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[CANCER RESEARCH 60, 4152–4160, August 1, 2000]

SU6668 Is a Potent Antiangiogenic and Antitumor Agent That Induces Regressionof Established Tumors1

A. Douglas Laird, Peter Vajkoczy, Laura K. Shawver, Andreas Thurnher, Congxin Liang, Moosa Mohammadi,Joseph Schlessinger, Axel Ullrich, Stevan R. Hubbard, Robert A. Blake, T. Annie T. Fong, Laurie M. Strawn, Li Sun,Cho Tang, Rachael Hawtin, Flora Tang, Narmada Shenoy, K. Peter Hirth, Gerald McMahon, andJulie M. Cherrington 2

SUGEN, Inc., San Francisco, California 94080 [A. D. L., L. K. S., C. L., R. A. B., T. A. T. F., L. M. S., L. S., C. T., R. H., F. T., N. S., K. P. H., G. M., J. M. C.]; Department ofNeurosurgery, Klinikum Mannheim, University of Heidelberg, D-68167 Mannheim, Germany [P. V., A. T.]; Department of Pharmacology, New York University Medical Center,New York, New York 10016 [M. M., J. S.]; Department of Molecular Biology, Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany [A. U.]; and Skirball Institute ofBiomolecular Medicine and Department of Pharmacology, New York University Medical Center, New York, New York 10016 [S. R. H.]

ABSTRACT

Vascular endothelial growth factor, fibroblast growth factor (FGF),and platelet-derived growth factor (PDGF) and their cognate receptortyrosine kinases are strongly implicated in angiogenesis associated withsolid tumors. Using rational drug design coupled with traditional screen-ing technologies, we have discovered SU6668, a novel inhibitor of thesereceptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor1, and PDGF receptor b kinases revealed that SU6668 has competitiveinhibitory properties with respect to ATP. Cocrystallographic studies ofSU6668 in the catalytic domain of FGF receptor 1 substantiated theadenine mimetic properties of its oxindole core. Molecular modeling ofSU6668 in the ATP binding pockets of the Flk-1/KDR and PDGF receptorkinases provided insight to explain the relative potency and selectivity ofSU6668 for these receptors. In cellular systems, SU6668 inhibited receptortyrosine phosphorylation and mitogenesis after stimulation of cells byappropriate ligands. Oral or i.p. administration of SU6668 in athymicmice resulted in significant growth inhibition of a diverse panel of humantumor xenografts of glioma, melanoma, lung, colon, ovarian, and epider-moid origin. Furthermore, intravital multifluorescence videomicroscopyof C6 glioma xenografts in the dorsal skinfold chamber model revealedthat SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668treatment induced striking regression of large established human tumorxenografts. Investigations of SU6668 activity in cancer patients are ongo-ing in Phase I clinical trials.

INTRODUCTION

The sustained growth of solid tumors is dependent on angiogenesis,the growth of new blood vessels from existing host vasculature (1, 2).Several families of RTKs3 have been implicated in this process. Theseinclude the VEGF and angiopoietin receptors (reviewed in Ref.3),which are largely dedicated to angiogenesis, and the FGFRs andPDGFRs (reviewed in Refs. 4 and 5), which are involved in diversedevelopmental and oncogenic processes.

Evidence for the direct role of VEGF and its receptor, Flk-1/KDR,in angiogenesis has been well documented. The temporal and spatialpatterns of expression of VEGF and its receptors, along with theresults of targeted mutagenesis, demonstrate that they are required forangiogenesis during development (3). Similarly, the role of ligand andreceptor in tumor angiogenesis has been clearly demonstrated using

tumor models in rodents, in which disruption of VEGF signaling usinganti-VEGF antibodies, soluble VEGF receptors, and regulatable ex-pression constructs can inhibit neovascularization and compromiseexisting tumor vasculature, resulting in inhibition of tumor growth(reviewed in Ref. 6). Elevated VEGF levels have been correlated withincreased microvessel counts and poor prognosis in many humantumor types (reviewed in Ref. 7). Due to its central role in angiogen-esis and its modest role in normal adults, VEGF signaling is anattractive therapeutic target. Several VEGF receptor-specific kinaseinhibitors have entered clinical trials for the treatment of humancancers. To date, these compounds have shown initial indications ofgood tolerability, and objective responses have been observed in somepatients (8).

FGF and PDGF also play critical roles in angiogenesis, sometimesin concert with VEGF. The prototype FGF family member, FGF2, isa potent mitogen of different cell types including vascular endothelialcells and fibroblasts (9). Although FGF2 knockout mice have noapparent defects related to impaired angiogenesis, FGF2 is clearly anangiogenic factorin vivo (10). Additionally, FGF2 has been reportedto be synergistic with VEGF and to induce the expression of VEGF(10). FGF is also a tumor cell mitogen and is expressed, along with itsreceptors, in a variety of human tumor types (11–16).

PDGF and PDGFRs are expressed in microvascular endotheliuminvivo when endothelial cell activation and angiogenesis occur. More-over, PDGF exerts growth-stimulatory effects on pericytes (17) andfibroblast-like cells (18, 19) that surround endothelial cells. Directevidence for a role of PDGF-B in vasculogenesis was demonstrated inmice deficient in PDGF-B; these mice lacked microvascular pericytes,which normally form part of the capillary wall and contribute to itsstability (20). PDGF has been reported to up-regulate other angiogenicfactors such as VEGF; thus, it has been postulated that it may alsoplay an indirect activating role in angiogenesis (21, 22).

