EP1212365A2 - Structures cristallines de domaines de proteines tyrosine kinases de recepteur et de leurs ligands - Google Patents

Structures cristallines de domaines de proteines tyrosine kinases de recepteur et de leurs ligands

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Publication number
EP1212365A2
EP1212365A2 EP00959577A EP00959577A EP1212365A2 EP 1212365 A2 EP1212365 A2 EP 1212365A2 EP 00959577 A EP00959577 A EP 00959577A EP 00959577 A EP00959577 A EP 00959577A EP 1212365 A2 EP1212365 A2 EP 1212365A2
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EP
European Patent Office
Prior art keywords
ofthe
tyrosine kinase
protein tyrosine
receptor protein
ligand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00959577A
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German (de)
English (en)
Inventor
Joseph Schlessinger
Stevan B. Hubbard
Moosa Mohammadi
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New York University NYU
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New York University NYU
New York University School of Medicine
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Publication of EP1212365A2 publication Critical patent/EP1212365A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • Receptor protein tyrosine kinases include a large and diverse family of enzymes.
  • the RPTK family contains multiple subfamilies, one of which is the fibroblast growth factor receptor (FGFR) subfamily.
  • Another subfamily is the type III receptor tyrosine kinase (RTK) subfamily whose members include platelet-derived growth factor receptors ⁇ and ⁇ ("PDGFR ⁇ " and “PDGFR ⁇ "), macrophage colony-stimulating factor receptor (M- CSFR), c-kit (also referred to as SCF receptor (“SCFR”)) and i eflt3 receptor.
  • FGFR fibroblast growth factor receptor
  • RTK type III receptor tyrosine kinase
  • PDGFR ⁇ platelet-derived growth factor receptor
  • M- CSFR macrophage colony-stimulating factor receptor
  • SCFR SCF receptor
  • the members of this RTK subfamily contain five immunoglobulin-like (Ig) domains in their extracelllular ligand binding domains followed by a single transmembrane domain and a cytoplasmic tyrosine kinase domain interrupted by a large kinase-insert.
  • Ig immunoglobulin-like
  • RPTKs enzymatically transfer a high energy phosphate from adenosine triphosphate to a tyrosine residue in a target protein. These phosphorylation events regulate certain cellular phenomena in signal transduction processes.
  • Cellular signal transduction processes contain multiple steps that convert an extracellular signal into an intracellular signal. The intracellular signal is then converted into a cellular response.
  • RPTKs are components in many signal transduction processes.
  • an RPTK regulates the flow of a signal in a particular step in the process by phosphorylating a downstream molecule. This phosphorylation modulates the downstream molecule's activity by turning it either "on” or “off,” causing excessive or deficient signalling by the downstream molecule.
  • Excessive signalling can lead to such abnormalities as uncontrolled cell proliferation, which is characteristic of such disorders as cancer, angiogenesis induced by various tumors, atherosclerosis, and arthritis.
  • cellular proliferation can be induced therapeutically, for example angiogenesis may be used to ameliorate coronary artery disease by inducing collateral vascularization.
  • RPTKs Ligand-induced dimerization of RPTKs is an important step in the RPTK-mediated signal transduction process.
  • Some growth factors for example platelet-derived growth factor (“PDGF”) and stem cell factor (“SCF”), are dimeric molecules that, by themselves, induce dimerization of their specific receptors.
  • PDGF platelet-derived growth factor
  • SCF stem cell factor
  • FGFs fibroblast growth factors
  • FGFs typically function in concert with soluble or cell surface-bound heparin sulfate-containing proteoglycans (HSPGs).
  • the FGFR subfamily consists of at least 21 structurally related polypeptides, designated FGFR1 through FGFR21 , that are expressed in embryonic, fetal, and adult vertebrates.
  • FGFR1 through FGFR4 are known as "high affinity FGFRs," due to their ability to bind appropriate fibroblast growth factors with a high affinity.
  • These high affinity FGFRs are characterized by an extracellular ligand-binding domain which comprises three immunoglobulin (IG)-like domains (known as Dl, D2, and D3), a single transmembrane helix, and a cytoplasmic domain containing tyrosine kinase activity. See Lee et ah, 1989,
  • the present invention relates to the three dimensional structures of receptor protein tyrosine kinases and/or their ligands. These molecular structures may include an RPTK or ligand thereof, alone or as a complex including one or more ligands.
  • this application relates to molecular structures comprising a polypeptide which includes the extracellular domain of a receptor protein tyrosine kinase, alone and in complexes comprising one or more ligands.
  • the application describes molecular structures comprising a polypeptide which includes the receptor binding core of a growth factor, such as stem cell facter, alone or in a complex with one or more ligands such as a receptor protein tyrosine kinase.
  • the present application concerns solving and using the three dimensional structures of receptor protein tyrosine kinases, and more particularly to structures including the extracellular domain of receptor protein tyrosine kinases, alone and in complexes comprising one or more ligands.
  • X-ray crystallograpic techniques are used herein to determine the three dimensional structure of certain RPTK extracellular domains bound to certain ligands, such as FGF molecules or SCF molecules, at atomic resolution.
  • the application also concerns solving and using the three dimensional structures of stem cell factor, and more particularly to structures including the receptor binding core of stem cell factor, alone and in complexes comprising one or more ligands.
  • the three dimensional structures described herein elucidate specific interactions between receptor protein tyrosine kinases and/or ligands bound to them.
  • the coordinates that define the three dimensional structures of receptor protein tyrosine kinases are useful for determining three dimensional structures of receptor RPTKs with unknown structure.
  • the coordinates are also useful for designing and identifying modulators of receptor protein tyrosine kinase function. These modulators are potentially useful as therapeutics for treating or preventing disease, including (but not limited to) cell proliferative diseases, such as cancer, tumorigenic angiogenesis, atherosclerosis, and arthritis.
  • the invention features a crystalline form of a polypeptide corresponding to all or a portion ofthe extracellular domain of an RPTK.
  • the invention features a crystalline form of an RPTK bound to a ligand or ligand analog.
  • the RPTK is an FGFR, such as FGFR1 or FGFR2
  • the ligand is an FGF, such as FGF1 or FGF2.
  • the polypeptide comprises residues 150-360 of FGFR1 or residues 150-360 of FGFR2, the sequences of which are shown in Figure 4.
  • the ligand can be a fibroblast growth factor, such as an FGF1 including the amino acid sequence as shown in Figure 17 or an FGF2 including the amino acid sequence as shown in Figure 17.
  • crystalline form in the context ofthe invention, refers to a crystal formed from an aqueous solution comprising a purified polypeptide.
  • a crystal is formed from an aqueous solution comprising all or part ofthe extracellular domain of an RPTK.
  • a crystalline form of a polypeptide is characterized as being capable of diffracting x-rays in a pattern defined by one ofthe crystal forms depicted in Blundel et al, 1976, Protein Crystallography, Academic Press, and in Hahn, 1996, The International Tables or Crystallography, Volume A, Fourth Edition, Kluwer Academic Publishers.
  • a crystalline form may also be formed from a purified polypeptide corresponding to all or part of the extracellular domain of an RPTK in a complex with one or more ligands or ligand analogs, as defined herein.
  • a crystalline form of an RPTK may also comprise a crystal formed from an aqueous solution comprising a purified polypeptide corresponding to all or part ofthe extracellular domain of an RPTK, with or without a complexed ligand or ligand analogue, into which one or more heavy atoms are introduced.
  • introduction of a heavy atom results in as minimal a change to the original crystalline structure as possible.
  • a heavy atom can be introduced into the protein crystal by well known techniques.
  • Preferred reagents for introduction of heavy atoms are platinum tetrachloride, mercuric acetate, ethyl mercury thiosalicylate, iridium hexachloride, gadolinium sulfate, samarium acetate, gold chloride, uranyl acetate, mercury chloride, and ethyl mercury chloride.
  • RPTK receptor protein tyrosine kinase
  • an enzyme comprising an intracellular catalytic domain capable of transferring the high energy phosphate of adenosine triphosphate to a tyrosine residue located on a protein target, an extracellular domain that serves as a receptor for a specific ligand or set of ligands, and a membrane-spanning domain linking the intracellular and extracellular domains.
  • the binding of a ligand to its receptor results in receptor dimerization and activation ofthe intracellular catalytic domain.
  • Preferred RPTKs ofthe invention are PDGFR, SCFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, or MUSK.
  • a receptor PTK ofthe invention is a member ofthe FGFR family, such as FGFRl , FGFR2, FGFR3, and FGFR4. Certain receptor PTKs have no known ligand, and are referred to as "orphan receptor PTKs.”
  • FGFRl refers to one member of multiple receptor PTKs that are homologous to one another, and which bind FGF.
  • the term “homologous” preferably refers to about 70% or greater amino acid identity between two members ofthe FGFR family, more preferably at least about 80% amino acid identity, and most preferably at least about 90% amino acid identity.
  • the term “FGFRl” includes human FGFRl which comprises or consists ofthe amino acid sequence of residues 150-360 of FGFRl as shown in Figure 4. "Homologous” in this and other contexts also includes molecules of similarity sufficient to indicate relation by a common origin or archetype.
  • extracellular domain refers to all or a portion ofthe region of an RPTK that exists outside the plasma membrane of a cell.
  • an extracellular domain is anchored to the plasma membrane by a polypeptide region that associates with the plasma membrane, and most preferably by a polypeptide region that is embedded within or crosses the plasma membrane.
  • An extracellular domain can also be a soluble domain that is not anchored to the plasma membrane of a cell.
  • an extracellular domain comprises one or more binding sites for one or more ligands.
  • RPTK extracellular domains can comprise one or more known structural motifs.
  • these structural motifs can be one or more ofthe following: cysteine-rich regions, fibronectin Ill-like domains, Ig-like domains, EGF-like domains, factor VHI-like domains, and Kringle domains.
  • RPTKs comprising one or more IG-like domains.
  • FGFRl , FGFR2, FGFR3, and FGFR4 each contain three IG-like domains, labeled Dl, D2, and D3.
  • ligand refers to a molecule that specifically binds to a receptor.
  • ligands are growth factors, cytokines, lymphokines, or hormones.
  • Preferred ligands include, but are not limited to, epidermal growth factors, insulin, platelet-derived growth factors, stem cell factors, vascular endothelial growth factors, hepatocyte growth factors, and neurotrophins.
  • Particularly preferred ligands are fibroblast growth factors.
  • fibroblast growth factor refers to a family of polypeptide growth factors that share extensive sequence homologies and a common structural fold. At the time ofthe invention, the FGF family contains about 21 known members, named FGF1 through FGF21.
  • FGFs bind to FGFRs, and to HSPGs.
  • Preferred FGFs are FGF1, FGF2, FGF3, and FGF4.
  • ligand analog refers to a molecule that is structurally or functionally similar to a ligand and that binds to the ligand binding site on a polypeptide.
  • a ligand analog may be structurally similar to a ligand if the analog results from the substitution, addition, or deletion of one or more atoms, functional groups, or amino acid residues of a ligand.
  • a ligand analog is functionally similar to a ligand if the ligand analog binds to the ligand binding site ofthe ligand receptor, or if binding ofthe ligand analog to the ligand receptor results in a similar biochemical event(s) to those resulting from ligand binding.
  • Such a ligand analog may also be referred to as a ligand "mimic.”
  • Binding of a ligand analog may also result in an inhibition of one or more biochemical events which result from ligand binding, or may act as a competitor of ligand binding. Such a ligand analog may also be referred to as an "inhibitor.”
  • a ligand analog may also bind to the putative ligand binding site of an orphan receptor PTK.
  • a ligand analog may preferably bind to its ligand receptor with lower, equal, or greater affinity than does the corresponding ligand.
  • a ligand analog may be a mutant ligand.
  • the term "mutant" is defined herein.
  • binding refers to a specific interaction of two or more molecules. Binding preferably refers to noncovalent binding. Such binding is typically mediated by one or more of hydrogen-bonding, van der Waals interactions, aromatic interactions, electrostatic interactions, and hydrophobic interactions. In certain embodiments, binding can refer to covalent binding of two or more molecules.
  • catalytic domain refers to a region of a protein that can exist as a separate entity from the protein, but that retains complete or partial catalytic function.
  • the catalytic domain of a protein tyrosine kinase is characterized as having considerable amino acid identity to the catalytic domain of other protein tyrosine kinases.
  • the catalytic domain of a protein tyrosine kinase is also characterized as being a polypeptide that is soluble in solution.
  • considered amino acid identity preferably refers to at least about 30% identity, more preferably at least about 35% identity, and most preferably at least about 40% identity. These degrees of amino acid identity refer to the identity between different protein tyrosine kinase families. Amino acid identity for members of a given protein tyrosine kinase family range from about 55% to about 90%.
  • identity refers to a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly, the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Two sequences may also be homologous to one another.
  • homologous is defined herein, and can include, but is not limited to molecules (e.g., proteins) of similarity sufficient to indicate relation by a common origin or archetype..
  • BLAST Altschul, etal, 1990, J. Mol. Biol. 215:403-410) and FASTA (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-2448).
  • FASTA Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-2448.
  • the term "functional" refers to the ability of a portion of a protein to retain all or partial function ofthe intact protein.
  • a functional RPTK catalytic domain may retain the ability to convert a substrate into a product by phosphorylating the substrate, while a functional RPTK extracellular domain may retain the ability to bind to its ligand.
  • a polypeptide can exist as an extracellular domain, even though it is not functional.
  • a polypeptide corresponding to an extracellular domain may not comprise all ofthe structures necessary for binding a ligand or ligand analog.
  • a measure of an RPTK extracellular domain can be a polypeptide that is homologous to other RPTK extracellular domains.
  • the crystal comprises a polypeptide, which includes an extracellular domain of a receptor protein tyrosine kinase, and a ligand bound to the extracellular domain.
  • the receptor protein tyrosine kinase can be a fibroblast growth factor receptor, such as FGFRl or FGFR2
  • the ligand can be a fibroblast growth factor, such as FGF1 or FGF2.
  • the crystal may also include a sulfated oligosaccharide bound to the receptor protein tyrosine kinase and/or a ligand bound thereto. The size (and thus molecular weight) ofthe sulfated oligosaccharide may vary.
  • Suitable sulfated oligosaccharide which may be contained in the crystal include a sulfated disaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides.
  • the sulfated oligosaccharide is sulfated mucooligosaccharide, such as heparin.
  • the crystal includes a FGF:FGFR:heparin ternary complex.
  • the crystal can include a FGF:FGFR:heparin ternary complex such as an FGF 1 :FGFR1 :heparin ternary complex, an FGF2:FGFR1 :heparin ternary complex, an FGF1 :FGFR2:heparin ternary complex, or an FGF2:FGFR2:heparin ternary complex.
  • a FGF:FGFR:heparin ternary complex such as an FGF 1 :FGFR1 :heparin ternary complex, an FGF2:FGFR1 :heparin ternary complex, an FGF1 :FGFR2:heparin ternary complex, or an FGF2:FGFR2:heparin ternary complex.
  • a crystal may comprise a polypeptide which includes the receptor binding core of a stem cell factor.
  • the receptor binding core generally has a three dimensional structure which includes a four-helix bundle and two ⁇ strands.
  • Stem cell factors and fragments containing the receptor binding core typically crystallize in a form which includes a homodimer ofthe polypeptide. While the monomers which make up the homodimer may be covalently linked, e.g., by one or more intermolecular disulfide bonds, the SCF crystals described herein include a noncovalent homodimer.
  • the SCF homodimer may also be crystallized (e.g., in the presence of to form monoclinic crystals.
  • Monoclinic crystals of a noncovalent SCF homodimer were used to obtain the atomic structural coordinates shown in Table 4. These atomic coordinates are for crystals formed from a homodimer of a polypeptide which contains amino acid residues 1- 141 of stem cell factor. These crystals have C2 symmetry.
  • Crystals of this type may also include an RPTK, such as c-kit (SCFR) bound to a stem cell factor or fragment thereof.
  • the crystal includes c-kit bound to the receptor binding core of a stem cell factor.
  • polypeptide refers to an amino acid chain representing a portion of, or the entire sequence of, amino acid residues comprising a protein.
  • association refers to a condition of proximity between a chemical entity or compound, or portions or fragments thereof, and RPTK, or portions or fragments thereof.
  • the association may be non-covalent, i.e., where the juxtaposition is energetically favored by, e.g., hydrogen-bonding, van der Waals, electrostatic or hydrophobic interactions, or it may be covalent.
  • a crystal ofthe invention can comprise one or more heavy metal atoms. Such a crystal is referred to herein as a "derivative crystal.”
  • the invention features a crystalline form of a polypeptide corresponding to the D2-D3 region of an RPTK extracellular domain.
  • the invention features a crystalline form ofthe D2-D3 region of a receptor PTK extracellular domain bound to a ligand or ligand analog.
  • the RPTK is an FGFR, such as FGFRl or FGFR2
  • the ligand is an FGF, preferably FGF1 or FGF2.
  • the polypeptide comprises residues 150-360 of FGFRl or residues 150-360 ofFGFR2 the sequences of which are shown in Figure 4.
  • the ligand may counterpart protein or a mimic thereof.
  • the ligand can be a fibroblast growth factor, such as an FGF1 including the amino acid sequence as shown in Figure 17 or an FGF2 including the amino acid sequence as shown in Figure 17.
  • D2-D3 region refers to the second and third Ig-like domains of an FGFR.
  • Ig-like domain is well known to those of skill in the art.
  • the D2-D3 region ofthe invention may not comprise the entire second and third Ig-like domains, but contain sufficient residues to provide a binding site for the ligand ofthe FGFR.
  • the term "D2-D3 region” refers to proteins which include residues 150-360 of human FGFRl.
  • mutant refers to a polypeptide which is obtained by replacing at least one amino acid residue in a native RPTK or polypeptide ligand with a different amino acid residue. Mutation can also be accomplished by adding and/or deleting amino acid residues within the native polypeptide or at the N- and/or C-terminus of a polypeptide. Preferably, a mutant polypeptide has substantially the same three-dimensional structure as the native polypeptide.
  • the term "having substantially the same three-dimensional structure" as used herein refers to a set of atomic structure coordinates that have a root mean square deviation (r.m.s.d.) of less than or equal to about 2 A when superimposed with the atomic structure coordinates ofthe native polypeptide from which the mutant is derived, when at least about 50% to 100% ofthe C ⁇ atoms ofthe native tyrosine kinase are included in the superposition.
  • the invention relates to a crystalline form of an RPTK extracellular domain bound to a ligand defined by the structural coordinates set forth in Table 1 or Table 2.
  • atomic structural coordinates refers to a data set that defines the three dimensional structure of a molecule or molecules. Structural coordinates can be slightly modified and still render nearly identical three dimensional structures. A measure of a unique set of structural coordinates is the root-mean-square deviation ofthe resulting structure. Structural coordinates that render three dimensional structures that deviate from one another by a root-mean-square deviation of less than about 1.5 A may be viewed by a person of ordinary skill in the art as identical. Hence, the structural coordinates set forth in Tables 1-4 and 6 are not limited to the values defined therein. The use of X-ray crystallography can elucidate the three dimensional structure of crystalline forms ofthe invention.
  • the first characterization of crystalline forms by X-ray crystallography can determine the unit cell shape and its orientation in the crystal.
  • unit cell refers to the smallest and simplest volume element of a crystal that is completely representative ofthe unit of pattern ofthe crystal.
  • the dimensions ofthe unit cell are defined by six numbers: dimensions a, b and c and angles ⁇ , ⁇ and ⁇ .
  • a crystal can be viewed as an efficiently packed array of multiple unit cells.
  • crystallographic terms are described in Hahn, 1996, The International Tables for Crystallography, Volume A, Fourth Edition, Kluwer Academic Publishers; and Shmueli, The International Tables for Crystallography, Volume B, First Edition, Kluwer Academic Publishers.
  • the invention features a crystalline form of a polypeptide corresponding to the D2-D3 region of a receptor PTK extracellular domain bound to a ligand or ligand analog, where the crystal is characterized by having tetragonal unit cells and space group symmetry P4j2 ⁇ 2.
  • the RPTK is an FGFR, preferably FGFRl
  • the ligand is an FGF, preferably FGF2.
