cold DMEM/BSA. To determine the degree
of receptor-bound 125I-labeled FGF2, the cells were incubated in cold PBS (pH 4) containing
1.6 M NaCl and 25 mM HEPES. The cell extracts were counted in a gamma counter.
Nonspecific binding was determined in the presence of 100-fold excess of unlabeled ligand
and did not exceed 20% of the total bound ligand.
inherent reduced affinity of the soluble FGFR to the FGF ligand
compared with that of cell-associated receptors (18).
Affinity Labeling of Cells by 125I-Labeled FGF2 Is Inhibited by
TMPP. To determine the specificity of this effect, chemical crosslinking of 125I-labeled FGF2 to cells was carried out in the absence
and presence of increasing concentrations of TMPP. As shown in Fig.
Fig. 2. Covalent cross-linking of 125I-labeled FGF2 to CHO cells expressing FGFR is
inhibited by TMPP. Binding of 125I-labeled FGF2 to confluent monolayers of FGFR1expressing cells was performed as described above in the presence of increasing concentrations of TMPP. After 90 min, disuccinylimidyl suberate (0.15 mM in PBS) was added.
The protein complexes were separated by electrophoresis on a 7.5% SDS polyacrylamide
gel and analyzed on X-ray film.
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
sacrificed. The extent of lung metastasis at this point was examined by
gross morphological examination and by determining the gain in lung
weight. Aggressive metastasis formation is noted in the lungs of
control mice (those not treated with TMPP). In mice treated with
TMPP (25 mg/kg body mass), the lungs were significantly less affected (Fig. 6) and in some cases indifferent from those of the
noninjected control mice weighing ⬃200 mg, similar to the lungs
from the noninjected control mice (Fig. 6).
Novel Porphyrin Derivatives Demonstrate Improved in Vitro
and in Vivo Activity. To elucidate the structural requirements needed
to achieve FGF and VEGF inhibitory activity, we have synthesized
and examined a series of porphyrin analogues. It became clear that
only cationic charged porphyrins, but not neutral or anionic charged
derivatives, are active. On the basis of the structure of TMPP, we have
synthesized novel meso-pyridylium-substituted porphyrins in which
the position of the N-methyl (the positive charge) was varied from the
para position to ortho and meta, as well as porphyrins with fewer than
Fig. 3. A, effect of TMPP on the binding of 125I-labeled VEGF to endothelial cells
transfected with the VEGF receptor. Confluent monolayers of endothelial cells expressing
the VEGF receptor (Flk-1/KDR) were incubated with 125I-labeled VEGF (2 ng/ml) for 90
min at 4°C in the presence of increasing concentrations of TMPP. The binding medium
was discarded, and the cells were washed with ice-cold DMEM/BSA. To determine the
degree of receptor-bound 125I-labeled VEGF, the cells were incubated in cold PBS (pH 4)
containing 1.6 M NaCl and 25 mM HEPES. The cell extracts were counted in a gamma
counter. Nonspecific binding was determined in the presence of increasing concentrations
of unlabeled ligand and did not exceed 20% of the total bound ligand. B, TMPP does not
inhibit covalent cross-linking of 125I-labeled EGF to EGF receptor. Binding of 125Ilabeled EGF to soluble EGF receptor-Fc immunoglobulin fusion protein was performed as
described by Tzahar et al. (19). Binding of 125I-labeled FGF2 to confluent monolayers of
FGFR1-expressing cells was performed as described above. Both binding experiments
were performed in the presence or absence of 10 ␮g/ml TMPP. After 90 min, chemical
cross-linking was performed by the addition of disuccinylimidyl suberate (0.15 mM in
PBS). The protein complexes were separated by electrophoresis on a 7.5% SDS polyacrylamide gel and analyzed on X-ray film.
FGF2 (2 ng/ml) dramatically increased the degree of cell growth and
vascularization. However, the addition of FGF2 together with 10 ␮M
TMPP dramatically reduced the extent of endothelial cell growth and
differentiation. When 100 ␮M TMPP was added in the presence of
FGF2, complete inhibition of microvascular tubule sprouting was
achieved, and no endothelial cell growth was observed (Fig. 4).
TMPP Inhibits Primary Tumor Progression in a Lewis Lung
Carcinoma Tumor Model. Lewis lung carcinoma D122 cells
(200,000 cells/mouse) were injected into the foot pads of 10-week-old
C57 black mice according to O’Reilly et al. (23). Mice that received
the TMPP (25 ␮g/g of body mass) by i.p. injections twice a week for
5 weeks showed a marked inhibition in primary tumor growth in
comparison with the control group (Fig. 5). The experiment was
repeated five times, and the findings were reproducible and statistically established. These results indicated that TMPP is not only active
in vitro but is capable of inhibiting tumor growth in vivo as well.
TMPP Inhibits Lung Metastasis in the Lewis Lung Carcinoma
Model. Mice were injected with Lewis lung carcinoma cells (2 ⫻ 105
cells/mouse) into the foot pad, and primary tumors were allowed to
develop over a period of ⬃3 weeks to a volume of ⬃8 mm3. Subsequently, primary tumors were removed through amputation, and lung
metastases were allowed to develop for 4 weeks before the mice were
Fig. 4. In vitro angiogenic assay using a rat aorta section embedded in a collagen gel.
