Introduction

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Protein tyrosine kinases are enzymes that phosphorylate proteins on specific tyrosine residues within the primary sequence of the proteins. Protein phosphorylation has been found to be a common mechanism for transmitting mitogenic signals and regulating a wide variety of cellular processes (Burke, 1992; Cadena and Gill, 1992; Fantl et al., 1993).

The FGF receptor family consists of a group of four homologous receptor tyrosine kinases transcribed either as distinct gene products or by alternative splicing of their primary transcripts. Each consists of an extracellular ligand-binding domain that possesses three immunoglobulin-like domains, a single transmembrane region and a cytoplasmic domain containing protein tyrosine kinase activity (Ullrich and Schlessinger, 1990; Jaye et al., 1992; Friesel and Maciag, 1995). Ligand binding to the FGF receptor results in autophosphorylation followed by subsequent tyrosine phosphorylation of various protein substrates (Friesel and Maciag, 1995; Mohammadi et al., 1996, 1997). The FGFs that bind to these receptors consist of nine members. In normal tissues, FGFs are involved in the regulation of cell growth and differentiation, embryogenesis and angiogenesis (Folkman, 1985; Friesel and Maciag, 1995).

Inappropriate expression of FGF and/or its receptor or altered function of the tyrosine kinase activity has been shown to contribute to diverse pathologies, including tumorigenesis, psoriasis, rheumatoid arthritis and diabetic retinopathy, as well as vascular proliferative diseases such as atherosclerosis and restenosis (Folkman and Klagsbrun, 1987; Libby et al., 1992; Brogi et al., 1993). Because of these observations, we have been interested in the FGF receptor tyrosine kinases as targets for such proliferative diseases as cancer, atherosclerosis and restenosis. The present study focuses specifically on the FGF-1 receptor tyrosine kinase because it is the most predominant FGF receptor subtype expressed in vascular cells and shows high affinity binding for both aFGF and bFGF.

The importance of protein tyrosine kinases in signal transduction and the association of aberrant protein tyrosine kinase receptor and ligand expression with proliferative disorders make agents that modulate the activity of protein tyrosine kinases attractive therapeutic targets.

We recently identified a new class of protein tyrosine inhibitors based on the parent 6-aryl pyrido[2,3-d]pyrimidine structure (Blankley et al., 1997) by screening a compound library. The substitution of 3,5-dimethoxy on the 6-phenyl group of the parent pyrido[2,3-d]pyrimidine resulted in significant FGFR tyrosine kinase selectivity and the identification of PD 166866. In the present study, we report on PD 166866, a highly selective and nanomolar potent inhibitor of the FGFR-1 tyrosine kinase. PD 166866 is uniquely distinguished from any previously reported protein tyrosine kinase inhibitors by possessing a high level of specificity for the FGFR-1 tyrosine kinase and by demonstrating low nanomolar inhibitor potency of FGF-mediated cellular phosphorylation events, cell proliferation and potent antiangiogenic activity.

	Methods

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Chemicals and reagents. Human recombinant PDGF-BB, EGF and bFGF, anti-phosphotyrosine monoclonal (clone 4G10), anti-human PDGFR- polyclonal and anti-human EGFR polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies raised to the human FGFR-1 (flg) were a kind gift from Dr. Moosa Mohammadi (Department of Pharmacology, NYU Medical Center, New York, NY). p44/p42 MAPK antibodies were purchased from New England Biolabs (Beverly, MA). Enhanced chemiluminescence (ECL) reagents were purchased from Amersham Life Science (Arlington Heights, Il). Selenium, transferrin and hydrocortisone were purchased from Sigma Chemical (St. Louis, MO). DMEM, DMEM/Ham's F12, RPMI, Medium 199, Dulbecco's PBS, Geneticin (G-418), 1% glutamine, fungazone and 1% penicillin/streptomycin were obtained from Life Technologies (Grand Island, NY). FBS was purchased from Hyclone (Provo, UT). FCS was obtained from Commonwealth Serum Laboratories (Melbourne, Australia). PD 166866 was prepared in DMSO to achieve consistency in the vehicle and to ensure compound solubility. Appropriate DMSO controls were simultaneously evaluated with the test compound.