  • the polypeptide includes residues 150-360 of FGFRl or residues 150-360 of FGFR2, the sequences of which are shown in Figure 4.
  • the invention features a crystalline form of a polypeptide corresponding to the D2-D3 region of a receptor PTK extracellular domain bound to a ligand or ligand analog, where the crystal is characterized by having tetragonal unit cells and space group symmetry PI.
  • the RPTK is an FGFR, preferably FGFRl
  • the ligand is an FGF, preferably FGF1.
  • the polypeptide comprises residues 150-360 of FGFRl or residues 150-360 of FGFR2, the sequences of which are shown in Figure 4.
  • the invention features a crystalline form of a polypeptide corresponding to the D2-D3 region of a receptor PTK extracellular domain bound to a ligand or ligand analog, where the crystal is characterized by having triclinic unit cells and space group symmetry PI .
  • the RPTK is an FGFR, such as FGFR2
  • the ligand is an FGF, such as FGF2.
  • the polypeptide comprises residues 150-360 of FGFR2, the sequence of which are shown in Figure 4, and FGF2 has the sequence set forth in Figure 17.
  • space group refers to the symmetry of a unit cell.
  • space group designation e.g., P4!2 ⁇ 2, or PI
  • the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the unit cell without changing its appearance.
  • lattice in reference to crystal structures refers to the array of points defined by the vertices of packed unit cells.
  • symmetry operations refers to geometrically defined ways of exchanging equivalent parts of a unit cell, or exchanging equivalent molecules between two different unit cells. Examples of symmetry operations are screw axes, centers of inversion, and mirror planes.
  • isolated in reference to a polypeptide is meant a polymer of, for example, 6, 12, 18 or more amino acids linked to each other by chemical (e.g., peptide) bonds, including polypeptides that are isolated from natural or recombinant sources or that are chemically synthesized.
  • the isolated polypeptides ofthe present invention are unique in the sense that they are not found in a pure or separated state in nature.
  • Use ofthe term “isolated” indicates that a naturally occurring sequence, or an analog thereof, has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90 - 95% pure at least) of other material.
  • enriched refers to a specific amino acid sequence constituting a significantly higher fraction ofthe total of polypeptides present in the cells or solution of interest than in the cells or solution from which the sequence was taken.
  • a polypeptide is enriched about 2-fold, about 3-fold, about 5- fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold.
  • Enrichment may be effected by preferential reduction in the amount of other polypeptides, or by a preferential increase in the amount ofthe specific polypeptide of interest, or by a combination ofthe two.
  • enriched does not imply that there are no other polypeptides present, just that the relative amount ofthe polypeptide of interest has been significantly increased.
  • significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably about 2-fold, about 3- fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, or more.
  • an amino acid sequence be in purified form.
  • purified as used herein in reference to a polypeptide does not refer to absolute purity (such as a homogeneous preparation); instead, it refers to a polypeptide that is relatively purer than in the natural environment.
  • a polypeptide is purified about 2- fold, about 3-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100- fold.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • the substance is free of contamination at a functionally significant level.
  • the invention features a method for creating crystalline forms described herein.
  • the method may utilize the polypeptides described herein to form a crystal.
  • the method comprises the steps of: (a) mixing a volume of polypeptide solution with a reservoir solution; and
  • step (b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container, under conditions suitable for crystallization.
  • the polypeptide solution comprises about 1 mg/ml to about 50 mg/ml of the polypeptide to be crystallized, and most preferably about 1 mg/ml, 2 mg/ml, 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, and about 50 mg/ml.
  • the polypeptide solution is preferably buffered to between about pH 6.5 and about pH 9.5, most preferably about pH 8.5.
  • the solution also comprises salt, preferably in the form of KC1 or NaCl, between about 1 mM and about 500 mM, most preferably about 150 mM.
  • the reservoir solution preferably comprises between about 0.5 and about 3 M ammonium sulfate, most preferably about 1.6 M ammonium sulfate, and between about 5% and about 50% glycerol, most preferably 20% glycerol.
  • the reservoir solution preferably comprises between about 5% and about 50% polyethylene glycol, and most preferably about 20%, and between 0.05 M and 0.5 M Li 2 SO 4 , most preferably about ' 0.2 M.
  • the reservoir solution is preferably buffered to between about pH 6.5 and about pH 9.5, and most preferably about pH 8.5.
  • the invention features a three dimensional representation of a structure of an RPTK extracellular domain, alone or in complex with a ligand or ligand analog.
  • the invention features a three dimensional representation of a structure ofthe D2-D3 region of a receptor PTK extracellular domain bound to a ligand or ligand analog.
  • the RPTK is an FGFR, such as FGFRl or FGFR2
  • the ligand is an FGF, preferably FGFl or FGF2.
  • the polypeptide comprises residues 150-360 of FGFRl or residues 150-360 of FGFR2, the sequences of which are shown in Figure 4.
  • the ligand can be a fibroblast growth factor, such as an FGFl including the amino acid sequence as shown in Figure 17 or an FGF2 including the amino acid sequence as shown in Figure 17.
  • three dimensional representation refers to any non-natural representation of one or more molecules which utilize a three dimensional coordinate space.
  • the skilled artisan will recognize that the atomic structural coordinates in Tables 1-4 and 6, for example, use a three dimensional coordinate space, and thus are three dimensional representations.
  • a three dimensional representation can be a model prepared from the atomic coordinates of one or more molecules.
  • a three dimensional representation can be a model prepared from the atomic coordinates of one or more molecules that exists in a computer's memory and/or that is displayed on a computer's screen.
  • the coordinates disclosed herein provide the skilled person with the information needed to study molecular structures and interactions.
  • Comparable data can be obtained by crystallizing the molecules in view ofthe teachings contained herein and conducting x-ray analysis in accordance with the teachings contaqined herein. Such data so obtained are within the scope ofthe present invention. Moreover, variations made to the data contained herein are within the scope ofthe present invention.
  • the invention features a recombinant DNA encoding an RPTK extracellular domain.
  • the recombinant DNA can include a coding strand which includes a nucleotide sequence coding for amino acid residues 150-360 of FGFRl or residues 150-360 of FGFR2, the sequences of which are shown in Figure 4.
  • the invention relates to methods of determining three dimensional structures of RPTK extracellular domains with unknown structure by utilizing known atomic structural coordinates of an RPTK extracellular domain. These methods can relate to homology modeling, molecular replacement, and nuclear magnetic resonance methods.
  • the invention relates to a method of determining three dimensional structures of RPTK extracellular domains with unknown structures by homology modelling. These methods use the known atomic structural coordinates of an RPTK extracellular domain in conjunction with the amino acid sequences of receptor PTKs having unknown three dimensional structures.
  • the methods comprise the steps of: (a) aligning an amino acid sequence of an RPTK with unknown structure with that of an RPTK with known atomic structural coordinates, where alignment is achieved by matching homologous regions of the amino acid sequences; (b) transferring the atomic structural coordinates of each of the homologous amino acids from the known atomic structural coordinates to a computer representation of a structure ofthe corresponding amino acids in the RPTK sequence with unknown structure; and (c) determining low energy conformations ofthe resulting RPTK structure.
  • the known atomic structural coordinates are of an RPTK extracellular domain bound to a ligand or ligand analog.
  • the known atomic structural coordinates are of an FGFR extracelluar domain, preferably FGFRl, bound to an FGF, preferably FGFl or FGF2.
  • the known atomic structural coordinates are the coordinates set forth in Table 1 or Table 2.
  • the term "amino acid sequence" describes the order of amino acids in the amino acid chain comprising a polypeptide corresponding to all or a portion of an RPTK. In preferred embodiments, the amino acid sequence describes the order of amino acids in all or a portion ofthe extracellular domain of an RPTK.
  • aligning describes matching the beginning and the end of two or more amino acid sequences. Homologous amino acid sequences are placed on top of one another during the alignment process.
  • homologous as used herein in reference to protein sequences describes amino acids in two sequences that are identical or have similar side-chain chemical groups (e.g., aliphatic, aromatic, polar, negatively charged, or positively charged). Thus, protein sequences of similarity sufficient to indicate relation by a common origin or archetype are considered to possess homology, for instance. Examples of homologous amino acids are provided below.
  • corresponding refers to an amino acid that is aligned with another in the sequence alignment mentioned above.
  • determining the low energy conformation describes a process of changing the conformation ofthe RPTK structure such that the structure is of low free energy.
  • the RPTK structure may or may not have a molecule(s), such as a ligand or ligand analog, bound to it.
  • low free energy describes a state where the molecules are in a stable state as measured by the process. A stable state is achieved when favorable interactions are formed within the complex.
  • favorable interactions refers to, among other things, hydrophobic, aromatic, and ionic forces, and hydrogen bonds.
  • the term "compound” refers to an organic molecule.
  • organic molecule refers to a molecule which has at least one carbon atom in its structure.
  • the compound can have a molecular weight of less than 6kDa. Both the geometry ofthe compound and the interactions formed between the compound and the polypeptide preferably govern high affinity binding between the two molecules. High affinity binding is preferably governed by a dissociation equilibrium constant on the order of 10 "6 M or less
  • binding site refers to a location on an enzyme or polypeptide chain to which one or more molecules may bind.
  • a binding site can be a ligand binding site, a HSPG binding site, or an interaction surface between two receptors which form a dimer upon ligand binding.
  • reactions refers to hydrophobic, aromatic, and ionic forces and hydrogen bonds formed between atoms. Such interactions can be “intramolecular,” or within the same molecule, or “intermolecular,” or between separate molecules.
  • cofactor refers to a compound that may, in addition to the substrate, bind to a protein and undergo a chemical reaction. Multiple co-factors are nucleotides or nucleotide derivatives, such as phosphate and nicotinamide derivatives of adenosine.
  • substrate refers to a compound that reacts with an enzyme. Enzymes can catalyze a specific reaction on a specific substrate. For example, RPTKs can phosphorylate specific protein and peptide substrates on tyrosine moieties. In addition, nucleotides can act as substrates for protein kinases.
  • substrate analog refers to a compound that is structurally similar, but not identical, to a substrate. The substrate analog may be a nucleotide analog. Examples of nucleotide analogs are described below.
  • allosteric effector refers to a compound that causes allosteric interactions in a protein.
  • allosteric interactions refers to interactions between separate sites on a protein. The sites can be different from the active site.
  • the allosteric effector can enhance or inhibit catalytic activity by binding to a site that may be different than the active site.
  • co-crystal refers to a crystal where the polypeptide is in association with one or more compounds.
  • ATP refers to the chemical compound adenosine triphosphate.
  • non-hydrolyzable refers to a compound having a covalent bond that does not readily react with water.
  • non-hydrolyzable analogs of ATP are AMP-PNP and AMP-PCP, whose structures are well known to those skilled in the art.
  • AMP-PNP refers to adenylyl imidodiphosphate, a non-hydrolyzable analog of ATP.
  • AMP-PCP refers to adenylyl diphosphonate, a non-hydrolyzable analogue of ATP.
  • Alkyl refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon.
  • the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
  • alkenyl refers to a straight-chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond.
  • the alkenyl group has 2 to 12 carbons. More preferably it is a lower alkenyl of from 2 to 7 carbons, more preferably 2 to 4 carbons.
  • “Alkynyl” refers to a straight-chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond.
  • the alkynyl group has 2 to 12 carbons. More preferably it is a lower alkynyl of from 2 to 7 carbons, more preferably 2 to 4 carbons.
  • Alkoxy refers to an "O-alkyl” group.
  • Aryl refers to an aromatic group which has at least one ring having a conjugated pi- electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups. The aryl group may preferably be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO 2 , amine, thioether, cyano, alkoxy, alkyl, and amino.
  • Alkaryl refers to an alkyl that is covalently joined to an aryl group. Preferably, the alkyl is a lower alkyl.
  • Carbocyclic aryl refers to an aryl group wherein the ring atoms are carbon.
  • Heterocyclic aryl refers to an aryl group having from 1 to 3 heteroatoms as ring atoms, the remainder ofthe ring atoms being carbon. Heteroatoms include oxygen, sulfur, and nitrogen.
  • heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N- lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.
  • Amide refers to -C(O)-NH-R, where R is alkyl, aryl, alkylaryl or hydrogen.
  • Thioamide refers to -C(S)-NH-R, where R is alkyl, aryl, alkylaryl or hydrogen.
  • Amide refers to a -N(R')R" group, where R' and R" are independently selected from the group consisting of alkyl, aryl, and alkylaryl.
  • Thioether refers to -S-R, where R is alkyl, aryl, or alkylaryl.
  • acyl denotes groups -C(O)R, where R is alkyl as defined above, such as formyl, acetyl, propionyl, or butyryl.
  • the invention relates to methods of determining three dimensional structures of RPTK extracellular domains with unknown structures by applying the known atomic structural coordinates of an RPTK extracellular domain to incomplete X- ray crystallographic data sets for RPTK extracellular domains having unknown three dimensional structures.
  • the methods comprise the steps of: (a) determining the positions of atoms in the unit cell by matching diffraction data from two crystals, where one data set is from a crystal comprising an RPTK of unknown structure and the other is from a crystal comprising an RPTK having known atomic structural coordinates; and (b) determining a low energy conformation ofthe resulting RPTK structure.
  • the complete diffraction data is from a crystal of an RPTK extracellular domain bound to a ligand or ligand analog. More preferably, the complete diffraction data is from a crystal of an FGFR extracelluar domain, preferably FGFRl , bound to an FGF, preferably FGFl or FGF2.
  • the diffraction data set from the crystal comprising an RPTK of unknown structure may be a complete data set or an incomplete data set.
  • incomplete data set as used herein relates to a X-ray crystallographic data set that does not have enough information to give rise to a three dimensional structure.
  • the invention relates to methods of determining three dimensional structures of receptor PTK extracellular domains with unknown structure by applying the known atomic structural coordinates of an RPTK extracellular domain to nuclear magnetic resonance (NMR) data of RPTK extracellular domains having unknown three dimensional structures.
  • the methods comprise the steps of: (a) determining the secondary structure of an RPTK extracellular domain of unknown three dimensional structure using NMR data; and (b) simplifying the assignment of through-space interactions of amino acids using the known atomic structural coordinates of an RPTK.
  • the RPTK extracellular domain of unknown three dimensional structure may or may not be complexed with compounds, ligands or modulators.
  • the known atomic structural coordinates are of an RPTK extracellular domain bound to a ligand or ligand analog. More preferably, the known atomic structural coordinates are of an FGFR extracelluar domain, preferably FGFRl, bound to an FGF, preferably FGFl or FGF2. Most preferably, the known atomic structural coordinates are the coordinates set forth in Table 1 or Table 2.
  • secondary structure describes the arrangement of amino acids in a three dimensional structure, such as in ⁇ -helix or ⁇ -sheet elements.
  • through-space interactions defines the orientation ofthe secondary structural elements in the three dimensional structure and the distances between amino acids from different portions ofthe amino acid sequence.
  • the term "assignment" defines a method of analyzing NMR data and identifying which amino acids give rise to signals in the NMR spectrum.
  • the invention features methods of identifying potential modulators of PTK function. By identifying one or more potential modulators from a larger group of molecules, it is possible to reduce the number of molecules that must be tested using costly and time-consuming biological assays. Thus, the methods described herein for identifying potential modulators of PTK function can provide increased efficiencies in identifying actual modulators of PTK function.
  • modulators are preferably identified by docking a three dimensional representation of a structure of a compound with a three dimensional representation ofthe RPTK extracellular domain.
  • the computer representation ofthe RPTK extracellular domain can be defined by atomic structural coordinates.
  • one or more modulators are docked into the ligand binding site ofthe RPTK extracellular domain, and/or into the binding site for heparin sulfate-containing proteoglycans (HSPGs) ofthe RPTK extracellular domain.
  • HSPGs heparin sulfate-containing proteoglycans
  • the method of identifying potential modulators of RPTK function comprises the steps of: (a) providing a three dimensional representation of the atomic structural coordinates of an RPTK and docking a three dimensional representation of a compound from a computer data base with the three dimensional representation ofthe RPTK; (b) determining a conformation ofthe resulting complex having a favorable geometric fit and favorable complementary interactions; and (c) identifying compounds that best fit the RPTK as potential modulators of RPTK function.
  • the initial RPTK structure may or may not have one or more compounds, ligands, or modulators bound to it.
  • the atomic structural coordinates are of an RPTK extracellular domain bound to a ligand or ligand analog. More preferably, the atomic structural coordinates are of an FGFR extracelluar domain, preferably FGFRl, bound to an FGF, preferably FGFl or FGF2. Most preferably, the atomic structural coordinates are the coordinates set forth in Table 1 or Table 2.
  • modulator of RPTK function refers to a compound or ligand analog which alters the catalytic activity of an RPTK.
  • a modulator of RPTK function can either stimulate or inhibit RPTK catalytic activity.
  • inhibitory modulators may be one or more compounds or ligand analogs that disrupt dimerization of an RPTK, prevent dimerization of an RPTK, or prevent binding of an RPTK to its ligand or to HSPGs.
  • a stimulatory modulator may be one or more compounds or ligand analogs that stabilize dimer formation, or mimic the activity ofthe ligand of an RPTK, or mimic the activity of HSPGs.
  • the term "chemical group” refers to moieties that can form hydrogen bonds, hydrophobic, aromatic, or ionic interactions.
  • the term “docking” refers to a process of placing a compound, ligand or ligand analog in close proximity with an RPTK.
  • docking can refer to placing a three dimensional representation ofthe compound, ligand, or ligand analog in close proximity with a three dimensional representation ofthe RPTK.
  • the term can also refer to a process of finding low energy conformations ofthe resulting compound RPTK, ligand/RPTK, or ligand analog/RPTK complex.
  • vorable geometric fit refers to a conformation ofthe compound/RPTK, ligand/RPTK, or ligand analog/RPTK complex where the surface area of die compound, ligand, or ligand analog is in close proximity with a surface ofthe RPTK-site without forming unfavorable interactions. Unfavorable interactions can be steric hindrances between atoms in the bound molecule and atoms in the RPTK.
  • vorable complementary interactions relates to hydrophobic, aromatic, ionic, and hydrogen bond donating, and hydrogen bond accepting forces formed between the compound, ligand, or ligand analog and the RPTK.
  • best fit describes compounds, ligands, or ligand analogs that complexed the most surface area and/or form the most favorable complementary interactions with the receptor PTK in a given experiment.
  • best fit can also refer to a subset of compounds, ligands, or ligand analogs from amongst a larger group of compounds, ligands, or ligand analogs which complex the most surface area and or form the most favorable complementary interactions with the receptor PTK.
  • a molecule which exhibits a best fit is in the 70 th percentile or better of molecules tested in terms of complexing the most surface area and/or forming the most favorable complementary interactions, more preferably a molecule which exhibits a best fit is in the 80 th percentile or better of molecules tested, and most preferably, a molecule which exhibits a best fit is in the 90 th percentile or better of molecules tested.
  • Other preferred embodiments ofthe invention are methods of identifying potential modulators of receptor PTK function. The method involves utilizing a three dimensional structure of a receptor PTK.
  • the method comprises the steps of: (a) modifying a three dimensional representation of a receptor PTK having one or more compounds, ligands, or ligand analogs bound to it, where the three dimensional representations ofthe compounds, ligands, or ligand analogs and the receptor PTK are defined by atomic structural coordinates; (b) determining a conformation ofthe resulting complex having a favorable geometric fit and favorable complementary interactions; and (c) identifying the compounds, ligands, or ligand analogs that best fit the receptor PTK active-site as potential modulators of receptor PTK function.
  • the atomic structural coordinates are of an RPTK extracellular domain bound to a ligand or ligand analog. More preferably, the atomic structural coordinates are of an FGFR extracelluar domain, preferably FGFRl, bound to an FGF, preferably FGFl or FGF2. Most preferably, the atomic structural coordinates are the coordinates set forth in Table 1 or Table 2.
  • modifying refers to replacing, deleting, or adding one or more chemical groups.
  • Computer representations ofthe chemical groups can be selected from a computer data base.
  • Yet another preferred embodiment ofthe invention is a method of identifying potential modulators of RPTK function by operating modulator construction or modulator searching computer programs on the compounds, ligands, or ligand analogs complexed with the RPTK.