PDGF and its receptors have been detected in diverse humancancers (23–30), and PDGFRs are expressed on tumor neovasculatureand up-regulated during tumor progression (23). Circulating PDGFhas been associated with metastases (31) and higher microvesselcounts (32). Again suggesting its direct and indirect roles in angio-genesis, PDGFR has been shown to be expressed on vascular endo-thelial cells as well as smooth muscle cells in the stroma of tumors (33).

The signaling cascades generated by these three ligands and theirrespective receptors are complex, directly and indirectly affectingtumor angiogenesis and tumor growth. Given the early promise dem-onstrated by compounds that inhibit VEGF signaling in the clinic andthe knowledge that additional players are important in angiogenesis,we developed a multipotent therapeutic agent that augmented favor-able anti-Flk-1/KDR properties with efficacy against other angiogenicsignaling molecules. Data presented here demonstrate that SU6668, asmall molecule synthetic kinase inhibitor, is a potent inhibitor of thetyrosine kinase activity of Flk-1/KDR, PDGFR, and FGFR; inhibits

Received 8/23/99; accepted 5/25/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 The intravital fluorescence microscopy studies were supported by the GermanResearch Foundation (DFG VA 151/4-1 and UL 60/4-1).

2 To whom requests for reprints should be addressed, at Preclinical Therapeutics,SUGEN, Inc., 230 East Grand Avenue, San Francisco, CA 94080.

3 The abbreviations used are: RTK, receptor tyrosine kinase; VEGF, vascular endo-thelial growth factor; FGF, fibroblast growth factor; FGFR, FGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; EGFR, epidermal growth factor receptor;HUVEC, human umbilical vein endothelial cell; GST, glutathioneS-transferase; TBST,10 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20; FBS, fetal bovine serum.

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tumor vascularization and growth of tumor xenografts of diverseorigin; and induces regression of large established tumors.

MATERIALS AND METHODS

SU6668 Chemical Synthesis

SU6668, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid (CAS Registry Number 210644-62-5), was pre-pared using a five-step synthesis from the commercially available 4-(2-methoxycarbonyl-ethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid benzyl ester(34). Briefly, 4-(2-methoxycarbonyl-ethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylicacid benzyl was hydrogenated over palladium on carbon to give 4-(2-methoxycarbonyl-ethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid, followed bydecarboxylation to give 3-(2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid methylester. It was then formulated using Vilsmeier reagent and hydrolyzed with sodiumhydroxide to give 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid. Thefinal step involved condensation of the oxindole and the above aldehyde byaldo-condensation in ethanol in the presence of piperidine to give 3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid,SU6668.

Biochemical Tyrosine Kinase Assays

Recombinant Protein Production. GST-fusion proteins of FGFR1 (ki-nase domain) and Flk-1 (cytoplasmic domain) were produced in the baculo-virus expression system. For both constructs, pFBG2T was used as the transfervector. This plasmid contains the GST coding sequence, which was amplifiedby PCR as aBamHI/BglII fragment and cloned into theBamHI site ofpFastBac-1 (Life Technologies, Inc., Rockville. MD). The portion of theFGFR1 cDNA encoding amino acids 459–757 was amplified by PCR as anEcoRI/HindIII fragment and ligated downstream of and in frame with the GSTcoding sequence in pFBG2T. The portion of Flk-1 cDNA encoding aminoacids 812-1346 was amplified by PCR as aNotI/SphI fragment and ligateddownstream of and in frame with the GST coding sequence in pFBG2T.Recombinant viruses containing the different recombinant transfer vectorswere produced following standard protocols (FastBac manual; Life Technol-ogies, Inc.). For protein production, Sf9 cells were infected following standardprocedures (35), and fusion proteins were purified by affinity chromatographyon glutathione-Sepharose (Sigma, St. Louis, MO). GST-fusion preparationswere determined to be of high quality, with no detectable breakdown products[as determined using Western blot analysis for the GST moiety followed byPonceau S staining (data not shown)].

trans-Phosphorylation Reactions.Biochemical tyrosine kinase assays toquantitate thetrans-phosphorylation activity of Flk-1 and FGFR1 were per-formed in 96-well microtiter plates precoated (20mg/well in PBS; incubatedovernight at 4°C) with the peptide substrate poly-Glu,Tyr (4:1). Excess proteinbinding sites were blocked with the addition of 1–5% (w/v) BSA in PBS.Purified GST-FGFR1 (kinase domain) or GST-Flk-1 (cytoplasmic domain)fusion proteins were produced in baculovirus-infected insect cells. GST-FGFR1 and GST-Flk-1 were then added to the microtiter wells in 23 con-centration kinase dilution buffer consisting of 100 mM HEPES, 50 mM NaCl,40 mM NaVO4, and 0.02% (w/v) BSA. The final enzyme concentration forGST-Flk-1 and GST-FGFR1 was 50 ng/ml. SU6668 was dissolved in DMSOat 1003the final required concentration and diluted 1:25 in H2O. Twenty-fiveml of diluted SU6668 were subsequently added to each reaction well toproduce a range of inhibitor concentrations appropriate for each enzyme. Thekinase reaction was initiated by the addition of different concentrations of ATPin a solution of MnCl2 so that the final ATP concentrations spanned theKm forthe enzyme, and the final concentration of MnCl2 was 10 mM. The plates wereincubated for 5–15 min at room temperature before stopping the reaction withthe addition of EDTA. The plates were then washed three times with TBST.Rabbit polyclonal antiphosphotyrosine antisera were added to the wells at a1:10,000 dilution in TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfatdry milk, and 100mM NaVO4 and incubated for 1 h at 37°C. The plates werethen washed three times with TBST, followed by the addition of goat antirabbitantisera conjugated with horseradish peroxidase (1:10,000 dilution in TBST).The plates were incubated for 1 h at 37°C and then washed three times withTBST. The amount of phosphotyrosine in each well was quantitated as

described previously (36) after the addition of 2,29-azino-di-[3-ethylbenzthia-zoline sulfonate] as substrate.