Recombinant kinases. cDNA coding for the full-length human FGFR-1 active tyrosine kinase (three IgG loop form) was kindly provided by Dr. Tom Maciag (American Red Cross, Rockville, MD) and was cloned into the baculovirus transfer vector pBacPAK8 (Clontech, Palo Alto, CA). Recombinant baculovirus bearing the FGFR-1 DNA was prepared, identified and purified using Spodoptera frugiperda (Sf9) insect cells as hosts according to the BaculoGold system (PharMingen, San Diego, CA) by following the instructions provided with the kit. A cDNA construct encoding the cytoplasmic domain of the human FGFR-1 (Lys³⁶³ to Arg⁷⁸⁵) was subcloned into a baculovirus transfer vector pBacPAK8 (Clontech, Palo Alto, CA). Transfection of Sf9 insect cells was performed according to methods provided with the BaculoGold transfection system (PharMingen). Baculovirus containing sequence for the full-length human PDGFR- receptor was obtained from Dr. William LaRochelle (National Institutes of Health, Bethesda, MD). Production of PDGFR- protein in infected Sf9 insect cells was performed as previously described (Jensen et al., 1992). Baculovirus containing sequence for the full-length EGFR, insulin receptor and c-Src kinases and PKC were prepared in a similar manner and have been previously described (Fry et al., 1994a, 1994b; Thompson et al., 1994). Assays for measuring these protein kinases were performed as previously described (Panek et al., 1997). The assay for MEK activity was performed as previously described by Dudley et al. (1995).

Tyrosine kinase assays. Assays using the full-length and cytoplasmic domain of the FGFR-1 tyrosine kinase were performed in a total volume of 100 µl containing 25 mM HEPES buffer (pH 7.4), 150 mM NaCl, 10 mM MnCl₂, 0.2 mM sodium orthovanadate, 750 µg/ml of a random copolymer of glutamic acid and tyrosine (4:1), various concentrations of inhibitor and 60 to 75 ng of enzyme as previously described (Panek et al., 1997). The reaction was initiated by the addition of [-³²P]ATP (50 µM ATP containing 0.4 µCi of [-³²P]ATP per incubation) and samples incubated at 25°C for 10 min. The reaction was terminated by the addition of 30% TCA and the precipitation of material onto glass-fiber filter mats. Filters were washed three times with 15% TCA, and the incorporation of [³²P] into the glutamate tyrosine polymer substrate was determined by counting the radioactivity retained on the filters in a Wallac 1250 betaplate reader. Nonspecific activity was defined as radioactivity retained on the filters after incubation of samples without enzyme. Specific activity was determined as total activity (enzyme plus buffer) minus nonspecific activity. The concentration of compound that inhibited specific enzymatic activity by 50% (IC₅₀) was determined graphically. For determination of ATP kinetics, assay conditions were the same as above except that varying concentrations of ATP were added in the absence or presence of a single concentration of PD 166866 to generate ATP concentration curves. K_i determinations for PD 166866 were obtained by a nonlinear regression analysis to fit the inhibition data to equations that describe different types of inhibition (Cleland, 1979). A comparison of the K_i (slope) vs. K_i (intercept) was then used to refine the curve-fit analysis. Kinetic analyses were performed using GraFit v 3.0 (Leatherbarrow, 1992).