  • the method comprises the steps of: (a) providing a three-dimensional representation of one or more compounds, ligands, or ligand analogs complexed with an RPTK, where the computer representations ofthe compounds, ligands, or ligand analogs and the receptor PTK are defined by atomic structural coordinates; and (b) searching a data base for compounds, ligands, or ligand analogs similar to the compounds, ligands, or ligand analogs using a compound searching computer program, or replacing portions ofthe compounds, ligands, or ligand analogs complexed with the RPTK with similar chemical structures from a data base using a compound construction computer program, where the representations ofthe compounds are defined by structural coordinates.
  • the atomic structural coordinates are of an RPTK extracellular domain bound to a ligand or ligand analog. More preferably, the atomic structural coordinates are of an FGFR extracelluar domain, preferably FGFRl, bound to an FGF, preferably FGFl or FGF2. Most preferably, the known atomic structural coordinates are the coordinates set forth in Table 1 or Table 2.
  • operating refers to utilizing the three-dimensional conformation of molecules defined by the processes described herein in various computer programs.
  • similar compound refers to a compound, ligand, or ligand analog that has a similar geometric structure as compounds, ligands, or ligand analogs that can bind to a receptor PTK.
  • the similar molecule can also have similar chemical groups as a molecule that is either bound to an RPTK or once bound to an RPTK.
  • the similar chemical groups can form complementary interactions with the RPTK.
  • compound searching computer program describes a computer program that searches computer representations of compounds, ligands, or ligand analogs from a computer data base that have similar three dimensional structures and similar chemical groups as a compound of interest.
  • similar chemical structures refers to one or more chemical groups that share similar a similar geometry with one or more portions of another molecule.
  • a similar chemical structure shares a similar geometry with a molecule that is in a complex with an RPTK, or shares a similar geometry with a molecule that has been removed from an RPTK structure.
  • Similar chemical structures can also refer to chemical groups that can form one or more complementary interactions with an RPTK that are similar to those formed between and an RPTK and a complexed molecule.
  • plating structures refers to removing one or more portions of a molecule that is in a complex with an RPTK, or removing one or more portions of a molecule that has been removed from an RPTK, and connecting the broken bonds to produce a similar molecule.
  • compound construction computer program describes a computer program that replaces computer representations of chemical groups in a compound, ligand, or ligand analog with groups from a computer data base.
  • similar three dimensional structure describes two molecules with nearly identical shape and volume.
  • the invention relates to an RPTK.
  • the RPTK is preferably PDGFR, EGFR, SCFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members ofthe FGFR family, such as FGFRl, FGFR2, FGFR3, and FGFR4, or an orphan receptor PTK.
  • the invention features a potential modulator of RPTK function identified by methods disclosed in the invention.
  • Another aspect ofthe invention is a method for synthesizing a potential modulator of RPTK function or its pharmaceutically acceptable salts, isomers, metabolites, esters, amides, or prodrugs by a standard synthetic method known in the art. Synthetic procedures are discussed below.
  • the invention features methods for identifying a modulator of RPTK function.
  • the method comprises the steps of: (a) administering a potential modulator of RPTK function, ligand, ligand analog, or compound to cells; (b) comparing the level of RPTK phosphorylation between cells not administered the potential modulator, ligand, ligand analog, or compound and cells administered the potential modulator; and (c) identifying the potential modulator, ligand, ligand analog, or compound as a modulator of RPTK function based on the difference in the level of receptor PTK phosphorylation.
  • cells refers to any type of cells either primary or cultured.
  • Primary cells can be extracted directly from an organism while cultured cells rapidly divide and can be cultured in many successive rounds.
  • Cells can be grown in a variety of containers including, but not limited to flasks, dishes, and well plates.
  • administer refers to a method of delivering a potential modulator, ligand, ligand analog, or compound to cells.
  • the compound can be prepared using a carrier such as dimethyl sulfoxide (DMSO) in an aqueous solution.
  • DMSO dimethyl sulfoxide
  • the aqueous solution comprising the compound also termed an “aqueous preparation” can be simply mixed into the medium bathing the layer of cells or microinjected into the cells themselves.
  • the compounds may be administered to the cells using a suitable buffered solution.
  • suitable buffered solution refers to an aqueous preparation ofthe compound that comprises a salt that can control the pH ofthe solution at low concentrations. Because the salt exists at low concentrations, the salt preferably does not alter the function of the cells.
  • RPTK phosphorylation refers to the presence of phosphate on the RPTK.
  • Phosphates on RPTKs can be identified by antibodies that bind them specifically with high affinity.
  • the invention features a method of identifying a potential modulator of RPTK function as a modulator of RPTK function.
  • the method comprises the steps of: (a) administering a potential modulator of RPTK function to cells; (b) comparing the level of cell growth between cells not administered the potential modulator and cells administered the potential modulator; and (c) identifying the potential modulator as a modulator of RPTK function based on the difference in cell growth.
  • cell growth refers to the rate at which a group of cells divides. Cell division rates can be readily measured by methods utilized by those skilled in the art.
  • Another aspect ofthe invention features a method of diagnosing a disease by identifying cells harboring a RPTK with inappropriate activity. The method comprises the steps of: (a) administering a modulator of RPTK function to cells; (b) comparing the rate of cell growth between cells not administered the modulator and cells administered the modulator; and (c) diagnosing a disease by characterizing cells harboring a RPTK with inappropriate activity from the effect ofthe modulator on the difference in the rate of cell growth.
  • the modulator can be identified by the methods ofthe invention.
  • the term "inappropriate activity” refers to an RPTK that regulates a step in a signal transduction process at a higher or lower rate than normal cells. Aberrations in the rate of signal transduction can be caused by alterations in the stimulation of an RPTK by a growth factor, alterations in the activity of RPTK -specific phosphatase, over-expression of a RPTK in a cell, or mutations in the catalytic region ofthe RPTK itself.
  • signal transduction process describes the steps in a cascade of events where an extracellular signal is transmitted into an intracellular signal.
  • RPTK -specific phosphatase describes an enzyme that dephosphorylates a particular RPTK and thereby regulates that RPTK's activity.
  • Another aspect ofthe invention is a method of treating a disease associated with a
  • RPTK with inappropriate activity in a cellular organism where the method comprises the steps of: (a) administering the modulator of RPTK function to the organism, where the modulator is in an acceptable pharmaceutical preparation; and (b) activating or inhibiting the RPTK function to treat the disease.
  • organ relates to any living being comprised of at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal.
  • administering in reference to an organism, refers to a method of introducing the compound to the organism.
  • the compound can be administered when the cells or tissues ofthe organism exist within the organism or outside ofthe organism. Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, ocular, subcutaneous, and rectal applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
  • composition refers to a preparation comprising the modulator of RPTK activity.
  • the composition is acceptable if it does not appreciably cause irritations to the organism administered the compound.
  • the receptor PTK is selected from the group consisting of PDGFR, SCFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members ofthe FGFR family, such as FGFRl, FGFR2, FGFR3, and FGFR4, and orphan receptor PTKs.
  • FIG. 1 provides a ribbon diagram ofthe structure of a dimer of FGFRl D2-D3. complexed with FGF2. Two views are related by a rotation of about 90° about the vertical axis. The D2 and D3 domains are shown in green and blue, respectively, the short linker connecting D2 and D3 is shown in gray, and the FGF2 molecules are shown in orange.
  • FIG. 2 provides a ribbon diagram ofthe structure of FGFRl D2-D3 complexed with FGFl .
  • the D2 and D3 domains are shown in green and blue, respectively, the short linker connecting D2 and D3 is shown in gray, and FGFl is shown in orange.
  • FIG. 3 provides a topology diagram ofthe Ig folds of FGFRl D2 and D3 in comparison to the Ig fold of telokin.
  • FIG. 4 provides a sequence alignment ofthe D2-D3 region of human FGFl, FGF2, FGF3, and FGF4.
  • Figure 5 shows ribbon diagrams ofthe FGFl -FGFRl and FGF2-FGFR2 complexes with the Ig-like domains 2 (D2) and 3 (D3) are shown in green and cyan, respectively.
  • the short linker that connects D2 and D3 is colored gray.
  • FGFl and FGF2 are shown in orange.
  • the secondary structure assignments for FGFRl and FGFR2 were obtained with the program PROCHECK (Laskowski et al., J. Appl. Cryst., 26, 283-291 (1993)).
  • the beta strands for D2 and D3 are labeled according to the strand nomenclature for the canonical I-set member telokin.
  • the helix between betaA and betaA 1 , gA is a 3JQ helix.
  • the betaC-betaC loops in D3 are disordered.
  • most of the segment between betaC and betaE in D3 of FGFl -FGFRl is disordered as well.
  • this segment is well ordered and is colored purple.
  • the amino- and carboxy-termini are denoted by NT and CT.
  • the disulfide bonds in D2 and D3 are shown in ball-and-stick rendering with sulfur atoms colored yellow.
  • the beta strands of FGFl are labeled from 1 to 12 according to published nomenclature (Faham et al., Curr. Opin. Struct. Biol. 8, 578-586 (1998)). This figure was created using the programs Molscript (Kraulis, J. Appl. Crystallogr. 24, 946-950 (1991)) and Raster3D (Merrit et al., Methods Enzymol. 277, 505-524 (1997)).
  • Figure 6 shows Space-filling models ofthe FGF1-FGFR1 and FGF2-FGFR2 complexes.
  • the view and the coloring for D2, D3, the linker and FGFs are the same as in Figure 5.
  • the molecules are pulled away from each other and rotated 90° about the vertical axis as indicated (left and right panels).
  • Residues in FGFl and FGF2 are colored with respect to the FGFR regions with which they interact.
  • FGFl and FGF2 residues that interact with D2 are colored green
  • residues that interact with the linker region are colored gray
  • residues that interact with D3 are colored cyan.
  • FGF2 residues that interact with the betaC'-betaE segment (shown in purple) of FGFR2 are colored red.
  • receptor residues in the betaC'-betaE segment that contact FGF2 are in red.
  • Ligand and receptor residues are considered to be in the FGF-FGFR interface if at least one pair of atoms (side chain or main chain) has an inter-atomic distance of 3.8 A or less. This figure was created using the programs Molscript and Raster3D.
  • Figure 7 shows a stereo view of detailed interactions in the hydrophobic interface between FGF2 and D2 of FGFR2
  • Figure 8 shows a stereo view of detailed interactions in the hydrophobic interface between between FGFl and D2 of FGFRl.
  • Figure 9 shows a stereo view of detailed interactions ofthe conserved network of hydrogen bonds between FGF2 and FGFR2 in the vicinity of Arg251 in the D2-D3 linker.
  • Figure 10 shows a stereo view of detailed interactions the network of hydrogen bonds between FGFl and FGFRl in the vicinity of Arg250 in the D2-D3 linker.
  • Figure 11 shows a stereo view of detailed interactions in the interface between FGF2 and the betaF-betaG loop of D3 in the FGF2-FGFR2 structure. At the right side of each stereo pair, a view ofthe whole structure in the exact orientation as in stereo views is shown and the region of interest is highlighted. Only side chains of interacting residues are shown. Color coding is the same as in Figure 7. Dotted lines represent hydrogen bonds.
  • Figure 12 shows a stereo view of detailed interactions in the interface between N- terminal sequences (prior to betal) of FGF2 and D3 in the FGF2-FGFR2 structure. Views and coding are the same as in Figure 11.
  • Figure 13 shows a stereo view of detailed interactions in the interface between FGF2 and the betaC'-betaE segment (shown in purple) of D3 in the FGF2-FGFR2 structure. Views and coding are the same as in Figure 11.
  • Figure 14 shows a stereo view of detailed interactions in the interface between FGFl and D3 in the FGFl -FGFRl structure. Views and coding are the same as in Figure 11.
  • Figure 15 shows Structure-based sequence alignment ofthe ligand binding domains of D2 and D2-D3 linker of human FGF receptors.
  • Figure 16 shows Structure-based sequence alignment ofthe ligand binding domains of D3 of human FGF receptors.
  • Figure 17 shows structure-based sequence alignment of FGFs performed using the
  • FGF residues are colored with respect to the region on FGFR with which they interact: FGF residues that interact with D2 are colored green, residues that interact with the linker region are colored gray, and residues that interact with D3 are colored cyan. FGF residues that interact with the betaC'-betaE segment in D3 are colored red.
  • a period indicates sequence identity to FGF2.
  • a dash represents a gap introduced to optimize the alignment.
  • a tilde at the C-terminus of FGF indicates that there are additional sequences down stream to the last amino acid shown.
  • a star indicates that numbering does not start at the initiation methionine. Residue numbering for FGF2 is according to Springer et., J. Biol. Chem.
  • FGFl Residue numbering for FGFl is according to Zhu et al., Science 251, 90-93 (1991).
  • a checkmark indicates FGF residues that have been shown by mutagenesis to be important for receptor binding.
  • Figure 18 depicts the locations ofthe mutations in the human FGFR2 gene that lead to skeletal disorders are mapped onto a ribbon representation the FGF2-FGFR2 structure. Side chains ofthe residues are colored with respect to the type of substitution. In yellow are mutations that substitute a cysteine with another amino acid or vice versa, resulting in the creation of unpaired cysteines. In red are mutations that are expected to destabilize the tertiary structure of D3 and thus disfavor the formation ofthe correct intra-domain disulfide bridge. In green are mutations that are predicted to affect ligand-binding affinity or specificity.
  • Figure 19 depicts the overall structure of SCF (constructed by Molscript and Raster3D
  • Figure 20 depicts the sequence alignment based on secondary structures of SCF, M- CSF and IL-5. Secondary structure assignments for M-CSF and IL-5 are from PDB databank. beta-Strands are yellow and helices are marked bright green.
  • Figure 21 shows a stereo view ofthe dimeric interface of SCF constructed by Molscript and Raster3D (Kraulis, J. Appl. Crystallogr. 24, 946-950 (1991); Merrit et al., Methods Enzymol. 277, 505-524 (1991)). For clarity, only side-chains of residues at the core ofthe interface are shown.
  • the coding ofthe secondary structures is the same as used in Fig. 19, the strands are rendered as arrows, the helices as ribbons, and the loop regions as tubes.
  • Figure 22 shows 2Fo-Fc electron density created by O (Jones et al., Acta Crystallogr. A 47, 110-119 (1991)), contoured at 1.2 , for the hydrogen bond circle of Tyr26 and Asp25' at the dimeric interface.
  • Figure 23 depicts a model of covalent SCF dimer constructed by Molscript and
  • Residues ofthe acidic patch are colored red and residues ofthe two basic patches are colored blue. Stars mark amino acid residues that are altered in rodents.
  • the secondary structures are marked below the sequences with ⁇ representing helices and 'E 1 representing beta strands
  • Figure 26 shows a proposed model ofthe SCF in complex with Ig-like domains 2-5 of the extracellular domain of c-kit (labeled D2 to D5) created by GRASP (Nicolls et al.,
  • FIG. 27 depicts an electron density map of decasaccharides soaked into preformed crystals of an FGF2-FGFR1 complex showing the location of decasaccharides in the dimeric assemblage. Only the C ⁇ traces of D2s (cyan) and FGFs (orange) are shown. The decasaccharides are rendered in white sticks.
  • Figure 28 depicts a stereoview of an FoFc electron density map of an FGF2-FGFR1 complex shown in Figure 27 computed after simulated annealing with decasaccharide omitted from the atomic model.
  • the map is computed at 3.0 A resolution and contoured at 1.8 ⁇ .
  • Sugar rings are labeled A through H starting at the non-reducing end ofthe decasaccharide.
  • Atom coloring is as follows: oxygens in red, sulfurs in yellow, nitrogens in blue, and carbons in gray. This figure was constructed using Bobscript (Esnouf, J. Mol. Graph. Model 15, 132-134 (1997).
  • Figure 29 shows a stereoview ofthe detailed interactions between ordered decasaccharide rings (A-F), FGF and FGFR. Only the side chains of interacting residues are shown.
  • the two D2s ofthe adjoining FGFRs are colored cyan and green respectively.
  • Atom coloring is the same as in Figure 27.
  • the carbon atoms in FGFRs have the same coloring as the D2 to which they belong. Dotted lines represent hydrogen bonds.
  • Figure 30 shows a schematic diagram of interactions between decasaccharide (heparrin), FGF and FGFR in the ternary complex. Only the relevant functional groups and backbone atoms ofthe interacting amino acids are shown. Dashed lines represent hydrogen bonds. Hashed lines represent hydrophobic interactions. The sugar rings of heparin are labeled A through F starting at the non-reducing end. The backbone carbon atoms of heparin are numbered according to IUPAC nomenclature. The type and the number of interacting residues are colored based on the molecule to which they belong.
  • Figure 31 shows the results of a separation on a Superdex 200 column (Pharmacia) of dimer formation for a set of mixtures of various ratios of homogeneously-sulfated hexasaccharide with purified 1 : 1 FGF1-FGFR2 complex.
  • the following reaction mixture were used: A, control (no hexasaccharide added); B, hexasaccharide:FGFl-FGFR2 complex molar ratio of 0.5: 1 ; C, , hexasaccharide:FGFl-FGFR2 complex molar ratio of 1 : 1 ; D, and hexasaccharide:FGFl-FGFR2 complex molar ratio of 2.85:1.
  • FIG. 32 depicts a molecular surface representation ofthe "two end” model ofthe dimeric 2:2:2 FGF2-FGFR1 -heparin ternary complex. The view is from the top (same view as Fig. 27) looking down into the heparin-binding canyon. The FGF2 surface is shown in orange and D2 in green. Only the first 6 sugar rings ofthe decasaccharides are rendered in ball-and-stick and the non-reducing and reducing ends are labeled.
  • Figure 33 shows a schematic illustration of a computer based system which can be used for displaying, studying, comparing, manipulating, interpreting and/or extrapolating data from the crystallographic analysis of molecular structures, such as the molecular structures of RPTKs, their ligands and related complexes.
  • Table 1 provides the atomic structure coordinates of crystals of FGFRl -D2-D3 complexed with FGF2 ofthe invention as determined by X-ray crystallography.
  • Table 2 provides the atomic structure coordinates of crystals of FGFRl -D2- D3 complexed with FGFl ofthe invention as determined by X-ray crystallography.
  • Table 4 provides the atomic structure coordinates of crystals of an SCF (1-141) non-covalent homodimer.
  • Table 6 provides the atomic structure coordinates of crystals of a dimeric 2:2:2 FGF2:FGFR1 :heparin ternary complex.
  • the present invention is directed to the determination and use of three dimensional structures of receptor protein tyrosine kinases.
  • the three dimensional structures of receptor PTKs can facilitate the design and identification of modulators of receptor PTK function.
  • PTKs Protein tyrosine kinases
  • the PTK family is subdivided into members that are receptors and those that are non-receptors.
  • the receptor PTK (RPTK) family contains multiple subfamilies, one of which is the fibroblast growth factor receptor (FGFR) PTK which is a molecule implicated in regulating angiogenesis a well as cellular proliferation and differentiation.
  • FGFRl through FGFR4 are known as "high affinity FGFRs," due to the ability to bind fibroblast growth factors with a high affinity.
  • FGFRs are characterized by an extracellular ligand-binding domain which comprises three immunoglobulin (IG)-like domains (known as Dl through D3), a single transmembrane helix, and a cytoplasmic domain containing tyrosine kinase activity.
  • IG immunoglobulin
  • Dl through D3 immunoglobulin-like domains
  • FGFRs can mediate cellular functions by their role in one or more cellular signal transduction processes.
  • Cellular signal transduction processes comprise a cascade of multiple steps that convert an extracellular signal into an intracellular signal.
  • RPTK-mediated signal transduction is initiated by binding of a specific extracellular ligand to the extracellular domain, followed by receptor dimerization, and subsequent autophosphorylation ofthe RPTK.
  • Preferred ligands are epidermal growth factors, insulin, platelet-derived growth factors, vascular endothelial growth factors, fibroblast growth factors, hepatocyte growth factors, and neurotrophins.
  • the FGF subfamily presently contains about 18 members, named FGFl through FGFl 8, which bind to FGFRs, and to HSPGs.