Autophosphorylation Reactions. Tyrosine kinase assays to quantitate theautophosphorylation activity of PDGFR or EGFR were performed in a similarmanner, except that the wells were precoated (0.5mg/well in 100ml of PBS)with PDGFRb- or EGFR-specific monoclonal antibodies (28D4C10 andSUMO1, respectively) to capture the respective kinase from lysates of NIH-3T3 cells engineered to overexpress PDGFRb or EGFR. The reaction bufferfor the autophosphorylation studies consisted of 25 mM Tris, 100 mM NaCl, 10mM MnCl2, 0.1% (v/v) Triton X-100, and 0.5 mM DTT.

The linear phase of each assay was determined, and reaction rates werecalculated from the linear phase of a series of reactions whose durationspanned the linear period. Assays were highly linear with respect to substrateconcentration and time (data not shown). Data were analyzed using theLineweaver-Burk inverse-reciprocal plot of 1/rateversus1/ATP concentration.Ki calculations were made using the assumption that in the case of competitiveinhibition, Km is increased by a factor of (11 [I]/K i), where [I] is theconcentration of inhibitor, and in the case of noncompetitive inhibition,Vmax

is decreased by a factor of (11 [I]/K i).

X-ray Crystallography and Molecular Modeling

Crystallographic studies of FGFR1-SU6668 complexes were performed asdescribed previously for a related molecule, SU5402 (37). Expression, purifi-cation, and crystallization of FGFR1 were performed as described previously(38). Crystals of unliganded FGFR1 were found to grow in space group C2with two molecules in the asymmetric unit and unit cell parameters whenfrozen with dimensions of a5 208.9 Å, b 5 57.5 Å, c 5 65.7 Å, andb 5 107.6 degrees. Unliganded crystals were soaked in 500ml of stabilizingsolution [25% polyethylene glycol 10000, 0.3M (NH4)2SO4, 0.1M bis-Tris (pH6.5), 5% ethylene glycol, and 2% DMSO] containing 2 mM SU6668 at 4°C for1 week. Data were collected on a Rigaku RU-200 rotating anode (Cu Ka)operating at 50 kV and 100 mA and equipped with double-focusing mirrorsand a R-AXIS IIC image plate detector. Crystals were flash-cooled in a drynitrogen stream at2175°C. Data were processed using DENZO and SCALE-PACK (39). Difference Fourier electron density maps were computed usingphases calculated from the structure of unliganded FGFR1 (40). The crystal-lography and NMR system (CNS) software suite (40) was used for simulatedannealing and positional/B-factor refinement, and O software suite (41) wasused for model building. Bulk solvent and anisotropic B-factor correctionswere applied during refinement. The average B-factor is 37.0 Å2 for all atoms,37.1 Å2 for protein atoms, and 43.0 Å2 for SU6668 atoms.

Homology models for the catalytic domains of Flk-1/KDR and PDGFRwere generated using the Modeler program (42), with the FGFR1/SU6668cocrystal structure as a reference. Sequence alignment was based on that ofHanks and Quinn (43), with slight modifications. Docking of SU6668 toFlk-1/KDR and PDGFR was performed manually, based on the FGFR1/SU6668 cocrystal structure, followed by simple energy minimization.

Cellular Assays

All cell lines were propagated as described previously (44, 45). For cellulartyrosine kinase experiments, parental NIH-3T3 mouse fibroblasts and NIH-3T3 cells overexpressing PDGFRb or EGFR were used. PDGFRb and EGFRwere highly overexpressed in the engineered NIH-3T3 cell lines relative tountransfected NIH-3T3 cells as assessed by Western blot analysis (data notshown). Cells were seeded (33 105 cells/35-mm well) in DMEM containing10% (v/v) FBS and grown to confluence and then quiesced in DMEM con-taining 0.1% serum for 2 h before drug treatment. HUVECs (seeded at 23 106

cells/10-cm plate) were grown to confluence in endothelial cell growth media[containing 12mg/ml bovine brain extract, 10mg/ml human epidermal growthfactor, 1mg/ml hydrocortisone, 2% (v/v) FBS, 50mg/ml gentamicin, and 50mg/ml amphotericin B in modified MCDB 131 (Clonetics Corp., Walkersville,MD)] and then quiesced in endothelial cell basal media (modified MCDB 131;Clonetics) containing 0.5% FBS for 24 h before drug treatment. All cell lineswere incubated with the indicated concentrations of SU6668 for 60 min beforeligand stimulation (100 ng/ml) for 10 min. Preparation of cell lysates, sepa-ration of cellular proteins (30mg from NIH-3T3 engineered cells, 100mg fromHUVECs), and immunoblotting with antiphosphotyrosine antibody were per-formed as described previously (36, 44). To determine receptor protein levels,

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REGRESSION OF ESTABLISHED TUMORS BY SU6668



membranes were stripped with elution buffer (Pierce, Rockford, IL) andreprobed with a polyclonal antibody directed against either KDR, PDGFRb,FGFR, or EGFR (all from Santa Cruz Biotechnology, Santa Cruz, CA)followed by donkey antirabbit IgG conjugated to peroxidase (AmershamPharmacia Biotech AB, Uppsala, Sweden). Immunoreactive proteins weredetected using an enhanced chemiluminescence detection reagent (AmershamPharmacia Biotech AB). To measure inhibition of ligand-stimulated mitogen-esis, HUVECs were treated with SU6668, followed by the addition of ligand,and processed as described previously (36).