Cell culture. L6 rat myoblasts, which lack FGF receptors, were transfected with the cDNA encoding the human three Ig-like disulfide loop form of the FGFR-1 (L6-pXZ106 cells) and were generously supplied by Dr. Tom Maciag (American Red Cross, Holland Research Labs, Rockville, MD). L6-pXZ106 cells were grown in DMEM containing 10% FBS supplemented with 0.1 mg/ml geneticin (G-418). NIH 3T3 cells expressing the endogenous FGF receptors were grown in DMEM containing 10% FBS with 1% penicillin/streptomycin. For PDGF receptor autophosphorylation experiments, smooth muscle cells were isolated from the thoracic aorta of adult male Sprague-Dawley rats (300-350 g; Charles River, Portage, MI) and explanted according to the method of Ross (1971). Cells were grown in DMEM containing 10% fetal bovine serum, 1% glutamine and 1% penicillin/streptomycin. Cells were identified as smooth muscle cells by their "hill-and-valley" growth pattern and by fluorescent staining with a monoclonal antibody specific for smooth muscle cell -actin. Cells were used between passages 8 and 20 for all experiments. For EGF receptor autophosphorylation experiments, A431 human epidermal carcinoma cells were obtained from Dr. David Fry (Parke-Davis, Cancer Research) and have previously been shown to endogenously express high levels of EGF receptors (Fry et al., 1994b). NIH 3T3 cells overexpressing the insulin receptor (NIHIR) were generously supplied by Dr. Cynthia Corley Mastick (Parke-Davis, Department of Cell Biology) and were maintained in DMEM (high glucose) containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin and 0.5 mg/ml G-418.

Immunoprecipitation and immunoblot analysis. To measure FGF receptor autophosphorylation, NIH 3T3 cells expressing endogenous FGF receptors or L6 myoblasts overexpressing FGF-1 receptors (L6 cells) were grown in 100-mm cultures dishes until 80% to 90% confluency. Growth medium was removed and replaced with serum-free medium consisting of DMEM/F12 (1:1), 30 nM selenium, 50 µg/ml transferrin, 10 nM hydrocortisone and 5 µg/ml insulin, and cells were incubated for an additional 24 hr. PD 166866 was then added directly to fresh serum free medium, and cells incubated for an additional 2 hr. Cells were then treated with bFGF (25 ng/ml) for 5 min at 37°C to stimulate FGF receptor autophosphorylation. The cells were washed briefly with cold PBS and lysed in 1 ml of lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 10% glycerol, 1% Triton-X 100, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin and 10 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride) containing phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate, 30 mM p-nitrophenyl phosphate and 10 mM sodium pyrophosphate). Lysates were centrifuged at 10,000 × g for 10 min. Supernatants of 0.7 ml each (800 µg of protein) from NIH 3T3 or L6 cell lysates were incubated for 2 hr with a 1:1000 dilution of anti-human FGFR-1 polyclonal antibody to immunoprecipitate FGF-1 receptors. Protein A-Sepharose beads were then added for 2 hr with continuous mixing followed by several 1-ml washes of the immune complexes bound to the beads. Immune complexes were solubilized in 40 µl of Laemmli's sample buffer and electrophoresed at 120 V for 2 hr in 8% SDS-polyacrylamide gels (Novex, San Diego, CA).

Cell growth assays. L6 cells were grown in DMEM containing 10% FBS supplemented with 0.1 mg/ml G-418 and plated at 10,000 cells/well onto 24-well plates in 0.5 ml of DMEM containing 10% FBS. After 24 hr, serum-supplemented medium was removed, and cells were washed thoroughly and then maintained in serum-free medium (as described above) for 24 hr to growth-arrest the cells. PD 166866 or vehicle (0.5% DMSO, final concentration) were added every day to triplicate cultures of cells together with 25 ng/ml bFGF to stimulate FGF-driven growth. In some experiments, PD 166866 was added every day to triplicate cultures of cells together with 30 ng/ml PDGF-BB to stimulate PDGF-driven growth. Cell number was measured by Coulter counting on days 1, 3, 6 or 8 after drug exposure.