  • FGFl through FGFl 8 which bind to FGFRs, and to HSPGs.
  • Those skilled in the art can identify presently unknown members ofthe FGF subfamily by sequence homology to known subfamily members, and/or by the presence of a common protein fold.
  • Each ofthe four high affinity FGFRs binds to a specific subset of FGFs. Ornitz et al, 1996, J. Biol. Chem. 211:
  • the phosphate groups are binding sites for intracellular signal transduction molecules which leads to the formation of protein complexes at the cell membrane. These complexes facilitate an appropriate cellular effect (e.g., cell division, metabolic effects to the extracellular microenvironment) in response to the ligand that began the cascade of events.
  • RPTKs function as binding sites for several intracellular proteins. Intracellular RPTK binding proteins are divided into two principal groups: (1) those which harbor a catalytic domain; and (2) those which lack such a domain but serve as adapters and associate with catalytically active molecules. Songyang etal., 1993, Cell 72:767-778. SH2 (src homology) domains are common adaptors found in proteins which directly bind to the RPTK. SH2 domains are harbored by RPTK binding proteins of both groups mentioned above. Fantl et al, 1992, Cell 69 ⁇ 13-423; Songyang et al, 1994, Mol. Cell. Biol. 74:2777-2785); Songyang et al, 1993, Cell 72:161-11%; and Koch et al, 1991, Science 252:668-678.
  • RPTKs The specificity ofthe interactions between RPTKs and the SH2 domains of their binding proteins is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities of SH2 domains is correlated with the observed differences in substrate phosphorylation profiles of downstream molecules in the signal transduction process. Songyang et al., 1993, Cell 72:161-11%. These observations suggest that the function of each RPTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, RPTKs provide a controlling regulatory role in signal transduction processes as a consequence of autophosphorylation.
  • RPTK-mediated signal transduction regulates cell proliferative, differentiation, and metabolic responses in cells. Therefore, inappropriate RPTK activity can result in a wide array of disorders and diseases. These disorders, which are described below, may be treated by the modulators of RPTK function designed or identified by the methods disclosed herein.
  • the present invention also relates to crystalline polypeptides corresponding to the extracellular domain of receptor tyrosine kinases.
  • Such receptor protein tyrosine kinases are not covalently cross-linked, but are understood to undergo ligand-induced dimerization.
  • the crystalline extracellular domains are of sufficient quality to allow for the determination of a three-dimensional X-ray diffraction structure to a resolution of about 1.5 A to about 3 A, and most preferably about 2.8 A.
  • the invention also relates to methods for preparing and crystallizing the polypeptides.
  • polypeptides themselves, as well as information derived from their crystal structures can be used to analyze and modify tyrosine kinase activity as well as to identify compounds that interact with the extracellular domain.
  • the polypeptides ofthe invention are most preferably designed on the basis ofthe structure of a region in the extracellular domain ofthe RPTKs that contains the ligand binding domain.
  • FIG. 4 shows the amino acid sequence alignment of the ligand binding D2-D3 domains of human FGFRl, FGFR2, FGFR3, and FGFR4.
  • the applicants have discovered and determined the boundaries ofthe extracellular domain required for crystallization ofthe resulting polypeptide. Surprisingly, these boundaries are very similar to a naturally occurring variant of FGFRl which retains approximatly full ligand binding capacity and specificity. See Johnson etal, Mol. Cell. Biol, 1990 10: 4728-4736.
  • the resulting crystal structures consists of a unit cell comprising a dimer of two FGFRl D2-D3 domains, each bound to an FGF molecule.
  • the dimeric structure is stabilized by interactions between the two D2 domains, and by interactions between the FGF molecule in one member ofthe dimer and the D2 domain ofthe other member ofthe dimer.
  • These contacts which stabilize the dimeric structure within the crystal are believed to be similar or identical to contacts which result in dimerization and activation of FGFRl in vivo.
  • the crystal structures ofthe invention provides for the first time a detailed view ofthe events leading to ligand-induced dimerization and activation of RPTKs.
  • the crystal structures also disclose a possible role for the acid box region ofthe extracellular domain of RPTKs in dimerization and activation.
  • the acid box is a continuous stretch of acidic residues in the linker between Dl and D2.
  • Models inferred from the crystal structures ofthe invention imply that the acid box may interact with the heparin binding region of D2, competing with heparin for binding.
  • these models imply that loss ofthe ofthe acid box / D2 interaction may permit heparin-induced dimerization and activation of FGFRl in the absence of FGF.
  • modulators of RPTK function for example molecules which contribute to or disrupt receptor/ligand binding or intradimer contacts. Such modulators may provide useful treatments for various RPTK diseases.
  • PTK-associated diseases and disorders include, but are not limited to, blood vessel proliferative disorders, fibrotic disorders, and mesangial cell proliferative disorders.
  • Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels.
  • the formation and spreading of blood vessels play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development, for example in Kaposi's sarcoma.
  • blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness, and von Hippel- Lindau disease (VHL), which is characterized by a predisposition for retinal angiomas, hemangioblastomas in the central nervous system, renal cell carcinomas, pheochromocytomas, and islet cell tumors ofthe pancreas.
  • VHL von Hippel- Lindau disease
  • disorders related to the shrinkage, contraction or closing of blood vessels are implicated in such diseases as restenosis.
  • Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis.
  • Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
  • Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies.
  • the PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al, 1993, Kidney International 43:47S-54S.
  • RPTKs are directly associated with the cell proliferative disorders described above. For example, some members ofthe RPTK family have been associated with the development of cancer. Some of these receptors, like EGFR (Tuzi et al, 1991, Br. J. Cancer 6 ' 3:227-233; Torp et al, 1992, APMIS 100:713-119) HER2/neu (Slamon et al, 1989, Science 244:707- 712) and PDGF-R (Kumabe et al, 1992, Oncogene 7:627-633) are over-expressed in many tumors and/or persistently activated by autocrine loops.
  • EGFR Tuzi et al, 1991, Br. J. Cancer 6 ' 3:227-233; Torp et al, 1992, APMIS 100:713-119
  • HER2/neu Semon et al, 1989, Science 244:707- 712
  • PDGF-R Kerabe et al
  • RPTK over-expression (Akbasak and Suner-Akbasak et al, 1992, J. Neurol. Sci. 777:119-133; Dickson et al, 1992, Cancer Treatment Res. 57:249-273; Korc et al, 1992, J. Clin. Invest. 90:1352-1360) and autocrine loop stimulation (Lee and Donoghue, 1992, J. Cell. Biol 118:1057-1070; Korc et al. , supra; Akbasak and Suner-Akbasak et al. , supra) account for the most common and severe cancers.
  • EGFR is associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer.
  • HER2 is associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.
  • PDGFR is associated with glioblastoma, lung, ovarian, and prostate cancer.
  • the RPTK c-met is generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma.
  • c-met is linked to malignant tumor formation. More specifically, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression ofthe c-met gene has been detected in patients with Hodgkin's disease, Burkitt's disease, and the lymphoma cell line.
  • the IGF-I RPTK in addition to being implicated in nutritional support and in type-II diabetes, is also associated with several types of cancers.
  • IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al. , 1989, J. Clin. Invest. 84: 1418- 1423) and small lung tumor cells (Macauley et al, 1990, Cancer Res. 50:2511-2517).
  • IGF-I integrally involved in the normal growth and differentiation ofthe nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al, 1993, Cancer Res. 53:2475-2478.
  • IGF-IR insulin growth factor-associated fibroblasts
  • fibroblasts epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells ofthe bone marrow
  • IGF-I insulin growth factor-associated fibroblasts
  • Goldring and Goldring 1991, Eukaryotic Gene Expression 7:301-326.
  • IGF-IR plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res. 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al, 1994, Mol. Cell. Biol. 7 :4588-4595.
  • RPTKs are associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases.
  • EGFR is indicated in corneal and dermal wound healing.
  • Defects in InsulinR and IGFIR are indicated in type-II diabetes mellitus.
  • the instant invention is directed in part towards designing modulators of RPTK function that could indirectly kill tumors by cutting off their source of sustenance.
  • Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy. Folkman and Shing, 1992, J. Biological Chem. 267: 10931-34.
  • many diseases are driven by persistent unregulated or inappropriate angiogenesis. For example, in arthritis, new capillary blood vessels invade the joint and destroy the cartilage. In diabetes, new capillaries in the retina invade the vitreous, bleed and cause blindness.
  • Ocular neovascularization is the most common cause of blindness and dominates approximately twenty (20) eye diseases.
  • vasculogenesis and/or angiogenesis can be associated with the growth of malignant solid tumors and metastasis.
  • a tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.
  • the new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and to metastasize to distant sites in the body.
  • VEGF vascular endothelial growth factor
  • placental growth factor a polypeptide with in vitro endothelial cell growth promoting activity. Examples include acidic and basic fibroblastic growth factor ( ⁇ FGF, ⁇ FGF), vascular endothelial growth factor (VEGF) and placental growth factor. Unlike ⁇ FGF and ⁇ FGF, VEGF has recently been reported to be an endothelial cell specific mitogen. Ferrara and Henzel, 1989, Biochem. Biophys. Res. Comm. 757:851-858; Vaisman et al, 1990, J. Biol. Chem. 255:19461-19566.
  • identifying the specific receptors that bind FGF or VEGF is important for understanding endothelial cell proliferation regulation.
  • Two structurally related receptor PTKs that bind VEGF with high affinity are identified: the flt-1 receptor (Shibuya et al, 1990, Oncogene 5:519-524; De Vries et al, 1992, Science 255:989-991) and the KDR/FLK-1 receptor (VEGFR2), discussed in the U.S. Patent Application No. 08/193,829.
  • a receptor that binds FGF is identified. Jaye et al, 1992, Biochem. Biophys. Acta 7735:185- 199). Consequently, these RPTKs most likely regulate endothelial cell proliferation.
  • FGFRs play important roles in angiogenesis, wound healing, embryonic development, and malignant transformation. Basilico and Moscatelli, 1992, Adv. Cancer Res. 59:115-165.
  • FGFRl -4 Four high affinity mammalian FGFRs (FGFRl -4) have been described and additional diversity is generated by alternative RNA splicing within the extracellular domains. Jaye et al, 1992, Biochem. Biophys. Acta 7735:185-199. Like other RPTKs, dimerization of FGF receptors is essential for their activation. Soluble or cell surface-bound heparin sulfate proteoglycans act in concert with FGF to induce dimerization (Schlessinger et al, 1995, Cell 53:357-360), which leads to autophosphorylation of specific tyrosine residues in the cytoplasmic domain. Mohammadi et al, 1996, Mol. Cell Biol. 75:977-989.
  • Apert FGFR2
  • Pfeiffer FGFRl and FGFR269-274
  • FGFR2 Jackson- Weiss
  • FGFR2 Crouzon
  • FGFR2 Crouzon
  • FGFR3 mutations in FGFR3 are implicated in long bone disorders and cause several clinically related forms of dwarfism including achondroplasia (Shiang et al., 1994, Cell 75:335-342), hypochondroplasia (Bellus et al., 1995, Nat. Genet. 70:357-359) and the neonatal lethal thanatophoric dysplasia
  • FGFs FGF-like growth factor receptors
  • angiogenesis Falkman and Klagsbrun, 1987, Science 235:442
  • inappropriate expression of FGFs or of their receptors or aberrant function ofthe tyrosine kinase activity could contribute to several human angiogenic pathologies such as diabetic retinopathy, rheumatoid arthritis, atherosclerosis and tumor neovascularization (Klagsbrun and Edelman, 1989,
  • FGFs are thought to be involved in malignant transformation. Indeed, the genes coding for the three FGF homologues int-2, FGF-5 and hst-1/K-fgf were originally isolated as oncogenes. Furthermore, the cDNA encoding FGFRl and FGFR2 are amplified in a population of breast cancers (Adnane et al., 1991, Oncogene 5:659-663). Over-expression of FGF receptors has been also detected in human pancreatic cancers, astrocytomas, salivary gland adenosarcomas, Kaposi's sarcomas, ovarian cancers and prostate cancers.
  • VEGF is not only responsible for endothelial cell proliferation, but also is a prime regulator of normal and pathological angiogenesis. See generally, Klagsburn and Soker, 1993, Current Biology 3:699-702; Houck et al, 1992, J. Biol. Chem. 257:26031-26037. Moreover, it has been shown that KDR/FLK-1 and flt-1 are abundantly expressed in the proliferating endothelial cells of a growing tumor, but not in the surrounding quiescent endothelial cells. Plate et al, 1992, Nature 359:845-848; Shweiki et al, 1992, Nature 359:843-845.
  • the invention is directed to designing and identifying modulators of RPTK functions that could modify the inappropriate activity of a RPTK involved with a clinical disorder.
  • the rational design and identification of modulators of RPTK functions can be accomplished by utilizing the structural coordinates that define a RPTK three dimensional structure.
  • PCT WO 94/03427 seleoindoles and selenides
  • PCT WO 92/21660 tricyclic polyhydroxylic compounds
  • PCT WO 91/15495 benzylphosphonic acid compounds
  • modulators of RPTK function are known, many of these are not specific for RPTK subfamilies and will therefore cause multiple side-effects as therapeutics.
  • Certain compounds ofthe oxindolinone/ thiolindolinone family are believed to be specific for the FGF receptor subfamily (U.S. Patent Application Serial No. 08/702,232, filed August 23, 1996, invented by Tang et al., entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease”).
  • compounds ofthe oxindolinone/thiolindolinone family are non-hydrolyzable in acidic conditions and can be highly bioavailable.
  • Crystalline RPTKs ofthe invention include native crystals, derivative crystals and co- crystals.
  • the native crystals ofthe invention generally comprise substantially pure polypeptides corresponding to the extracellular domain of an RPTK in crystalline form.
  • the crystals ofthe invention comprise polypeptides corresponding to the extracellular domain of an RPTK in a complex with a ligand.
  • the crystalline extracellular domains ofthe invention are not limited to naturally occurring or native extracellular domains. Indeed, the crystals ofthe invention include mutants of native extracellular domains. Mutants of native extracellular domains are obtained by replacing at least one amino acid residue in a native extracellular domain with a different amino acid residue, or by adding or deleting amino acid residues within the native polypeptide or at the N- or C-terminus ofthe native polypeptide, and have substantially the same three-dimensional structure as the native extracellular domain from which the mutant is derived.
  • the crystals ofthe invention include mutants of native extracellular domains and mutant ligands.
  • mutant ligands can be obtained by replacing at least one amino acid residue in a polypeptide ligand with a different amino acid residue, or by adding or deleting amino acid residues within the native polypeptide or at the N- or C-terminus of the native polypeptide, and have substantially the same three-dimensional structure as the native ligand from which the mutant is derived.
  • Fig. 3 shows that 68 common C ⁇ atoms in the D2 and D3 regions of FGFRl and telokin, a canonical IG-fold polypeptide, can be superimposed with a rms deviation of 0.8 A.
  • Amino acid substitutions, deletions and additions which do not significantly interfere with the three-dimensional structure of a polypeptide will depend, in part, on the region ofthe polypeptide where the substitution, addition or deletion occurs. In highly variable regions of the molecule, non-conservative substitutions as well as conservative substitutions may be tolerated without significantly disrupting the three-dimensional structure ofthe molecule. In highly conserved regions, or regions containing significant secondary structure, conservative amino acid substitutions may be preferred. Conservative amino acid substitutions are well-known in the art, and include substitutions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature ofthe amino acid residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • Other conservative amino acid substitutions are well known in the art.
  • the selection of amino acids available for substitution or addition is not limited to the genetically encoded amino acids. Indeed, the mutants described herein may contain non- genetically encoded amino acids. Conservative amino acid substitutions for many ofthe commonly known non-genetically encoded amino acids are well known in the art. Conservative substitutions for other amino acids can be determined based on their physical properties as compared to the properties ofthe genetically encoded amino acids.
  • a native extracellular domain in order to provide convenient cloning sites in a DNA, such as a cDNA, encoding the polypeptide, to aid in purification ofthe polypeptide, and for crystallization ofthe polypeptide.
  • a DNA such as a cDNA
  • Such substitutions, deletions and/or additions which do not substantially alter the three dimensional structure of the native tyrosine kinase domain will be apparent to those of ordinary skill in the art. It should be noted that the mutants contemplated herein need not exhibit ligand binding activity.
  • amino acid substitutions, additions or deletions that interfere with the ligand binding activity ofthe RPTK extracellular domain but which do not significantly alter the three-dimensional structure ofthe domain are specifically contemplated by the invention.
  • Such crystalline polypeptides, or the atomic structure coordinates obtained therefrom, can be used to identify compounds or molecules that bind to the native domain. These compounds or molecules may affect the activity ofthe native domain.
  • the derivative crystals ofthe invention generally comprise a crystalline RPTK extracellular domain polypeptide in covalent association with one or more heavy metal atoms.
  • the polypeptide may correspond to a native or a mutated tyrosine kinase domain.
  • Heavy metal atoms useful for providing derivative crystals include, by way of example and not limitation, gold, mercury, etc.
  • the co-crystals ofthe invention generally comprise a crystalline RPTK extracellular domain polypeptide in association with one or more compounds or other molecules.
  • the association may be covalent or non-covalent.
  • molecules include, but are not limited to, ligands, ligand analogs, cofactors, substrates, substrate analogues, inhibitors, activators, allosteric effectors, polypeptides, etc.
  • X-ray crystallography is a method of solving the three dimensional structures of molecules.
  • the structure of a molecule is calculated from X-ray diffraction patterns using a crystal as a diffraction grating.
  • Three dimensional structures of protein molecules arise from crystals grown from a concentrated aqueous solution of that protein.
  • the process of X-ray crystallography can include the following steps:
  • the native and mutated tyrosine kinase domain polypeptides described herein may be chemically synthesized in whole or part using techniques that are well-known in the art (see. e.g.. Creighton, 1983).
  • methods which are well known to those skilled in the art can be used to construct expression vectors containing the native or mutated tyrosine kinase domain polypeptide coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1989 and Ausubel et al, 1989.
  • a variety of host-expression vector systems may be utilized to express the RPTK extracellular domain coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the RPTK extracellular domain coding sequence; yeast transformed with recombinant yeast expression vectors containing the RPTK extracellular domain coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the RPTK extracellular domain coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g..).
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the RPTK extracellular domain coding sequence
  • yeast transformed with recombinant yeast expression vectors containing the RPTK extracellular domain coding sequence yeast transformed with recombinant yeast expression vector
  • plasmid expression vectors e.g.. Ti plasmid
  • the expression elements of these systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g..
  • heat shock promoters may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g..
  • the adenovirus late promoter may be used; when generating cell lines that contain multiple copies ofthe receptor PTK extracellular domain DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • Methods describing methods of DNA manipulation, vectors, various types of cells used, methods of incorporating the vectors into the cells, expression techniques, protein purification and isolation methods, and protein concentration methods are disclosed in detail with respect to the protein PYK-2 in U.S. Patents Nos. 5,837,524, 5,837,815, and PCT publication WO 96/18738, each of which is incorporated herein by reference in its entirety, including all claims, figures, and drawings. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.
  • Crystals are grown from solutions containing the purified and concentrated polypeptide by a variety of techniques. These techniques include batch, liquid, bridge, dialysis, vapor diffusion, and hanging drop methods. McPherson, 1982, John Wiley, New York; McPherson, 1990, Eur. J. Biochem. 759:1-23; Webber, 1991, Adv. Protein Chem. 47:1-36, inco ⁇ orated by reference herein in its entirety, including all figures, tables, and drawings.
  • the crystals of the invention are grown by adding precipitants to the concentrated solution ofthe polypeptide corresponding to the RPTK extracellular domain, with or without bound compound, modulator, ligand, or ligand analog. The precipitants are added at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
  • crystals ofthe invention For one ofthe exemplary crystals ofthe invention, it has been found that hanging drops containing about 2.0 ⁇ L of RPTK extracellular domain polypeptide with a bound ligand provide crystals suitable for high resolution X-ray structure determination.