Sections of rat aorta were immobilized in a collagen gel. After the addition of FGF2 to the
medium, in the presence or absence of TMPP, the extent of endothelial cell growth and
microvascular tubules sprouting from the vessel tissue embedded in the gel was measured.
Basal tubule formation can be detected even when no additional factors were added. The
results are expressed as the percentage of microvascular tubules sprouting in comparison
with the control experiment where FGF2 alone was added. Bars, SE.
Fig. 5. Inhibition of primary tumor growth in the Lewis lung carcinoma murine tumor
model by TMPP. Lewis lung carcinoma cells were injected into the foot pads of
10-week-old C57 black mice. TMPP (25 ␮g/g of body mass) was injected i.p. twice a
week over a period of 7 weeks, during which time the primary tumor volume was
monitored. Bars, SE.
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
four 4-pyridylium substituents. In this series, the most beneficial
effect, as judged by the inhibition of FGF2 binding (Fig. 7A), was
obtained with P1016, a nonsymmetric porphyrin with three positive
charges. The activity of P1016 in vitro was ⬃50 times higher than that
of TMPP, as can be seen in Fig. 7A. Another derivative, P1020, which
contains four positive charges at more remote positions, was also
significantly more active than TMPP (Fig. 7A). In contrast, P1012 was
10-fold less active in inhibiting FGF2 binding, with an IC50 of 10 ␮M
(data not shown). In the Lewis lung carcinoma tumor model, however,
only P1020, but not P1016, was more active than TMPP and P1012,
which demonstrated only residual capacity to inhibit FGF2 binding
and had no effect whatsoever in vivo (Fig. 7B).
Finally, we tested a novel water-soluble corrole analogue of TMPP
(P1021; Mr 1311.3) for its activity for both in vitro inhibition of FGF2
binding and for the inhibition of tumor growth. Indeed, this derivative,
having three positive charges as in P1016 but with the same side
groups as in P1020, displayed the best of both P1016 and P1020
characteristics, because it was ⬃10-fold more active than TMPP in
vitro and 5-fold more potent in the in vivo tumor models, inhibiting
lung metastasis formation in vivo at a concentration of only 5 mg/kg
body weight (Fig. 7B). These results suggest that rationally modified
porphyrin analogues can serve as highly potent inhibitors of growth
factor activity in vitro and in vivo.
DISCUSSION
We have identified TMPP, a member of the porphyrin family, as a
potent inhibitor of FGF2 and VEGF receptor binding in cells and
cell-free systems. TMPP also dramatically reduced the extent of
FGF2-induced endothelial cell growth and differentiation in an in
vitro angiogenesis model and efficiently blocks Lewis lung carcinoma
murine primary tumor growth and lung metastasis. Novel, rationally
designed TMPP porphyrin analogues demonstrate improved potency
in inhibiting receptor binding in vitro and tumor progression and
metastasis in vivo. Taken together, we have identified TMPP and its
analogues as a novel class of potent inhibitors of FGF2 and VEGF
activity in vitro and in vivo.
The exact mechanism by which TMPP and other related porphyrinlike molecules inhibit growth factor-receptor binding and activation is
not clear. However, preliminary results suggest that TMPP interferes
Fig. 6. Effect of TMPP on lung metastasis in the Lewis lung carcinoma murine tumor
model. Lewis lung carcinoma cells were injected into the foot pads of 10-week-old C57
black mice, and primary tumors were allowed to develop over a period of 4 weeks to a
volume of ⬃8 mm3. Subsequently, the primary tumors were removed by amputation, and
metastases were allowed to develop for an additional 4 weeks before mice were sacrificed.
Twenty-five ␮g/g body mass TMPP were injected i.p. twice a week during the 4-week
period. The mice were sacrificed and dissected, and the lungs were removed. The average
extent of lung metastasis was measured by weighing the lungs for gain of mass. Bars, SE.
Fig. 7. A, inhibition of FGF2 binding to the FGFR by porphyrin analogues. Effect
of porphyrin analogues on the binding of radiolabeled FGF2 to soluble FGFR1.
I-Labeled FGF2 (2 ng/ml) was incubated (90 min at 4°C) with immobilized
FGFR1. Incubations were performed in the presence of 100 ng/ml heparin and
increasing concentrations of porphyrin analogues TMPP, P1016, P1020, and P1021.
Nonspecific binding was determined in the presence of 100-fold excess of unlabeled
FGF2 and did not exceed 10% of the total binding. Results represent the means in one
of at least two independent experiments. B, inhibition of metastasis growth in the
Lewis lung carcinoma tumor model by porphyrin analogues. Lewis lung carcinoma
cells were injected into the foot pads of 10-week-old C57 black mice, and primary
tumors were allowed to develop over a period of 4 weeks to a volume of ⬃8 mm3.