In vitro human angiogenesis assay. Human placenta arterial vessel fragments were prepared as previously described by Brown et al. (1996). Briefly, superficial vessels, ~2 mm in diameter and 2 to 5 cm in length, were excised from the apical surface of human placentas within 24 hr of an elective cesarian birth. The vessels were placed in Hank's balanced salt solution and cut into 2-mm fragments. Assays were performed in 48-well culture plates containing 15 µl of thrombin (50 units/ml in 0.15 M NaCl; bovine plasma) plus 0.5 ml/well of fibrinogen in Medium 199. The thrombin and fibrinogen were mixed rapidly, and one vessel fragment (2 mm) was quickly placed in the center of the well before clot formation. Fibrin gel formation usually occurred within 30 sec, and the vessel fragment remained suspended in the gel. After gel formation, 0.5 ml/well of Medium 199 supplemented with 20% FCS, 0.1% -aminocaproic acid, L-glutamine, gentamycin and fungazone was added. Immediately after imbedding of vessel fragments in the fibrin gels, 0.5 ml of medium containing PD 166866 was added to each well, and each treatment was performed in quadruplicate. Fresh medium containing PD 166866 was added twice weekly. Control cultures received medium without test compound. Vessels were cultured at 37°C in a humidified environment for 14 to 21 days. Digital images of microvessels were obtained with a Dycam 3.04 digital camera (Dycam, Chatsworth, CA). Angiogenesis was quantified by tracing the edges of microvessels and calculating a mean pixel intensity of the outlined image using Image 1.41 software (National Institutes of Health, Bethesda, MD). The outgrowths have been previously verified by Brown et al. (1996) as composed of endothelial cells by staining positively for von Willebrand's factor and anti-CD-34.

Statistics. Data are expressed as the mean ± S.E.M., except where indicated. Linear regression analysis was used to generate IC₅₀ numbers. An analysis of variance with Dunnett's test was used to compare different kinase IC₅₀ values with those of the FGFR-1 kinase values. Statistical significance was defined as P < .05.

	Results

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Effect of PD 166866 on protein tyrosine kinase activity. PD 166866 (fig. 1) was identified as a potent and selective inhibitor of the human FGFR-1 tyrosine kinase with a half-maximal inhibitory potency (IC₅₀) of 52.4 ± 0.1 nM for the full-length receptor (n = 6) and 55.0 ± 2.8 nM (n = 12) for the cytoplasmic domain. In contrast, PD 166866 had no effect on c-Src, PDGFR-, EGFR or insulin receptor tyrosine kinases or MEK, PKC and CDK₄ at concentrations as high as 50 µM. Additional biochemical characterization of kinase inhibition was accomplished by analysis of reaction kinetics as a function of inhibitor concentration effects on ATP utilization by the enzyme. Table 1 shows representative inhibitory constants (K_i) and IC₅₀ determinations for PD 166866 against the various protein kinases. The K_i values obtained via nonlinear regression analysis for full-length and cytoplasmic domain forms of the FGFR-1 tyrosine kinase were similar to their respective IC₅₀ values. In figure 2, Lineweaver-Burke plots for inhibition of FGFR-1 by PD 166866 with respect to ATP concentration showed all curves intersecting the y-intercept at zero, indicative of a competitive mechanism of inhibition.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structure of PD 166866.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Representative double reciprocal plots showing the kinetics of inhibition of FGFR-1 cytoplasmic domain and full-length tyrosine kinase activity by PD 166866. Kinase activity was measured as described in the text. Kinase reactions were measured in the presence of varying concentrations of ATP. Initial reaction velocity was expressed as the number of picomoles of [-32P] incorporated into the glutamate tyrosine substrate. All x,y data sets were multiplied by 1000 for purposes of graphical presentation. The symbols indicate the concentrations of PD 166866 tested. Points represent the mean of triplicate determinations.