  • crystals are grown by mixing equal volumes of protein solution (10 mg/mL in 25 mM Tris- HCl, pH 8.5, and 150 mM NaCl) and reservoir buffer (1.6 M (NH 4 ) 2 SO 4 , 20% v/v glycerol and 100 mM Tris-HCl, pH 8.5), and suspending a hanging drop ofthe resulting solution over 0.5 mL reservoir buffer at 20°C.
  • the protein solution comprises 10 mg/mL FGFRl D2-D3 domain bound to an FGF2 molecule.
  • crystals are grown by mixing one volume of protein solution (1 mg/mL in 25 mM Tris-HCl, pH 8.5, and 150 mM NaCl) with four volumes of reservoir buffer (20% PEG 4000, 0.2 M Li2SO4, and 0.1 M Tris-HCl, pH 8.5), and suspending a hanging drop ofthe resulting solution over 0.5 mL reservoir buffer at 20°C.
  • the protein solution comprises 1 mg/mL FGFRl D2-D3 domain bound to an FGFl molecule.
  • crystallization conditions can be varied. Such variations may be used alone or in combination, and include polypeptide solutions containing polypeptide concentrations between about 1 mg/mL and about 50 mg/mL, Tris-HCl concentrations between about 10 mM and about 200 mM, dithiothreitol concentrations between about 0 mM and about 20 mM, pH ranges between about 5.5 and about 9.5; and reservoir solutions containing polyethylene glycol concentrations between about 10% and about 50% (w/v), polyethylene glycol molecular weights between about 1000 and about 20,000, (NH ) 2 SO concentrations between about 0.1 M and about 2.5 M, ethylene glycol or glycerol concentrations between about 0% and about 20% (v/v), bis-Tris concentrations between about 10 mM and about 200 mM, pH ranges between about 5.5 and about 9.5 and temperature ranges between about 0°C and about 25°C.
  • Other buffer solutions may be used such as HEP
  • Derivative crystals ofthe invention can be obtained by soaking native crystals in mother liquor containing salts of heavy metal atoms. It has been found that soaking a native crystal in a solution containing about 0.1 mM to about 5 mM thimerosal, 4- chloromeruribenzoic acid or KAu(CN) 2 for about 2 hr to about 72 hr provides derivative crystals suitable for use as isomorphous replacements in determining the X-ray crystal structure ofthe RPTK extracellular domain polypeptide.
  • Co-crystals ofthe invention can be obtained by soaking a native crystal in mother liquor containing one or more compounds, ligands, or ligand analogs that bind the receptor PTK extracellular domain, as described above, or can be obtained by co-crystallizing the RPTK extracellular domain polypeptide in the presence of one or more binding compounds, ligands, or ligand analogs.
  • Crystals comprising a polypeptide corresponding to a RPTK extracellular domain complexed with a compound, ligand, or ligand analog can be grown by one of two methods.
  • the compound, ligand, or ligand analog is added to the aqueous solution containing the polypeptide corresponding to the RPTK extracellular domain before the crystal is grown.
  • the compound, ligand, or ligand analog is soaked into an already existing crystal of a polypeptide corresponding to a RPTK extracellular domain.
  • the overall structures ofthe FGFl -FGFRl and FGF2-FGFR2 complexes are similar to the previously determined FGF2-FGFR1 structure (Fig. 5) (Plotnikov et al., 1999).
  • the FGFR ligand-binding domain consists of two Ig-like domains connected by a short linker.
  • the three-dimensional folds of D2 and D3 in both the FGFl -FGFRl and FGF2-FGFR2 structures resemble that ofthe I-set prototype member telokin, in which a ⁇ sandwich is formed by two layers of ⁇ sheets (Holden et al., 1992).
  • a highly conserved disulfide bond is buried in the hydrophobic core of D2 and D3 and bridges the two ⁇ sheets.
  • the ⁇ C- ⁇ C* loop in D3 is disordered (Fig. 5).
  • FGFl -FGFRl The main difference between the structures of FGFl -FGFRl and FGF2-FGFR2 is the conformation ofthe segment connecting ⁇ C and ⁇ E in D3 (Fig. 5).
  • this segment In the FGF2-FGFR2 structure, this segment is well ordered and interacts with FGF2, while in the FGFl -FGFRl structure, this segment is disordered and is not included in the atomic model. In the previously determined structure of FGF2-FGFR1, this segment is also well ordered and interacts with the ligand (Plotnikov et al., 1999).
  • a short ⁇ helix ( ⁇ D) has been assigned by PROCHECK (Laskowski et al., 1993).
  • the polypeptide chain at the C-terminal end adopts a very similar conformation, but is not assigned as a ⁇ helix.
  • the invention provides crystals of FGFRl D2-D3 domain bound to an FGF2 molecule.
  • the D2-D3 domain of this embodiment consists of residues 142-365, and thus is missing the Dl domain, the acid box, and the linker between D3 and the transmembrane helix.
  • Each D2-D3 domain is bound to a single FGF2 molecule.
  • the crystals were obtained by the methods provided in the Examples.
  • space group symmetry P4j2 ⁇ 2 space group symmetry
  • the interface between FGF and D2 in both complexes is mainly hydrophobic (Fig. 7 and 8).
  • a solvent-exposed hydrophobic surface in FGF packs against a highly conserved hydrophobic surface at the bottom of D2 in FGFR.
  • Tyr24, Leul40, and Metl42 in FGF2 make hydrophobic contacts with Alal68 in FGFR2.
  • Leul40, Tyr 103 and the aliphatic portion ofthe Asnl02 side chain in FGF2 make hydrophobic contacts with Pro 170 in FGFR2.
  • Phe31 of FGF2 is engaged in hydrophobic interactions with Leul 66 of FGFR2.
  • Leul 66, Alal 68 and Pro 170 of FGFR2 are located in ⁇ A' at the bottom of D2.
  • Val249, located in the C-terminal end of ⁇ G in D2 is also in the FGF2-D2 interface and interacts with Leul40 and Metl42 in FGF2.
  • FGF2 interacts extensively with D2, D3, and the linker between the two domains. While a single hydrogen bond is noted between Tyr-24 in FGF2 and Leu- 165 in FGFRl, the majority of interactions between D2 and FGF2 are hydrophobic. For example, hydrophobic contacts can be seen between Tyr- 24 and Met-142 of FGF2 and Ala 167 of D2, between Asn-102, Tyr-103, and Leu-140 of FGF2 and Pro-169 of D2, and between Leu-140 of FGF2 and Val-248 of FGFRl. It is noteworthy that Ala 167, Pro- 169, and Val-248 are conserved amongst FGFRs 1-4, and thus may represent a therapeutically important site in members ofthe FGFR subfamily.
  • FGF2 and FGFl differ in only two positions in the FGF-D2 interface: Met 142 in FGF2 is replaced by a homologous hydrophobic residue Leul35 in FGFl, and Asnl02 in FGF2 is substituted by His93 in FGFl. However, this latter substitution may not affect the binding of FGFl to D2, since only the aliphatic portion of this residue interacts with FGFR and not the actual functional group.
  • the FGF-Linker Interface The D2-D3 linker is highly conserved among FGFRs (Fig. 15).
  • FGF8 FGF 17 and FGFl 8 have a threonine residue at this position, whose side chain is shorter than asparagine. It is predicted that these FGFs will not form a direct hydrogen bond with the key linker arginine residue, and therefore will exhibit lower binding affinity towards FGFRs.
  • FGFl 1, FGF12, FGF13, and FGF14 have a valine in place of Asnl04 of FGF2 (Fig. 17). This substitution is expected to cause a strong decrease in FGFR binding.
  • the hydrogen bond between Arg251 and FGF2 takes place in a hydrophobic pocket composed ofthe aliphatic side chains of highly conserved Val249 and Pro253 in FGFR2 and Leu98 and Pro 141 in FGF2 (Fig. 9). The proximity to this hydrophobic environment will most likely stabilize this hydrogen bond. Moreover, the intramolecular hydrogen bonds between Arg251 and the invariant Asp283 in FGFR2 and Asnl04 and Tyr 106 in FGF2 serve to restrict the rotational freedom ofthe guanidium group of Arg251 and amide group of Asnl04 (Fig. 9). These interactions may increase the ligand-binding affinity by lowering the entropy of FGF-FGFR complex formation.
  • FGF-D2 and FGF-linker interfaces described above represents a general conserved binding interface for all FGF -FGFR complexes.
  • Interactions between FGF2 and the linker between D2 and D3 in the illustrative embodiment include hydrogen bonds between Asn-102 and Asn-104 of FGF2 with Arg-250 of FGFRl, and a hydrophobic interaction between Leu-98 of FGF2 and Val-248 of FGFRl.
  • Arg-250 is invariant in the FGFR subfamily, and thus may also represent a therapeutically important site.
  • FIG. 11-14 depict the interactions between FGF and the upper part ofthe D3 module. These interactions are mediated mainly by the ⁇ B'- ⁇ C, ⁇ C'- ⁇ E, and ⁇ F- ⁇ G loops of FGFRs. While residues in the ⁇ B'- ⁇ C are highly conserved, the amino acid sequences ofthe ⁇ C'- ⁇ E and ⁇ F- ⁇ G loops are significantly divergent among FGFRs (Fig. 16).
  • This loop is disordered in the crystal structure of FGFl -FGFRl (Fig. 5).
  • This difference reflects the lack of interaction between this loop and FGFl and is not the result of crystal packing; this segment is ordered in all the four FGF2-FGFR2 complexes in the unit cell and disordered in both FGFl -FGFRl complexes in the unit cell.
  • the total accessible surface area buried in the FGFl -FGFRl complex is 2200A 2 as compared to 2700A 2 in the FGF2-FGFR2 complex.
  • a total of five hydrogen bonds are formed at the interface between FGF2 and the ⁇ C- ⁇ E segment in the FGF2-FGFR2 structure (Fig. 13).
  • Two hydrogen bonds are formed between the side chain of Gln56 of FGF2 and Asp321 of FGFR2, and two hydrogen bonds are made between the side chain of Glu58 of FGF2 and backbone atoms of Val317 and Asn318 in FGFR2.
  • a fifth hydrogen bond is made between the backbone of Ala57 in FGF2 and the side chain of Asp321 via an ordered water molecule. Hydrophobic contacts between the side chain of Val317 in FGFR2 and the side chains of Tyr73, Val88, and Phe93 in FGF2 fortify this interface (Fig. 13).
  • FGFl does not engage in any specific contacts with the ⁇ C'- ⁇ E loop, providing a potential explanation for why FGFl binds indiscriminately to most FGFRs including the various alternatively- spliced forms, thus functioning as a universal ligand for all known FGFRs.
  • the crystal structure of an FGF 1 -FGFR2 complex was recently reported (Stauber et al., 2000).
  • the ⁇ C'- ⁇ E loop is ordered and makes several contacts with FGFl. Based on this structure changes in the primary sequence ofthe ⁇ C'- ⁇ E loop (as a result of alternative splicing) would clearly affect FGFl binding.
  • This structural feature does not agree with the well-documented universal binding characteristics of FGF 1.
  • the ⁇ C'- ⁇ E loop of all known FGFRs contains a highly conserved potential N-glycosylation site (Asn318 in FGFR2).
  • Asn318 is glycosylated in the extracellular domain of FGFR2 when expressed in insect cells.
  • the side chain of Asn318 makes two hydrogen bonds with FGF 1. This peculiarity along with the specificity conundrum led us to consider whether the interactions between the ⁇ C'- ⁇ E loop and FGFl , observed in this structure, could be due to crystal packing and thus not reflect the situation in vivo.
  • the ⁇ C'- ⁇ E loop in the FGF1-FGFR2 structure is closer to the ligand, and it is conceivable that the interactions between the ⁇ C'- ⁇ E loop and FGFl in this structure are the result of crystal packing.
  • the ⁇ C'- ⁇ E loop is disordered in both complexes in the asymmetric unit, providing two independent instances in which the ⁇ C'- ⁇ E loop does not engage FGFl.
  • Analysis of the lattice contacts in FGF 1 -FGFR2 structure provides a plausible mechanism by which crystal packing might have contributed to the observed interactions between the ⁇ C'- ⁇ E loop and FGFl.
  • the D2s of two symmetry mates insert into the space between the two D3s ofthe primary dimer, and appear to push the D3s closer to the FGFl molecules. Interactions between FGFR and FGF which Stabilize Dimerization
  • the D2-D3 / FGF2 dimer observed in the crystal structure of one illustrative embodiment is stabilized by interactions between each FGFR in the dimer, and by interactions between the FGF bound to one FGFR and the other receptor in the dimer.
  • the ligand-receptor contacts which stabilize dimerization are largely weak van der Waals interactions between residues Asp-99, Ser-100, Asn-101, Pro-132, Gly-133, and Leu-138 of FGF2 and Pro-199, Asp-200, Ile-203, Gly-204, Gly-205, Ser-219, and Val-221 of FGFRl.
  • Also noted are hydrogen bonds between Pro-132 of FGF2 andGly-204 of FGFRl, and Lys-26 of FGF2 and Asp-218 of FGFRl.
  • the receptor-receptor contacts which stabilize dimerization include a hydrophobic contact between Ala- 171 residues of each receptor, hydrogen bonds between Lys 172, Thr-173, and Asp-218 of each receptor, and van der Waals interactions between Ala-171 and Lys- 172 of each receptor.
  • the present invention describes a receptor-receptor interface which was not postulated previously, involving residues conserved in the FGFR subfamily. Disruption ofthe contacts which stabilize dimerization, for example by a molecule(s) which prevents the formation of one or more contacts, may provide a means of inhibiting RPTK function. Alternatively, a molecule(s) which further stabilize dimer formation may provide a means of stimulating RPTK function.
  • KGFR/FGFR2(IIIb) is two amino acids shorter than the corresponding loop in FGFRl -3 (Fig. 16). Based on the results described herein, it is predicted that this loop in FGFR4 can not efficiently interact with FGF2. This will result in reduced FGF2 binding affinity to FGFR4 as compared to FGFRl.
  • the ⁇ C'- ⁇ E loop of FGFR3 differs from that of FGFRl by two amino acid substitutions (Fig. 16). Significantly, in FGFR3 the residue corresponding to
  • Val317 of FGFR2 is an alanine, an amino acid with a smaller side chain than valine (Fig. 16). This will result in a weaker hydrophobic interaction with FGF2, affecting the affinity of FGF2 towards FGFR3. Indeed, when these residues in FGFR3 were replaced with the corresponding residues in FGFRl, the resultant FGFR3 mutant exhibited comparable binding affinity towards FGF2 (Chellaiah et al., 1999).
  • the interactions between FGF and the ⁇ C- ⁇ E loop in the crystal structure (Fig. 13) provide a molecular explanation for how specificity is regulated by the primary sequence composition ofthe ⁇ C'- ⁇ E loop.
  • the ⁇ F- ⁇ G loop in D3 also plays an important role in the modulation of FGF binding specificity.
  • Ser347 of FGFR2 located in ⁇ F- ⁇ G loop, makes two water mediated-hydrogen bonds with Glu96 and Leu98 of FGF2 (Fig. 11).
  • a water- mediated hydrogen bond between Gly345 in FGFR2 and Gly61 in FGF2 and a direct hydrogen bond between the backbone of Asn346 in FGFR2 and the side chain of Arg60 in FGF2 provide additional contacts in this region (Fig. 11).
  • Residues Arg60 and Gly61 are located in the ⁇ 4- ⁇ 5 loop in FGF2 (Fig. 17).
  • the amino acids 7 NYKKPKL 13 located at the junction between the N-terminal segment and ⁇ l in FGFl have been proposed to signal the nuclear accumulation of FGFl that occurs during sustained exposure of cells to FGFl (Imamura et al., 1990).
  • Tyr8 located in this amino acid stretch inserts into a shallow hydrophobic pocket formed by the side chains of Val279, Pro285 and Ile287.
  • the structural data described herein provide a direct role for this region in receptor binding. Deletion mutagenesis experiments support our structural finding. FGFl molecules lacking this amino acid stretch have 250-fold reduced ability to bind FGFR (Imamura et al., 1990).
  • Trp290 is located in the core of D3, adjacent to the disulfide bridge, and replacement of this residue with either ofthe two amino acids will likely reduce the stability of D3 (Fig. 18).
  • Mutations of two highly conserved residues Ser252 and Pro253 in the D2-D3 linker of FGFR2 are responsible for all the known cases of Apert syndrome. Mutation ofthe equivalent proline in FGFRl (Pro252) has been reported in some cases of Pfeifer syndrome. Based on our structural data we predict that these mutations introduce specific interactions between FGFR and FGF. Indeed, Anderson et al. (1998) have shown that, compared with wild type FGFR2, mutant FGFR2 molecules bearing the Apert mutations exhibit a selective increase in affinity towards FGF2, leading to enhanced signaling where availability of ligand is limiting (Anderson et al., 1998).
  • Residue Asp321 makes three hydrogen bonds with FGF2 (Fig. 13). Replacement of Asp321 with alanine, which is detected in some cases of Pfeifer syndrome, will therefore reduce the affinity of FGF2 towards FGFR2. It is conceivable, that this amino acid substitution will increase the affinity of FGFR2 for other members ofthe FGF family. Substitution of Ala315, also located in the ⁇ C'- ⁇ E loop, with a serine is also associated with Pfeifer syndrome. Residue Ala315 participates in the formation ofthe hydrophobic ⁇ C'- ⁇ E plug. This substitution can destabilize the hydrophobic plug and may affect ligand binding specificity. Heparin-binding Canyon
  • a highly positively charged "canyon" that continues onto the top side of both ligands is formed by the interaction ofthe two D2 regions in the dimer.
  • the canyon receives its positive potential from ly sines 160, 163, 172, 175, and 177 of FGFRl.
  • This canyon may represent the site of heparin binding.
  • FGF2 also contains a high affinity heparin binding site, consisting of Asn-27, Lys-125, Gln-134, and Arg-120, and heparin increases the apparent affinity of FGF2 for FGFRl.
  • these residues may represent a useful therapeutic target, for example using a molecule(s) which affects the affinity of a receptor PTK for its ligand.
  • heparin oligosaccharide is an octasaccharide and that an increase in heparin length parallels an increase in biological activity up to a dodecasaccharide.
  • hexasaccharides are biologically active and that even disaccharides possess biological activity.
  • FGF2 requires 2-O-sulfate for heparin binding but not 6-O-sulfate.
  • FGFl requires both sulfate groups to bind to heparin (Ishihara, 1994).
  • Pericellular HSPGs from different cells exhibit significant heterogeneity in sulfation patterns, carbohydrate content and length. These variations could have a profound effect on FGF-FGFR interactions.
  • remodeling ofthe extracellular matrix during development may be a means to regulate the biological activities of FGFs.
  • the heparin binding mode in the present structure disputes the previous findings regarding the minimal length requirement for heparin to promote FGF-FGFR dimerization as well as the stoichiometry of FGF:FGFR:heparin interactions.
  • the tripartite interactions between FGF, FGFR and heparin observed in the crystal structure suggest that heparin hexasaccharides are sufficient to promote receptor dimerization. Therefore, we decided to test the ability of a hexasaccharide to promote dimerization of FGF-FGFR complexes in vitro.
  • Homogeneously-sulfated hexasaccharide was mixed at various molar ratios with a purified 1 : 1 FGFl :FGFR2 complex and the reaction mixtures were analyzed by size exclusion chromatography to quantitate dimerization (Fig. 31).
  • Addition of hexasaccharide at a molar ratio of 0.5:1 hexasaccharide xomplex dimerized half of the FGF1-FGFR2 complexes Fig. 31, Panel B
  • Hexasaccharide at a molar ratio of 1 :1 hexasaccharidexomplex led to the quantitative dimerization of all the FGF1-FGFR2 complexes (Fig. 31, Panel C).
  • heparin Based upon the crystal structure and supporting biochemical experiments described herein, a new "two end" model by which heparin induces FGF-dependent FGFR dimerization (see Fig. 32) is proposed. According to this model, heparin interacts via its non-reducing ends with both FGF and FGFR and promotes the formation of a stable 1:1:1 FGF:FGFR:heparin ternary complex. A second 1:1:1 FGF:FGFR:heparin ternary complex is then recruited to the first complex via direct FGFR:FGFR contacts, secondary interactions between FGF in one ternary complex and FGFR in the other ternary complex, and indirect heparin-mediated FGFR-FGFR contacts.