In Vivo Tumor Xenograft Experiments

SUGEN, Inc. has an animal facility that is fully accredited by the Associ-ation for Assessment and Accreditation of Laboratory Animal Care Interna-tional. All procedures are conducted in accordance with the Institute ofLaboratory Animal Resources (NIH)Guide for the Care and Use of Labora-tory Animalsand with SUGEN Animal Care and Use Committee guidelines.Female athymic mice (BALB/c,nu/nu) were obtained from Charles RiverLaboratories. Animals were maintained under clean room conditions in sterileMicro-isolator cages (Lab Products) with Alpha-Dri bedding and provided freeaccess to sterile rodent chow and water. They received sterile rodent chow andwaterad libitum.

A375, Calu-6, A431, C6, and SF763T tumor cells were obtained andcultured as described previously (44). Colo205 and H460 cells were obtainedfrom American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 (Life Technologies, Inc.) supplemented with 10% FBS and 2 mM gluta-mine. SKOV3 cells were obtained from American Type Culture Collection andpassaged five times through mice to yield SKOV3TP5 cells. These cells werecultured in DMEM supplemented with 10% FBS and 2 mM glutamine. Tumorcells (3–103 106 cells/animal) were implanted s.c. into the hind flank of miceon day 0 as described previously (45). Daily treatment with SU6668 or vehiclecommenced 1 day after implantation of cells (to test efficacy against newlyimplanted tumors) or when tumors had reached a predetermined average size(to test efficacy against established tumors). SU6668 was delivered i.p. bybolus injection in DMSO or p.o. by gavage in a cremophor-based vehicleaccording to the specifics stated in figure and table legends. Tumor growth wasmeasured twice a week using vernier calipers for the duration of treatment.Tumor volumes were calculated as a product of length3 width 3 height.Pswere calculated using the two-tailed Student’st test.

Intravital Multifluorescence Videomicroscopy

Fluorescence-labeled C6 glioma cells (53 105 cells; labeled with Fast Blue;Sigma) were implanted into the dorsal skinfold chamber model of nude miceas described previously (46, 47). Animals (n5 5) were treated daily withSU6668 i.p. in DMSO (75 mg/kg/day in 50ml of DMSO), starting on the dayof glioma cell implantation. Control animals received DMSO alone (n5 4; 50ml). Intravital microscopic studies of tumor angiogenesis were performed ondays 10 and 22 after tumor cell implantation as described previously (46, 47).The vascular compartment including angiogenic sprouts, newly formed mi-crovessels, and the tumor microvasculature was visualized after contrast en-hancement with 2% FITC-conjugated dextran (0.1 ml of FITC-conjugateddextran 150 i.v.;Mr 150,000; Sigma) under blue light epi-illumination. Meas-urements of tumor vessel density (cm/cm2) were performed by means of a

computer-assisted image analysis system (46, 47). For quantitative analysis,tumor vessel densities were measured in six to nine randomly assigned regionsof interest per animal and per observation time point. Data are given as meanvalues6 SD. Mean values were calculated from the average values in eachanimal. For analysis of differences between the groups,post hocunpairedBonferroni t test was used, followed by one-way ANOVA. Results withP , 0.05 were considered significant, and results withP , 0.01 wereconsidered highly significant.

RESULTS

We have previously reported the synthesis and characterization ofa series of 3-substituted indolin-2-ones with potent and selectiveinhibitory activity toward different RTKs (48). The selectivity of thesecompounds against particular RTKs depended on the substituents onthe indolin-2-one core, especially at the C-3 position. Of specialinterest, 3-[(substituted pyrrol-2-yl)methylidenyl]indolin-2-ones showedselective inhibitory activity against VEGF receptor tyrosine autophos-phorylation at the cellular level. SU5416 is a potent inhibitor of Flk-1/KDR (46), whereas SU5402 (Fig. 1), another compound from this series,was found to inhibit tyrosine phosphorylation of Flk-1/KDR and FGFR(37, 48). SU5416 and SU5402 were used as prototype compounds forfurther modifications to develop an inhibitor active against Flk-1, FGFR,and PDGFR.