Effect of PD 166866 on FGF-stimulated FGFR-1 tyrosine kinase autophosphorylation in cells. The inhibitory effects of PD 166866 on bFGF-stimulated FGFR-1 tyrosine kinase autophosphorylation was evident in viable cells. NIH 3T3 and L6 cells were pretreated with varying concentrations of PD 166866 for 2 hr and then exposed to bFGF (25 ng/ml) for 5 min to induce autophosphorylation of FGF receptors. Figure 3 shows the effect of PD 166866 on FGFR-1 autophosphorylation in NIH 3T3 cells expressing endogenous FGF receptors (A) and L6 myoblasts overexpressing the human full-length FGFR-1 (B). In both cell types, bFGF elicited a robust stimulation of FGFR-1 autophosphorylation as identified by anti-phosphotyrosine immunoblotting of immunoprecipitated FGF receptors, the two bands representing FGFR-1 isoforms derived from alternative mRNA splicing of the FGFR-1 gene. PD 166866 inhibited FGFR-1 autophosphorylation in NIH 3T3 cells by 50% at a concentration of 10.8 nM, whereas FGFR-1 autophosphorylation in L6 cells was inhibited with an IC₅₀ value of 3.1 nM.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Representative immunoblots showing the effects of PD 166866 on tyrosine phosphorylation of FGFR-1 induced by bFGF in NIH 3T3 cells (A) and L6 myoblasts overexpressing the human full-length FGFR-1 (B). C, Effects of PD 166866 on bFGF-stimulated tyrosine phosphorylation of MAPKs in L6 cells. The cells were exposed to various concentrations of PD 166866 for 2 hr and then stimulated for 5 min with bFGF (25 ng/ml). The two phosphorylated FGFR-1 bands represent isoforms of the FGFR-1. Immunoprecipitation and western blotting with antiphosphotyrosine antibodies was performed as described in the text. C, Control, unstimulated cells.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Representative immunoblots showing a lack of effect of PD 166866 on tyrosine phosphorylation of PDGFR, EGFR and insulin receptor (beta subunit and receptor substrate IRS-1) induced by PDGF-BB in rat aortic smooth muscle cells (A), EGF in A431 cells (B) and insulin in NIHIR cells (C). The cells were exposed to various concentrations of PD 166866 for 2 hr. A, Rat aortic smooth muscle cells (A) were stimulated with PDGF-BB (30 ng/ml), A431 cells (B) with EGF (20 ng/ml) and NIHIR cells (C) with insulin (100 ng/ml) for 5 min. Immunoprecipitation and Western blotting with antiphosphotyrosine antibodies were performed as described in methods. C, Control, unstimulated cells.

Effect of PD 166866 on FGF-driven cell proliferation. To determine whether inhibition of FGF-stimulated FGFR-1 tyrosine kinase activity by PD 166866 would ultimately lead to interruption of the ability of the cells to replicate in response to FGF, L6 cells overexpressing FGF receptors were treated for 8 consecutive days with concentrations of PD 166866 from 1 to 100 nM. Figure 5 shows that L6 cells exposed to bFGF alone (25 ng/ml) elicit a pronounced growth response in culture. The addition of PD 166866 together with bFGF produced a potent, concentration-related inhibition of bFGF-stimulated cell growth with an IC₅₀ value of 24.1 ± 8.6 nM (n = 3) by the eighth day. At a concentration of 100 nM PD 166866, bFGF-driven growth of L6 cells was almost suspended (fig. 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of PD 166866 on bFGF-stimulated growth of L6 cells. Quiescent cells were treated every day for 8 consecutive days with varying concentrations of PD 166866 together with 25 ng/ml bFGF to stimulate growth. Cell number was measured by coulter counting on days 1, 3, 6 and 8 after drug exposure. Values represent the mean ± S.E.M. of three separate experiments performed in triplicate.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of PD 166866 on EGF- and PDGF-stimulated growth of L6 cells. Quiescent cells were treated every day for 6 consecutive days with varying concentrations of PD 166866 together with 30 ng/ml PDGF-BB to stimulate growth. EGF added at 20 ng/ml/day failed to elicit a growth response in L6 cells. Cell number was measured by coulter counting on days 1, 3 and 6 after drug exposure. Values represent the mean of three separate experiments performed in triplicate. Error bars were omitted for clarity.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of PD 166866 on serum-stimulated growth of rat aortic smooth muscle cells. Quiescent cells were treated every day for 8 consecutive days with varying concentrations of PD 166866 together with 10% FBS to stimulate growth. Cell number was measured by Coulter counting on days 1, 3, 6 and 8 after drug exposure. Values represent the mean of three separate experiments performed in triplicate. Error bars were omitted for clarity.

Effect of PD 166866 on microvessel outgrowth from human placental vessels. PD 166866 was evaluated for its ability to inhibit microvessel outgrowth from fragments of human placental arteries using a novel, physiologically relevant in vitro assay for human angiogenesis developed by Brown et al. (1996). Figure 8 (top) shows extensive microvessel outgrowth arising from fragments of human placental artery embedded in a fibrin gel and cultured in medium containing 20% serum for 20 days. Continuous exposure of PD 166866 for 20 days resulted in a concentration-related inhibition of microvessel outgrowth with ~50% of microvessel growth inhibited at 470 ± 8.7 nM (n = 3) with almost complete inhibition at 25 µM (figs. 8 and 9).