  • heparin augments direct FGFR-FGFR and secondary FGF-FGFR interactions.
  • the proposed "two end" model presented in this report is consistent with the chemical architecture of heparan sulfate chains, which are linked by the reduced end (01) to the protein core of HSPG.
  • heparan sulfate can be roughly divided into low and high sulfate regions (Gambarini et al., Mol. Cell Biochem. 124, 121-129 (1993)). The low sulfate region is proximal to the protein core.
  • the high sulfate region is located towards the non-reducing end (O4) that corresponds to the non-reducing ends ofthe decasaccharides bound in the center ofthe canyon in our structure.
  • the chemical nature ofthe highly sulfated non-reducing ends resemble heparin and are made up of tri-sulfated disaccharide units (IdoA,2S-GlcNS,6S) considered to be the building block of HSPG (Gambarini et al., Mol. Cell Biochem. 124, 121-129 (1993)).
  • the invention provides crystals of FGFRl D2-D3 domain bound to an FGFl molecule.
  • the D2-D3 domain of this embodiment again consists of residues 142-365, and each D2-D3 domain is bound to a single FGFl molecule.
  • the crystals were obtained by the methods provided in the Examples.
  • the FGFRl D2-D3 / FGFl crystals which may be native crystals, derivative crystals or co-crystals, have triclinic unit cells, and space group symmetry PI.
  • FGFl interacts extensively with D2, D3, and the linker between the two domains.
  • a single hydrogen bond is noted between Tyr-15 in FGFl and Leu-165 in FGFRl, but the majority of interactions between D2 and FGFl are hydrophobic.
  • hydrophobic contacts can be seen between Tyr-15 and Leu-133, and Leu-135 of FGFl and Ala 167 of D2, between Tyr-94, Leu-133, and His-93 of FGFl and Pro-169 of D2, and between Phe-22 of FGFl and Val-248 of FGFRl. These contacts are similar to the contacts described herein for the D2-D3/FGF2 crystal.
  • Interactions between FGFl and the linker between D2 and D3 in the illustrative embodiment include hydrogen bonds between His-93 and Asn-95 of FGFl with Arg-250 of FGFRl . Again, these contacts are similar to the contacts described herein for the D2- D3/FGF2 crystal. Additionally, several regions of FGFl interact with D3, including Tyr-8, which inserts into a hydrophobic pocket in D3 formed by Val 279, Pro-285, and Ile-287. Additionally, Tyr- 8 participates in a hydrogen bond with Gln-284 of FGFRl .
  • residues 46, 48-51, and 54 of FGFl form van der Waals contacts with Gln-284, Pro-285, His-286, Gly-344, and Asn-345 of FGFRl Ala-57 of FGF2, and Glu-49 in FGFl forms a hydrogen bond with His-286 of FGFRl .
  • this latter region may be important in defining the binding specificity of FGFRs, and thus may be a therapeutically important site.
  • the crystal can be placed in a glass capillary tube and mounted onto a holding device connected to an X-ray generator and an X-ray detection device. Collection of X-ray diffraction patterns are well documented by those in the art. Ducruix and Geige, 1992, IRL Press, Oxford, England, and references cited therein. A beam of X-rays enter the crystal and then diffract from the crystal. An X-ray detection device can be utilized to record the diffraction patterns emanating from the crystal. Although the X-ray detection device on older models of these instruments is a piece of film, modern instruments digitally record X- ray diffraction scattering.
  • the unit cell dimensions and orientation in the crystal can be determined. They can be determined from the spacing between the diffraction emissions as well as the patterns made from these emissions.
  • the unit cell dimensions are characterized in three dimensions in units of
  • the symmetry ofthe unit cell in the crystals is also characterized at this stage.
  • the symmetry ofthe unit cell in the crystal simplifies the complexity ofthe collected data by identifying repeating patterns. Application ofthe symmetry and dimensions ofthe unit cell is described below.
  • Each diffraction pattern emission is characterized as a vector and the data collected at this stage ofthe method determines the amplitude of each vector.
  • the phases ofthe vectors can be determined using multiple techniques. In one method, heavy atoms can be soaked into a crystal, a method called isomorphous replacement, and the phases ofthe vectors can be determined by using these heavy atoms as reference points in the X-ray analysis.
  • the isomorphous replacement method usually requires more than one heavy atom derivative.
  • the amplitudes and phases of vectors from a crystalline polypeptide with an already determined structure can be applied to the amplitudes ofthe vectors from a crystalline polypeptide of unknown structure and consequently determine the phases of these vectors.
  • This second method is known as molecular replacement and the protein structure which is used as a reference must have a closely related structure to the protein of interest. Naraza, 1994, Proteins 77:281-296.
  • the vector information from a receptor PTK of known structure such as those reported herein, are useful for the molecular replacement analysis of another receptor PTK with unknown structure.
  • the vector amplitudes and phases, unit cell dimensions, and unit cell symmetry can be used as terms in a Fourier transform function.
  • the Fourier transform function calculates the electron density in the unit cell from these measurements.
  • the electron density that describes one of the molecules or one ofthe molecule complexes in the unit cell can be referred to as an electron density map.
  • the amino acid structures ofthe sequence or the molecular structures of compounds complexed with the crystalline polypeptide may then be fit to the electron density using a variety of computer programs. This step ofthe process is sometimes referred to as model building and can be accomplished by using computer programs such as TOM/FRODO. Jones, 1985, Methods in Enzymology 775:157-171.
  • a theoretical electron density map can then be calculated from the amino acid structures fit to the experimentally determined electron density.
  • the theoretical and experimental electron density maps can be compared to one another and the agreement between these two maps can be described by a parameter called an R-factor.
  • a low value for an R-factor describes a high degree of overlapping electron density between a theoretical and experimental electron density map.
  • the R-factor is then minimized by using computer programs that refine the theoretical electron density map.
  • a computer program such as X-PLOR can be used for model refinement by those skilled in the art. Brunger, 1992, Nature 355:472-475. Refinement may be achieved in an iterative process.
  • a first step can entail altering the conformation of atoms defined in an electron density map. The conformations ofthe atoms can be altered by simulating a rise in temperature which will increase the vibrational frequency ofthe bonds and modify positions of atoms in the structure.
  • a force field which typically defines interactions between atoms in terms of allowed bond angles and bond lengths, Van der Waals interactions, hydrogen bonds, ionic interactions, and hydrophobic interactions, can be applied to the system of atoms.
  • Favorable interactions may be described in terms of free energy and the atoms can be moved over many iterations until a free energy minimum is achieved.
  • the refinement process can be iterated until the R-factor reaches a minimum value.
  • the three dimensional structure ofthe molecule or molecule complex is described by atoms that fit the theoretical electron density characterized by a minimum R-value.
  • a file can then be created for the three dimensional structure that defines each atom by coordinates in three dimensions. Examples of such structural coordinate files are defined in Tables 1-4 and 6.
  • SCF Stem cell factor
  • MCGF mast cell growth factor
  • SI steel
  • KL kit ligand
  • SCF is believed to be critical for mast cell production and function and to play an important role in the development of melanocytes, germ cells, and intestinal pacemaker cells.
  • SCF is believed to mediate its biological effects by binding to and activating a receptor protein tyrosine kinase designated c-kit (also referred to as SCF receptor (“SCFR").
  • SCFR receptor protein tyrosine kinase
  • SCF Like other RPTK ligands, SCF induces dimerization of c-kit followed by trans-autophosphorylation ofthe cytoplasmic protein tyrosine kinase domain leading to subsequent recruitment of signaling proteins, tyrosine phosphorylation of substrates and activation of multiple signaling pathways.
  • SCF stem cell factor
  • SCF has also been proven to be effective in enhancing the ability of G-CSF to mobilize peripheral blood hematopoietic progenitor and stem cells. It is believed that these cells can be transplanted to reconstitute the hematopoietic system in patients receiving bone marrow ablative therapy (Nicola et al., Protein Chem. 52, 1-65 (1998)).
  • SCF exist naturally as membrane anchored and soluble isoforms as a result of alternative RNA splicing and proteolytic processing.
  • the soluble form of SCF has 165 amino acids, but its receptor binding core has been mapped to the first 141 residues (Langley et al., Arch. Biochem. Biophys.311, 55-61 (1994)).
  • SCF functions as a non-covalent homodimer, but under physiological conditions, the majority of SCF is reported to exist as a monomer. Dimerization of SCF is a dynamic process and it may play a regulatory role in the control of SCFR binding affinity and receptor activation. Comparison of SCF with other Growth Factors
  • SCF belongs to the short-chain helical cytokine family (Bazan, 1991 ; Rozwarski et al., 1994), but its resemblance to the other cytokines is limited only to the overall fold.
  • the primary structures exhibit very weak similarity and sequences can be aligned only by comparison ofthe secondary structures (Fig. 20).
  • the structure of SCF is most similar to the structure of M-CSF (Pandit et al., Science 258, 1358-1362 (1992)).
  • the core four helix bundles ofthe two proteins superimpose relatively well, with r.m.s. deviation of 1.98 A for the alpha-C atoms.
  • the two beta-strands deviate significantly.
  • fit 3 ligand (Hannum et al., Nature 368, 643-648 (1994)) can be predicted with reasonable confidence based upon the crystal structures of SCF described herein together with the previously described crystal structure of M-CSF (Pandit et al., Science 258, 1358- 1362 (1992)).
  • SCF functions as a non-covalent homodimer (Pandit et al., Science 258, 1358-1362 (1992)). It has been shown that the bivalency of SCF is the sole driving force responsible for dimerization ofthe extracellular ligand binding domain of c-kit. Hence analysis ofthe molecular interactions that control SCF-dimer formation are critical for understanding the mechanism of activation of c-kit.
  • Recombinant SCF is expressed in E. coli as inclusion bodies in a denatured form and an active SCF protein is produced by a procedure involving refolding and oxidation. It has previously been reported that a small fraction ofthe refolded-oxidized protein is a covalent disulfide linked form of SCF. Interestingly, the covalent SCF dimer has bben reported to bind to c-kit with slightly reduced affinity but was more potent in stimulation of hematopoietic cells. Comparison ofthe secondary and tertiary structures by spectroscopic methods demonstrated that the covalent dimer is indistinguishable from the non-covalent dimer (Lu et al., J. Biol. Chem.
  • the disulfide linkages ofthe covalent dimer were found to be identical to those in the non-covalent dimer except that the disulfide linkages in the variant protein were intermolecular. That is, Cys4 and Cys43 from one protomer form disulfide bonds with Cys89 and Cys 138, respectively, of the second protomer. It was thus proposed that the covalent dimer could be formed by a three-dimensional domain swapping of helices alphaA and alphaD between the two monomers (Lu et al, J. Biol. Chem. 277, 11309-11316 (1996)).
  • helix alphaD and strand 2 of one protomer together with helices alphaA, alphaB, alphaC and strand 1 from the other protomer, form one domain ofthe two-domain dimer.
  • helices alphaA, alphaB, alphaC and strand 1 from the other protomer form one domain ofthe two-domain dimer.
  • SCF monomers and dimers may be linked to the physiological requirement for activation of c-kit expressed on target cells in vivo.
  • the more potent disulfide-linked dimer is generally preferred because it can be administered at low doses to avoid significant mast cell activation while stimulating hematopoietic recovery (Nocka et al., Blood 90, 3874-3883 (1997)).
  • SCF dimer are known to bind soluble or membrane forms of c-kit with high affinity and specificity.
  • the binding of SCF to c-kit was analyzed by biochemical methods, by employing site-directed mutagenesis and by epitope mapping with site-specific anti-c-kit antibodies. It was reported that deletions of residues 1 to 3 from the N-terminus reduced the binding of SCF to c-kit by approximately 50%. Deletion of Cys4 inactivated SCF, whereas deletion of Cysl38 and additional residues form the C-terminus only compromised SCF activity. Moreover, an SCF double mutant at Cys43Ala and Cysl38Ala, which eliminate one pair of disulfide bonds, resulted in a partially active SCF as well. These experiments demonstrated that the N-terminus of SCF and the integrity ofthe Cys4-Cys89 disulfide bond are crucial for full CSF activity.
  • This region contains a deep crevice at the end of alphaC formed by side chains ofthe hydrophobic residues Phel02, Leu98, Pro34, Tyr32, and by the Cys43-Cysl38 disulfide bridge (see Fig. 24).
  • a positively charged patch (Arg5, Arg7, and Lysl27) followed by a negatively charged patch (Asp84, Asp85, Glu88, and Glu92) and then by an additional positively charged patch (Lys91, Lys99, LyslOO and Lysl03).
  • Figure 24 shows the locations ofthe positively charged and negative charged patches as well as the hydrophobic crevice.
  • This surface may function as a receptor binding site with the charged interactions providing anchor and specificity for ligand receptor interactions and the hydrophobic interactions providing enthalpy to complex formation. While human and rodent SCF are highly conserved, the charged patches that may function as part of receptor binding regions are quite divergent (see Fig. 25). Residues Arg5 and Arg7 in the first positively charged patch ofthe human SCF are replaced by glycine and proline residues in rodents, respectively. In the second positively charged patch, residues LyslOO and Lys91 are substituted by glutamate residues in both mouse and rat. These changes could account for the difference in the binding affinity of human and murine SCF to the human c-kit that has been reported.
  • Natural and CHO-cell derived recombinant SCF are glycosylated on multiple asparagine, serine and threonine residues.
  • the receptor binding properties of glycosylated SCF are consistent with the assignment of SCFR binding region shown in Fig. 24.
  • Asn72 is not glycosylated probably because its side chain is buried in the dimer interface.
  • the side chains of Asnl20, Asn65, and Asn93 remain accessible to the solvent in the structure and are indeed glycosylated to different extent.
  • Asn 120 is always glycosylated but this does not affect the binding of SCF to c-kit.
  • Asn65 and Asn93 are glycosylated in some, but not all, SCF molecules.
  • glycosylation of these asparagine residues has been reported to have an adverse effect on SCF binding to SCFR.
  • the structure described herein provides possible explanations for the adverse effect of glycosylation of these residues on the activity of SCF.
  • the glycosylation of Asn93 may hinder SCF binding to c-kit as this residue is located very close to the acidic patch and to the hydrophobic crevice.
  • Asn65 is located close to the dimer interface and glycosylation of this residue may interfere with SCF dimerization.
  • the extracellular ligand binding domains of several receptor tyrosine kinases contain multiple Ig-like domains.
  • the extracellular domains of FGF receptors contain three Ig-like domains while the extracellular domain of PDGF-receptor family to which c-kit belongs is composed of five Ig-like domains.
  • VEGF-receptor has been reported to contain seven Ig-like domains. Although the ligands of these receptors are very diverse, the ligand binding regions in these three families of receptors have been mapped to Ig-like domains two and three (see, e.g., Plotnikov et al., Cell 98, 641-650 (1999)). The determination ofthe structures ofthe ligand binding domains of FGF and VEGF receptors demonstrated that FGF and VEGF bind differently to their respective receptors. In the FGF:FGFR complex the two receptors are packed side by side to one face and the ligands occupy the second face.
  • the two VEGFR bind to the far ends ofthe VEGF-dimer creating an inverted "A" shaped complex with the ligand representing the cross bar in the "capital A”. Since SCF functions as a dimer, SCF binding to c-kit would be expected to resemble the structure which has been reported for the VEGF :VEGFR complex.
  • the x-ray crystal structure ofthe SCF dimer was used to build a model of SCF:c-kit complex formation and dimerization.
  • a model for Ig-like domains 2-3 as well as 4-5 of c-kit was developed.
  • Ig-like domains 2 and 3 were then docked to the proposed SCF binding surface adopting the mode of FGFR binding to FGF2 (Plotnikov et al., Cell 98, 641-650 (1999)).
  • the orientation of Ig-like domains 4 and 5 was adjusted to allow for interactions between domain 4 in the complex as suggested by previous biochemical studies (Blechman et al., Cell 80, 103-113 (1995)); see Fig. 26).
  • c-kit belongs to the same family of RTKs that also includes M-CSFR, PDGFR alpha, PDGFR alpha and lt3. Comparison of their primary structures shows that these RTKs are much more conserved than their ligands. Indeed, the structures of PDGF-A and PDGF-B are dramatically different from the structures of M-CSF and SCF and probably also flt3 ligand. The similarity ofthe RTKs is also reflected in the chromosomal localizations of their human and murine genes (Kondo et al., Gene 208, 297-305 (1998)). It is thought that this family of RTKs has evolved from a common ancestral gene that undergone several gene-duplication events.
  • RTKs that bind to and are activated by ligands with structures of four-bundle helix are primarily involved in the control of hematopoeisis, whereas other members of this family of RTKs exhibit broader expression pattern and are involved in the regulation of growth and development of several tissues and organs. Determination ofthe three dimensional structure of SCF would facilitate the determination ofthe structure of SCF in complex with the extracellular domain of c-kit, and enable the design and production of more potent forms of therapeutic SCF analogues.
  • the crystals ofthe invention and particularly the atomic structure coordinates obtained therefrom, have a wide variety of uses.
  • the crystals described herein can be used as a starting material in any ofthe art-known methods of use for RPTKs.
  • Such methods of use include, for example, identifying molecules that bind to the native or mutated extracellular domain of RPTKs.
  • the crystals and structure coordinates are particularly useful for identifying compounds which are modulators of RPTK function as an approach towards developing new therapeutic agents (see, e.g., Levitzki and Gazit, 1995, Science 257:1782-8).
  • the structure coordinates described herein can also be used as phasing models for determining the crystal structures of additional native or mutated receptor PTK extracellular domains, as well as the structures of co-crystals of such domains complexed with molecules such as ligands, ligand analogs, inhibitors, activators, agonists, antagonists, polypeptides, and other molecules.
  • the structure coordinates, as well as models ofthe three-dimensional structures obtained therefrom, can also be used to aid the elucidation of solution-based structures of native or mutated receptor PTK extracellular domains, such as those obtained via NMR.
  • the crystals and atomic structure coordinates ofthe invention provide a convenient means for elucidating the structures and functions of receptor tyrosine kinases.
  • the crystals ofthe invention will be described by reference to specific FGFRl D2-D3 / FGF2 and FGFRl D2-D3 / FGFl exemplary crystals.
  • receptor PTKs such as are PDGFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members of the FGFR family, such as FGFRl, FGFR2, FGFR3, and FGFR4, and orphan receptor PTKs.
  • PTKs such as are PDGFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members of the FGFR family, such as FGFRl,
  • Structural coordinates such as those set forth in Tables 1 -4 and 6 can be used to determine the three dimensional structures of RPTKs with unknown structure.
  • the methods described below can apply structural coordinates of a polypeptide with known structure to another data set, such as an amino acid sequence, X-ray crystallographic diffraction data, or nuclear magnetic resonance (NMR) data.
  • Preferred embodiments ofthe invention relate to determining the three dimensional structures of receptor PTKs and related polypeptides.
  • receptor PTKs such as are PDGFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members ofthe FGFR family, such as FGFRl, FGFR2, FGFR3, and FGFR4, and orphan receptor PTKs. Structures Using Amino Acid Homology
  • Homology modeling is a method of applying structural coordinates of a polypeptide of known structure to the amino acid sequence of a polypeptide of unknown structure. This method is accomplished using a computer representation ofthe three dimensional structure of a polypeptide or polypeptide complex, the computer representation of amino acid sequences ofthe polypeptides with known and unknown structures, and standard computer representations ofthe structures of amino acids. Homology modeling comprises the steps of
  • step (b) transferring the coordinates ofthe conserved amino acids in the known structure to the corresponding amino acids ofthe polypeptide of unknown structure; (c) constructing structures ofthe rest ofthe polypeptide; and (d) refining the subsequent three dimensional structure.
  • conserved amino acids between two proteins can be determined from the sequence alignment step in step (a).