After the formation of primary tumors, they were removed by amputation, and
metastases were allowed to develop for 4 weeks before mice were sacrificed. Porphyrin analogues TMPP, P1012, P1016, P1020, and P1021 were injected i.p. twice a
week. The mice were sacrificed and dissected, and the lungs were removed. The
average extent of lung metastasis was measured by weighing the lungs. The graph
demonstrates the concentration (mg/kg body mass) of porphyrin analogue required to
achieve inhibition of metastasis growth. Bars, SE.
125
with the formation of the trimolecular complex of growth factorheparin and the tyrosine kinase receptor (11), thus abrogating
receptor signaling. It is interesting to note that TMPP does not
inhibit the binding of EGF, which is not a heparin-binding or
heparin-dependent growth factor (25), to its high-affinity tyrosine
kinase receptor,4 suggesting that interfering with heparin binding
may play a key role in the inhibitory effect of TMPP. Both the FGF
ligand and the FGFR contain heparin-binding domains critical for
FGFR activation (26 –29), and several heparin mimetics have been
described as potent inhibitors of FGFR binding and activation (24,
30). TMPP, however, does not resemble in its structure any of the
known heparin mimetics. Nevertheless, the requirement for positively charged groups and their spatial distribution may mimic a
restricted highly sulfated domain in heparin, thus serving as a
heparin mimetic. Several other inhibitors of FGF and VEGF that
have been shown to inhibit angiogenesis were designed to inhibit
4
A. Segev, D. Aviezer, M. Safran, Z. Gross, and A. Yayon, manuscript in preparation.
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PORPHYRIN INHIBITORS OF FGF BINDING AND TUMOR PROGRESSION
the intrinsic tyrosine kinase activity of the FGF and VEGF growth
factor receptors (31, 32). This novel class of FGF and VEGF
inhibitors, however, most likely works via a different molecular
mechanism involving direct interference with growth factor receptor interaction, thus inhibiting their biological responses.
Porphyrin derivatives are widely used for the treatment of tumors and malignant tissues in combination with electromagnetic
radiation or radioactive emissions. Because they strongly absorb
light in the 690 – 880 nm region, many porphyrins were suggested
for use as photosensitizers in photodynamic therapy (16). Some
porphyrin derivatives are used in combination with electromagnetic radiation or radioactive emissions for inhibiting angiogenesis
(33). It has been suggested that the activity of porphyrin derivatives as antitumor agents in the absence of electromagnetic radiation or radioactive emission may be based on their ability to cleave
DNA because of their capacity to bind to DNA and because they
must always include an excitable central Fe or Mn metal atom.
Here we find that porphyrin-like compounds that do not contain a
metal atom can directly interfere with growth factor receptor
tyrosine kinase interactions.
The potent antiproliferative effect of TMPP is of potential clinical
application not only in blocking growth factor-mediated tumor proliferation but also in other processes of pathological proliferation such
as restenosis, accelerated atherosclerosis, and pathological angiogenesis as in diabetic retinopathy and arthritis. In support is the observation that TMPP markedly inhibited the outgrowth of microvessels
from aortic rings embedded in a collagen gel. Furthermore, TMPP and
its analogues are potent inhibitors of vascular smooth muscle cell
proliferation in vitro.4 Studies are under way to elucidate the inhibitory effect of TMPP on angiogenesis and restenosis in experimental
animal models.
The fact that we were able to improve the potency of the TMPP
lead compound for its FGF and VEGF inhibitory activity, by
rationally modifying specific groups on the porphyrin backbone, is
of great importance. On the basis of the TMPP blueprint, we found
that only cationic charged porphyrins, but not neutral or anionic
charged derivatives, were active. When meso-pyridylium-substituted porphyrins, as well as porphyrins with fewer than four
4-pyridylium substituents, were synthesized and tested, the most
beneficial effect, as judged by the inhibition of FGF2 binding (Fig.
7A), was obtained with P1016, a nonsymmetric porphyrin with
three positive charges. Another derivative, P1020, which contains
four positive charges at more remote positions, was also significantly more active (Fig. 7A). The corrole (P1021), which contains
three positive charges as in P1016 but with the same side groups as
in P1020, displayed the best of both P1016 and P1020 characteristics. Furthermore, P1021 synthesis takes a much more straightforward approach than P1016 (20). The key to developing highly
potent and specific antitumor agents relies on the ability to perform
chemical modifications along the course of the development process. The vast knowledge accumulated with regard to the biological and chemical properties of porphyrins is therefore of great
advantage for any potential medicinal chemistry approach. This
fact, along with their capacity to block growth factor-mediated
tumor progression and angiogenesis, makes these porphyrins
highly attractive candidates for the development of anticancer
drugs in the future.
ACKNOWLEDGMENTS
soluble EGF receptor; Ezra Vadai and Lea Eisenbach for help with the Lewis
lung carcinoma assay; and Andrew Seddon and Peter Bohlen for helpful
discussions.
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Porphyrin Analogues as Novel Antagonists of Fibroblast
Growth Factor and Vascular Endothelial Growth Factor
Receptor Binding That Inhibit Endothelial Cell Proliferation,
Tumor Progression, and Metastasis
David Aviezer, Sara Cotton, Magda David, et al.
Cancer Res 2000;60:2973-2980.
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