View larger version (65K): [in this window] [in a new window]   Fig. 8.   Effect of PD 166866 on microvessel outgrowths arising from fragments of human placental arteries embedded in a fibrin gel and cultured in Medium 199 containing 20% FCS and various concentrations of inhibitor. Representative photomicrographs are of a 20-day response to PD 166866.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Effect of PD 166866 on microvessel outgrowths arising from fragments of human placental arteries embedded in a fibrin gel and cultured in Medium 199 containing 20% FCS and various concentrations of inhibitor. PD 166866 was tested in serum-containing medium at 0.25, 2.5 and 25 µM. Data represent angiogenesis (microvessel outgrowth) as quantified by digital image analysis after 20 days of culture. Bars represent the mean ± S.E.M. of three separate experiments performed in quadruplicate.

	Discussion

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The growth factor receptor families along with their array of ligands represent a complex network of receptor tyrosine kinases involved in growth, mitogenesis, migration and differentiation (Fantl et al., 1993; Panayotou and Waterfield, 1993). Consequently, interruption of protein tyrosine kinase signaling has been considered a potential strategy for inhibiting such vascular pathologies as angiogenesis, tumor growth and restenosis. We recently identified a new class of protein tyrosine inhibitors based on the parent 6-aryl pyrido[2,3-d]pyrimidine structure (Blankley et al., 1997) by screening a library of synthetic compounds. A series of key substitutions around the parent pyrido[2,3-d]pyrimidine structure became apparent for determination of selectivity of various kinases, including PDGFR, FGFR and c-Src. A 2,6-dichloro substitution on the 6-phenyl group was found in broadly active and c-Src active compounds (Blankley et al., 1997; Panek et al., 1997). Changing this substitution pattern to 3,5-dimethoxy resulted in significant FGFR tyrosine kinase selectivity and the identification of PD 166866.

This report describes the biological characteristics of PD 166866, a novel, highly selective inhibitor of the FGFR-1 tyrosine kinase. PD 166866 exhibits several characteristics that are distinct from any previously reported protein tyrosine kinase inhibitors, including (1) the most potent and selective FGFR-1 tyrosine kinase inhibitor thus far identified, (2) a novel 3,5-dimethoxy pyrido[2,3-d]pyrimidine bicyclic structure, (3) ATP competitive for FGFR-1 tyrosine kinase and (4) a nanomolar potent inhibitor of FGF-mediated cellular functions, including FGFR autophosphorylation, proliferation and in vitro human angiogenesis.

A number of inhibitors of protein tyrosine kinases have been reported (Burke, 1992; Fry et al., 1994a; Traxler and Lydon, 1995). However, suppression of intracellular tyrosine phosphorylation by most of the existing compounds has been demonstrated mainly against EGFR tyrosine kinase activity and include such structures as tyrphostins (Lyall et al., 1989), lavindustin (Onoda et al., 1990), dianilinonapthanlimides (Trinks et al., 1994) and phenylamino quinazolines (Ward et al., 1994; Fry et al., 1994b) or PDGFR tyrosine kinase activity, including inhibition by tyrphostins (Bilder et al., 1991), substituted quinolines (Dolle et al., 1994), phenylaminopyrimidines (Buchdunger et al., 1995; Zimmermann et al., 1996) and biarylhydrazones (Sawutz et al., 1996). In addition, Mohammadi et al. (1996) recently described SU 4984 and SU 5402 as two members of a family of tyrosine kinase inhibitors referred to as the indolinones, which displayed preferential selectivity toward the FGF-1 receptor tyrosine kinase. Both compounds were poor inhibitors of FGF-1 receptor autophosphorylation in NIH 3T3 cells, with IC₅₀ values ranging from 10 to 40 µM. These values are ~100,000-fold less potent than the potency of PD 166866 for inhibition of FGF-1 receptor autophosphorylation.