Effect of SU6668 on Biochemical Tyrosine Kinase Activity.Theeffect of SU6668 on biochemical tyrosine kinase activity was inves-tigated in enzyme kinetic experiments. Data in Table 1 demonstratethat SU6668 was a competitive inhibitor, with regard to ATP, of Flk-1trans-phosphorylation (Ki 5 2.1 mM), FGFR1trans-phosphorylation(Ki 5 1.2 mM), and PDGFR autophosphorylation (Ki 5 0.008mM).The respective ATPKm values for each kinase are also shown inTable 1; consideration of both of these values suggests that SU6668has greatest potency against PDGFR autophosphorylation but alsostrongly inhibits inhibits Flk-1 and FGFR1trans-phosphorylation. Incontrast, SU6668 did not inhibit EGFR kinase activity at concentra-tions up to 100mM (data not shown). Moreover, the biochemical IC50sof SU6668 against the EGFR, insulin-like growth factor I receptor,Met, Src, Lck, Zap70, Abl, and cyclin-dependent kinase 2 are at least10 mM (data not shown), indicating that SU6668 shows a high level ofselectivity against other tyrosine and serine/threonine kinases.

Crystallographic and Modeled Structures of SU6668 in Recep-tors. To investigate the structural basis for the observation thatSU6668 is more potent against PDGFR than Flk-1/KDR or FGF, thethree-dimensional structure of SU6668 cocrystallized within the cat-alytic domain of FGFR1 was determined. Table 2 shows the crystal-lographic data collection and refinement statistics used to generate thecocrystal model illustrated in Fig. 2,left panel. This structure was thenused to construct homology models of SU6668 bound within the ATPbinding domains of Flk-1/KDR and PDGFR. Consistent with the highdegree of amino acid homology between the kinase domain of FGFR1and Flk-1/KDR (62%) or PDGFR (51%), the modeled structures ofFlk-1/KDR (data not shown) and PDGFR (Fig. 2,right panel) werefound to be very similar to the cocrystal structure of FGFR1.

The binding of the oxindole core of SU6668 in the active sites ofFGFR1 and PDGFR is similar and comparable to what has been

Fig. 1. Chemical structures of SU6668, SU5416, and SU5402.

Table 1 Ki and Km values of SU668 versus Flk-1, FGFR1, and PDGFRb

The kinetics of inhibition of SU6668 were determined in tyrosine kinase assays asdescribed in “Materials and Methods.” Values are reported inmM.

Flk-1 trans-phosphorylation

FGFR1trans-phosphorylation

PDGFRb auto-phosphorylation

Ki Km (ATP) Ki Km (ATP) Ki Km (ATP)

SU6668 2.1 0.53 1.2 4.61 0.008 2.2

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described previously in detail for the binding of SU5402 in FGFR1(37). In both FGFR1 and PDGFR, as in Flk-1/KDR (data not shown),the oxindole core structure of SU6668 forms hydrogen bonds with thereceptor backbone at the hinge region. However, the interactionsbetween the proprionic acid side chain of SU6668 and the receptorbackbone differ between the receptors. In the SU6668/FGFR1 coc-rystal structure, the proprionic acid side chain can occupy severalpositions, with the primary one being anchored by the interactionbetween the terminal carboxylate of the side chain and the asparagineat receptor residue 568 (Fig. 2,left panel, Asn568). A similar bindingmode would be expected for Flk-1, which also has an asparagine

(Asn-921) at this position. However, this interaction is unlikely whenSU6668 is bound in the PDGFR active site because the equivalentresidue in PDGFR is an aspartic acid (Fig. 2,right panel, Asp688).Instead, when bound in the active site of PDGFR, the carboxylate ofthe proprionic acid side chain of SU6668 likely forms a more favor-able interaction with the side chain of the arginine at residue 604 (Fig.2, right panel, Arg604). Thus, from a structural viewpoint, the greaterpotency of SU6668 against PDGFR can be explained by the morefavorable interaction of the proprionic acid side chain with the recep-tor backbone.

Effect of SU6668 on Cellular Tyrosine Kinase Activity. Toconfirm the measured biochemical activity of SU6668 in a cell based-assay, tyrosine phosphorylation of receptors after ligand stimulationwas determined. HUVECs stimulated by VEGF exhibit an increase intyrosine phosphorylation of KDR. Treatment of cells with SU6668inhibited this increase in a dose-dependent manner (Fig. 3A). SU6668also inhibited PDGF-stimulated PDGFRb tyrosine phosphorylation inNIH-3T3 cells overexpressing PDGFRb at a minimum concentrationof 0.03–0.1mM (Fig. 3B). SU6668 inhibited acidic FGF-inducedphosphorylation of the FGFR1 substrate 2 (FRS-2) at concentrationsof 10 mM and higher (Fig. 3C). However, SU6668 had no detectableeffect on epidermal growth factor-stimulated EGFR tyrosine phos-phorylation in NIH-3T3 cells overexpressing EGFR at concentrationsof up to 100 mM (Fig. 3D). These cellular data demonstrate thatSU6668 inhibits Flk-1/KDR, PDGFR, and FGFR but has no activityagainst EGFR at the concentrations tested.

Effect of SU6668 on Endothelial Cell Mitogenesis.To determinewhether inhibition of purified receptors translated into a biologicaleffect in cells, the ability of SU6668 to modulate VEGF- and acidicFGF-induced mitogenesis of endothelial cells was examined. SU6668

Table 2 Crystallographic data collection and refinement statistics for theFGFR1/SU6668 structure

A. Data collection

Resolution(Å)

Observations(N)

Completeness(%) Redundancy

Rsyma

(%)Signal^I/sI&

30.0–2.3 65852 97.9 (93.5)b 3.7 4.3 (24.3)b 12.4

B. Refinementc

Resolution(Å)

Reflections(N)

Rcrystd

(%)

Root-mean-square deviations

bonds(Å)

angles(°)

B-factorse

(Å2)

25.0–2.3 29215 21.1 (24.3)f 0.007 1.2 1.5a Rsym 5 100 3 (hkl(iuIi(hkl) 2 ^I(hkl)&u/(hkl(iIi(hkl). Data are from one crystal.b Value in parentheses is for the highest resolution shell.c Atomic model includes 550 residues (two kinase molecules), two SU6668 molecules,

199 water molecules, and one sulfate ion (4569 atoms).d Rcryst 5 100 3 (hkliFo(hkl)u 2 uFc(hkl)i/(hkluFo(hkl)u, where Fo and Fc are the

observed and calculated structure factors, respectively (Fo . 2s).e For bonded protein atoms.f Value in parentheses is the free Rcryst determined from 5% of the data.

Fig. 2. Crystal structure of SU6668 in FGFR1 (left panel) and homology model of SU6668 in PDGFR (right panel).Left panel, the region of the SU6668/FGFR1 cocrystal structurecorresponding to the ATP binding site is shown. The receptor is represented byturquoise ribbons. The backbone/side chains of residues of particular interest with respect to theirinteraction with SU6668 (Asn568andLys482) are shown as stick figures with carbon atoms coloredgray. SU6668 is also shown in stick representation, with carbon atoms coloredyellow. Hydrogen bonds/close contacts between SU6668 and FGFR1 are indicated bydotted lines.Right panel, SU6668 docked into a homology model of the ATP binding site ofPDGFR. Representation and color schemes are the same as those described above for theleft panel. The interaction between the terminal carboxylate of the propionic acid side chainof SU6668 and Arg-604 of PDGFR is highlighted. The position of the side chain of Asp-688, the residue corresponding to Asn-568 in FGFR1, is also indicated.

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inhibited VEGF-driven mitogenesis of HUVECs in a dose-dependentmanner with a mean IC50 of 0.346 0.05mM (Fig. 4). In comparison,FGF-driven mitogenesis of HUVECs was inhibited with a mean IC50

of 9.6 6 0.4 mM. These data demonstrate that, consistent with thebiochemical data, SU6668 inhibits mitogenesis of HUVECs inducedby both VEGF and FGF. PDGF did not elicit a mitogenic response inHUVECS; thus, the effect of SU6668 could not be examined in thissetting. In contrast, SU6668 did not potently inhibit the proliferationof tumor cells grown in culture (IC50 . 15 mM; data not shown).

Effect of SU6668 on Tumor Xenograft Growth. Given the po-tency of SU6668versusFlk-1/KDR, PDGFR, and FGFRin vitro, itsantitumor properties were determined. p.o. administered SU6668 in-duced dose-dependent inhibition of A431 tumor growth in the s.c.xenograft model in athymic mice (Fig. 5). No mortality was observedin any treatment group. SU6668 was also efficacious when adminis-tered i.p. or p.o. in additional xenograft models, including A375,Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 cells (Table 3).Where tested, for the models in which p.o. data are shown, SU6668administered i.p. at either 75 or 100 mg/kg exhibited statisticallysignificant efficacy (data not shown). Thesein vivo data demonstratethat SU6668 readily induced.75% growth inhibition against a broadrange of tumor types.

Effect of SU6668 on Tumor Angiogenesis.To test the hypothesisthat inhibition of angiogenesis contributed to the observed effect on

tumor growth, the effect of SU6668 on tumor angiogenesis wasassessed by intravital multifluorescence videomicroscopy of C6 gli-oma xenografts implanted into dorsal skinfold chambers in nude mice.As illustrated in Fig. 6, daily treatment with SU6668 significantlysuppressed tumor angiogenesis and vascularization throughout theentire 22-day observation period. The dense network of tumor mi-crovessels in animals treated with vehicle alone (Fig. 6A) contrastswith the isolated microvessels (indicated byarrows) seen in animalstreated with SU6668 (Fig. 6B). Compared with controls, tumor vesseldensity in treated tumors was reduced by 65–95% on days 22 and 10,respectively (Fig. 6C). These results clearly demonstrate that SU6668inhibits tumor-induced microvascular proliferationin vivo and areconsistent with SU6668-directed inhibition of at least two processes,VEGF- and FGF-induced endothelial cell mitogenesis (Fig. 4) andFGF- and PDGF-stimulated proliferation of angiogenesis-promotinghost pericytes and fibroblasts.

Effect of SU6668 on Established Tumor Xenografts.Finally,given the antiangiogenic and antitumor effects demonstrated above,we challenged established tumors with SU6668. SU6668 treatmentwas initiated in groups of A431 tumor-bearing mice after tumors hadreached average group sizes of approximately 200, 400, and 800 mm3.

Fig. 4. Inhibition of endothelial cell proliferation stimulated by either VEGF or FGF.HUVECs were plated and treated as described in “Materials and Methods.” The meanabsorbance readings6 SE values are plotted from triplicate determinations.

Fig. 5. Efficacy of SU6668 on s.c. A431 xenograft growth in athymic mice. A431 cells(5 3 106; n 5 10 animals in each SU6668-treated group;n 5 20 animals in the controlgroup) were implanted s.c. into the hind flank of female athymic mice on day 0. Daily oraladministration of SU6668 (4, 40, 75, and 200 mg/kg/day in a cremophor-based formula-tion) or cremophor-based vehicle began 1 day after implantation. Tumor growth wasmeasured using vernier calipers, and tumor volumes were calculated as the product oflength3 width 3 height. Values plotted are mean tumor volume6 SE.

Fig. 3.A, HUVECs;B, NIH-3T3 cells overexpressing PDGFRb; C, NIH-3T3 cells;D,NIH-3T3 cells overexpressing EGFR. In each case, cells were cultured and serum-deprived as described in “Materials and Methods.” Cells were treated with the indicatedconcentration (mM) of SU6668 for 60 min, followed by ligand-stimulation (100 ng/ml) for10 min. After treatments, lysates were prepared and analyzed by SDS-PAGE and immu-noblotting as described in “Materials and Methods.” Thirtymg (NIH-3T3 cells andengineered cells) or 100mg (HUVECs) of lysate protein were analyzed. In all panels,lysates were initially probed using an antiphosphotyrosine antibody (top blots) andsubsequently reprobed using antibodies directed against peptide antigens to visualizeprotein levels (bottom blots).

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SU6668 induced dramatic and uniform regression in all groups, re-gardless of initial tumor size (Fig. 7A). In 20 of 39 treated animals(approximately half of the animals in all three groups), tumors re-gressed completely, leaving a vestigial scar. After discontinuation oftreatment on day 40, all 20 animals remained tumor free for anadditional 133 days (one tumor-free animal died on day 91), withthree exceptions showing regrowth (Fig. 7B). Resumption of SU6668treatment (indicated byarrows) resulted in regression of a largeregrown tumor (approximately 900 mm3; Fig. 7B). This is consistentwith observations made in the remaining 19 of 39 animals that hadtumors that did not completely regress after initial treatment withSU6668. Tumors in some of those animals regrew during the 50 daysafter cessation of treatment at day 40. After resumption of SU6668treatment, regression of tumor growth was observed for all of theseanimals, with rare exceptions (data not shown). These data demon-strate that SU6668 regresses even large tumor xenografts. Further-more, when sustained regression was not obtained after initial SU6668treatment, it was achieved in a second round of treatment.

DISCUSSION

It has become apparent over the last decade that RTKs are attractivetargets for pharmacological intervention. For example, a monoclonalantibody (Herceptin) that targets the RTK HER2 has been approvedfor the treatment of advanced breast cancer. The elucidation of thethree-dimensional structures of kinase domains has led to a greaterunderstanding of the similarities and differences among the variouskinase families and has provided insights into the structural featuresthat may be necessary for intervention by small molecules (48).Consequently, numerous small molecule, adenine mimetic inhibitors

have been developed that target different RTKs. Several inhibitors arenow being further investigated in clinical trials (8, 48, 49), withseveral more likely to progress to Phase I studies in the near future.

As shown in Table 1, SU6668 has broad activity in biochemicalassays. SU6668 is a potent inhibitor of PDGFR kinase activity with aKi value at least 503lower than theKm value of ATP. SU6668 alsoinhibited Flk-1/KDR and FGFR1 kinase activity. Cell-based assaysincluding HUVEC proliferation and inhibition of tyrosine phospho-rylation of these target kinases confirmed the activity of SU6668against these RTKs (Figs. 3 and 4). However, it is interesting that thelow Ki value of SU6668 for PDGFR relative to Flk-1 did not result ina significantly greater ability to inhibit the receptor in cells. It isnoteworthy that although theKi values of SU6668versusFlk-1 andFGFR are very similar, the inhibition of HUVEC mitogenesis isapproximately 20-fold more potent when using VEGF as a ligand ascompared with FGF (Fig. 5). Similarly, the inhibition of KDR andFlk-1 phosphorylation in cells is achieved at a lower concentration ofSU6668 than inhibition of FRS-2, a substrate phosphorylated byFGFR (Fig. 3). Although we do not fully understand these results,these data illustrate that inhibitory constants derived in the context ofpurified receptor proteins may not be uniformly translated to receptorsreplete with additional associated signaling molecules in living cells.

Analysis of the interactions of SU6668 and RTKs by X-ray crys-tallography and modeling has provided some insight into the differ-ences in SU6668 potency against PDGFR and FGFR. The proprionicacid moiety of SU6668 is in a perfect position to interact with theArg-604 side chain located at the N-lobe of the entrance of theATP-binding site on PDGFR. In contrast, the corresponding residueon FGFR and Flk-1 is a lysine. Because the lysine side chain is shorter

Table 3 Effect of daily SU6668 administration on the growth of s.c. tumor xenografts in athymic mice

Daily administration of SU6668 at the indicated does (mg/kg/day) began 1 day after implantation. SU6668 was administered i.p. in DMSO or p.o. in a cremophor-based vehicle(with the exception of the C6 experiment, where an aqueous labrasol vehicle was used). Tumor growth was measured twice a week using vernier calipers. Tumor volumes werecalculated as the product of length3 width 3 height. The percentage of inhibition compared to the vehicle-treated group was calculated on the last day of experiment.P values werecalculated by comparing mean tumor size of the untreated group with the mean tumor size of the vehicle-treated control group using the Student’st test (two tailed).n 5 8–20animals/group. Number of cells implanted per animal: A375, C6, and SF763T, 33 106; A431, Calu-6, H460, and Colo205, 53 106; SKOV3TP5, 13 107.

Cell line Tumor type Dose (mg/kg) Route of administration % Inhibition P

A375 Human melanoma 75 i.p. 91 0.03Calu-6 Human lung 100 i.p. 86 0.001A431 Human epidermoid 200 p.o. 97 0.0001Colo205 Human colon 200 p.o. 98 ,0.0001H460 Human lung 200 p.o. 94 0.02C6 Rat glioma 200 p.o. 81 ,0.0001SF763T Human glioma 200 p.o. 79 ,0.0001SKOV3TP5 Human ovarian 200 p.o. 75 0.001

Fig. 6. Effect of SU6668 on tumor xenograft angiogenesis. Fluorescence-labeled C6 glioma cells (53 105) were implanted into dorsal skinfold chamber preparations in athymicmice. Daily i.p. administration of SU6668 (75 mg/kg/day in 50ml of DMSO) or vehicle alone (50ml of DMSO) was initiated on day 0.A andB, C6 glioma microvasculature on day22 after tumor cell implantation of animals treated with DMSO (A) or SU6668 (B). Intravital multifluorescence videomicroscopy, with contrast enhancement provided by 2%FITC-conjugated dextran 150 injected i.v., is shown.Arrows, individual tumor microvessels.Scale bar, 50mm. C, tumor vessel density in C6 gliomas on day 10 and 22 after tumorcell implantation in nude mice with control tumors (n5 4; M) and in nude mice with SU6668-treated tumors (n5 5; f). Microcirculatory parameters were analyzed off-line usinga computer-assisted image analysis system. The mean6 SD values are represented. Statistical analysis was performed using ANOVA followed by unpaired Student’st test.pp, P , 0.01versuscontrol.

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than the arginine side chain, the interaction between SU6668 onFGFR and Flk-1 would be weaker than that with PDGFR (Fig. 2).

As would be expected of an inhibitor of Flk-1, FGFR1, and PDGFRkinase activity, SU6668 demonstrated significant antitumor activityagainst a wide range of xenografts (Table 3; Fig. 5). Of particularinterest are the tumor types that were poorly inhibited by SU5416,such as the human glioma cell line SF763T (37) and the humanovarian cell line SKOV3TP5. Given its target profile, SU6668 mayinfluence tumor growth by multiple mechanisms including inhibitionof endothelial cell proliferation and/or survival as well as tumor celland stromal cell proliferation. In addition, we cannot preclude thepossibility that activity against kinases (as yet unidentified) other thanFlk-1/KDR, PDGFR, and FGFR contributes to the biological activityof SU6668.

Strikingly, SU6668 has the ability to induce regression of largeestablished tumors (Fig. 7A). Whereas the mechanism(s) underlying

this capability is unknown, the anti-Flk-1/KDR activity of SU6668 islikely to be pertinent, given data implicating VEGF/Flk-1 signaling inthe survival of immature blood vessels and cultured endothelial cells(6, 50). Additionally, SU6668 may also impact other host-derivedtumor-associated cells such as pericytes and fibroblasts. Pericytesexpress VEGF and play an indispensable, PDGF-dependent, mechan-ical role in stabilizing immature blood vessels (51, 52). Fibroblastsmay support tumor growth by producing VEGF and are a potentialtarget for PDGF- and FGF-mediated proliferation (53). Consistentwith this proposed activity against host-derived cells, SU6668 exhib-ited potent antiangiogenic activity in glioma xenografts implanted intodorsal skinfold chambers (Fig. 6). In contrast, SU6668 did not po-tently inhibit the growth of cancer cells in culture (data not shown).

The activity of SU6668 on multiple members of the split RTKfamily has provided the opportunity to study some key questionsconcerning inhibitors that target several tyrosine kinases compared

Fig. 7. Efficacy of SU6668 against establishedA431 s.c. xenografts in athymic mice.A, SU6668regresses established tumors in athymic mice.A431 cells (53 106) were implanted s.c. into thehind flank of female athymic mice on day 0. Dailyoral administration of SU6668 at 200 mg/kg/day ina cremophor-based formulation was initiated forgroups of animals as they attained average tumorsizes of approximately 200 (day 13;n 5 10), 400(day 18;n 5 10), or 800 (day 29;n 5 19) mm3. Allanimals received either SU6668 or the cremophor-based vehicle alone from day 13 onward untilSU6668 treatment began.B, tumor regression wassustained in 17 of 20 mice with completely re-gressed tumors in the absence of further treatment.Arrows, resumption of treatment in three mice atday 147. Tumor growth was measured using ver-nier calipers, and tumor volumes were calculated asthe product of length3 width 3 height. Valuesplotted are mean tumor volume6 SE.

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with inhibitors that target one kinase specifically, such as SU5416.The attractive and validated targets of SU6668, coupled with itsbroad, remarkable, activity in tumor xenograft models, have moti-vated its entry into clinical development. Accordingly, SU6668 hasrecently entered Phase I clinical trials, and its safety and efficacyprofile in humans will emerge in the near future.

ACKNOWLEDGMENTS

We thank Terence Hui, William R. Kuchler, Miloe McCall, and Dr. AudieRice for protein purification and development of the kinase assays; DannyTam, James Rodda, Brian Dowd, and Dr. Stefan Vasile for performingin vitrokinase assays; Rachel Harnish, Brian Sutton, Jeremy Carver, and SheilaTanciongco for performing thein vivo xenograft studies; Ginny Li for per-forming the cellular tyrosine kinase assays; Randy Schreck for performing theHUVEC assays; Dr. Dirk Mendel for insightful comments on the manuscript;and Dr. Donna P. Schwartz for technical assistance.

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