  • Alignment ofthe amino acid sequence is accomplished by first placing the computer representation ofthe amino acid sequence of a polypeptide with known structure above the amino acid sequence ofthe polypeptide of unknown structure. Amino acids in the sequences are then compared and groups of amino acids that are homologous (e.g., amino acid side chains that are similar in chemical nature - aliphatic, aromatic, polar, or charged) are grouped together. This method will detect conserved regions ofthe polypeptides and account for amino acid insertions or deletions. Once the amino acid sequences ofthe polypeptides with known and unknown structures are aligned, the structures ofthe conserved amino acids in the computer representation ofthe polypeptide with known structure are transferred to the corresponding amino acids ofthe polypeptide whose structure is unknown. For example, a tyrosine in the amino acid sequence of known structure may be replaced by a phenylalanine, the corresponding homologous amino acid in the amino acid sequence of unknown structure.
  • a tyrosine in the amino acid sequence of known structure may
  • the structures of amino acids located in non-conserved regions are to be assigned by either using standard peptide geometries or molecular simulation techniques, such as molecular dynamics.
  • the final step in the process is accomplished by refining the entire structure using molecular dynamics and/or energy minimization.
  • the homology modeling method is well known to those skilled in the art and has been practiced using different protein molecules.
  • the three dimensional structure of the polypeptide corresponding to the catalytic domain of a serine/threonine protein kinase, myosin light chain protein kinase was homology modeled from the cAMP-dependent protein kinase catalytic subunit. Knighton et /., 1992, Science 255:130-135.
  • Molecular replacement is a method of applying the X-ray diffraction data of a polypeptide of known structure to the X-ray diffraction data of a polypeptide of unknown sequence. This method can be utilized to define the phases describing the X-ray diffraction data of a polypeptide of unknown structure when only the amplitudes are known.
  • X-PLOR is a commonly utilized computer software package used for molecular replacement. Briinger, 1992, Nature 355:472-475.
  • AMORE is another program used for molecular replacement. Navaza, 1994, Acta Crystallogr. A50: 157-163.
  • the resulting structure does not exhibit a root-mean-square deviation of more than 3 A.
  • a goal of molecular replacement is to align the positions of atoms in the unit cell by matching electron diffraction data from two crystals.
  • a program such as X-PLOR can involve four steps. A first step can be to determine the number of molecules in the unit cell and define the angles between them. A second step can involve rotating the diffraction data to define the orientation ofthe molecules in the unit cell. A third step can be to translate the electron density in three dimensions to correctly position the molecules in the unit cell. Once the amplitudes and phases ofthe X-ray diffraction data are determined, an R-factor can be calculated by comparing electron diffraction maps calculated experimentally from the reference data set and calculated from the new data set.
  • a fourth step in the process can be to decrease the R-factor to roughly 20% by refining the new electron density map using iterative refinement techniques described herein and known to those or ordinary skill in the art. Structures Using NMR Data
  • Structural coordinates of a polypeptide or polypeptide complex derived from X-ray crystallographic techniques can be applied towards the elucidation of three dimensional structures of polypeptides from nuclear magnetic resonance (NMR) data. This method is used by those skilled in the art. Wuthrich, 1986, John Wiley and Sons, New York: 176- 199; Pflugrath et al, 1986, J. Molecular Biology 759:383-386; Kline et al, 1986, J. Molecular Biology 759:377-382. While the secondary structure of a polypeptide is often readily determined by utilizing two-dimensional NMR data, the spatial connections between individual pieces of secondary structure are not as readily determinable. The coordinates defining a three-dimensional structure of a polypeptide derived from X-ray crystallographic techniques can guide the NMR spectroscopist to an understanding of these spatial interactions between secondary structural elements in a polypeptide of related structure.
  • Structure-based modulator design and identification methods are powerful techniques that can involve searches of computer data bases containing a wide variety of potential modulators and chemical functional groups.
  • the computerized design and identification of modulators is useful as the computer data bases contain more compounds than the chemical libraries, often by an order of magnitude.
  • For reviews of structure-based drug design and identification see Kuntz et al, 1994, Ace. Chem. Res. 27:117; Guida, 1994, Current Opinion in Struc. Biol. 4: 777; Colman, 1994, Current Opinion in Struc. Biol. 4: 868.
  • the three dimensional structure of a polypeptide defined by structural coordinates can be utilized by these design methods.
  • the structural coordinates of Table 1 or Table 2 can be utilized by this method.
  • the three dimensional structures of RPTKs determined by the homology, molecular replacement, and NMR techniques described herein can also be applied to modulator design and identification methods.
  • receptor PTKs such as are PDGFR, EGFR, VEGFR, HGFR, neurotrophinR, HER2, HER3, HER4, InsulinR, IGFR, CSFIR, FLK, KDR, VEGFR2, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, MUSK, members ofthe FGFR family, such as FGFRl, FGFR2, FGFR3, and FGFR4, and orphan receptor PTKs, can be utilized by the methods described herein.
  • One such data base (ACD distributed by Molecular Designs Limited Information Systems) contains, for example, 200,000 compounds that are synthetically derived or are natural products. Methods available to those skilled in the art can convert a data set represented in two dimensions to one represented in three dimensions. These methods are enabled by such computer programs as CONCORD from Tripos Associates or DB-Converter from Molecular Simulations Limited. Multiple methods of structure-based modulator design are known to those in the art. Kxmtz et aL, 1982, J. Mol Biol. 162: 269; Kuntz et ⁇ /., 1994, Ace. Chem. Res. 27: 117; Meng et ⁇ /., 1992, J. Compt. Chem. 13: 505; Bohm, 1994, J Comp.
  • a computer program widely utilized by those skilled in the art of rational modulator design is DOCK from the University of California in San Francisco. The general methods utilized by this computer program and programs like it are described in three applications below. More detailed information regarding some of these techniques can be found in the Molecular Simulations User Guide, 1995.
  • a typical computer program used for this purpose can comprise the following steps:
  • search libraries for molecular fragments which (i)can fit into the empty space between the compound and the active-site, and (ii) can be linked to the compound;
  • Part (e) link the fragments found above to the compound and evaluate the new modified compound.
  • Part (c) refers to characterizing the geometry and the complementary interactions formed between the atoms ofthe RPTK and the compound, ligand, or ligand analog. A favorable geometric fit is attained when a significant surface area is shared between the compound and RPTK atoms without forming unfavorable steric interactions.
  • the method can be performed by skipping parts (d) and (e) and screening a data base of many compounds.
  • Structure-based design and identification of modulators of RPTK function can be used in conjunction with assay screening. As large computer data base of compounds (around 10,000 compounds) can be searched in a matter of hours, the computer based method can narrow the compounds tested as potential modulators of RPTK function in cellular assays.
  • Another way of identifying compounds, ligands, or ligand analogs as potential modulators is to modify an existing modulator in the polypeptide active-site.
  • the computer representation of modulators can be modified within the computer representation of a RPTK ligand binding site. Detailed instructions for this technique can be found in the Molecular Simulations User Manual, 1995 in LUDI.
  • the computer representation ofthe modulator is modified by changing, deleting, or adding one or chemical groups.
  • the atoms ofthe modified compound, ligand, or ligand analog and the RPTK can be shifted in conformation, and the distance between the compound, ligand, or ligand analog and the RPTK atoms may be scored along with any complimentary interactions formed between the two molecules. Scoring can be complete when a favorable geometric fit and favorable complementary interactions are attained. Compounds that have favorable scores are potential modulators of RPTK function.
  • a third method of structure-based modulator design is to screen compounds designed by a modulator building or modulator searching computer program. Examples of these types of programs can be found in the Molecular Simulations Package, Catalyst. Descriptions for using this program are documented in the Molecular Simulations User Guide (1995). Other computer programs used in this application are ISIS/HOST, ISIS/BASE, ISIS/DRAW) from Molecular Designs Limited and UNITY from Tripos Associates.
  • a modulator construction computer program is a computer program that may be used to replace computer representations of chemical groups in a compound, ligand, or ligand analog complexed with a RPTK with groups from a computer data base.
  • a modulator searching computer program is a computer program that may be used to search computer representations of compounds from a computer data base that have similar three dimensional structures and similar chemical groups as compounds bound to a receptor PTK.
  • a typical program can operate by using the following general steps: (a) map the compounds, ligands, or ligand analogs by chemical features such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites;
  • important chemical features include, but are not limited to, a hydrogen bond donor, a hydrogen bond acceptor, and/or two hydrophobic points of contact.
  • Those skilled in the art also recognize that not all ofthe possible chemical features ofthe compound need be present in the model of (b).
  • the versatility of computer-based modulator design and identification lies in the diversity of structures screened by the computer programs.
  • the computer programs can search data bases that contain 200,000 molecules and can modify modulators already complexed with a polypeptide, using a wide variety of chemical functional groups.
  • a consequence of this chemical diversity is that a potential modulator of RPTK function may take a chemical form that is not predictable.
  • One example of such a reference is March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. New York, McGraw Hill.
  • the techniques required to synthesize a potential modulator of RPTK function identified by computer-based methods are readily available to those skilled in the art of organic chemical synthesis.
  • Cellular assays Measuring the Effect of a Receptor PTK Modulator in Signal Transduction Pathways
  • Cellular assays can be used to test the activity of a potential modulator of RPTK function as well as diagnose a disease associated with inappropriate RPTK activity.
  • a potential modulator of RPTK function can be tested for activity in vitro by assays that measure the effect of a potential modulator on the autophosphorylation of a particular RPTK over-expressed in a cell line.
  • a modulator that acts as a potent inhibitor of ligand binding to the extracellular domain corresponding to a RPTK would decrease the amount of autophosphorylation catalyzed by that RPTK.
  • Potential modulators could also be tested for activity in cell growth assays in vitro as well as in animal model assays in vivo.
  • Patent No. 5,792,783, and WO 96/40116 published on December 19, 1996, entitled “Indolinone Compounds for the Treatment of Disease,” each of which is incorporated herein by reference in its entirety, including all drawings, figures, and tables.
  • FIG. 33 An illustrative computer based system 10 is depicted in Figure 33 for displaying, studying, comparing, manipulating, inte ⁇ reting and/or extrapolating data from the crystallographic analysis of molecular structures which include molecules, portion(s) of molecules and/or molecular interactions, such as the molecular structures of RPTKs, their ligands and related complexes depicted in Figs. 1, 2, 5, 19, 26 and 27.
  • Exemplary molecules are proteins and/or complexes of proteins with ligands.
  • Exemplary molecular portions are catalytic domains of proteins, ligand receptor binding sites of proteins, signaling regions of proteins and transport regions of proteins.
  • Exemplary molecular interactions include binding between an enzyme and its substrate, factor/co-factor relationships, antibody/antigen binding, and protein/receptor recognition and binding, such as that occurring in signal transduction.
  • One or more ofthe above types of studies are useful for elucidating and understanding natural biochemical processes and to design and screen mimetics, agonists, inhibitors, and antagonists.
  • this aspect ofthe invention permits the skilled person to understand and practice molecular modeling processes and provides the skilled person with the necessary hardware and software to create and display images that represent the multidimensional structure of a molecule, molecular portion or molecular interaction, as desired.
  • these undertakings are greatly facilitated by employing computer technology.
  • the system 10 includes data storage entity(ies) 20, such as a memory (e.g, as archival memory and/or video memory and computer-readable medias), that retrievably stores information representing molecule, molecule portions and/or molecular interactions.
  • the memory typically has a first-type storage region or capability 22, having recorded thereon or contained therein structural data, like a set of spatial (atomic) coordinates, specifying a location in a three dimensional space, as disclosed herein or obtained in accordance with the teachings contained herein.
  • the memory also can have a second-type storage region or capability 24, which contains information. This information typically represents a property, characteristic or attribute of one of a plurality of amino acids, or other chemical moiety, for example.
  • a second-type storage region or capability can be associated with the first-type storage regions in the storage entity 20 to represent a geometric and or spatial arrangement of at least one characteristic, property or attribute of a molecule, molecule portion or molecular interaction, preferably one that represents three dimensional space.
  • the memory can take the form of any type recognizable by the skilled person such as RAM and ROM, and other computer-readable mediums like magnetic media, optical media, magnetic-optical media, floppy disks, hard disks, mini-disks, servers, web-based systems, CD, DVD, tape, etc.
  • Memory 22 (a type of storage region or capability) can include or contain, for example, the coordinate data shown in Tables 1, 2, 3, 4 or 6, or other coordinates obtained in accordance with the present teachings
  • memory 24 (a type of storage region or capability) can include or contain associated charge or electron density data, for example.
  • the system includes a plurality ofthe first-type and second-type storage regions.
  • the storage entities 20, namely the first-type storage regions or capabilities 22, said second-type storage regions or capabilities 24, and the storage devices or capabilities 34 can be regions of, for example, a shared semiconductor memory, cache, RAM, ROM, regions of a shared optical disk, regions of a shared magnetic memory, and/or be server based to be accessible by intranet and the internet, including the world wide web.
  • the systems ofthe present invention include unitary systems, network-based systems, satellite communications, and internet-based systems, which can be interactively connected regardless of geography.
  • the system 10 also includes a processor and/or is interactively associated with a processor, interactively coupled to the data storage entity(ies) to access the first-type storage regions 22 and optionally the second-type storage regions 24, to generate image signals for depicting a visual three dimensional image of at least one characteristic ofthe molecule, molecule portion or molecular interaction in the three dimensional space based on data from the storage entity 20.
  • the processor can be a general or special pu ⁇ ose processor with a CPU, register, memory and the like. Software, and logic architecture and circuitry, can be employed as desired.
  • processor 26 and storage entity 20 can be in the form of a UNIX or VAX computer, such those available from Silicon Graphics, Sun Microsystems, and IBM.
  • processor 26 and storage entity 20 can be in the form of a UNIX or VAX computer, such those available from Silicon Graphics, Sun Microsystems, and IBM.
  • VAX computer such those available from Silicon Graphics, Sun Microsystems, and IBM.
  • the invention is not limited to use of these types of hardware and software systems.
  • a display 28 is commonly interactively coupled to the processor 26 via lines or a wireless connection 30 to receive the image signals in order to depict a visual three dimensional image of at least one characteristic of a molecule, molecule portion or molecular interaction in the three dimensional space based on the data.
  • Suitable displays for use in the system include a computer screen 32 (e.g., CRT, LCD, active and passive matrix, etc.), printer, plotter or film.
  • the image data includes data for depicting a visual three dimensional image of a structure of molecule, molecule portion or molecular interaction in three dimensional space, such as shown in Figs. 1, 2, 5, 19, 26 and 27.
  • the image data includes data for depicting a visual three dimensional image of a solid model representation of molecule, molecule portion or molecular interaction in three dimensional space.
  • the image data includes data for depicting a visual three dimensional image of electrostatic surface potential of molecule, molecule portion or molecular interaction in three dimensional space.
  • the image data includes data for depicting a visual three dimensional stereo image of molecule, molecule portion or molecular interaction in three dimensional space.
  • the system 10 of the present invention may further comprise a storage device, structure, region or capability 34 that stores data representing a geometric and/or spatial arrangement of a characteristic of a composition in addition to the molecule, molecule portion or molecular interaction, such as shown in Figs. 9, 10, 22 and 24.
  • Storage devices or capabilities 34 can include or contain, for example, the three-dimensional X-ray coordinate data for other chemical entities, including other proteins for comparison pu ⁇ oses.
  • the storage devices and capabilities 34 can take the form of any type recognizable by the skilled person such as RAM and ROM, and other computer-readable mediums like magnetic media, optical media, magnetic-optical media, floppy disks, hard disks, mini-disks, servers, CD, DVD, tape, etc.
  • the processor 26 can be interactively coupled to the storage device or capability 34 and the display 28, and generates additional image data for depicting the geometric arrangement ofthe characteristic ofthe composition relative to said visual three dimensional image of said at least one characteristic ofthe molecule, molecule portion or molecular interaction on the screen 32 based on instructions.
  • the storage device or capability 34 is shown as part ofthe storage entity 20, although other arrangements are available to the skilled person.
  • the computer system includes or employs instructions, which can be software or hardware based.
  • the instructions such as those in logic circuits and software program(s), permit the computer system to, among other things, input, handle, analyze and output data. Exemplary programs are identified herein, although the skilled person is not limited to such programs in the practice ofthe invention.
  • the system 10 also includes an operator interface 36, such as a mouse, tracker ball, touch pad, projector (including multi-dimensional projector systems), touch screen, joy stick, pointer, keyboard, modem, card and/or voice recognition system, or docking system for receiving instructions from a operator, which is interactively connected with the display 28, processor 26 and storage entity(ies) 20.
  • operator interface 36 such as a mouse, tracker ball, touch pad, projector (including multi-dimensional projector systems), touch screen, joy stick, pointer, keyboard, modem, card and/or voice recognition system, or docking system for receiving instructions from a operator, which is interactively connected with the display 28, processor 26 and storage entity(ies) 20.
  • operator interface 36 such as a mouse, tracker ball, touch pad, projector (including multi-dimensional projector systems), touch screen, joy stick, pointer, keyboard, modem, card and/or voice recognition system, or docking system for receiving instructions from a operator, which is interactively connected with the display 28, processor 26 and storage entity(ies) 20.
  • the computer systems according to the invention can be programmed and contain data to undertake the analyses discussed in Sections VI-IX above, for example, including the use of x-ray crystallographic data in conjunction with other analytical techniques, such as NMR.
  • the invention also includes computer-readable media containing various data structures and the information disclosed herein. For example, magnetic media, optical media, magnetic-optical media, floppy disks, hard disks, mini-disks, servers, CD, DVD, tape, etc. containing the coordinate data set forth in the accompanying tables and figures, when computer analyzed according to set(s) of instructions and rules provided by hardware and/or software, are useful for ascertaining the three-dimensional structures of molecules, molecular portions and molecular interactions.
  • Tables 1-3 provide the atomic structural coordinates for a number of ligand / FGFR complex dimers.
  • Table 5 provides the atomic structural coordinates for a SCF dimer.
  • Table 6 provides the atomic structural coordinates for a the ternary FGF2-FGFR1 -heparin complex. The following abbreviations are used in the Tables:
  • Atom Type refers to the element whose coordinates are provided. The first letter in the column defines the element. "A. A.” refers to amino acid.
  • B is a thermal factor that measures movement ofthe atom around its atomic center.
  • OCC refers to occupancy, and represents the percentage of time the atom type occupies the particular coordinate. OCC values range from 0 to 1, with 1 being 100%.
  • PRT1 or “PRT2” relate to occupancy, with PRT1 designating the coordinates ofthe atom when in the first conformation and PRT2 designating the coordinates ofthe atom when in the second or alternate conformation.
  • the structural coordinates for the dimers may be modified by mathematical manipulation. Such manipulations include, but are not limited to, crystallographic permutations ofthe raw structure coordinates, fractionalization ofthe raw structure coordinates, integer additions or subtractions to sets ofthe raw structure coordinates, inversion ofthe raw structure coordinates and any combination ofthe above.
  • the structural coordinates can be slightly modified and still render nearly identical three dimensional structures. Therefore, a measure of a unique set of structural coordinates is the root-mean-square deviation ofthe resulting structure. Structural coordinates that render three dimensional structures that deviate from one another by a root- mean-square deviation of less than 1.5 A may be viewed as identical.
  • D2-D3 A DNA fragment encoding residues 142-365 of human FGFRl (“D2-D3”) was subcloned into bacterial expression vector pET-23a using Ncol and Hindlll restriction sites using techniques well known to the skilled artisan.
  • Bacterial strain BL21(DE3) was used for expression of D2-D3, and was induced with IPTG for 5 hours. Following induction of expression, the cells were collected by centrifugation, and lysed using a French press in a buffer containing 25 mM potassium phosphate, 150 mM NaCl, 2 mM EDTA, and 10% glycerol.
  • a pellet containing D2-D3 was collected by centrifugation, and dissolved in 6M guanidium hydrochloride, 100 mM Tris-HCl, pH 8.0. D2-D3 was allowed to refold by dialyzing for 48 hours against a buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, and 1 mM L-cysteine. The refolded D2-D3 was chromatographed on a heparin sepharose column on which FGF2 had been previously immobilized.
  • the resulting D2-D3 / FGF2 complex was eluted from the heparin sepharose column with a buffer containing 25 mM Tris-HCl, pH 7.5, and 1.5 M NaCl.
  • the D2-D3 / FGF2 complex was concentrated by ultrafiltration using a Centricon
  • Crystals of purified D2-D3 / FGF2 complex were grown at 20°C by vapor diffusion in hanging drops by mixing equal volumes of protein solution (10 mg/mL D2-D3 / FGF2 complex in 25 mM Tris-HCl, pH 8.5, and 150 mM NaCl) and reservoir buffer (1.6 M (NH 4 ) 2 SO 4 , 20% v/v glycerol and 100 mM Tris-HCl, pH 8.5), and suspending a 2.0 ⁇ l hanging drop ofthe resulting solution over 0.5 mL reservoir buffer at 20°C.
  • protein solution 10 mg/mL D2-D3 / FGF2 complex in 25 mM Tris-HCl, pH 8.5, and 150 mM NaCl
  • reservoir buffer 1.6 M (NH 4 ) 2 SO 4 , 20% v/v glycerol and 100 mM Tris-HCl, pH 8.5
  • Diffraction data were collected from a crystalline specimen, which had been flash frozen in a dry nitrogen stream, at beamline X-4A at the National Synchrotron Light Source, Brookhaven National Laboratory. Synchrotron data were collected on a CCD detector. All data were processed using DENZO and SCALEPACK. Otwinowski, 1993, "Oscillation data reduction program," Proceedings ofthe CCP4 Study Weekend, Sawyer et al., eds. (Daresbury, United Kingdom: SERC Daresbury Laboratory), 56-62.
  • the structure ofthe D2-D3 / FGF2 complex was determined by molecular replacement using the program AmoRe (Navaza, 1994, Acta Cryst. A 50: 157-163) using the structures of FGF2 (2FGF, Zhang et al, 1991, Proc. Natl. Acad. Sci. 88: 3446-3450) and telokin (1TLK, Holden et al, 1992, J. Mol. Biol 227: 840-851) as search models. Homology models were constructed from the telokin structure for the FGFRl D2 and D3 domains.
  • a molecular replacement solution was determined for both FGF2 molecules and one copy of D2 and D3 in the dimer, and the second copy of D2 and D3 was determined by rigid body rotation and translation ofthe first copy of D2 and D3 onto the second FGF2 molecule.
  • the placement ofthe second copy of D2 and D3 was confirmed by rigid body refinement techniques using CNS (Br ⁇ nger et al, 1998, Acta Cryst. D 54: 905-921).
  • the atomic model ofthe D2-D3 / FGF2 complex includes FGF2 residues 16-144 and FGFRl residues 149-359, except in one ofthe FGFRl receptors in the dimer, where residues 293-307 are disordered.
  • the average B-factor for all atoms is 38.7 A 2 for all atoms, 37.6/38.9 A 2 for FGF2, and 38.3/39.1 A 2 for FGFRl.
  • Atomic Structural Coordinates Table 1 provides the atomic structural coordinates ofthe FGFRl (D2-D3VFGF2 complex dimer complex dimer.
  • the structure ofthe FGFRl (D2-D3)/FGF2 complex has been described in Plotnikov et al., Cell 98, 641-650 (1999) and the coordinates for the FGFRl (D2- D3VFGF2 complex are available on the internet through the Protein Data Bank (assigned Protein Data Bank ID code 1CVS), the disclosures of which are herein inco ⁇ orated by reference.
  • D2-D3 A DNA fragment encoding residues 142-365 of human FGFRl (“D2-D3”) was subcloned into bacterial expression vector pET-23a using Ncol and Hindlll restriction sites using techniques well known to the skilled artisan.
  • Bacterial strain BL21(DE3) was used for expression of D2-D3, and was induced with IPTG for 5 hours. Following induction of expression, the cells were collected by centrifugation, and lysed using a French press in a buffer containing 25 mM potassium phosphate, 150 mM NaCl, 2 mM EDTA, and 10% glycerol.
  • a pellet containing D2-D3 was collected by centrifugation, and dissolved in 6M guanidium hydrochloride, 100 mM Tris-HCl, pH 8.0.
  • D2-D3 was allowed to refold by dialyzing for 48 hours against a buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, and 1 mM L-cysteine.
  • the refolded D2-D3 was chromatographed on a heparin sepharose column on which FGFl had been previously immobilized.
  • the resulting D2-D3 / FGFl complex was eluted from the heparin sepharose column with a buffer containing 25 mM Tris-HCl, pH 7.5, and 1.5 M NaCl.
  • the D2-D3 / FGFl complex was concentrated by ultrafiltration using a Centricon 10TM (Amicon) centrifugal concentrator, and further purified by size exclusion chromatography on a SuperdexTM 200 column (Pharmacia) using a buffer containing 25 mM Tris-HCl, pH 7.5, and 1.5 M NaCl. Prior to crystallization, the D2-D3 / FGFl complex was concentrated to 1 mg/mL in a buffer containing 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl.
  • Crystal Growth Crystals of purified D2-D3 / FGFl complex were grown at 20°C by vapor diffusion in hanging drops by mixing one volume of protein solution (1 mg/mL in 25 mM Tris-HCl, pH 8.5, and 150 mM NaCl) with four volumes of reservoir buffer (20% PEG 4000, 0.2 M L.2SO4, and 0.1 M Tris-HCl, pH 8.5), and suspending a 2.0 ⁇ l hanging drop ofthe resulting solution over 0.5 mL reservoir buffer at 20°C.
  • Diffraction data were collected from a crystalline specimen, which had been flash frozen in mother liquor containing 10% glycerol using a dry nitrogen stream, at beamline X- 4 A at the National Synchrotron Light Source, Brookhaven National Laboratory. Synchrotron data were collected on a CCD detector. All data were processed using DENZO and SCALEPACK. Otwinowski, 1993, "Oscillation data reduction program," Proceedings ofthe CCP4 Study Weekend, Sawyer et al., eds. (Daresbury, United Kingdom: SERC Daresbury Laboratory), 56-62.
  • the structure ofthe D2-D3 / FGFl complex was determined by molecular replacement using the program AmoRe (Navaza, 1994, Acta Cryst. A 50: 157-163) using the structures of FGFl (2AFG, Blaber et al, 1996, Biochemistry 35: 2086-2094) and telokin (1TLK, Holden et al, 1992, J. Mol. Biol. 227: 840-851) as search models. Homology models were constructed from the telokin structure for the FGFRl D2 and D3 domains. A molecular replacement solution was determined for two copies each of FGFl, D2, and D3 in the dimer.
  • the atomic model ofthe D2-D3 / FGFl complex includes FGFl residues 8-138 and FGFRl residues 147-359 except residues 294- 305 and 315-323, which are disordered.
  • the average B-factor for all atoms is 30.4 A 2 for all atoms, 31.2/33.0 A 2 for FGFl, and 29.1/28.7 A 2 for FGFRl.
  • Table 2 provides the atomic structural coordinates ofthe FGFRl (D2-D3VFGF1 complex dimer.
  • the residue number is preceded by a 1, e., residue number 464 ofthe first FGFRl molecule ofthe dimer is denoted by "1464".
  • the structure ofthe FGFR1(D2-D3)/FGF1 complex has been described in Plotnikov et al., Cell 101, 413-424 (2000) and the coordinates for the FGFRl (D2-D3VFGF1 complex are available on the internet through the Protein Data Bank (assigned Protein Data Bank ID code 1EVT), the disclosures of which are herein inco ⁇ orated by reference.
  • DNA fragments encoding residues 147 to 366 of FGFR2 were amplified by polymerase chain reaction (PCR) and subcloned into the bacterial expression vector pET-28a using Ncol and Hindlll cloning sites and transfected into the bacterial strain BL21(DE3).
  • PCR polymerase chain reaction
  • the refolded FGFR2 protein was loaded onto heparin sepharose columns on which FGF2 (basic FGF) had previously been immobilized.
  • the FGF2-FGFR2 complex was then eluted from the heparin sepharose column with a buffer containing 25mM Tris-HCl (pH 7.5) and 1.5M NaCl.
  • the FGF2-FGFR2 complex was concentrated using Centricon 10 (Amicon) filters and further purified by size exclusion chromatography (Pharmacia, Superdex 200) with a buffer containing 25mM Tris-HCl (pH 7.5) and 1.5M NaCl.
  • the complex migrated at a position consistent with the formation of a 1:1 FGF:FGFR complex.
  • Crystals were grown by vapor diffusion at 20°C using the hanging drop method.
  • 2 microliters of protein solution lOmg/ml, 25mM Tris-HCl (pH 7.5), 150mM NaCl) were mixed with 2 microliters ofthe crystallization buffer containing 10-15% PEG 4000, 10% isopropanol, and 0.1M HEPES-NaOH (pH 7.5).
  • the structure ofthe FGF2-FGFR2 complex was determined by molecular replacement using the program AmoRe (Navaza, Acta Cryst. A 50, 157-163 (1994)) and the structure of FGF2-FGFR1 (1CVS; Plotnikov et al.,Cell 98, 641-650 (1999)) as the search model. A molecular replacement solution was found for four copies of FGF2-FGFR2 complexes. Model building and refinement were performed with the programs O (Jones et al., Acta Crystallogr. A 47, 110-119 (1991)) and CNS (Brunger et al, Acta. Crystallogr.
  • the atomic model for FGF2-FGFR2 consists of four FGF2 molecules, four FGFR2 molecules, four sulfate ions, and 263 water molecules.
  • the structure of FGF2-FGFR2 was refined at 2.2 A with an R value of 24.8% (free R value of 27.3%).
  • Data collection and refinement statistics are given in Table 7.
  • the atomic model includes FGF2 residues 16-145 and FGFR2 residues 148-365, 4 sulfate ions, and 263 water molecules. In all four FGFR2 molecules residues 295-306 (bC-bC loop in D3) are disordered.
  • the average B-factor is 40.5 A 2 for FGF2 molecules, 37.7 A 2 for FGFR2 molecules, 73 A 2 for sulfate ions, and 32.6 A 2 for water molecules.
  • Table 3 provides the atomic structural coordinates ofthe FGF2/FGFR2 complex dimer.
  • the structure ofthe FGFR2/FGF2 complex has been described in Plotnikov et al., Cell 101, 413-424 (2000) and the coordinates for the FGFR2/FGF2 complex are available on the internet through the Protein Data Bank (assigned Protein Data Bank ID code 1EV2), the disclosures of which are herein inco ⁇ orated by reference.
  • FGF1-FGFR1 25.0 - 2.8 21539 24.9 / 30.0 0.009 1.5 2.3
  • Rcryst/free 100 x ⁇ n ki l
  • where F 0 (>0 ⁇ ) and F c are the observed and calculated structure factors, respectively. 5% of the reflections were used for calculation of Rfree. e For bonded protein atoms.
  • a molecular replacement attempt with the data collected from the orthorhombic crystals using a model built from the alpha C atom positions ofthe human colony stimulating factor was not successful.
  • Data used for the structure determination were collected from the monoclinic crystals at wavelengths 1.01 A and 1.5 A that are not at the abso ⁇ tion edge of Sm.
  • the anomalous signal was clear from Patterson difference maps.
  • the heavy metal position refinement and phasing was done with PHAESE (Furey et al., Methods Enzymol. 277, 590- 620 (1997)). A total of three Sm sites were used for phasing while four Sm atoms were placed in the final model.
  • the structure was determined by using anomalous scattering differences of samarium ions in the crystal at two wavelengths and refined to 2.3 A (Table 4). There are four molecules in each asymmetric unit and the initial experimental electron density clearly showed the four-helix bundle and two beta strands in the molecules. The connecting loops, as well as the N-terminal and C-terminal regions, were built from 2Fo-Fc maps. Table 4 gives the statistics ofthe final model, which contains 120 solvent molecules, four samarium ions, two calcium ions and one Tris molecule. The structure ofthe human stem cell factor homodimer has been described in Zhang et al., Proc.Nat.Acad.Sci.
  • the biological dimer is unmistakably recognizable.
  • the four protomers are superimposable except for the N-terminal and C-terminal loop regions. These loops are flexible and adopt multiple conformations in the four molecules in the asymmetric unit.
  • the protomers in the biological dimer are packed head-to-head in a manner of almost perfect C2 symmetry (see Fig. 19).
  • the dimer bends approximately 30° toward the side ofthe beta strands, resulting in an elongated shape with approximate dimensions of 87A x 32A x 25 A.
  • SCF is a non-covalent homodimer composed of two slightly wedged protomers.
  • the overall topology of a SCF protomer displays an antiparallel four-helix bundle fold (see Fig. 19), in a manner similar to other short-chain helix cytokines (Roswarski et al., Structure 2, 159-173 (1994)).
  • the helices run up-up-down-down, with two crossing beta strands wrapped on one side.
  • the structure ofthe dimer interface shows that dimerization is mediated by extensive polar and non-polar interactions between the two protomers with a large buried surface area.
  • the structure includes a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor binding site.
  • the X-ray structure of SCF shows that there are extensive interactions between the two SCF protomers, with approximately 1700 A 2 surface area buried upon dimerization (calculated with a probe of radius 1.4 A). This buried surface area accounts for about 20% ofthe total surface of each individual protomer, and is twice that reported for the 850 A 2 buried surface area ofthe disulfide linked M-CSF dimer.
  • Cys4 and Cys89 as well as Cys43 and Cysl38 form two intramolecular disulfide pairs. Both disulfide bonds are located at one end (tail) of each protomer away from the dimer interface.
  • the Cys4-Cys89 disulfide bond is more exposed than Cys43-Cysl38, a disulfide bond wrapped by the side chains of Val39, Leu98, Pro40 and His42. This probably explains why the Cys4-Cys89 bond is more susceptible to chemical reduction than the Cys43-Cysl38 disulfide bond (Lu et al, J. Biol. Chem. 271, 11309-1131 (1996)).
  • the SCF dimer interface is composed of loops between alphaA and betal, alphaB and alphaC, and can also be divided into three layers (see Fig. 21).
  • the bottom layer at the side of the beta strands is composed of hydrophobic interactions.
  • Side chains from Tyr26, Pro23, Phe63 and Leu22 from one protomer pack against corresponding side chains from the other protomer, with Tyr26-Asp25' and Tyr26'-Asp25 forming a hydrogen bond circle as the ca ⁇ et (see Fig. 2B).
  • These intermolecular hydrogen bond pair replace the intermolecular disulfide bond between the two M-CSF protomers (Bazan, Cell 65, 9-10 (1991); Broudy, Blood 90, 1345-1364 (1997)).
  • Example 5 Structure Determination Ternary FGF2-FGFR1 -Heparin complex
  • the expression, purification and crystallization of FGF2-FGFR1 complexes were carried out as described previously (Plotnikov et al., Cell 98, 641-650 (1999)). Crystals ofthe native FGF2-FGFR1 complex were incubated in 10 ⁇ l of stabilizing solution (40 % PEG 8000,
  • Table 6 provides the atomic structural coordinates ofthe the ternary FGF2-FGFR1- heparin complex.
  • the heparin can be approximated as a helix generated by repeating disaccharide units of D-glucosamine (GlcN) and L-iduronic acid (IdoA) joined by ⁇ -1-4 linkages. Each disaccharide unit is sulfated at three positions; one at the 2-hydroxyl group of IdoA and two at the 2-amino and 6-hydroxyl groups of GlcN. Sulfate and carboxylate groups form the negatively-charged edges ofthe heparin helix and appear on a given side ofthe helix every 17-19 A on average.
  • GlcN D-glucosamine
  • IdoA L-iduronic acid
  • Heparin polysaccharides are polar entities with a non- reducing end (04) and a reducing end (01). In the crystal structure, the decasaccharides bind with their non-reducing ends in the center of canyon and run out onto the high-affinity heparin binding sites ofthe ligands. Consequently, the symmetry ofthe dimeric assembly is maintained. Traversing ofthe canyon by one polar heparin molecule disrupts the two-fold symmetry ofthe system.
  • Each decasaccharide makes a total of 30 hydrogen bonds with FGF and both FGFRs (see Figs. 29 and 30).
  • 25 hydrogen bonds are made with heparin.
  • the remaining 5 hydrogen bonds with heparin originate from the FGFR ofthe adjoining 1:1 FGF :FGFR complex.
  • Lysines 160, 163, 172, 175 and 177 located on the heparin-binding surface of D2, form 7 hydrogen bonds between FGFR and heparin in the context of a 1:1 FGF:FGFR complex.
  • Hydrophobic contacts between Ile-216 and the non-reduced ring A of heparin further fortify this interface.
  • the hydrogen bonds between Lys-207 and heparin are mediated via carboxylate, linker and ring oxygens of heparin.
  • Arg-209 makes hydrogen bonds with the 6-O-sulfate group of ring D, thereby emphasizing the critical dual role of 6-O-sulfate in promoting 1:1 FGF2:FGFR interaction and inducing 2:2 FGF:FGFR dimer formation.
  • the crystal structure provides a plausible explanation for the well-documented inability of 6-O-desulfated heparin oligosaccharides to promote mitogenic activities by failing to induce receptor dimerization.
  • Rcryst/free 00 x ⁇ hk
  • the invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
  • the structure ofthe FGFRl (D2-D3)/FGF2 complex has been described in Plotnikov et al., Cell 98, 641-650 (1999).
  • the structures ofthe FGFRl (D2-D3)/FGF1 complex and the FGFR2/FGF2 complex are described in Plotnikov et al., Cell 101, 413-424 (2000).
  • the structure ofthe human stem cell factor homodimer is described in Zhang et al., Proc.Nat.Acad.Sci. 97(14), 7732-7737 (2000).
  • the disclosures of these three references are herein inco ⁇ orated by reference.
  • Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate. Mol. Cell Biochem. 124, 121-
  • FGF-1 FGF-2
  • FGF-4 J. Biol. Chem. 268, 23906-23914.
  • FGF-21 preferentially expressed in the liver. Biochim. Biophys. Acta, 1492, 203-206.
  • Heparin is required for cell-free binding of bFGF to a soluble receptor and for mitogenesis in whole cells. Mol. Cell. Biol. 12, 240-247.
  • Rusnati M., Coltrini, D., Caccia, P., Dell'Era, P., Zoppetti, G., Oreste, P., Valsasina, B., and
  • ATOM 33 C ASP A 19 65, .520 30, .193 131 .765 1 .00 38 .86
  • ATOM 249 CA LYS A 46 71. .461 38, .254 120. .193 1, .00 31, .44
  • ATOM 252 CD LYS A 46 73. .931 37, .596 123. .067 1, .00 37, .72
  • ATOM 258 CA SER A 47 68. .649 40, .804 120. .550 1, .00 30, .89
  • ATOM 269 C ASP A 48 65. .287 38 .383 119, .277 1 .00 31, .67
  • ATOM 272 CD PRO A 49 63, .576 39, .618 117, .907 1 .00 28, .94
  • ATOM 273 CA PRO A 49 62, .923 38, .467 119, .933 1, .00 30, .97
  • ATOM 1050 CA HIS B 16 100. 953 9. ,958 141. 508 1. 00 49. 06
  • ATOM 1052 CA PHE B 17 102. ,763 12. ,726 143. ,423 1. ,00 45. ,92
  • ATOM 1063 CA LYS B 18 105. ,415 10, .325 144, .772 1. .00 47. .59
  • ATOM 1076 CD PRO B 20 109. .144 9. .140 139. .612 1. .00 41, .29
  • ATOM 1083 CA LYS B 21 107. .581 13, .877 136. .656 1, .00 33, .82
  • ATOM 1232 CA ARG B 39 123. .969 22. ,666 133, ,809 1. .00 39. .37
  • ATOM 1250 CA ASP B 41 118 .607 23 .430 129 .669 1 .00 39, .12
  • ATOM 1258 CA GLY B 42 115 .860 21 .849 127 .590 1 .00 35, .38

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Abstract

La présente invention concerne la détermination et l'utilisation de structures tridimensionnelles de protéines tyrosine kinases de récepteur (RPTK) et/ou de leurs ligands. Un groupe particulier de ces structures comprend des structures tridimensionnelles du domaine extracellulaire des RPTK. Les structures tridimensionnelles des RPTK peuvent faciliter la conception et l'identification de modulateurs de la fonction RPTK. D'autres structures de ce type peuvent comprendre des ligands RPTK, tels qu'un facteur de cellules souches ou un fragment de ce dernier. Les modulateurs de la fonction RPTK peuvent être utilisés pour traiter des maladies induites par une activité inappropriée de RPTK.
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