In the present study, kinase inhibition data showed PD 166866 to be a highly selective and nanomolar potent inhibitor of the human full-length FGFR-1 tyrosine kinase. A similar inhibitory potency against the human FGFR-1 cytoplasmic domain demonstrated that the inhibitory effects of PD 166866 were due to direct interactions of this molecule with the catalytic domain of the FGFR-1, not an extracellular ligand binding site. The ATP competitive nature of PD 166866 activity further confirmed an intracellular kinase binding site. In contrast, PD 166866 had no effect on c-Src, PDGFR-, EGFR or insulin receptor tyrosine kinases or MEK, PKC and CDK₄ activities at concentrations as high as 50 µM. The inhibitory potency of PD 166866 was also apparent in its effects on cells. PD 166866 was a nanomolar potent inhibitor of bFGF-mediated FGF receptor autophosphorylation in L6 cells overexpressing the human FGFR-1 tyrosine kinase and in NIH 3T3 cells expressing endogenous FGFR-1, confirming a tyrosine kinase mechanism of action in cells in culture. PD 166866 did not inhibit PDGF, EGF or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or insulin receptor-overexpressing NIHIR cells, respectively, confirming its selectivity for the FGFR in cells. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa MAPK isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1, connecting cell surface FGF receptor activation with downstream intracellular signal transduction through MAPK. PD 166866 was also examined in cells growing in culture to determine whether inhibition of FGFR-1 tyrosine kinase activity in cells would lead to an inhibition of FGF-mediated cell proliferation. Exposure of PD 166866 to L6 cells stimulated to replicate with bFGF resulted in a potent, specific and concentration-related inhibition of cell growth with PD 166866, showing little effect on PDGF-stimulated growth of L6 cells or serum-stimulated growth of vascular smooth muscle cells.

FGFs are thought to play important roles in embryonic development, angiogenesis, wound healing and some malignancies. Inappropriate expression of FGFs or activation of FGF receptors could contribute to several human angiogenic pathologies such as diabetic retinopathy, rheumatoid arthritis, atherosclerosis and tumor neovascularization (Klagsbrun and D'Amore, 1991). Therefore, we examined PD 166866 for its ability to inhibit in vitro angiogenesis and found it to be a potent inhibitor of microvessel outgrowth from human placental blood vessel segments. The lower potency for inhibition of angiogenesis by PD 166866 (470 nM) relative to FGF-driven cellular autophosphorylation or cell growth (IC₅₀ = 10-20 nM) may be related to the ability of PD 166866 to affect endogenously produced growth factors stimulating microvessel outgrowth via autocrine/paracrine mechanisms. With static cell culture systems, FGF is supplied exogenously to modulate the response (cell autophosphorylation or growth). These results suggest that FGF plays a role in this angiogenic response and are supported by the recent findings of Brown et al. (1996), who have shown that mRNA for both bFGF and FGFR-1 is readily detected in both parent vessel and new microvessel outgrowths of the human placenta. Moreover, neutralizing antibodies against bFGF inhibited microvessel outgrowth by 66%. These data are also supported by the work of Villaschi and Nicosia (1993), who showed that bFGF is released by wounded aortic explants during angiogenesis in vitro, which implicates bFGF in autocrine regulation of angiogenesis in vitro. In addition, Brown et al. (1996) also observed the presence of VEGF and VEGF receptors (Flt-1 and KDR) in both parent vessel and new microvessel outgrowths of the human placenta. VEGF is a mitogen that is specific for endothelial cells and is thought to induce angiogenesis by acting both as an endothelial cell mitogen and by enhancing microvascular permeability. Further experimentation is in progress to determine whether PD 166866 is an inhibitor of VEGF receptor tyrosine kinases.

This study described the biological characteristics of PD 166866, a newly discovered, highly selective inhibitor of the FGFR-1 tyrosine kinase. PD 166866 is uniquely distinguished from any previously reported protein tyrosine kinase inhibitors because it demonstrates a high level of specificity for the FGFR-1 tyrosine kinase and low nanomolar inhibitory potency of FGF-mediated cellular phosphorylation and cell proliferation. Moreover, PD 166866 is the first selective FGF receptor tyrosine kinase inhibitor that we are aware of that demonstrates antiangiogenic activity. The present study highlights the discovery of PD 166866, a novel small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as an antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques.