WO1998007835A2 - Crystal structures of a protein tyrosine kinase - Google Patents

Abstract

The present invention relates to the three-dimensional structures of a protein tyrosine kinase optionally complexed with one or more compounds. The atomic coordinates that define the structures of the protein tyrosine kinase and any of the compounds bound to it are pertinent to methods for determining the three-dimensional structures of protein tyrosine kinases with unknown structure and to methods that identify modulators of protein tyrosine kinase functions.

Description

DESCRIPTION

CRYSTAL STRUCTURES OF A PROTEIN TYROSINE KINASE

RELATED APPLICATIONS

This application is related to U.S. Application Serial No. 08/701,191, by Moham adi , et al . , entitled "Crystals of the Tyrosine Kinase Domain of Non- Insulin Receptor Tyrosine Kinases," filed August 21, 1996 (Lyon & Lyon Docket No. 227/088) and U.S. Application Serial No. 60/034,168, by McMahon, et al . , entitled "Crystal Structures of a Protein Tyrosine Kinase Complexed with Compounds of the Oxindolinone/Thiolindolinone Family, " filed December 19, 1996 (Lyon & Lyon Docket No. 221/282), which are hereby incorporated herein by reference in their entirety including any drawings, tables, and figures.

INTRODUCTION The present invention relates to the three dimensional structures of protein kinases.

BACKGROUND OF THE INVENTION The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

Protein tyrosine kinases (PTKs) comprise a large and diverse class of enzymes (for a review, see Schlessinger and Ullrich, 1992, Neuron 9 : 383-391) . The PTK family contains multiple subfamilies, one of which is the fibroblast growth factor receptor (FGF-R) subfamily (for a review, see Givol and Yayon, 1992, FASEB J. 6 (15 ) : 3362-3369) .

All PTKs enzymatically transfer a high energy phosphate from adenosine triphosphate to a tyrosine residue in a target protein. These phosphorylation events regulate 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. PTKs are components in many signal transduction processes. A PTK regulates the flow of a signal in a particular step in the process by phosphorylating a downstream molecule. The addition of a phosphate can either modulate the activity of the downstream molecule by turning it "on" or "off". Thus, aberrations in a particular PTK's activity can either cause overflow or underflow of the signal. Overflow of a signal can lead to such abnormalities as uncontrolled cell proliferation, which is representative of such disorders as cancer and angiogenesis .

Scientists in the biomedical community are searching for PTK inhibitors that down-regulate overflow signal transduction pathways. In particular, small molecule PTK inhibitors are sought that can traverse the cell membrane and not become hydrolyzed in acidic environments. These small molecule PTK inhibitors can be highly bioavailable and can be administered orally to patients.

Some small molecule PTK inhibitors have already been discovered. For example, bis (monocyclic) , bicyclic or heterocyclic aryl compounds (PCT WO 92/20642) , vinylene-azaindole derivatives (PCT WO 94/14808) , 1- cyclopropyl-4-pyridyl-quinolones (U.S. Patent No. 5,330,992) , styryl compounds (U.S. Patent No.

5,217,999) , styryl-substituted pyridyl compounds (U.S. Patent No. 5,302,606) , certain quinazoline derivatives (EP Application No. 0 566 266 Al) , seleoindoles and selenides (PCT WO 94/03427) , tricyclic polyhydroxylic compounds (PCT WO 92/21660) , and benzylphosphonic acid compounds (PCT WO 91/15495) are described as PTK inhibitors .

Although many PTK inhibitors are known, many of these are not specific for PTK subfamilies and will therefore cause multiple side-effects as therapeutics. Compounds of the indolinone family, however, are specific for the FGFR subfamily and are non- hydrolyzable . WO 96/40116, "Indolinone Compounds for the Treatment of Disease," published December 19, 1996, inventors Tang et al . Although the use of X-ray crystallography has provided three dimensional structures of other PTKs, they are not complexed with PTK subfamily specific, hydrolysis resistant, small molecules . Despite recent advances, the need remains in the art for crystallographic analysis of protein kinases, so that improved therapeutic molecules can be designed and synthesized.

SUMMARY OF THE INVENTION

The present invention relates to the three dimensional structures of protein tyrosine kinases. The use of X-ray crystallography can define the three dimensional structure of protein tyrosine kinase at atomic resolution. The three dimensional structures described herein elucidate specific interactions between protein tyrosine kinases and compounds bound to them. The coordinates that define the three dimensional structures of protein tyrosine kinases are useful for determining three dimensional structures of PTKs with unknown structure. In addition, the coordinates are also useful for designing and identifying modulators of protein tyrosine kinase function. These modulators are potentially useful as therapeutics for diseases, including (but limited to) cell proliferative diseases, such as cancer, angiogenesis, atherosclerosis, and arthritis.

Thus in a first aspect, the invention features a crystalline form of a polypeptide corresponding to the catalytic domain of a protein tyrosine kinase. The term "crystalline form," in the context of the invention, is a crystal formed from an aqueous solution comprising a purified polypeptide corresponding to the catalytic domain of a PTK. A crystalline form of a protein tyrosine kinase is characterized as being capable of diffracting x-rays in a pattern defined by one of the crystal forms depicted in Blundel et al . , 1976, Protein Crystallography. Academic Press. A crystalline form of a protein kinase is not characterized as being capable of diffracting x-rays in a pattern analogous to a crystalline form consisting of primarily salt or primarily a compound, for example. The term "protein tyrosine kinase," or PTK, refers to an enzyme that transfers the high energy phosphate of adenosine triphosphate to a tyrosine residue located on a protein target. A protein tyrosine kinase catalytic domain of the invention can originate from receptor protein tyrosine kinases that bind fibroblast growth factor (FGF) . These protein tyrosine kinases are known as "FGFR" herein, and can relate to one member of the FGFR family, such as FGFR1.

The term "catalytic domain" refers to the region of a protein that can exist as a separate entity from the protein. 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. Considerable amino acid identity preferably refers to at least 30% identity, more preferably at least 35% identity, and most preferably at least 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 55% to 90%. The catalytic domain may be functional as a separate entity. The catalytic domain of a protein tyrosine kinase is also characterized as a polypeptide that is soluble in solution.

The term "identity" identity as used herein 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. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.

The term "functional" refers to the ability of a catalytic domain to convert a substrate into a product by phosphorylating the substrate. The term "functional" also relates to the ability of a catalytic domain to bind natural binding partners. The catalytic region may comprise an N-terminal tail, a catalytic core, and a C-terminal tail. The catalytic core is a polypeptide that can be functional in terms of catalysis. N- and C- terminal tails are polypeptide regions that may not confer appreciable functionality in terms of catalysis, but may confer functionality in terms of modulator specificity. A polypeptide can exist as a catalytic domain eventhough it is not functional. For example, a polypeptide corresponding to a catalytic domain may not be functional if it does not harbor phosphate moieties in key areas. Multiple examples of phosphorylation- state dependent function are well documented in the art. Therefore, a catalytic domain can also exist without being functional. A measure of a protein kinase catalytic domain is a polypeptide that is homologous to other protein kinase catalytic domains. The term "polypeptide" refers to an amino acid chain representing a portion of, or the entire sequence of, amino acids comprising a protein.

A preferred embodiment of the invention includes a crystalline form of a PTK that is a receptor PTK.

Receptors are proteins that straddle the inside and outside of the cell membrane. Receptor PTKs comprise an extracellular region, a transmembrane region, and an intracellular region comprising a catalytic domain.

Another preferred embodiment of the invention is the crystalline form of a receptor PTK selected from the group consisting of FGF-R, PDGF-R, FLK, CCK4 , MET, TRKA,

AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, R0R1 , and MUSK. Yet another preferred embodiment of the invention is the crystalline form of a PTK that is a non-receptor

PTK. Non-receptor PTKs are located inside the cell and do not harbor extracellular or membrane-spanning polypeptides attached to the polypeptide corresponding to the catalytic domain. Non-receptor PTKs may harbor fatty acids or lipids, which can impart a membrane associated character to a PTK. In preferred embodiments of the invention, crystalline forms of non-receptor PTKs are selected from the group consisting of SRC, BRK, BTK,

CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

In still another preferred embodiment, the invention features a crystalline form of a PTK that comprises a heavy metal atom. These types of crystals can be referred to as derivative crystals.

The term "derivative crystal" refers to a crystal where the polypeptide is in association with one or more heavy-metal atoms . The term "association" refers to a condition of proximity between a chemical entity or compound, or portions or fragments thereof, and tyrosine kinase domain protein, 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 .

The term "heavy metal atom" refers to an atom that is a transition element, a lanthanide metal, or an actinide metal. Lanthanide metals include elements with atomic numbers between 57 and 71, inclusive. Actinide metals include elements with atomic numbers between 89 and 103, inclusive.

In a preferred embodiment, the invention features a crystal of an FGF receptor tyrosine kinase domain protein. The FGF receptor tyrosine kinase domain protein can relate to FGFR1.

The term "FGFR1" refers to one member of multiple receptor PTKs that are homologous to one another and bind FGF. In this context, the term "homologous" refers to at least 70% amino acid identity between two members of the FGFR family.

The term "FGFR1" can also refer to a mutant of human FGFR1 which is characterized by the amino acid sequence of SEQ ID NO: 2. As compared to human FGFR1 , FGFR1 contains the following amino acid substitutions: Cys-488 → Ala, Cys-584 - Ser, Leu-457 - Val, and has an additional five amino acid residues at the N- terminus (Ser-Ala-Ala-Gly-Thr) .

The term "human FGFR1" refers to the tyrosine kinase domain of human fibroblast growth factor receptor 1 ("FGFR1") having the amino acid sequence of SEQ ID NO:l. Generally, human FGFR1 comprises a 310 amino acid residue fragment (residues 456 to 765) of human FGFR1.

The term "mutant" refers to a polypeptide which is obtained by replacing at least one amino acid residue in a native tyrosine kinase domain with a different amino acid residue. Mutation can 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 corresponding to a native tyrosine kinase domain having substantially the same three-dimensional structure as the native tyrosine kinase domain from which it is derived. By having substantially the same three-dimensional structure is meant having 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 of the native tyrosine kinase domain from which the mutant is derived when at least about 50% to 100% of the Cα atoms of the native tyrosine kinase are included in the superposition. A mutant may have, but need not have, PTK activity.

In another preferred embodiment, the invention relates to a crystalline form defined by the structural coordinates set forth in Table 1. The term "atomic structural coordinates" as used herein 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 of the 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 by a person of ordinary skill in the art as identical. Hence, the structural coordinates set forth in Table 1, Table 2, Table 3, and Table 4 are not limited to the values defined therein.

In other preferred embodiments, the invention features a crystalline form of the polypeptide in association with a compound. These types of crystalline forms can be referred to as co- crystals. The compound may be a cofactor, substrate, substrate analog, inhibitor, or allosteric effector.

The term "compound" refers to an organic molecule . The term "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 of the 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 IO^-6 M or less. The compound is preferably a modulator that alters the function of a PTK. The term "function," in reference to the effect of a modulator on PTK function, refers to the ability of a modulator to enhance or inhibit the catalytic activity of a PTK.

The term "catalytic activity", in the context of the invention, defines the ability of a PTK to phosphorylate a substrate polypeptide. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. The conversion of the substrate to a product occurs at the active-site of the PTK. The term "active-site" refers to a cavity located in the PTK in which one or more substrate molecules may bind. Addition of a modulator to cells expressing a PTK may enhance (activate) or lower (inhibit) the catalytic activity of the PTK. A small number of inhibitors of PTK catalytic activity are known in the art. Small molecule inhibitors may modulate PTK function by blocking the binding of substrates. Indolinone compounds, for example, may bind to the active-site of PTK catalytic domains and inhibit them effectively, as measured by inhibition constants on the order of IO^-6 M or less.

Activators of PTK intracellular regions can enhance PTK function by interacting with both the PTK catalytic domain and the substrate. Activators may also promote dimerization of PTKs and thus activate them by bringing them into close proximity with one another. In addition, activators may operate by promoting a conformational change in the intracellular region of the PTK such that the catalytic region modifies substrates at a faster rate in the presence of the activator.

The term "function" can also refer to the ability of a modulator to enhance or inhibit the association between a PTK and a natural binding partner.

The term "natural binding partner" refers to a polypeptide that normally binds to a PTK in a cell. These natural binding partners can play a role in propagating a signal in a PTK signal transduction process. The natural binding partner can bind to a PTK with high affinity. High affinity represents an equilibrium binding constant on the order of IO^-6 M or less. However, a natural binding partner can also transiently interact with a PTK and chemically modify it . PTK natural binding partners are chosen from a group consisting of, but not limited to, src homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, nucleotide exchange factors, and other protein kinases or protein phosphatases .

The term "interactions" refers to hydrophobic, aromatic, and ionic forces and hydrogen bonds formed between atoms in the modulator and the enzyme active- site.

The term "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 .

The term "substrate" refers to a compound that reacts with an enzyme. Enzymes can catalyze a specific reaction on a specific substrate. For example, PTKs can phosphorylate specific protein and peptide substrates on tyrosine moieties. In addition, nucleotides can act as substrates for protein kinases.

The term "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. The term "inhibitor" refers to a compound that decreases the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner and more preferably catalytic activity.

The term "allosteric effector" refers to a compound that causes allosteric interactions in a protein. The term "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.

The term "co-crystal" refers to a crystal where the polypeptide is in association with one or more compounds.

In preferred embodiments, a co-crystal of the invention can be in association with a heavy metal atom. Examples of heavy metal atoms are described above.

In other preferred embodiments, the invention features a co-crystal comprising the crystalline form of the polypeptide in association with a compound, where the compound is a non-hydrolyzable analog of ATP. These analogs can be referred to as nucleotide analogs.

The term "ATP" refers to the chemical compound adenosine triphosphate .

The term "non-hydrolyzable" refers to a compound having a covalent bond that does not readily react with water. Examples of non-hydrolyzable analogs of ATP are AMP-PNP and AMP-PCP, whose structures are well known to those skilled in the art.

The term "AMP-PNP" refers to adenylyl imidodiphosphate, a non-hydrolyzable analog of ATP.

The term "AMP-PCP" refers to adenylyl diphosphonate , a non-hydrolyzable analogue of ATP.

In another preferred embodiment, the invention relates to a crystalline form defined by the structural coordinates set forth in Table 2.

In preferred embodiments, the invention relates to crystalline forms, where the compound in association with the polypeptide is an indolinone. Certain indolinones are specific modulators of PTK function. A preferred embodiment of the invention is the crystalline form of a PTK complexed with an indolinone of formula I or II:

( i :

or a pharmaceutically acceptable salt, isomer,

metabolite, ester, amide, or prodrug thereof, where:

(a) A_lf A₂ , A₃, and A₄ are independently carbon or nitrogen;

(b) R_x is hydrogen or alkyl;

(c) R₂ is oxygen in the case of an oxindolinone or sulfur in the case of a thiolindolinone ;

(d) R₃ is hydrogen; (e) R₄, R₅, R₆, and R₇ are optionally present, and are either (i) independently selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0₂NRR ' , S0₃R, SR, N0₂, NRR', OH, CN, C(0)R, 0C(0)R, NHC(0)R, (CH₂)_nC0_?R, and CONRR' or (ii) any two adjacent R₄ , R₅, R₆, and R₇ taken together form a fused ring with the aryl portion of the indole-based portion of the indolinone;

(f) R₂ ' , R₃', R₄ ' , R_b ' , and R₆ ' are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0₂NR ' , S0₃R, SR, N0_?, NRR', OH, CN, C(0)R, 0C(0)R, NHC(0)R, (CH )_nC0₂R, and CONRR ' ;

(g) n is 0, 1, 2, or 3; (h) R is hydrogen, alkyl or aryl; (i) R' is hydrogen, alkyl or aryl; and (j) A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1 , 2 , 3 - triazole , 1 , 2 , 4 -triazole, oxazole, isoxazole, thiazole, isothiazole, furan, 1,2,3- oxadiazole, 1 , 2 , 4 -oxadiazole, 1 , 2 , 5-oxadiazole , 1,3,4- oxadiazole, 1 , 2 , 3 , -oxatriazole , 1 , 2 , 3 , 5-oxatriazole,

1, 2 , 3-thiadiazole, 1 , 2 , 4 -thiadiazole , 1 , 2 , 5-thiadiazole, 1 , 3 , 4 -thiadiazole, 1, 2 , 3 , 4- thiatriazole , 1,2,3,5- thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethy] , S(0)R, S0₂NRR', S0₃R, SR, N0₂, NRR', OH, CN, C(0)R, 0C(0)R, NHC(0)R, (CH₂)_nC0₂R or CONRR'.

The term "pharmaceutically acceptable salt" refers to those salts which retain the biological activity and properties of the free bases. Pharmaceutically acceptable salts can be obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid and the like. The term "prodrug" refers to an agent that is converted into the parent drug in vi vo . Prodrugs may be easier to administer than the parent drug in some situations. For example, the prodrug may be bioavailable by oral administration but the parent is not, or the prodrug may improve solubility to allow for intravenous administration.

"Alkyl" refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon. Preferably, 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. The alkyl group may be optionally substituted with one or more substituents are selected from the group consisting of hydroxyl , cyano, alkoxy, =0, =S, N0₂, halogen, N(CH₃)₂ amino, and SH.

"Alkenyl" refers to a straight -chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond. Preferably, 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. The alkenyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, =0, =S, N0₂, halogen, N(CH₃)₂ amino, and SH.

"Alkynyl" refers to a straight -chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond. Preferably, 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. The alkynyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, =0, =S, N0₂, halogen, N(CH₃)_? amino, and SH .

"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 be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO_?, 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 of the ring atoms being carbon. Heteroatoms include oxygen, sulfur, and nitrogen. Thus, heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N- lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.

"Amide" refers to -C(0)-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. "Amine" refers to a -N(R')R'' group, where R' and

R¹' are independently selected from the group consisting of alkyl, aryl, and alkylaryl.

"Thioether" refers to -S-R, where R is alkyl, aryl, or alkylaryl . "Sulfonyl" refers to -S(0)₂-R, where R is aryl,

C(CN) =C-aryl, CH₂CN, alkyaryl, sulfonamide, NH-alkyl, NH- alkylaryl, or NH-aryl.

The term "acyl" denotes groups -C(0)R, where R is alkyl as defined above, such as formyl , acetyl, propionyl, or butyryl .

It is understood by those skilled in the art that when A_{l t} A₂, A₃ , and A₄ are nitrogen or sulfur that the corresponding R₄, R₅, R₆, and R₇, as well as the corresponding bond, do not exist.

Examples of indoles having such fused rings (as described in (e) (ii) above include the following:

The six membered rings shown above exemplify possible A rings in compound II. Other preferred embodiments of the invention are crystalline forms comprising 3 - [ (3 - (2-carboxyethyl) -4 - methylpyrrol-5-yl) methylene] -2 -indolinone as well as 3- [4- (4-formylpiperazine-1-yl- ) benzylidenyl] -2 - indolinone . The polypeptide of these crystalline forms can be FGFR, and specifically, FGFR1.

In preferred embodiments, the crystalline forms of the invention can be defined by the structural coordinates set forth in Table 3 or Table 4. The use of X-ray crystallography can elucidate the three dimensional structure of crystalline forms of the invention. The first characterization of crystalline forms by X-ray crystallography can determine the unit cell shape and its orientation in the crystal. In other preferred embodiments, the invention features a crystal of an FGF receptor tyrosine kinase domain protein, where the crystal is characterized by having monoclinic unit cells. The crystal may also be characterized by having space group symmetry C2. The term "unit cell" refers to the smallest and simplest volume element (i.e., parallelpiped-shaped block) of a crystal that is completely representative of the unit of pattern of the crystal. The dimensions of the unit cell are defined by six numbers: dimensions a, b and c and angles α, β and γ . A crystal can be viewed as an efficiently packed array of multiple unit cells. Detailed descriptions of crystallographic terms are described in, which is hereby incorporated herein by reference in its entirety, including any drawings, figures, and tables.

The term "monoclinic unit cell" refers to a unit cell where a ≠ b ≠ c ; a = γ = 90° ; and β > 90°.

The term "space group" refers to the symmetry of a unit cell. In a space group designation (e.g., C2) 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 .

The term "lattice" in reference to crystal structures refers to the array of points defined by the vertices of packed unit cells.

The term "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.

In a preferred embodiment, the invention features a crystalline form, where the monoclinic unit cells have dimensions of about a=208.3 A, b=57.8 A, c=65.5 A and β=107.2°.

In a preferred embodiment, the invention features a FGFR1 crystal, where the monoclinic unit cells have dimensions of about a=211.6 A, b=51.3 A, c=66.1 A and β=107.7°. In another aspect the invention features a polypeptide corresponding to the catalytic domain of a protein tyrosine kinase, containing at least about 20 amino acid residues upstream of the first glycine in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues downstream of the conserved arginine located at the C-terminal boundary of the catalytic domain.

The polypeptides of the invention can be isolated, enriched or purified. In addition, the crystalline forms of the invention can be formed from polypeptides that are isolated, enriched, or purified.

By "isolated" in reference to a polypeptide is meant a polymer of 6 , 12, 18 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature . Use of the term "isolated" indicates that a naturally occurring sequence 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 material naturally associated with it.

By the use of the term "enriched" in reference to a polypeptide it is meant that the specific amino acid sequence constitutes a significantly higher fraction (2 - 5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that "enriched" does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term 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 at least 5 to 10 fold or even more. The term also does not imply that there are no amino acids from other sources. The other source amino acids may, for example, comprise amino acids encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.

It is also advantageous for some purposes that an amino acid sequence be in purified form. The term "purified" in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e . g. , in terms of mg/ml) . 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 preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure. In a preferred embodiment, the invention features a polypeptide corresponding to the catalytic domain of a receptor PTK. The receptor PTK may have a three- dimensional structure substantially similar to that of the insulin receptor, even though the amino acid content may be different. In a preferred embodiment, the invention features a polypeptide corresponding to the catalytic domain of a non-receptor PTK, where the non-insulin receptor tyrosine kinase is a cytoplas ic tyrosine kinase.

In a preferred embodiment, the invention features a polypeptide corresponding to the catalytic domain of a receptor PTK, selected from the group consisting of FGF- R, PDGF-R, KDR, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , or MUSK.

In a preferred embodiment, the invention features a polypeptide corresponding to the catalytic domain of a non-receptor PTK, selected from the group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, or ACK.

In a preferred embodiment, the invention features a polypeptide corresponding to the catalytic domain of a PTK, having the amino acid sequence shown in Table 1 or Table 2.

In another aspect, 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

(b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container, under conditions suitable for crystallization.

These processes are described in detail in the section entitled "Detailed Description of the Invention. "

In another aspect, the invention features a method of obtaining FGF receptor tyrosine kinase domain polypeptide in crystalline form, comprising the steps of: (a) mixing a volume of polypeptide solution with an equal volume of reservoir solution, where the polypeptide solution comprises 1 mg/mL to 60 mg/mL FGF- type tyrosine kinase domain protein, 10 mM to 200 mM buffering agent, 0 mM to 20 mM dithiothreitol and has a pH of about 5.5 to about 7.5, and where the reservoir solution comprises 10% to 30% (w/v) polyethylene glycol, 0.1 M to 0.5 M ammonium sulfate, 0% to 20% (w/v) ethylene glycol or glycerol, 10 M to 200 mM buffering agent and has a pH of about 5.5 to about 7.5; and (b) incubating the mixture obtained in step (a) over said reservoir solution in a closed container at a temperature between 0° and 25°C until crystals form.

In a preferred embodiment, the invention features a method of obtaining FGF receptor tyrosine kinase domain polypeptide in crystalline form, where the polypeptide solution comprises about 10 mg/mL FGF receptor tyrosine kinase domain, about 10 mM sodium chloride, about 2 mM dithiothreitol, about 10 mM Tris-HCl and has a pH of about 8; the reservoir buffer comprises about 16% (w/v) polyethylene glycol (MW 10000), about 0.3 M ammonium sulfate, about 5% ethylene glycol or glycerol, about 100 mM bis-Tris and has a pH of about 6.5; and the temperature is about 4°C. In another preferred embodiment, the invention features a method of obtaining FGF receptor tyrosine kinase domain polypeptide in crystalline form, where the polypeptide solution includes a compound such as a cofactor, substrate, substrate analog, inhibitor or allosteric effector. In still another preferred embodiment, the invention features a method of obtaining FGF receptor tyrosine kinase domain polypeptide in crystalline form, where the compound is a nucleotide analog, such as a non-hydrolyzable analog of ATP, or an indolinone. Indolinone compounds have the general structural formula as described herein.

In another aspect, the invention features a cDNA encoding an FGF receptor tyrosine kinase domain protein, where a coding strand of the cDNA has the nucleotide sequence of SEQ ID NO : 5.

Another aspect of the invention relates to a method of determining three dimensional structures of PTKs with unknown structure by utilizing the structural coordinates of Table 1, Table 2, Table 3, and Table 4. These methods can relate to homology modeling, molecular replacement, and nuclear magnetic resonance methods.

In a preferred embodiment, the invention relates to a method of determining three dimensional structures of PTKs with unknown structures by utilizing the coordinates of Table 1, Table 2, Table 3, or Table 4 in conjunction with the amino acid sequences of PTKs. This method of homology modeling comprises the steps of: (a) aligning the computer representation of an amino acid sequence of a PTK with unknown structure with that of a PTK with known structure, where alignment is achieved by matching homologous regions of the amino acid sequences; (b) transferring the computer representation of an amino acid structure in the PTK sequence of known structure to a computer representation of a structure of the corresponding amino acid in the PTK sequence with unknown structure; and (c) determining low energy conformations of the resulting PTK structure.

The term "amino acid sequence" describes the order of amino acids in the amino acid chain comprising a polypeptide corresponding to the catalytic domain of a PTK .

The term "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. The term "homologous" 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) .

The term "corresponding" refers to an amino acid that is aligned with another in the sequence alignment mentioned above.

The term "determining the low energy conformation" describes a process of changing the conformation of the PTK structure such that the structure is of low free energy. The PTK structure may or may not have molecules, such as modulators bound to it .

The term "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.

The term "favorable interactions" refers to hydrophobic, aromatic, and ionic forces, and hydrogen bonds .

Another preferred embodiment of the invention relates to a method of determining three dimensional structures of PTKs with unknown structure. This method is accomplished by applying the structural coordinates of Table 1, Table 2, Table 3, or Table 4 to an incomplete X-ray crystallographic data set for a PTK. The method comprises the steps of: (a) aligning the positions of atoms in the unit cell by matching electron diffraction data from two crystals, where one data set is complete and the other is incomplete; and (b) determining a low energy conformation of the resulting PTK structure. The term "incomplete data set" relates to a X-ray crystallographic data set that does not have enough information to give rise to a three dimensional structure .

In another preferred embodiment, the invention relates to a method of determining three dimensional structures of PTKs with unknown structure by applying the structural coordinates of Table 1, Table 2, Table 3, or Table 4 to nuclear magnetic resonance (NMR) data of a PTK. This method comprises the steps of: (a) determining the secondary structure of a PTK structure using NMR data; and (b) simplifying the assignment of through-space interactions of amino acids. The PTK structure may not be complexed with compounds or modulators . The term "secondary structure" describes the arrangement of amino acids in a three dimensional structure, such as in α-helix or β-sheet elements.

The term "through-space interactions" defines the orientation of the secondary structural elements in the three dimensional structure and the distances between amino acids from different portions of the 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. In another aspect, the invention features a method of identifying potential modulators of PTK function. These modulators are identified by docking a computer representation of a structure of a compound with a computer representation of a cavity formed by the active-site of a PTK. The computer representation of the PTK active-site structure can be defined by structural coordinates.

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 in close proximity with a PTK. The term can also refer to a process of finding low energy conformations of the co pound/PTK complex. A preferred embodiment of the invention is a method of identifying potential modulators of PTK function. The method involves utilizing the structural coordinates or a PTK three dimensional structure . The structural coordinates set forth in Table 1, Table 2, Table 3, and Table 4 can be utilized. The method comprises the steps of: (a) removing a computer representation of a PTK structure and docking a computer representation of a compound from a computer data base with a computer representation of the active-site of the PTK; (b) determining a conformation of the complex with a favorable geometric fit and favorable complementary interactions; and (c) identifying compounds that best fit the PTK active-site as potential modulators of PTK function. The initial PTK structure may or may not have compounds bound to it. The term "favorable geometric fit" refers to a conformation of the compound-PTK complex where the surface area of the compound is in close proximity with the surface area of the active-site without forming unfavorable interactions. Unfavorable interactions can be steric hindrances between atoms in the compound and atoms in the PTK active-site.

The term "favorable complementary interactions" relates to hydrophobic, aromatic, ionic, and hydrogen bond donating, and hydrogen bond accepting forces formed between the compound and the PTK active-site.

The term "potential" qualifies the term "modulator of PTK function" because the potential modulator or PTK function has not yet been tested for activity in vi tro or in vivo. The term "best fit" describes compounds that complexed the most surface area in the complex and/or form the most favorable complementary interactions with the PTK in the screen in a given experiment .

Another preferred embodiment of the invention is a method of identifying potential modulators of PTK function. The method involves utilizing a three dimensional structure of a PTK, with or without compounds bound to it . The method comprises the steps of: (a) modifying a computer representation of a PTK having one or more compounds bound to it, where the computer representations of the compound or compounds and PTK are defined by structural coordinates; (b) determining a conformation of the complex with a favorable geometric fit and favorable complementary interactions; and (c) identifying the compounds that best fit the PTK active-site as potential modulators of PTK function.

The term "modifying" relates to deleting a chemical group or groups or adding a chemical group or groups. Computer representations of the chemical groups can be selected from a computer data base.

Yet another preferred embodiment of the invention is a method of identifying potential modulators of PTK function by operating modulator construction or modulator searching computer programs on the compounds complexed with the PTK. The method comprises the steps of: (a) removing a computer representation of one or more compounds complexed with a PTK; and (b) searching a data base for compounds similar to the removed compounds using a compound searching computer program, or replacing portions of the compounds complexed with the PTK with similar chemical structures from a data base using a compound construction computer program, where the representations of the compounds are defined by structural coordinates. The term "operating" as used herein refers to utilizing the three-dimensional conformation of molecules defined by the processes described herein in various computer programs.

The term "similar compound" refers to a compound in a computer data base that has a similar geometric structure as compounds that can bind to a PTK. The similar compound can also have similar chemical groups as the compounds that are either bound to the PTK or once bound to the PTK. The similar chemical groups can form complementary interactions with the PTK. The term "compound searching computer program" describes a computer program that searches computer representations of compounds from a computer data base that have similar three dimensional structures and similar chemical groups as a compound of interest. The compound of interest is preferably an indolinone compound.

The term "similar chemical structures" refers to chemical groups that share similar geometry as portions of the compounds in complex with the PTK or compounds removed from the PTK structure. Similar chemical structures can also refer to chemical groups that may form similar complementary interactions as portions of the compounds in complex with the PTK or compounds removed from the PTK structure. The term "replacing structures" refers to removing a portion of the compounds in complex with the PTK or compounds removed from the PTK structure and connecting the broken bonds to a similar chemical structure.

The term "compound construction computer program" describes a computer program that replaces computer representations of chemical groups in a compound with groups from a computer data base. The compound is preferably an indolinone compound.

The term "similar three dimensional structure" describes two molecules with nearly identical shape and volume .

In another preferred embodiment of the invention, the PTK structures used in the modulator design or identification method of the invention are defined by the structural coordinates of Table 1, Table 2, Table 3, or Table 4.

The methods for using the crystalline forms and three dimensional structures of the invention can relate to a broad range of protein kinases. Thus, in preferred embodiments, the invention relates to a receptor PTK. The receptor PTK can be selected form the group consisting of FGF-R, PDGF-R, FLK, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, R0R1 , and MUSK. The PTK may also exist as a non-receptor PTK. The non- receptor PTK can be selected from the group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and

ACK.

In another aspect, the invention features a potential modulator of PTK function identified by methods disclosed in the invention. A preferred embodiment of the invention is that the potential modulator of PTK function is an oxindolinone or a thiolindolinone of formula I or II disclosed above.

Another aspect of the invention is a method for synthesizing a potential modulator of PTK 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.

In another aspect, the invention features a method of identifying a potential modulator of PTK function as a modulator of PTK function. The method comprises the steps of: (a) administering a potential modulator of PTK function to cells; (b) comparing the level of PTK phosphorylation between cells not administered the potential modulator and cells administered the potential modulator; and (c) identifying the potential modulator as a modulator of PTK function based on the difference in the level of PTK phosphorylation.

The term "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. The term "administer" refers to a method of delivering a compound to cells. The compound can be prepared using a carrier such as dimethyl sulfoxide (DMSO) in an aqueous solution. 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.

The term "suitable buffered solution" refers to an aqueous preparation of the compound that comprises a salt that can control the pH of the solution at low concentrations. Because the salt exists at low concentrations, the salt preferably does not alter the function of the cells.

The term "PTK phosphorylation" refers to the presence of phosphate on the PTK. Phosphates on PTKs can be identified by antibodies that bind them specifically with high affinity.

In another aspect, the invention features a method of identifying a potential modulator of PTK function as a modulator of PTK function. The method comprises the steps of: (a) administering a potential modulator of PTK 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 PTK function based on the difference in cell growth.

The term "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 of the invention features a method of diagnosing a disease by identifying cells harboring a PTK with inappropriate activity. The method comprises the steps of: (a) administering a modulator of PTK 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 PTK with inappropriate activity from the effect of the modulator on the difference in the rate of cell growth. The modulator can be identified by the methods of the invention .

The term "inappropriate activity" refers to a PTK 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 a receptor PTK by a growth factor, alterations in the activity of PTK-specific phosphatase, over-expression of a PTK in a cell, or mutations in the catalytic region of the PTK itself.

The term "signal transduction process" describes the steps in a cascade of events where an extracellular signal is transmitted into an intracellular signal.

The term "PTK-specific phosphatase" describes an enzyme that dephosphorylates a particular PTK and thereby regulates that PTK's activity.

Another aspect of the invention is a method of treating a disease associated with a PTK with inappropriate activity in a cellular organism, where the method comprises the steps of: (a) administering the modulator of PTK function to the organism, where the modulator is in an acceptable pharmaceutical preparation; and (b) activating or inhibiting the PTK function to treat the disease. The term "organism" 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.

The term "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 of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, and injection applications. For cells outside of the patient, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.

The term "pharmaceutically acceptable composition" refers to a preparation comprising the modulator of PTK activity. The composition is acceptable if it does not appreciably cause irritations to the organism administered the compound.

Preferred embodiments of the of the invention are that the PTK is a receptor PTK selected from the group consisting of FGF-R, PDGF-R, FLK-1, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , and MUSK. Other preferred embodiments of the invention are that the PTK is a non-receptor PTK selected from the group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

The summary of the invention described above is non- limiting and other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 provides a ribbon diagram of the structure of FGFR1 showing the side chains of tyrosines Tyr-653 and Tyr-654 and the helical ( C, αD, E, EF, αF-αl), β strand (βl-β5, β7, β8), nucleotide-binding loop, catalytic loop, activation loop and kinase insert regions of the molecule. The termini are denoted by N and C. The loop between β2 and β3 is disordered, indicated by a break in the chain in this region.

FIG. 2 provides a stereo view of a C_α trace of FGFRl shown in the same orientation as FIG. 1, with every tenth amino acid residue marked with a filled circle and every twentieth amino acid residue labeled with a residue number.

FIG. 3 provides a structure-based sequence alignment of human fibroblast growth factor receptor 1 (FGFRl), human fibroblast growth factor receptor 2 (FGFR2), human fibroblast growth factor receptor 3 (FGFR3), human fibroblast growth factor receptor 4 (FGFR4) , a D. malanogaster homolog (DFGFR1), a C. elegans homolog (EGL-15) and insulin receptor tyrosine kinase (IRK) . FIGS. 4A and 4B provide ribbon diagrams of the

N-terminal lobes (4A) and C-terminal lobes (4B) of FGFRl and IRK in which the C_α atoms of the β sheets (4A) or α- helices (4B) of the two proteins have been superimposed. FIG. 5 illustrates the side-chain positions of the tyrosine autophosphorylation sites of FGFRl on the backbone representation of FGFRl.

FIGS. 6A and 6B are amino acid sequence alignments of the catalytic domains of PTKs, including receptor and non-receptor type PTKs. FIG. 6A depicts one representative member from each of the eighteen subfamilies of receptor tyrosine kinases. FIG. 6B depicts one representative member from each of the subfamilies of cytoplasmic tyrosine kinases. In FIGS. 6A and 6B highly conserved residues are boxed. The position of the glycine-rich domain, kinase insert, catalytic loop, and activation loop are indicated. The numbering is for human FGF-receptor .

BRIEF DESCRIPTION OF THE CRYSTALLOGRAPHIC ATOMIC STRUCTURAL COORDINATES The crystallographic structural coordinates are located at the end of the section entitled "Examples" and before the claims. Three sets of coordinates can be found in the Protein Data Bank under accession names 1FGK, 1AGW, and 1FGI . The 1FGK coordinates correspond to those listed in Table 1, the 1AGW coordinates correspond to those listed in Table 4, and the 1FGI coodinates correspond to those listed in Table 3. The 1AGW and 1FGI coordinate sets will be publically available in March 1998. Table 1 provides the atomic structure coordinates of native FGFRl crystals of the invention as determined by X-ray crystallography; and

Table 2 provides the atomic structure coordinates of FGFRl :AMP-PCP co-crystals of the invention as determined by X-ray crystallography.

Table 3 lists crystallographic coordinates defining the three dimensional structure of FGF-Rl complexed with 3- [ (3- (2-carboxyethyl) -4 -methylpyrrol- 5 -yl) methylene] -2- indolinone. The columns (from left to right) are descriptions of the atoms by number and type, amino acid and number containing the atom, the x coordinate, y coordinate, z coordinate, bond connectivity, and temperature factor. All of these parameters are well defined in the art.

Table 4 is a file of crystallographic coordinates defining the three dimensional structure of FGF-Rl complexed with 3 - [4 - (4 -formylpiperazine-1-yl) benzylidenyl] -2 -indolinone . The columns are as described in Table 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and identification of modulators of protein tyrosine kinase function that are PTK subfamily specific, non- hydrolyzable under acidic conditions, and highly bioavailable. The three dimensional structures of a PTK optionally complexed with compounds can facilitate design and identification of modulators of PTK function.

Protein tyrosine kinases (PTKs) comprise a large and diverse class of enzymes. Schlessinger and Ullrich, 1992, Neuron 9 : 383-391. The PTK family is subdivided into members that are receptors and those that are non- receptors. The PTK receptor family contains multiple subfamilies, one of which is the fibroblast growth factor receptor (FGF-R) PTK which is a molecule implicated in regulating angiogenesis a well as cellular proliferation and differentiation. Givol and Yayon, 1992, FASEB J. 6 (15) : 3362-3369.

FGF-Rl can mediates cellular functions by its role in one or more cellular signal transduction processes. Cellular signal transduction processes comprise multiple steps that convert an extracellular signal into an intracellular signal.

Receptor PTK mediated signal transduction is initiated by binding a specific extracellular ligand, followed by receptor dimerization, and subsequent autophosphorylation of the receptor PTK. 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 .

Receptor PTKs function as binding sites for several intracellular proteins , Intracellular PTK 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 eϋ al . , 1993, Cell 72:767-778. SH2 ( src homology) domains are common adaptors found in proteins which directly bind to the receptor PTK. SH2 domains are harbored by PTK binding proteins of both groups mentioned above. Fantl eϋ al . , 1992, Cell 69 :413 -423 ; Songyang et al . , 1994, Mol . Cell . Biol . 14:2777-2785); Songyang et al . , 1993, Cell 72:767-778; and Koch et al . , 1991, Science 252:668-678.

The specificity of the interactions between receptor PTKs 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 72:767-778. These observations suggest that the function of each receptor PTK 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, PTKs provide a controlling regulatory role in signal transduction processes as a consequence of autophosphoryla ion .

PTK-mediated signal transduction regulates cell proliferative , differentiation, and metabolic responses in cells. Therefore, inappropriate PTK activity can result in a wide array of disorders and diseases. These disorders, which are described below, may be treated by the modulators of PTK function designed or identified by the methods disclosed herein.

The present invention also relates to crystalline polypeptides corresponding to the catalytic domain of receptor tyrosine kinases . Such tyrosine kinases include receptors of a class that are not covalently cross -linked but are understood to undergo ligand- induced dimerization, as well as cytoplasmic tyrosine kinases. Preferably, the crystalline catalytic 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 2.5 A. The invention also relates to methods for preparing and crystallizing the polypeptides. The 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 catalytic domain.

The polypeptides of the invention are designed on the basis of the structure of a region in the cytoplasmic domain of the receptor tyrosine kinase that contains the catalytic domain. By way of illustration, FIG. 6A shows the amino acid sequence alignment of the catalytic domains of eighteen human receptor tyrosine kinases; one representative member from each of the eighteen subfamilies is shown. FIG. 6B shows the alignment for cytoplasmic kinases. The applicants have discovered and determined the boundaries of the domain required for crystallization of the resulting polypeptide. Surprisingly, these boundaries differ from that required for catalytic activity. For example, referring to FIG. 6A, the domain required for catalytic activity is generally believed to span about 7 amino acid residues upstream of the first glycine (FIG. 6A residue number 485) of the N-terminal glycine-rich region through about 10 residues beyond the C-terminal conserved arginine (FIG. 6A, residue number 744) . However, the additional sequence upstream of the N- terminal glycine-rich region and downstream of the C- terminal conserved arginine can be required for crystallization. In particular, at least about 20 amino acid residues (+/- 5 amino acid residues) upstream of the first glycine (i.e.. FIG. 6A, residue number 485) in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues (+/- 5 amino acid residues) downstream of the conserved arginine (i.e. , FIG. 6A, residue number 744) located at the C- terminal boundary of the catalytic domain can be required to engineer a polypeptide suitable for crystallization. In those situations where the resulting polypeptide contains cysteine residues that interfere with crystallization (e.g. , cysteine residue numbers 488 and 584 in the FGF-Rl sequence shown in FIG. 6A) , such cysteine residues can be substituted with an appropriate amino acid that does not readily form covalent bonds with other amino acid residues under crystallization conditions; e.g., by substituting the cysteine with Ala, Ser or Gly. Any cysteine located in a non-helical or non-β- stranded segment, based on secondary structure assignments, are good candidates for replacement. For example, cysteines located in regions corresponding to the glycine-rich-loop, the kinase insert, the juxtamembrane region or the activation loop are prime candidates for replacement. However, substitutions of cysteine residues that - are conserved among the kinases (e.g.. FIG. 6A at positions 725 and 736) are preferably avoided.

I. PTK Associated Diseases 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. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, 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 In terna tional 43 :47S-54S. PTKs are directly associated with the cell proliferative disorders described above. For example, some members of the receptor PTK family have been associated with the development of cancer. Some of these receptors, like EGFR (Tuzi et al . , 1991, Br . J. Cancer 63 : 227-233 ; Torp et al . , 1992, APMIS 100 : 713 - 719) 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. In fact, PTK over- expression (Akbasak and Suner-Akbasak et al . , 1992, J^". Neurol . Sci . 111:119-133 ; Dickson et al . , 1992, Cancer Trea tment Res . 61:249-273 ; Korc et al . , 1992, J. Clin . Inves t . 50:1352-1360) and autocrine loop stimulation (Lee and Donoghue , 1992, J. Cel l . Biol . 118 : 1057 - 1070 ; Korc et al . , supra ; Akbasak and Suner-Akbasak et al . , supra) account for the most common and severe cancers. For example, 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. PDGF-R is associated with glioblastoma, lung, ovarian, melanoma and prostate cancer. The receptor PTK c-met is generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, 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 of the c-met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cell line. The IGF- I receptor PTK, in addition to being implicated in nutritional support and in type-II diabetes, is also associated with several types of cancers. For example, 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 . Inves t . 54:1418-1423) and small lung tumor cells (Macauley et al . , 1990, Cancer Res . 50:2511- 2517). In addition, IGF- I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al . , 1993, Cancer Res . 53:2475- 2478. The importance of the IGF- IR and its modulators in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts , epithelial cells, smooth muscle cells, T- lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow) are stimulated to grow by IGF- I. Goldring and Goldring, 1991, Eukaryotic Gene Expression 1:301- 326. In a series of recent publications suggest that 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 75:927-930; Coppola et al . , 1994, Mol . Cell . Biol . 14:4588-4595.

The association between abnormalities in receptor PTKs and disease are not restricted to cancer, however. For example, receptor PTKs are associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. EGF-R is indicated in corneal and dermal wound healing. Defects in Insulm-R and IGF-IR are indicated type- II diabetes mellitus. A more complete correlation between specific receptor PTKs and their therapeutic indications is set forth in Plowman et al . , 1994, DN&P 7.334-339 Non-receptor PTKs, including src, abl, fps , yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al . , 1992, FASEB J. 6 : 3403-3409 ) , are involved in the proliferative and metabolic signal transduction pathways also associated with receptor PTKs. Therefore, the present invention is also directed towards designing modulators against this class of PTKs For example, mutated src (v-src) is an oncoprotein (pp60^{v &rc}) m chicken. Moreover, its cellular homolog, the proto- oncogene pp60^c~src transmits oncoge ic signals of many receptors. For example, over-expression of EGF-R or

HER2/neu m tumors leads to the constitutive activation of pp60^c~src, which is characteristic of the malignant cell but absent in the normal cell. On the other hand, mice deficient for the expression of c-src exhibLt an osteopetrotic phenotype, indicating a key participation of c-src osteoclast function and a possible involvement related disorders. Similarly, Zap 70 s implicated in T-cell signaling. Both receptor PTKs and non-receptor PTKs are connected to hyperimmune disorders.

The instant invention is directed in part towards designing modulators of PTK function that could indirectly kill tumors by cutting off their source of sustenance. Normal vasculogenesis and angiogenesis play important roles m 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. Folk an and Shing, 1992, J. Bi ologi cal Chem. 267 : 10931-34. However, 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. Folkman, 1987, in: Congress of

Thrombosis and Haemostasis (Verstraete, et. al , eds . ) , Leuven University Press, Leuven, pp.583-596. Ocular neovascularization is the most common cause of blindness and dominates approximately twenty (20) eye diseases. Moreover, 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. Furthermore, 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. Folkman, 1990, J. Natl . Cancer Inst . 82 : 4. - 6 ; Klagsbrunn and Soker, 1993, Current Biology 3:699-702; Folkman, 1991, J. Na tl . , Cancer Inst . 82:4-6; Weidner et al . , 1991, New Engl . J. Med . 324 : 1 - 5 .

Several polypeptides with in vi tro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (αFGF, βFGF) , vascular endothelial growth factor (VEGF) and placental growth factor. Unlike αFGF and βFGF, VEGF has recently been reported to be an endothelial cell specific mitogen. Ferrara and Henzel, 1989, Biochem. Biophys . Res . Comm . 161 : 851 - 858 ; Vaisman et al . , 1990, J. Biol . Chem . 265 :19461-19566.

Thus, 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, discussed in the U.S. Patent Application No. 08/193,829. In addition, a receptor that binds αFGF and βFGF is identified. Jaye et al . , 1992, Biochem . Biophys . Acta 1135:185-199). Consequently, these receptor PTKs 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 . 55:115-165. Four mammalian FGFR (FGFR1-4) have been described and additional diversity is generated by alternative RNA splicing withm the extracellular domains. Jaye et al . , 1992, Biochem . Biophys . Acta 1135 : 185 - 199 . Like other receptor PTKs, 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 (Schlessmger et al . , 1995, Cell 83 : 357 - 360) , which leads to autophosphorylation of specific tyrosine residues the cytoplasmic domain. Mohammadi et al., 1996, Mol . Cell Biol . 16:977-989.

Mutations in three human FGF receptor genes, FGFRl, FGFR2, and FGFR3 , have been implicated in a variety of human genetic skeletal disorders. Mutations in FGFRl and FGFR2 result in the premature fusion of the flat bones of the skull and cause the craniosynostosis syndromes, such as Apert (FGFR2) (Wilkie et al . , 1994, Na t . Genet. 8:269-274), Pfeiffer (FGFRl and FGFR2 ) (Muenke et al . , 1994, Nat. Genet. 8:269-274), Jackson-Weiss (FGFR2) (Jabs et al . , 1994, Na t . Genet. 8:275-279) and Crouzon (FGFR2) (Jabs et al . , 1994, Na t . Genet . 8:275-279) syndromes. In contrast mutations in FGFR3 are implicated in long bone disorders and cause several clinically related forms of dwarfism including achondroplasia (Shiang et al . , 1994, Cell 78:335-342), hypochondroplasia (Bellus et al . , 1995, Na t . Genet. 10:357-359) and the neonatal lethal thanatophoric dysplasia (Tavormina et al., 1995, Na t . Gene t . 9 : 321 - 328) . It has been shown that these mutations lead to constitutive activation of the tyrosine kinase activity of FGFR3 (Webster et al . , 1996, EMBO J. 15:520-527). Furthermore gene-targeting experiments in mice have revealed an essential role for FGFR3 in developmental bone formation (Deng et al . , 1996, Cell 84:911-921).

Another major role proposed for FGFs in vivo is the induction of angiogenesis (Folkman and Klagsbrun, 1987, Science 236:442) . Therefore, inappropriate expression of FGFs or of their receptors or aberrant function of the tyrosine kinase activity could contribute to several human angiogenic pathologies such as diabetic retinopathy, rheumatoid arthritis, atherosclerosis and tumor neovascularization (Klagsbrun and Edelman, 1989, Arterioscl erosis 5:269) . Moreover, FGFs are thought to be involved in malignant transformation. Indeed, the genes coding for the three FGF homologues int-2, FGF-5 and hst-l/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 6:659-663). Over-expression of FGF receptors has been also detected in human pancreatic cancers, astrocytomas, salivary gland adenosarco as , Kaposi sarcomas, ovarian cancers and prostate cancers. Evidence, such as the disclosure set forth in copending U.S. Application Serial No. 08/193,829, strongly suggests that 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 . 267:26031-26037. 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, Na ture 355:845-848; Shweiki et al . , 1992, Na ture 355:843-845.

The invention is directed to designing and identifying modulators of receptor and non-receptor PTK functions that could modify the inappropriate activity of a PTK involved with a clinical disorder. The rational design and identification of modulators of PTK functions can be accomplished by utilizing the structural coordinates that define a PTK three dimensional structure. II . Modulators of PTK functions as Therapeutics for Disease As a consequence of the disorders discussed above, scientists in the biomedical community are searching for modulators of PTK functions that down-regulate signal transduction pathways associated with inappropriate PTK activity .

In particular, small molecule modulators of PTK functions are sought as some can traverse the cell membrane and do not hydrolyze in acidic environments. Some compounds have already been discovered. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642) , vinylene-azaindole derivatives (PCT WO 94/14808) 1-cyclopropyl -4 -pyridyl - quinolones (U.S. Patent No. 5,330,992), styryl compounds (U.S. Patent No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al) , seleoindoles and selenides (PCT WO 94/03427) , tricyclic polyhydroxylic compounds (PCT WO 92/21660) , and benzylphosphonic acid compounds (PCT WO 91/15495) are described as PTK inhibitors.

Although some modulators of PTK function are known, many of these are not specific for PTK subfamilies and will therefore cause multiple side-effects as therapeutics. Compounds of the oxindolinone/ thiolindolinone family, however, are 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," Attorney Docket No. 221/187). In addition, compounds of the oxindolinone/thiolindolinone family are non-hydrolyzable in acidic conditions and can be highly bioavailable . The invention provides information regarding the specific interactions between a PTK and compounds of the oxindolinone/thiolindolinone family. Although the use of X-ray crystallography has provided three dimensional structures of other PTKs, the PTKs in these structures are not complexed with PTK subfamily specific, hydrolysis resistant, highly bioavailable small molecules. The X-ray crystallography techniques used in the current invention resolve interactions between a PTK and compounds in complex with it at the atomic level, which provides detailed information regarding the orientation of chemical groups defining an effective modulator of PTK function.

Ill . Crystalline Tyrosine Kinases Crystalline PTKs of the invention include native crystals, derivative crystals and co-crystals. The native crystals of the invention generally comprise substantially pure polypeptides corresponding to the tyrosine kinase domain in crystalline form. It is to be understood that the crystalline tyrosine kinase domains of the invention are not limited to naturally occurring or native tyrosine kinase domains. Indeed, the crystals of the invention include mutants of native tyrosine kinase domains. Mutants of native tyrosine kinase domains are obtained by replacing at least one amino acid residue in a native tyrosine kinase 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 of the native polypeptide, and have substantially the same three- dimensional structure as the native tyrosine kinase domain from which the mutant is derived.

By having substantially the same three-dimensional structure is meant having a set of atomic structure coordinates that have a root -mean-square deviation of less than or equal to about 2A when superimposed with the atomic structure coordinates of the native tyrosine kinase domain from which the mutant is derived when at least about 50% to 100% of the Cα atoms of the native tyrosine kinase domain are included in the superposition.

Amino acid substitutions, deletions and additions which do not significantly interfere with the three- dimensional structure of the tyrosine kinase domain will depend, in part, on the region of the tyrosine kinase domain where the substitution, addition or deletion occurs. In highly variable regions of the molecule, such as those shown in FIG. 6, non-conservative substitutions as well as conservative substitutions may be tolerated without significantly disrupting the three- dimensional structure of the molecule. In highly conserved regions, or regions containing significant secondary structure, such as those regions shown in FIG. 6, conservative amino acid substitutions are 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 of the amino acid residues involved. For example, 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.

For tyrosine kinase domains obtained in whole or in part by chemical synthesis, the selection of amino acids available for substitution or addition is not limited co the genetically encoded amino acids. Indeed, the mutants described herein may contain non-genetically encoded amino acids. Conservative amino acid substitutions for many of the 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 of the genetically encoded amino acids .

In some instances, it may be particularly advantageous or convenient to substitute, delete and/or add amino acid residues to a native tyrosine kinase domain in order to provide convenient cloning sites in cDNA encoding the polypeptide, to aid in purification of the polypeptide, and for crystallization of the polypeptide. 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 PTK activity. Indeed, amino acid substitutions, additions or deletions that interfere with the kinase activity of the tyrosine kinase domain but which do not significantly alter the three-dimensional structure of the domain are specifically contemplated by the invention. Such crystalline polypeptides, or the atomic structure coordinates obtained therefrom, can be used to identify compounds that bind to the native domain. These compounds may affect the activity or the native domain. The derivative crystals of the invention generally comprise a crystalline tyrosine kinase 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 of the invention generally comprise a crystalline tyrosine kinase domain polypeptide in association with one or more compounds. The association may be covalent or non-covalent . Such compounds include, but are not limited to, cofactors, substrates, substrate analogues, inhibitors, allosteric effectors, etc. IV. Three Dimensional Structure Determination Using X- ray Crystallography 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:

(a) synthesizing and isolating a polypeptide;

(b) growing a crystal from an aqueous solution comprising the polypeptide with or without a modulator; and

(c) collecting X-ray diffraction patterns from the crystals, determining unit cell dimensions and symmetry, determining electron density, fitting the amino acid sequence of the polypeptide to the electron density, and refining the structure.

Production of Polypeptides

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) . Alternatively, 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 vi tro 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 tyrosine kinase 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 tyrosine kinase domain coding sequence; yeast transformed with recombinant yeast expression vectors containing the tyrosine kinase domain coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g.. baculovirus) containing the tyrosine kinase domain coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.. Ti plasmid) containing the tyrosine kinase domain coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities.

Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, 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; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g. , the 35S RNA promoter of CaMV; the coat protein promoter of TMV) 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; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the tyrosine kinase 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 PCT publication WO 96/18738. This publication is incorporated herein by reference in its entirety, including any drawings. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it . Crystal Growth

Crystals are grown from an aqueous solution 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 . 189:1-23; Webber, 1991, Adv. Protein Chem . 41 : 1 - 36 , incorporated by reference herein in its entirety, including all figures, tables, and drawings.

Generally, the native crystals of the invention are grown by adding precipitants to the concentrated solution of the polypeptide corresponding to the PTK catalytic domain. 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.

For crystals of the invention, it has been found that hanging drops containing about 2.0 μL of tyrosine kinase domain polypeptide (10 mg/mL in lOmM Tris-HCl, pH 8.0, 10 mM NaCl and 2 mM dithiothreitol) and 2.0 μL reservoir solution (16% w/v polyethylene glycol MW 10000, 0.3 M (NH₄)₂S0₄, 5% v/v ethylene glycol or glycerol and 100 mM bis-Tris, pH 6.5) suspended over 0.5 mL reservoir buffer for about 3-4 weeks at 4°C provide crystals suitable for high resolution X-ray structure determina ion .

Those of ordinary skill in the art will recognize that the above-described 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 60 mg/mL, Tris-HCl concentrations between about 10 mM and about 200 M, dithiothreitol concentrations between about 0 M and about 20 mM, pH ranges between about 5.5 and about 7.5; and reservoir solutions containing polyethylene glycol concentrations between about 10% and about 30% (w/v), polyethylene glycol molecular weights between about 1000 and about 20,000, (NH₄)₂S0₄ concentrations between about 0.1 M and about 0.5 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 7.5 and temperature ranges between about 0° C and about 25°C. Other buffer solutions may be used such as HEPES buffer, so long as the desired pH range is maintained.

Derivative crystals of the 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 M 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 of the tyrosine kinase domain polypeptide .

Co-crystals of the invention can be obtained by soaking a native crystal in mother liquor containing compound that bind the kinase domain, or described above, or can be obtained by co-crystallizmg the kinase domain polypeptide in the presence of one or more binding compounds .

For co-crystals of tyrosine kinase domain polypeptide in co-complex with AMP-PCP, it has been found that co-crystallizing the kinase domain polypeptide in the presence of AMP-PCP using the above- described crystallization conditions for obtaining native crystals with a polypeptide solution additionally containing 10 mM AMP-PCP and 20 mM MgCl₂ yields co- crystals suitable for the high resolution structure determination by X-ray crystallography. Of course, those having skill in the art will recognize that the concentrations of AMP-PCP and MgCl₂ in the polypeptide solution can be varied, alone or in combination with the variations described above for native crystals. Such variations include polypeptide solutions containing AMP- PCP concentrations between 0.1 mM and 50 mM and MgCl₂ concentrations between 0 mM and 50 mM.

Crystals comprising a polypeptide corresponding to a PTK catalytic domain complexed with a compound can be grown by one of two methods. In the first method, the modulator is added to the aqueous solution containing the polypeptide corresponding to the PTK catalytic domain before the crystal is grown. In the second method, the modulator is soaked into an already existing crystal of a polypeptide corresponding to a PTK catalytic domain. Crystalline FGFR

In one illustrative embodiment, the invention provides crystals of FGFRl. The crystals were obtained by the methods provided in the Examples. The FGFRl crystals, which may be native crystals, derivative crystals or co-crystals, have monoclinic unit cells (i.e.. unit cells wherein a/b^c; α=γ=90°; and β>90°) and space group symmetry C2. There are two FGFRl molecules in the asymmetric unit, related by an approximate two- fold axis.

Two forms of crystalline FGFRl were obtained. In one form (designated "C2-A form"), the unit cell has dimensions of a=208.3 A, b=57.2 A, c=65.5 A and β=107.2°. In another form (designated "C2-B form"), the unit cell has dimensions of a=211.6 A, b=51.3 A, c=66.1 A and β=107.7°.

Three distinct two-fold related FGFRl dimers are observed in both the C2-A and C2-B forms of the FGFRl crystal, one non-crystallographically related dimer and two crystallographically related dimers . The non- crystallographically related dimer comprises the two molecules in the asymmetric unit. The residues making up the dimer interface are located in C-terminal lobe. In this dimer, the C-terminal lobes abut with the N- terminal lobes distal to one another. The total amount of surface area buried in the surface is about 950 A². Very few of the interactions in the interface are of a specific nature, e.g., hydrogen-bonding or close packing of hydrophobic residues. There are two crystallographically-related dimers in the C2 lattice. In the first dimer, the residues that constitute the dimer interface are limited to those in the β-sheet of the N-terminal lobe (amino acid residues 477, 479, 498, 506, 508 and 496) . The total surface area buried in this interface is about 670 A². The interactions are rather specific. Three hydrophobic residues which are partially solvent -exposed in the monomer, Val-479, Ile-498 and Val-508, come together with their two-fold-related residues to form a compact hydrophobic plug. This plug is capped on either side by a salt bridge between Arg-477 and Glu-496. In addition, two main-chain hydrogen-bonds connect the β-sheets of the two monomers at the start of β3 (amino acid residues 506 and 508) . The residues in this dimer interface, or their residue character, are generally conserved in the mammalian FGF receptors, but not in the invertebrate homologues .

The other crystallographically-related dimer buries about 1650 A² in its interface. In this dimer, the αC helices of the two monomers are nearly parallel and contact each other at their C-terminal ends. Met-534 and Met-537 are in van der Waals contact with their twofold-related residues. Other hydrophobic contacts involve Pro-466 with Ile-648 and Pro-469 with Ile-676 and Thr-678. In addition, hydrogen bonds (side-chain to main-chain) are made between Arg-470 and Lys-618 and between His-649 and Glu-464, and there are several water molecules that bridge the two monomers through hydrogen bonding.

In the C2-B form of the crystal, the monomers of this second crystallographically-related dimer are shifted slightly with respect to one another (6° rotation) , indicating that this interface is somewhat fluid.

In both of the crystallographically-related dimers, the N-termmi of the two molecules comprising the dimer point the same direction and are reasonably close to one another.

Determining Unit Cell Dimensions and the Three Dimensional Structure of a Polypeptide or Polypeptide Complex

Once the crystal is grown, it 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. Methods for obtaining the three dimensional structure of the crystalline form of a peptide molecule or molecule complex are well known in the art Ducruix and Geige, 1992, IRL Press, Oxford, England, and references cited therein. The following are steps in the process of determining the three dimensional structure of a molecule or complex from X-ray diffraction data. After the X-ray diffraction patterns are collected from the crystal, 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 Angstroms (one A = 10^"10 meters) and by angles at each vertices. The symmetry of the unit cell in the crystals is also characterized at this stage. The symmetry of the unit cell in the crystal simplifies the complexity of the collected data by identifying repeating patterns. Application of the symmetry and dimensions of the unit cell is described below. Each diffraction pattern emission is characterized as a vector and the data collected at this stage of the method determines the amplitude of each vector. The phases of the 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 of the vectors can be determined by using these heavy atoms as reference points in the X-ray analysis. Otwinowski, 1991, Daresbury, United Kingdom, 80-86. The isomorphous replacement method usually requires more than one heavy atom derivative. In another method, the amplitudes and phases of vectors from a crystalline polypeptide with an already determined structure can be applied to the amplitudes of the 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 11:281-296. Thus, the vector information from a PTK of known structure, such as those reported herein, are useful for the molecular replacement analysis of another PTK with unknown structure .

Once the phases of the vectors describing the unit cell of a crystal are determined, 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 of the molecule complexes in the unit cell can be referred to as an electron density map. The amino acid structures of the sequence or the molecular structures of compounds complexed with the crystalline polypeptide may then fit to the electron density using a variety of computer programs. This step of the 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 115 : 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. Br nger, 1992, Na ture 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 of the atoms can be altered by simulating a rise in temperature which will increase the vibrational frequency of the bonds and modify positions of atoms in the structure. At a particular point in the atomic perturbation process, 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 of the 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 Table 1, Table 2, Table 3, and Table 4. V. Structures of FGFRl

The present invention provides high-resolution three-dimensional structures and atomic structure coordinates of crystalline FGFRl and crystalline FGFRl :AMP-PCP co-complex as determined by X-ray crystallography. The specific methods used to obtain the structure coordinates are provided in the examples. The atomic structure coordinates of crystalline FGFRl, obtained from the C2-A form of the crystal to 2.0 A resolution, are listed in Table 3; the coordinates of crystalline FGFRl :AMP-PCP co-complex, obtained from the C2-A form of the crystal to 2.3 A resolution are listed in Table 4. Those having skill in the art will recognize that atomic structure coordinates as determined by X-ray crystallography are not without error. Thus, it is to be understood that any set of structure coordinates obtained for crystals of FGFRl, whether native crystals, derivative crystals or co-crystals, that have a root mean square deviation ("r.m.s.d.") of less than or equal to about 1.5 A when superimposed, using backbone atoms (N, C_α C and 0) , on the structure coordinates listed in Table 3 or Table 4 are considered to be identical with the structure coordinates listed in the Tables when at least about 50% to 100% of the backbone atoms of FGFRl are included in the superposition.

Referring now to FIG. 1, the overall structure of FGFRl is bi-lobate. The N- terminal lobe of FGFRl spans amino acid residues 456-567 (FIG. 3) and comprises a curled β-sheet of five anti-parallel strands (βl-β5) and one α-helix (αC) . The C-terminal lobe spans amino acid residues 568-765 (FIG. 3) and comprises two β-strands (β7, β8) and seven α-helices (αD, αE, αEF, αF-αl) . The secondary structure nomenclature follows that used for IRK (Hubbard et al . , 1994) which in turn is based on the assignments for cAPK (Knighton et al . , 1991). FIG. 2 shows a stereo view of a C_α trace of FGFRl in the same orientation as FIG. 1.

A structure-based sequence alignment of the tyrosine kinase domains of human fibroblast growth factor receptor 1 (human FGFRl; labelled FGFRl), human fibroblast growth factor receptors 2, 3 and 4 (labelled FGFR2, FGFR3 and FGFR4 , respectively), a D . melanogaster homologue (labelled DFDFR1) , a C elegans homologue (labelled EGL-15) and insulin receptor kinase (labelled IRK), is shown in FIG. 3. The sequence of FGFRl, which is not shown in FIG. 3 is identical to the sequence of FGFRl except that FGFRl has the following amino acid substitutions and additions: Cys-488 → Ala, Cys-584 - Ser, Leu-457 → Val and an additional five N-terminal amino acids (Ser-Ala-Ala-Gly-Thr) . The secondary structure assignments for FGFRl and IRK were obtained using the Kabsch and Sander algorithm (Kabsch and Sander, 1983) as implemented in PROCHECK (Laskowski et al . , 1993). In the FGF receptor sequences, a period represents sequence identity to FGFRl. In the IRK sequence, residues that are identical to FGFRl are highlighted. A hyphen denotes an insertion.

The numbers under the EGL-15 sequence represent the fractional solvent accessibility (FSA2) of the residue in the FGFRl structure. The FSA ratio is the ratio of the solvent-accessible surface area of a residue in a Gly-X-Gly tripeptide compared to that in the FGFRl structure. A value of 0 represents an FSA between 0.00 and 0.09; 1 represents an FSA between 0.10 and 0.19, etc. The higher the value, the more solven -exposed the residue. An asterisk or pound sign in the FSA line indicates that the residue (asterisk) or side chain (pound sign) is not included in the atom model due to disorder. The numbers below the FSA line are the FSAs for those residues that form part of a dimer interface. The amino acid residue numbers for FGFRl, and hence FGFRl, and IRK provided in FIG. 3 are used in the discussion that follows. Significant differences in the N-terminal lobe of FGFRl as compared to IRK are found in the loops between β strands and in αC. Residues from the end of βl through the beginning of β2 (amino acid residues 485-490) form the nucleotide-binding loop, named because of its role in ATP coordination. This residue stretch contains the protein kinase-conserved GXGXXG sequence motif, where X is any amino acid. This loop is poorly ordered in one FGFRl molecule in the asymmetric unit and disordered (i.e. , not included in the atomic model) in the other FGFRl molecule in the asymmetric unit. The loop between βl and β3 is disordered in both FGFRl molecules comprising the asymmetric unit.

Referring now to FIG. 4A, which provides a ribbon diagram of the N-terminal lobes of FGFRl and IRK in which the C_α atoms of the β-sheets have been superimposed, it can be seen that in FGFRl αC is longer by one helical turn than in IRK and is oriented such that residues Lys-514 and Glu-531, which are conserved in protein kinases, form a salt bridge (represented by a black line) . While not intending to be bound by theory, this salt bridge is believed to be important for proper positioning of the conserved lysine side chain, which coordinates two phosphate oxygens of ATP. The salt bridge is observed in the structures of cAPK (Knighton et al . , 1991) and mitogen-activated protein kinase (MAPK) (Zhang et al . , 1994). Referring now to FIG. 4B, which provides a ribbon diagram of the C-terminal lobes of FGFRl and IRK in which the C_α atoms of the α-helices have been superimposed, a significant difference is found in the C-terminal helix of FGFRl when compared to IRK; helix αl of FGFRl is longer by seven residues (two helical turns) than its counterpart in IRK. The extended length of αl is presumably important in the biological functioning of FGF receptors, since the tyrosine autophosphorylation site to which an SH2 domain of PLCy binds is six residues C-terminal to this helix.

The structure of FGFRl displays an open disposition of the N- and C-terminal lobes. Despite having different sets of lattice contacts, the two FGFRl molecules in the asymmetric unit have only a 2° difference in relative lobe orientation. It appears as though the stearic interaction between residues in αC (Glu-531 and Met-534) with Phe-642 and Gly-643 of the protein kinase-conserved DFG sequence at the beginning of the activation loop accounts for the open conformation of FGFRl.

The active site of FGFRl is characterized by at least amino acid residues spanning the catalytic loop, activation loop and nucleotide binding loop. Unlike the structure of IRK, in which Tyr-1162 occupies the active site of the molecule, the active sites of both FGFRl molecules in the asymmetric unit are unoccupied. The activation loop, which regulates phosphorylation, is characterized by at least resides 640 to 663. Quite surprisingly, while the activation loops of FGFRl and IRK contain the same number of amino acid residues and share greater than 50% sequence homology, the paths of the polypeptide chains are strikingly dissimilar, diverging at Ala-640 (Gly-1149 in IRK) and reconverging at Val-664 (Val-1173 in IRK) . Tyr-653 and Tyr 564 are not bound in the active site. Instead, these residues point away from it. Tyr-653 is in van der Waals contact with several hydrophobic residues (Val-664, Leu-672 and Phe-710) and is hydrogen- bonded via its hydroxyl group to a backbone carbonyi oxygen (Leu-672) . Tyr- 654 is more solvent exposed than Tyr-653, and its only van der Waals contact is with Val- 706. Temperature factor data suggest that the activation loop is relatively mobile and adopts multiple conformations .

The catalytic loop of protein kinases lies between secondary structure elements αE and β7 and contains an invariant aspartic acid residue (Asp-623 in FGFRl) which serves as the catalytic base in the phosphotransfer reaction, abstracting the proton from the hydroxyl group of the substrate tyrosine, serine or threonine . The catalytic loop sequence of FGFRl comprises at least residues His-621 to Asn-628 (amino acid sequence HRDLAARN) , and is identical to that for IRK and most receptor and non-receptor PTKs.

In addition to the two tyrosine autophosphorylation sites in the activation loop (Tyr-653 and Tyr-654), there are four other autophosphorylation sites present in the FGFRl crystals of the invention: one in the juxtamembrane region (Tyr-463), two in the kinase insert (Tyr-583 and Tyr-585) and one in the C-terminal lobe (Tyr-730) (Mohammadi et al . , 1996). They exhibit varying degrees of conservation in mammalian FGF receptors: Tyr-463 and Tyr-585 in FGFRl and 2; Tyr-583 in FGFRl, 2 and 3; and Tyr- 730 in FGFR 1, 2, 3 and 4 (FIG. 3) .

Referring now to FIG. 5, the positions of the autophosphorylation sites are mapped onto the FGFRl structure. The juxtamembrane site (Tyr-463) and the residues N-terminal to it are disordered in one of the FGFRl molecules in the asymmetric unit . In the other molecule in the asymmetric unit Tyr-463 is involved in a lattice contact.

The kinase insert region (the region between helices αD and αE) contains autophosphorylation sites Tyr-583 and Tyr-585 and is disordered in both FGFRl molecules in the asymmetric unit of the C2-A form of the crystal. In the C2-B form, several lattice contacts partially pin down this region in one of the two FGFRl molecules in the asymmetric unit, allowing a trace of the polypeptide chain to be made. There is no well- defined secondary structure for these residues. Tyr- 730, situated in αH in the C-terminal lobe, is nearly buried and the side-chain hydroxyl group makes two hydrogen-bonds . The side chains of neighboring Me -732 and Met-733 are both buried. Therefore, phosphorylation of Tyr- 730 would presumably require prior unfolding of αH. Aside from Tyr-730, the five other autophosphorylation sites (including Tyr-653 and Tyr- 654) are found in relatively mobile segments of the FGFRl molecule . While not intending to be bound by theory, the spatial positions of the autophosphorylat on sites relative to the active site suggest that autophosphorylation occurs by a trans mechanism between two kinase domains, supporting the hypothesis that ligand- induced receptor dimerization is critical for the initiation of autophosphorylation events. The structure of crystalline FGFRl : AMP-PCP co- complex is essentially similar to that observed for crystalline FGFRl. There are no significant changes in the structure of FGFRl induced by AMP-PCP binding. In particular, binding of AMP-PCP, and by extension ATP, does not by itself promote lobe closure under the crystallization conditions used. Furthermore, complexation did not result in any noticeable changes in the conformations of the activation and nucleotide- binding loops . The crystalline FGFRl :AMP-PCP co-complex contains hydrogen bonds that are present between Nl of adenine and the amide nitrogen of Ala- 564 and between N6 of adenine and the carbonyi oxygen of Glu-562. The adenine ring is flanked on one side by Leu-484 and Val-492 (N- terminal lobe) and on the other side by Leu-630

(C-terminal lobe) . The ribose hydroxyl groups make no direct hydrogen bonds with protein atoms. Lys-514 is hydrogen-bonded to oxygens of the β- and γ-phosphates . There is no unambiguous electron density that would indicate the positions of Mg^2f ions. Generally, AMP-PCP appears to be coordinated rather loosely to unphosphorylated FGFRl, being bound to the "roof" of the cleft rather than being tightly sandwiched between the two kinase lobes .

Structural Differences Between FGF-R and IRK

Several features distinguish the FGF-receptor structure from that of the insulin-receptor tyrosine kinase. These distinctions are likely to be important in signaling by FGF-receptors, and other monomeric receptors that are believed to undergo ligand- induced dimerization.

The most significant difference between the structures of FGFRl and IRK is the conformation of the activation loop. In FGFRl, the activation loop is disposed such that the binding site for substrate peptides is blocked not by an activation loop tyrosine, as in IRK, but by Arg-661 and PTK-invariant Pro-663, while the ATP binding site is accessible. This represents another molecular mechanism by which a receptor PTK may be autoinhibited. The observed autoinhibition in FGFRl would appear to be weaker than that in IRK because of fewer specific interactions made by residues in the FGFRl activation loop (manifested in the relatively higher B-values) and the accessibility of the ATP site. One obvious distinction between the insulin and FGF receptor families is that in the former, receptors are covalently linked heterotetramers (α₂β₂) , whereas in the latter, receptor dimerization is ligand dependent . Receptors whose kinase domains are always in close proximity may require a stronger autoinhibition mechanism than those receptors that associate only upon ligand binding (Taylor et al . , 1995). Since most growth factor receptors undergo ligand-dependent dimerization and activation, the FGF receptor autoinhibition mechanism appears to be a more general one .

VI . Uses of the Crystals and Atomic Structure Coordinates The crystals of the invention, and particularly the atomic structure coordinates obtained therefrom, have a wide variety of uses. For example, the crystals described herein can be used as a starting material in any of the art -known methods of use for receptor and non-receptor tyrosine kinases. Such methods of use include, for example, identifying molecules that bind to the native or mutated catalytic domain of tyrosine kinases. The crystals and structure coordinates are particularly useful for identifying compounds that inhibit receptor and non-receptor tyrosine kinases as an approach towards developing new therapeutic agents (see . e.g.. Levitzki and Gazit, 1995) .

The structure coordinates described herein can be used as phasing models for determining the crystal structures of additional native or mutated tyrosme kinase domains, as well as the structures of co-crystals of such domains with ligands such as inhibitors, agonists, antagonists, and other molecules The structure coordinates, as well as models of the three- dimensional structures obtained therefrom, can also be used to aid the elucidation of solution-based structures of native or mutated tyrosine kinase domains, such as those obtained via NMR. Thus, the crystals and atomic structure coordinates of the invention provide a convenient means for elucidating the structures and functions of receptor and non-receptor tyrosine kinases. For purposes of clarity and discussion, the crystals of the invention will be described by reference to specific FGFRl exemplary crystals. Those skilled in the art will appreciate that the principles described herein are generally applicable to crystals of the tyrosine kinase domain of any cytoplasmic tyrosine kinase that undergoes ligand- induced dimerization or receptor tyrosine kinase, including but not limited to the tyrosine kinases of FIG. 6.

VII. Structure Determination for PTKs with Unknown Structure Using Structural Coordinates

Structural coordinates, such as those set forth in

Table 1, Table 2, Table 3, and Table 4, can be used to determine the three dimensional structures of PTKs 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 of the invention relate to determining the three dimensional structures of PTKs and related polypeptides. These include receptor PTKs such as FGF- R, PDGF-R, KDR, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , and MUSK. Non-receptor PTKs such as SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK can also be used in the methods described herein.

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 of the three dimensional structure of a polypeptide or polypeptide complex, the computer representation of amino acid sequences of the polypeptides with known and unknown structures, and standard computer representations of the structures of amino acids. Homology modeling comprises the steps of (a) aligning the amino acid sequences of the polypeptides with and without known structure; (b) transferring the coordinates of the conserved amino acids in the known structure to the corresponding amino acids of the polypeptide of unknown structure; refining the subsequent three dimensional structure; and (d) constructing structures of the rest of the polypeptide. One skilled in the art recognizes that conserved amino acids between two proteins can be determined from the sequence alignment step in step (a) .

The above method is well known to those skilled in the art. Greer, 1985, Science 228 , 1055. Blundell et a . , 1988, Eur . J. Biochem . 172 , 513. A computer program currently utilized for homology modeling by those skilled in the art is the Homology module in the Insight II modeling package distributed by Molecular Simulations Inc.

Alignment of the amino acid sequence is accomplished by first placing the computer representation of the amino acid sequence of a polypeptide with known structure above the amino acid sequence of the 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 of the polypeptides and account for amino acid insertions or deletions. Once the amino acid sequences of the polypeptides with known and unknown structures are aligned, the structures of the conserved amino acids in the computer representation of the polypeptide with known structure are transferred to the corresponding amino acids of the 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. The structures of amino acids located in non- conserved regions are to be assigned manually 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 al . , 1992, Sci ence 258 : 130 - 135 .

Structures Using Molecular Replacement

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. Brύnger, 1992, Na ture 355:472-475. AMORE is another program used for molecular replacement. Navaza, 1994, Acta Crystallogr. A50 -. 157-163. Preferably, 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 of the 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 of the X-ray diffraction data is 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. An R-factor between 30-50% indicates that the orientations of the atoms in the unit cell are reasonably determined by this method. 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 185:383-386; Kline et al . , 1986, J. Mol ecular Biology 185:377-382. 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. The knowledge of spatial interactions between secondary structural elements can greatly simplify Nuclear Overhauser Effect (NOE) data from two- dimensional NMR experiments. Additionally, applying the crystallographic coordinates after the determination of secondary structure by NMR techniques only simplifies the assignment of NOEs relating to particular amino acids in the polypeptide sequence and does not greatly bias the NMR analysis of polypeptide structure. Conversely, using the crystallographic coordinates to simplify NOE data while determining secondary structure of the polypeptide would bias the NMR analysis of protein structure.

As the analysis of polypeptide structure by NMR methods is a relatively new technique, the use of structural coordinates defining a PTK structure will most likely be utilized more frequently in the near f ture. As the method progresses, the three dimensional structure analysis of polypeptides of the same size as a PTK catalytic domain will become more frequent.

VIII. Structure-Based Design of Modulators of PTK Function Utilizing Structural Coordinates 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, Table 2, Table 3, and Table 4 can be utilized by this method. In addition, the three dimensional structures of receptor and non-receptor PTKs determined by the homology, molecular replacement, and NMR techniques described herein can also be applied to modulator design and identification methods. Thus, the structures of receptor PTKs, FGF-R, PDGF-R, FLK, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, R0R1 , and MUSK, can be utilized by the methods described herein. The structures of non-receptor PTKs, SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK, can also be utilized by the rational modulator design method.

Desiσn by Searching Molecular Data Bases

One method of rational modulator design searches for modulators by docking the computer representation of compounds from a data base of molecules. Publicly available data bases include:

ACD from Molecular Designs Limited b) NCI from National Cancer Institute c) CCDC from Cambridge Crystallographic Data Center d) CAST from Chemical Abstract Service e) Derwent from Derwent Information Limited f) Maybridge from Maybπdge Chemical Company LTD g) Aldrich from Aldrich Chemical Company h) Directory of Natural Products from Chapman & Hall

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 m the art can convert a data set represented two dimensions to one represented m 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. Kuntz et al . , 1982, J^". Mol . Biol . 162 : 269; Kuntz et al . , 1994,

Ace . Chem . Res . 27: 117; Meng et al . , 1992, J. Compt . Chem . 13 : 505; Bohm, 1994, J. Comp . Aided Mol ec . Design 8 : 623.

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 m three applications below. More detailed information regarding some of these techniques can be found the Molecular Simulations User Guide, 1995. A typical computer program used for this purpose can comprise the following steps:

(a) remove the existing compound from the protein;

(b) dock the structure of another compound into the active-site using the computer program (such as

DOCK) or by interactively moving the compound into the active-site;

(c) characterize the space between the compound and the active -site atoms; (d) 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; and

(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 of the active-site and the compounds. A favorable geometric fit is attained when a significant surface area is shared between the compound and active-site atoms without forming unfavorable steric interactions. One skilled in the art would note that 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 PTK 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 PTK function in cellular assays. The above descriptions of structure-based modulator design are not all encompassing and other methods are reported in the literature:

(1) CAVEAT: Bartlett et al . , 1989, in "Chemical and Biological Problems in Molecular Recognition", Roberts,

S.M.; Ley, S.V.; Campbell, M.M. eds . ; Royal Society of Chemistry: Cambridge, ppl82-196.

(2) FLOG: Miller et al . , 1994, J . Comp . Aided Molec . Design 8:153. (3) PRO Modulator: Clark et al . , 1995, J. Comp .

Aided Mol ec . Design 9:13.

(4) MCSS : Miranker and Karplus, 1991, Pro teins : Structure, Function, and Geneti cs 11 : 29 .

(5) AUTODOCK: Goodsell and Olson, 1990, Pro teins : Structure, Function, and Genetics 8:195.

(6) GRID: Goodford, 1985, J. Med . Chem . 28:849.

Design by Modifying Compounds in Complex with PTKs

Another way of identifying compounds as potential modulators is to modify an existing modulator in the polypeptide active-site. For example, the computer representation of modulators can be modified within the computer representation of a PTK active-site. Detailed instructions for this technique can be found in the Molecular Simulations User Manual, 1995 in LUDI . The computer representation of the modulator is modified by the deletion of a chemical group or groups or by the addition of a chemical group or groups.

Upon each modification to the compound, the atoms of the modified compound and active-site can be shifted in conformation and the distance between the modulator and the active-site 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 PTK function.

Design by Modifying the Structure of Compounds that Bind PTKs 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 . These programs can be operated on the structure of a compound that has been removed from the active-site of the three dimensional structure of a compound-PTK complex. Operating the program on such a compound is preferable since it is in a biologically active conformation.

A modulator construction computer program is a computer program that may be used to replace computer representations of chemical groups in a compound complexed with a PTK 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 compound bound to a PTK. A typical program can operate by using the following general steps:

(a) map the compounds by chemical features such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites; (b) add geometric constraints to the mapped features; and

(c) search data bases with the model generated in (b) .

Those skilled in the art recognize that for indolinones, the important chemical features include, but are not limited to, a hydrogen bond donor, a hydrogen bond acceptor, and two hydrophobic points of contact. Those skilled in the art also recognize that not all of the possible chemical features of the compound need be present in the model of (b) . One can use any subset of the model to generate different models for data base searches.

IX. Organic Synthetic Techniques

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 the enzyme with a wide variety of chemical functional groups. A consequence of this chemical diversity is that a potential modulator of PTK function may take a chemical form that is not predictable. A wide array of organic synthetic techniques exist in the art to meet the challenge of constructing these potential modulators of PTK function. Many of these organic synthetic methods are described in detail in standard reference sources utilized by those skilled in the art. One example of such a reference is March, 1994, Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, New York, McGraw Hill. Thus, the techniques required to synthesize a potential modulator of PTK function identified by computer-based methods are readily available to those skilled in the art of organic chemical synthesis .

X. Cellular Assays Measuring the Effect of a PTK Modulator in Signal Transduction Pathways

Cellular assays can be used to test the activity of a potential modulator of PTK function as well as diagnose a disease associated with inappropriate PTK activity. A potential modulator of PTK function can be tested for activity in vi tro by assays that measure the effect of a potential modulator on the autophosphorylation of a particular PTK over-expressed in a cell line. Thus, a modulator that acts as a potent inhibitor of the catalytic domain corresponding to a PTK would decrease the amount of autophosphorylation catalyzed by that PTK. Potential modulators could also be tested for activity in cell growth assays in vi tro as well as in animal model assays in vivo . In vi vo assays are also useful for testing the bioactivity of a potential modulator designed by the methods of the invention.

Materials, methods, and experimental data for these assays are fully described in WO 96/40116 published on December 19, 1996, entitled "Indolinone Compounds for the Treatment of Disease" . This application is incorporated herein by reference in its entirety, including all drawings, figures, and tables.

XI . Administration of Modulators of PTK Function as Therapeutics for Disease Methods of administering compounds to organisms as therapeutics for disease are fully described in WO 96/40116 published on December 19, 1996, entitled

"Indolinone Compounds for the Treatment of Disease" . This application is incorporated herein by reference in its entirety, including all drawings, figures, and tables .

EXAMPLES The examples below are non- limiting and are merely representative of various aspects and features of the present invention. The examples provide illustrative methods for obtaining crystalline forms of protein kinase polypeptides, methods for determining three dimensional structures of these protein kinase polypeptides, and methods for identifying modulators of protein kinases using the three dimensional structures of the protein kinases. EXAMPLE 1 : X-ray Crystallographic Structure Determination of FGFRl

Polypeptide Synthesis and Isolation A recombinant baculovirus was engineered to encode residues 456-765 of human FGFRl. A cleavable N-terminal histidine tag was incorporated to aid in protein purification. Three amino acid substitutions were introduced: Cys-488 to Ala, Cys-584 to Ser and Leu-457 to Val. The two cysteine substitutions were made to prevent the formation of disulfide- linked oligomers, which occurs for the native protein. The substitution Leu-457 to Val introduced a Ncol cloning site near Met- 456. The codon for Tyr-766 (TAC) was changed to a stop codon (TAG) and a Hin lXI-cloning site was generated following this stop codon. These substitutions were introduced into the full length human cDNA of FGFRl in ml3MPI9 by site-directed mutagenesis according to the manufacturer's protocol (Amersham) . The resulting construct was digested with Ncol and Hindlll and was ligated into appropriately digested pBlueBac HistagB ( Invitrogen) . Transfection of insect cells (Sf9) was performed with the BaculoGold transfection system according to the manufacturer's protocol (Phar ingen) . Following identification of positive plaques, the recombinant baculovirus was amplified to high titer (5xl0⁷ virus particles/ml) . Sf9 cells were grown in 175-cm² flasks to a density of 2- 3xl0^; per flask and infected with recombinant baculovirus with a multiplicity of infection (MOI) of 10.

After 48 hr , cells were harvested by centrifugation at 3,000g for 35 min at 4°C and then lysed in 25 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl_2> 1 % Triton X-100, 10 μg/ml aprotonin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF) . Lysates were centrifuged in a Sorval RC 5C (Dupont) for 1 hr at 4°C at 40,000g followed by ultracentrifugation in an XL-80 (Beckman) at 100,000g for 1 hr . After centrifugation, the clarified lysate was passed over a Ni^2t -chelating column (Pharmacia) , and the bound histidine-tagged fusion protein was eluted with 100 mM imidazole (pH 7.5) . Pooled fractions were loaded onto a Mono Q anion exchange column (Pharmacia) and eluted with a NaCl gradient from 0 to 500 M .

The fractions containing the fusion protein were concentrated in a Centricon-30 (Amicon) , and the histidine tag was removed by overnight digestion with enterokinase (Biozyme) at 20°C. The digestion was terminated by the addition of aprotonin, leupeptin, PMSF, TPCK, and bovine pancreatic trypsin inhibitor (BPTI) . The cleaved kinase domain was then separated from the histidine tag on a Superose 12 size-exclusion column (Pharmacia) . The eluted kinase domain was further purified on a Mono Q column. The purified kinase domain was analyzed by N-terminal sequencing and mass spectrometry . Five amino acids (SAAGT) remained from the histidine tag. The predicted molecular mass was confirmed by mass spectrometry.

Crystal Growth Purified FGFRl was concentrated to 20-50 mg/ml and exchanged into 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 2 mM DTT using a Centricon-30. Crystals were grown at 4°C by vapor diffusion in hanging drops containing 2.0 μl of 10 mg/ml protein solution and 2.0 μl of reservoir solution: 16% polyethylene glycol (PEG) 10000, 0.3 M (NH₄),S0, , 5% ethylene glycol, and 100 mM bis-Tris (pH 6.5) .

Crystals of native FGFRl were soaked in 500 ml stabilizing solution [25% PEG 10000, 0.3 M (NH4)₂S0₄, 0.1 M Bis-Tris (pH 6.5), 5% ethylene glycol] containing 3- [ (3 - (2-carboxyethyl) -4-methylpyrrol-5-yl) methylene] -2- indolinone (1-5 mM) or 3 - [4 - (4-formylpiperazine-1-yl ) - benzylidenyl] -2-indolinone (1 mM) at 4°C for 24 to 48 hours. The final soaking concentration of DMSO was between 1 to 5%. The crystals cracked at higher concentrations of DMSO.

Co-crystals of FGFRl with the inhibitors could also be obtained by vapor diffusion in hanging drops containing 2.0 μl of 10 mg/ml protein solution and 2.0 μl of reservoir solution containing 1 mM 3- [(3- (2- carboxyethyl) -4 -methylpyrrol-5-yl) methylene] -2- indolinone and 3- [4- (4 -formylpiperazine-1-yl- ) benzylidenyl] -2 -indolinone .

Co-crystals of FGFRl complexed with AMP-PCP were obtained as described for the creation of native crystals, except that the protein solution additionally contained 10 mM AMP-PCP and 20 mM MgCl₂.

Preparation Of Heavy Atom Derivative Crystals

Heavy atom derivative crystals were obtained by soaking FGFRl native crystals (C2-A form) in a solution containing ethylmercurithiosalicylic acid (thimerosal) , KAu(CN)₂ or 4 -chloromercuribenzoic acid, as provided in Table 1, infra , , and containing 25% PEG 10000, 0.3M (NH₄)₂S0₄ , 5% ethylene glycol or glycerol, and 100 mM bis-Tris (pH 6.5), and were flash-cooled either in liquid nitrogen directly (Synchrotron) or in a dry nitrogen stream at -175°C (rotating anode).

Data Collection and Structure Determination

For native crystals and crystals comprising the nucleotide analog AMP-PCP, data were collected either on a Rigaku RU-200 rotating anode operated at 50 kV and 100 mA (Cu Kα) and equipped with double-focusing mirrors and an R-AXIS IIC image plate detector, or at beamline X-4A at the National Synchrotron Light Source, Brookhaven National Laboratory. Synchrotron data (λ=1.07A) were collected on Fuji image plates and read with a Fuji scanner. One cryo-cooled crystal was used for each of the data sets. To obtain cryo-cooled crystals, crystals were soaked in a cryo-protectant solution containing 25% PEG 10000, 0.3 M (NH₄)₂S0₄, 5% ethylene glycol or glycerol and 100 mM bis-Tris (pH 6.5), and were flash- cooled either in liquid nitrogen directly (synchrotron data) or in a dry nitrogen stream at -175°C (rotating anode data) . All data were processed using DENZO and SCALEPACK. Otwinowski , 1993, "Oscillation data reduction program," Proceedings of the CCP4 Study Weekend, Sawyer et al . , edε . (Daresbury, United Kingdom: SERC Daresbury Laboratory), 56-62.

For native crystals and crystals comprising the nucleotide analog AMP-PCP, a molecular replacement solution was found initially for the C2-B crystal form using an IRK search model that consisted of polyalanine with the common side chains for residues 993-1263 (FGFRl residues 475-754), excluding residues 1094-1105 (kinase insert) and 1153-1170 (activation loop) . With AMORE (Navaza, 1994, AmoRe : an automated package for molecular replacement," Acta Crystallogr . A50 : 157-163), using 80% of the structure factor amplitudes between 15.0 and 3.5 A, one of the two molecules in the asymmetric unit was located. The correlation coefficient { c . c . ) for the correct 1-molecule solution was 0.23 (versus 0.20 for the highest incorrect solution) . This molecule was rigid body-refined in X-PLOR (Brunger, 1992, X-PLOR (Version 3.1) Manual (New Haven, Conneticut: The Howeard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale Uiversity) ) , first as one rigid body unit, then as two units each comprising a lobe of the kinase. Rigid body refinement (12.0-3.5 A, F>3σ) resulted in a relative rotation of the two lobes of -10° and an increase of the c.c. from 0.20 to 0.25. The rigid body-refined molecule was then used as a new search model in AMORE, and this time both molecules in the asymmetric unit were located. The c.c. for the correct 2-molecule solution was 0.35 (versus 0.27 for the highest incorrect solution) . Multiple cycles of model building and refinement against 6.0-2.4 A data resulted in the addition to the model of many of the side chains and some of the missing polypeptide chain. Model building was performed using TOM/FRODO (Jones, 1985, "Diffraction methods for biological macromolecules . Interactive computer graphics: FRODO," Methods in Enzymology 115 : 157-171) and conjugate-gradient minimization and simulated annealing were performed using X-PLOR. Brunger, supra . At this stage, the R-value was 30% (free R-value of 36%). To help expedite model building and refinement, experimental phases were obtained. Because crystals grown in the presence of ethylene glycol were easier to manipulate than those grown in glycerol, several heavy- atom derivative data sets were collected from C2-A crystals that had been soaked in various heavy atom solutions. The C2-B structure was subsequently refined against 6.0-2.4 A data to an R-value of 23.8% (free R- value of 30.4%) with r.m.s.d. values of 0.008 A for bond distances and 1.4° for bond angles.

Molecular replacement was used to locate the two FGFRl molecules (designated FLGK-A and FLGK-B) in the asymmetric unit of the C2-A crystal form. Using AMORE with 80% of structure factor amplitudes between 15.0 and 3.5 A and the C2-B model, the c.c. for the correct 2- molecule solution was 0.62 (versus 0.35 for the highest incorrect solution) . Heavy atom positions were determined from difference Fourier maps using the calculated phases from the partial model. Refinement of heavy atom parameters and phase determination were performed with MLPHARE (Otwinowski, 1991, "Maximum likelihood refinement of heavy atom parameters,"

Isomorphous replacement and ano olous Ssattering, Evans and Leslie eds . (Darsbury, United Kingdom: SERC Daresbury Laboratory) , 56-62) ) . An initial molecular isomorphous replacement (MIR) -phased electron density map was calculated with data between 2.0. and 2.8 A resolution. This map was improved by solvent flattening, histogram matching, and non-crystallographic symmetry (NCS) averaging using DM (Cowtan, 1994, "Protein Crystallography," CCP4 and ESF-EACBM Newsl etter

Refinement of the C2-A FGFRl structure against 6.0- 2.0 A data proceeded by conjugate-gradient minimization and simulated annealing using X-PLOR. Tight NCS restraints were imposed until data to 2.0 A resolution were included in the refinement, at which point the restraints were lifted. An overall anisotropic B-value was calculated using X-PLOR and applied to the observed structure factors, reducing the R-value by -3%. Water molecules whose B-values refined to ≥70 A² were omitted from the subsequent refinement round. The average B- value is 37.5 A² for all protein atoms, 35.4 A^? for protein atoms in FLGK-A, 39.7 A² for protein atoms in FLGK-B, and 40.2 A² for water molecules. The side chains for Cys-603 in FLGK-A and FLGK-B and for Met-534 in FLGK-B have been modeled in two different conformations. Residues that are not included m the atomic model due to poor supporting electron density are for FLGK-A: 456- 463, 486-490, 501-504, 580-591, 763-765; and for FLG-B: 456-460, 501-504, 578-593, 646-651, 657-659, 762-765.

The positions of the two AMP-PCP molecules (one per FGFRl molecule) were easily identified in 2F_obs(co__COT,_plex, - FcaiciFGFRD difference Fourier maps. The AMP-PCP molecule bound to FLGK-B is less tightly bound and has been modeled with an occupancy of 0.5.

Table A summarizes the X-ray crystallography data sets of FGFRl derivative crystals that were used to determine the structures of crystalline FGFRl and crystalline FGFRl : AMP-PCP co-complex of the invention

TABLE 5

Data Collection and MIR Phasing Summarv

^aThι- l , Thι-2, ethylmercuπthiosalicylic acid (thimerosal), PCMB 4-chloromercuπben7oιc acid ^bR,_%in = 100 x Σ_hΣ, | I,(h)-<l(h)> |/Σ_hΣ,I,(h)

'Value in parentheses is for the highest resolution shell ^dI(+h) and I(-h) processed as independent reflections Anomalous scattering contributions were included ^eR„„ = 100 λ ∑_h I |F-(h)±F_p(h) |- |F_P„(h) | |/Σ,,|F_p(h)| , where F_p and F_PH are the native and derivative structure factors, respectively ^rPhasιng power r m s heavy atom structuie factor / r m s lack of closure (for acentric reflections from 20 0 to 2 8A) sR^,,, = 100 \ ∑_h I |F_PH(h)|-F_J1(calc)(h) | Σ_h|Fp_H(h)±F_p(h)| (for centric reflections from 20 0 to 2 8A) ^hFιgure of merit JP(φ)exp(ιφ)dφ/ jP(φ)d(φ), where P is the probability distribution of the phase angle φ For crystals comprising FGFRl and compounds 1 and 2, data were collected on a Rigaku RU-200 rotating anode

(Cu Kα) operating at 50 kV and 100 mA and equipped with double- focusing mirrors and an R-AXIS IIC image plate detector One cryo-cooled crystal was used for each of the data sets Crystals were soaked m a cryo-protectant [25% PEG 10000, 0.3 M (NH,)₂S0₄, 5% ethylene glycol, 100 mM bis-Tris (pH 6.5), and 1 mM 3-

[ (3 - (2-carboxyethyl) -4 -methylpyrrol-5-yl) methylene] -2 - indolinone (hereafter referred to as compound 1) or 3 -

[4- (4-formylpιperazme-1-yl- ) benzylidenyl] -2-mdolmone

(hereafter referred to as compound 2) and flash-cooled in a dry nitrogen stream at -175°C. Data were processed using DENZO and SCALEPACK. Otwmowski, 1993, Proceedings of the CCP4 Study Weekend (Daresbury, United Kingdom: SERC Daresbury Laboratory) pp 56-62

A summary of the data collection parameters are included in the following Table 6 :

TABLE 6

compound 1 structure 550 residues, 252 water molecules, 2 compound 1 molecules (4589 atoms) compound 2 structure 550 residues, 248 water molecules, 2 compound 2 molecules (4646 atoms)

Structure Analyses

Atomic superpositions were performed with TOSS (Hendrickson, 1979) . Per residue solvent accessible surface calculations were done with X-PLOR The surface area buried in a dimer interface was calculated with GRASP (Nicholls et al . , 1991) using a probe radius of 1.4 A. The stereochemical quality of the atomic model was monitored using PROCHECK (Laskowski et al . , 1993, PROCHECK: a computer program to check the stereochemical quality of protein structures," J. Appl Crys t . 26 283- 291) As defined in PROCHECK, 93% of the residues in the model have main-chain torsion angles in the most favored Ramachandran regions There are no residues in disallowed regions, and three residues generously allowed regions: Arg-622 in FLGK-A and FLGK-B and Arg- 554 in FLGK-A. The overall G-factor score is 0 42. Table 7 summarizes the X-ray crystallography refinement parameters of the structures of crystalline FGFRl and crystalline FGFRl :AMP-PCP co-complex of the invention. Table 8 summarizes the X-ray crystallography refinement parameters for the FGFRl/compound complexes.

TABLE 7

Refinement Parameters

FGFRl 550 residues, 252 water molecules (4589 atoms)

FGFRl AMP-PCP 550 residues, 238 water molecules, 2 AMP-PCP molecules (4638 atoms) Model d-spacings Reflection R-value^a R ra s d s

(A) (N) (%) bonds (A) angles (°) B-values^b

(A¹)

"R-value = 100 x ∑_h | |F_obs(h)| - |F_calc(h)| | / Σ F^h)! for reflections with F_ob >2σ ^bFor bonded protein atoms. ^cValue in parentheses is the free R-value (Brunger, 1993) determined from 5% of the data.

TABLE 8

^■R,„. - 100 x S„S, |I,(h) - -I(h)°| / S_hS, I_s(h) ^cValue in parentheses is for the highest resolution shell.

'R-value = 100 x S_h ||F₀(h)| - |F_c(h)|| / S„ |F₀(h)|, where F₀ and F_c are the observed and calculated structure factors, respectively (F₀ > 2s). For bonded protein atoms. ^kValue in parentheses is the free R-value determined from 5% of the data.

Atomic Structural Coordinates

Tables 1 and 2 provide the atomic structural coordinates of unphosphorylated FGFRl and unphosphorylated FGFRl :AMP-PCP co-complex, respectively. In the Tables, coordinates for both of the FGFRl molecules of the dimer comprising the asymmetric unit are provided. The amino acid residue numbers coincide with those used in FIG. 3. In the first FGFRl molecule of the dimer the residue number is preceded by a 1 , i.e., residue number 464 of the first FGFRl molecule of the dimer is denoted by "1464". Tables 3 and 4 provide the atomic structural coordinates of FGFRl in complex with indolinone compounds found to inhibit FGFRl function . 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.

"X, Y and Z" provide the Cartesian coordinates of the element .

"B" is a thermal factor that measures movement of the 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 of the atom when in the first conformation and PRT2 designating the coordinates of the atom when in the second or alternate conformation .

Structural coordinates for FGFRl may be modified by mathematical manipulation. Such manipulations include, but are not limited to, crystallographic permutations of the raw structure coordinates, fractionalization of the raw structure coordinates, integer additions or subtractions to sets of the raw structure coordinates, inversion of the raw structure coordinates and any combination of the above.

In addition, 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 of the 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.

EXAMPLE 2 : Computer-Based Design of Modulators of

PTK Function

Potential modulators of PTK function were designed and identified by operating the program Catalyst on the structure of 3 - [ (3 - (2-carboxyethyl) -4-methylpyrrol-5- yl) methylene] -2 -indolinone . The chemical features constraining the search model include a hydrogen bond donor, a hydrogen bond acceptor, and two hydrophobic points of contact. Approximately 40 compounds were identified as potential modulators of PTK function using this method.

The compounds identified by the method as potential modulators of PTK function were commercially available. These compounds were then tested for their ability to inhibit the FLK PTK in an enzyme linked immunosorbant assay (ELISA) . The method of performing this assay is taught in WO 96/40116, entitled "Indolinone Compounds for the Treatment of Disease," published on December 19, 1996, invented by Tang et al . , incorporated by reference herein in its entirety, including all figures, drawings, and tables. Flk-1 specific antibodies can be prepared from the following protocol:

1. Prepare a Tresyl-Activated Agarose/Flk-1-D column by incubating 10 ml of Tresyl -Activated Agarose with 20 mg of purified GST-Flk-1-D fusion protein in lOOmM sodium bicarbonate (pH 9.6) buffer overnight at 4°C.

2. Wash the column once with PBS.

3. Block the excess sites on the column with 2 M glycine for 2 hours at 4°C.

4. Wash the column with PBS.

5. Incubate the column with Rabbit anti-Flk-lD production bleed for 2 hours at 4°C.

6. Wash the column with PBS. 7. Elute antiserum with 100 mM Citric Acid, pH3.0 and neutralize the eluate immediately with 2 M Tris, pH 9.0.

8. Dialyize the eluate against PBS overnight at 4oC with 3 changes of buffer (sample to buffer ratio is 1:100) .

9. Adjust the dialyized antiserum to 5% glycerol and store at -80°C in small aliquotes.

The Flk-1 ELISA can include a 2 , 2-azino-bis (3- ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution, which can comprise lOOmM citric acid (anhydrous) , 250 mM Na₂HP0₄ (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A- 1888) . The solution is most appropriately stored in dark at 4°C until ready for use. The FLK-1 specific antibodies can also be purchased from Santa Cruz Biotechnology (Catalog No. SC-504) .

Four of the forty compounds identified as potential modulators of PTK function were potent modulators of FLK function. These molecules have the following structures:

The modulators inhibit the FLK protein kinase with the following IC_5Q values:

TABLE 9

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 terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled m the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. Those references not previously incorporated herein by reference, mcluding both patent and non-patent references, are expressly incorporated herein by reference for all purposes. Other embodiments are within the following claims.

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(1) GENERAL INFORMATION:

(i) APPLICANT: SUGEN, INCORPORATED 351 Galveston Drive Redwood City, CA 94063

[ii) TITLE OF INVENTION: CRYSTAL STRUCTURES OF A PROTEIN TYROSINE KINASE

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(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

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(A) NAME: Warburg, Richard J.

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(C) TELEX: 67-3510

(2) INFORMATION FOR SEQ ID NO : 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 310 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iil) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Met Leu Ala Gly Val Ser Glu Tyr Glu Leu Pro Glu Asp Pro Arg Trp 1 5 10 15

Glu Leu Pro Arg Asp Arg Leu Val Leu Gly Lys Pro Leu Gly Glu Gly 20 25 30

Cys Phe Gly Gin Val Val Leu Ala Glu Ala lie Gly Leu Asp Lys Asp 35 40 45

Lys Pro Asn Arg Val Thr Lys Val Ala Val Lys Met Leu Lys Ser Asp 50 55 60

Ala Thr Glu Lys Asp Leu Ser Asp Leu lie Ser Glu Met Glu Met Met 65 70 75 80

Lys Met lie Gly Lys H s Lys Asn lie lie Asn Leu Leu Gly Ala Cys 85 90 95

Thr Gin Asp Gly Pro Leu Tyr Val lie Val Glu Tyr Ala Ser Lys Gly 100 105 110

Asn Leu Arg Glu Tyr Leu Gin Ala Arg Arg Pro Pro Gly Leu Glu Tyr 115 120 125

Cys Tyr Asn Pro Ser His Asn Pro Glu Glu Gin Leu Ser Ser Lys Asp 130 135 140 Leu Val Ser Cys Ala Tyr Gin Val Ala Arg Gly Met Glu Tyr Leu Ala 145 150 155 160

Ser Lys Lys Cys lie His Arg Asp Leu Ala Ala Arg Asn Val Leu Val 165 170 175

Thr Glu Asp Asn Val Met Lys lie Ala Asp Phe Gly Leu Ala Arg Asp 180 185 190 lie His His lie Asp Tyr Tyr Lys Lys Thr Thr Asn Gly Arg Leu Pro 195 200 205

Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg lie Tyr Thr His 210 215 220

Gin Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu He Phe Thr 225 230 235 240

Leu Gly Gly Ser Pro Tyr Pro Gly Val Pro Val Glu Glu Leu Phe Lys 245 250 255

Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro Ser Asn Cys Thr Asn 260 265 270

Glu Leu Tyr Met Met Met Arg Asp Cys Trp His Ala Val Pro Ser Gin 275 280 285

Arg Pro Thr Phe Lys Gin Leu Val Glu Asp Leu Asp Arg He Val Ala 290 295 300

Leu Thr Ser Asn Gin Glu 305 310

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH : 315 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ll) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Ser Ala Ala Gly Thr Met Val Ala Gly Val Ser Glu Tyr Glu Leu Pro 1 5 10 15

Glu Asp Pro Arg Trp Glu Leu Pro Arg Asp Arg Leu Val Leu Gly Lys 20 25 30 Pro Leu Gly Glu Gly Ala Phe Gly Gin Val Val Leu Ala Glu Ala He 35 40 45

Gly Leu Asp Lys Asp Lys Pro Asn Arg Val Thr Lys Val Ala Val Lys 50 55 60

Met Leu Lys Ser Asp Ala Thr Glu Lys Asp Leu Ser Asp Leu He Ser 65 70 75 80

Glu Met Glu Met Met Lys Met He Gly Lys His Lys Asn He He Asn 85 90 95

Leu Leu Gly Ala Cys Thr Gin Asp Gly Pro Leu Tyr Val He Val Glu 100 105 110

Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Gin Ala Arg Arg Pro 115 120 125

Pro Gly Leu Glu Tyr Ser Tyr Asn Pro Ser His Asn Pro Glu Glu Gin 130 135 140

Leu Ser Ser Lys Asp Leu Val Ser Cys Ala Tyr Gin Val Ala Arg Gly 145 150 155 160

Met Glu Tyr Leu Ala Ser Lys Lys Cys He H s Arg Asp Leu Ala Ala 165 170 175

Arg Asn Val Leu Val Thr Glu Asp Asn Val Met Lys He Ala Asp Phe 180 185 190

Gly Leu Ala Arg Asp He His His He Asp Tyr Tyr Lys Lys Thr Thr 195 200 205

Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp 210 215 220

Arg He Tyr Thr His Gin Ser Asp Val Trp Ser Phe Gly Val Leu Leu 225 230 235 240

Trp Glu He Phe Thr Leu Gly Gly Ser Pro Tyr Pro Gly Val Pro Val 245 250 255

Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg Met Asp Lys Pro 260 265 270

Ser Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg Asp Cys Trp His 275 280 285

Ala Val Pro Ser Gin Arg Pro Thr Phe Lys Gin Leu Val Glu Asp Leu 290 295 300

Asp Arg He Val Ala Leu Thr Ser Asn Gin Glu 305 310 315 (2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iil) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr

1 5 10 15

Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30

Pro Ser Ser Arg Ser Ala Ala Gly Thr Met Val Ala Gly Val Ser Glu 35 40 45

Tyr Glu Leu Pro Glu Asp Pro Arg Trp Glu Leu Pro Arg Asp Arg Leu 50 55 60

Val Leu Gly Lys Pro Leu Gly Glu Gly Ala Phe Gly Gin Val Val Leu 65 70 75 80

Ala Glu Ala He Gly Leu Asp Lys Asp Lys Pro Asn Arg Val Thr Lys 85 90 95

Val Ala Val Lys Met Leu Lys Ser Asp Ala Thr Glu Lys Asp Leu Ser 100 105 110

Asp Leu He Ser Glu Met Glu Met Met Lys Met He Gly Lys His Lys 115 120 125

Asn He He Asn Leu Leu Gly Ala Cys Thr Gin Asp Gly Pro Leu Tyr 130 135 140

Val He Val Glu Tyr Ala Ser Lys Gly Asn Leu Arg Glu Tyr Leu Gin 145 150 155 160

Ala Arg Arg Pro Pro Gly Leu Glu Tyr Ser Tyr Asn Pro Ser His Asn 165 170 175

Pro Glu Glu Gin Leu Ser Ser Lys Asp Leu Val Ser Cys Ala Tyr Gin 180 185 190

Val Ala Arg Gly Met Glu Tyr Leu Ala Ser Lys Lys Cys He His Arg 195 200 205 Asp Leu Ala Ala Arg Asn Val Leu Val Thr Glu Asp Asn Val Met Lys 210 215 220

He Ala Asp Phe Gly Leu Ala Arg Asp He His His He Asp Tyr Tyr 225 230 235 240

Lys Lys Thr Thr Asn Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu 245 250 255

Ala Leu Phe Asp Arg He Tyr Thr His Gin Ser Asp Val Trp Ser Phe 260 265 270

Gly Val Leu Leu Trp Glu He Phe Thr Leu Gly Gly Ser Pro Tyr Pro 275 280 285

Gly Val Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His Arg 290 295 300

Met Asp Lys Pro Ser Asn Cys Thr Asn Glu Leu Tyr Met Met Met Arg 305 310 315 320

Asp Cys Trp His Ala Val Pro Ser Gin Arg Pro Thr Phe Lys Gin Leu 325 330 335

Val Glu Asp Leu Asp Arg He Val Ala Leu Thr Ser Asn Gin Glu 340 345 350

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 933 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

ATGCTAGCAG GGGTCTCTGA GTATGAGCTT CCCGAAGACC CTCGCTGGGA GCTGCCTCGG 60

GACAGACTGG TCTTAGGCAA ACCCCTGGGA GAGGGCTGCT TTGGGCAGGT GGTGTTGGCA 120

GAGGCTATCG GGCTGGACAA GGACAAACCC AACCGTGTGA CCAAAGTGGC TGTGAAGATG 180

TTGAAGTCGG ACGCAACAGA GAAAGACTTG TCAGACCTGA TCTCAGAAAT GGAGATGATG 240

AAGATGATCG GGAAGCATAA GAATATCATC AACCTGCTGG GGGCCTGCAC GCAGGATGGT 300

CCCTTGTATG TCATCGTGGA GTATGCCTCC AAGGGCAACC TGCGGGAGTA CCTGCAGGCC 360

CGGAGGCCCC CAGGGCTGGA ATACTGCTAC AACCCCAGCC ACAACCCAGA GGAGCAGCTC 420 TCCTCCAAGG ACCTGGTGTC CTGCGCCTAC CAGGTGGCCC GAGGCATGGA GTATCTGGCC 480

TCCAAGAAGT GCATACACCG AGACCTGGCA GCCAGGAATG TCCTGGTGAC AGAGGACAAT 540

GTGATGAAGA TAGCAGACTT TGGCCTCGCA CGGGACATTC ACCACATCGA CTACTATAAA 600

AAGACAACCA ACGGCCGACT GCCTGTGAAG TGGATGGCAC CCGAGGCATT ATTTGACCGG 660

ATCTACACCC ACCAGAGTGA TGTGTGGTCT TTCGGGGTGC TCCTGTGGGA GATCTTCACT 720

CTGGGCGGCT CCCCATACCC CGGTGTGCCT GTGGAGGAAC TTTTCAAGCT GCTGAAGGAG 780

GGTCACCGCA TGGACAAGCC CAGTAACTGC ACCAACGAGC TGTACATGAT GATGCGGGAC 840

TGCTGGCATG CAGTGCCCTC ACAGAGACCC ACCTTCAAGC AGCTGGTGGA AGACCTGGAC 900

CGCATCGTGG CCTTGACCTC CAACCAGGAG TAG 933

(2) INFORMATION FOR SEQ ID NO : 5 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1056 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

ATGCGGGGTT CTCATCATCA TCATCATCAT GGTATGGCTA GCATGACTGG TGGACAGCAA 60

ATGGGTCGGG ATCTGTACGA CGATGACGAT AAGGATCCGA GCTCGAGATC TGCAGCTGGT 120

ACCATGGTAG CAGGGGTCTC TGAGTATGAG CTTCCCGAAG ACCCTCGCTG GGAGCTGCCT 180

CGGGACAGAC TGGTCTTAGG CAAACCCCTG GGAGAGGGCG CCTTTGGGCA GGTGGTGTTG 240

GCAGAGGCTA TCGGGCTGGA CAAGGACAAA CCCAACCGTG TGACCAAAGT GGCTGTGAAG 300

ATGTTGAAGT CGGACGCAAC AGAGAAAGAC TTGTCAGACC TGATCTCAGA AATGGAGATG 360

ATGAAGATGA TCGGGAAGCA TAAGAATATC ATCAACCTGC TGGGGGCCTG CACGCAGGAT 420

GGTCCCTTGT ATGTCATCGT GGAGTATGCC TCCAAGGGCA ACCTGCGGGA GTACCTGCAG 480

GCCCGGAGGC CCCCAGGGCT GGAATACTCC TACAACCCCA GCCACAACCC AGAGGAGCAG 540

CTCTCCTCCA AGGACCTGGT GTCCTGCGCC TACCAGGTGG CCCGAGGCAT GGAGTATCTG 600

GCCTCCAAGA AGTGCATACA CCGAGACCTG GCAGCCAGGA ATGTCCTGGT GACAGAGGAC 660 AATGTGATGA AGATAGCAGA CTTTGGCCTC GCACGGGACA TTCACCACAT CGACTACTAT 720

AAAAAGACAA CCAACGGCCG ACTGCCTGTG AAGTGGATGG CACCCGAGGC ATTATTTGAC 780

CGGATCTACA CCCACCAGAG TGATGTGTGG TCTTTCGGGG TGCTCCTGTG GGAGATCTTC 840

ACTCTGGGCG GCTCCCCATA CCCCGGTGTG CCTGTGGAGG AACTTTTCAA GCTGCTGAAG 900

GAGGGTCACC GCATGGACAA GCCCAGTAAC TGCACCAACG AGCTGTACAT GATGATGCGG 960

GACTGCTGGC ATGCAGTGCC CTCACAGAGA CCCACCTTCA AGCAGCTGGT GGAAGACCTG 1020

GACCGCATCG TGGCCTTGAC CTCCAACCAG GAGTAG 1056

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SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145 v01

SSSD/55145. vϋ1

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. vOI

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

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SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145 v01 cn cn p p p p p p p P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Ln ^μ3 H ^μ3 H . H H H ^μ- ^μ3 ^μ3 ^μ- ^{μ μ μ} H ^μ3 ^μ3 ^μ3 ^μ3 H ^μ3 H 3 ^μ3 H H ^{μ μ} H H , ^μ3 ^μ3 H ^μ-

D o 3 3oo33o3oo3o2o2o2o2o2o3o33o3oo3o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o2o22o ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft co oo oo co co co co co oo co ∞ co co co co c-i co co cD ω m co cσ ω ∞

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< ui ft UJ to o co co σi Lπ ft Lo tO H O OO si cn Lπ ft co tO H co oo si cn cπ ft co H o co σ cn ft co M O CD O

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Ωd Hκ ^μw3 ^μκ Hκ ^μro3κv Hκ < < < < < d d d d d d d d < < < < < < <. P P P P P P P P P

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UI UI Ul U U W UI UI U UJ W U U IJ W W W UI W UJ U U N IO W tO tJ M t IO W M I M IO IO tO I

CO tO t t tO tO tO 'tO H H H H H H H o o o o o o o o co co co co cD co co co co oo co oo co co co si

m σi σi σi σi σ σi cn cn cn cn σ σi σ σi cn cn cn σi σi σi σi cπ ui cn cn iπ ui iπ σi iπ cπ cπ cπ ui ui ui cπ m i in in i iΛ ij ft u u μ o t μ M to μ ft w t μ μ B φ si φ iii io ii o . i iD ii iii oi si iiJ ι μ w a w ιn ui M uι uι oι θ - μ co kJ θ ι μ oι cθ si u μ co a μ ιi^c ιii ai M u⁾ uι aι ui ιn oi u o co co si to to cπ o co o si ft H O si m cD Co co to u^j cD ft tO H to co H si co co ω cn ui cD σi co m ft cn

CD Cπ to J m co ft cπ cD co ft si M ft σi o σ co H H cD co o ft cπ o co ui ft si to tO H Co co si H si oo

sl sl Ol ft Ol Ul Ul ft Uⁱ ft Ul cn ft UJ tO H H tO tJ O O O O O H O O H t ft Uⁱ CO sl sl On cπ ft ft

H Ui s ft s σi ft co Cv to co o σ co io co cπ cπ s H -n o u o^ co tO si ui ft si o co ft si ui tO H Co cn

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SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145 v01

SSSD/55145. v01 ATOM 5405 CD1 ILE 693 52.663 -3.747 19.194 1.00 25.37 ATOM 5406 C ILE 693 48.603 -5.023 16.452 1.00 29.21 ATOM 5407 O ILE 693 48.568 -5.807 15.512 1.00 27.89 ATOM 5408 N PHE 694 47.623 -4.942 17.336 1.00 31.33 ATOM 5410 CA PHE 694 46.523 -5.888 17.279 1.00 34.41 ATOM 5411 CB PHE 694 45.958 -6.114 18.687 1.00 35.37 ATOM 5412 CG PHE 694 46.978 -6.717 19.621 1.00 35.60 ATOM 5413 CD1 PHE 694 47.606 -5.942 20.586 1.00 37.23 ATOM 5414 CD2 PHE 694 47.424 -8.024 19.426 1 00 35.59 ATOM 5415 CE1 PHE 694 48.669 -6.460 21.333 1 00 36.39 ATOM 5416 CE2 PHE 694 48.484 -8.546 20.170 1 00 35.34 ATOM 5417 CZ PHE 694 49.110 -7.762 21.118 1 00 35.71 ATOM 5418 C PHE 694 45.481 -5.715 16.176 1 00 34.41 ATOM 5419 O PHE 694 44.623 -6.579 15.982 1 00 34.48 ATOM 5420 N THR 695 45.617 -4.637 15.404 1 00 33.03 ATOM 5422 CA THR 695 44.742 -4.379 14.263 1 00 31.81 ATOM 5423 CB THR 695 44.113 -2.957 14.278 1.00 29.75 ATOM 5424 OG1 THR 695 45.142 -1.961 14.218 1.00 30.72 ATOM 5426 CG2 THR 695 43.254 -2.759 15.524 1.00 29.40 ATOM 5427 C THR 695 45.596 -4.533 13.011 1.00 31.44 ATOM 5428 O THR 695 45.153 -4.241 11.906 1.00 33.00 ATOM 5429 N LEU 696 46.832 -4.987 13.209 1.00 31.24 ATOM 5431 CA LEU 696 47.799 -5.199 12.134 1.00 31.36 ATOM 5432 CB LEU 696 47.421 -6.418 11.291 1.00 33.53 ATOM 5433 CG LEU 696 47.270 -7.741 12.042 1.00 33.00 ATOM 5434 CD1 LEU 696 47.010 -8.838 11.052 1.00 35.50 ATOM 5435 CD2 LEU 696 48.515 -8.061 12.830 1.00 36.09 ATOM 5436 C LEU 696 48.066 -3.976 11.249 1 00 30.84 ATOM 5437 O LEU 696 48.135 -4.067 10.024 1 00 28.23 ATOM 5438 N GLY 697 48.302 -2.839 11.890 1.00 31.54 ATOM 5440 CA GLY 697 48.591 -1.632 11.141 1.00 33.87 ATOM 5441 C GLY 697 47.375 -0.765 10.924 1.00 32.77 ATOM 5442 O GLY 697 47.322 0.042 9.994 1.00 33.90 ATOM 5443 N GLY 698 46.392 -0.921 11.797 1.00 33.29 ATOM 5445 CA GLY 698 45.187 -0.122 11.681 1.00 32.66 ATOM 5446 C GLY 698 45.408 1.368 11.877 1.00 30.57 ATOM 5447 O GLY 698 46.336 1.803 12.553 1. 00 27.36 ATOM 5448 N SER 699 44.517 2.148 11.285 1.00 30.92 ATOM 5450 CA SER 699 44.552 3.595 11.376 1.00 32.19 ATOM 5451 CB SER 699 44.062 4 .202 10.058 1, 00 34.24 ATOM 5452 OG SER 699 44.019 5 ..616 10.123 1.00 38.67 ATOM 5454 C SER 699 43.644 4 ..014 12.538 1.00 31.81 ATOM 5455 O SER 699 42.431 3 .759 12.525 1.00 31.39 ATOM 5456 N PRO 700 44.228 4 .597 13.594 1.00 31.82 ATOM 5457 CD PRO 700 45.645 4 .842 13.919 1.00 28.82 ATOM 5458 CA PRO 700 43.353 4 .992 14.697 1.00 31.31 ATOM 5459 CB PRO 700 44.345 5. .341 15.809 1.00 31.31 ATOM 5460 CG PRO 700 45.552 5. .800 15.061 1.00 30.41 ATOM 5461 C PRO 700 42.484 6.170 14.295 1.00 31.19 ATOM 5462 o PRO 700 42.899 7.021 13.510 1.00 29.93 ATOM 5463 N TYR 701 41.235 6.144 14.736 1.00 32.69 ATOM 5465 CA TYR 701 40.291 7.223 14.445 1.00 32.54 ATOM 5466 CB TYR 701 40.650 8.416 15.323 1.00 34.47 ATOM 5467 CG TYR 701 40.512 8.141 16.794 1.00 39.16

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145 v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. vϋl

SSSD/55145. v01

SSSD/55145 vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. 01 TABLE 2

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. 01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vϋ1

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. 01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/551 5. v01

SSSD/551 5. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01 PRT1

SSSD/55145. v01

SSSD/55145. 01

SSSD/55145. v01

SSSD/55145 v01

SSSD/55145. vOI

SSSD/55145. vOI

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SSSD/55145. vϋ1

SSSD/55145. vOI

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SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01 ATOM 6000 O GLU 752 55.548 -9.261 32.328 1.00 46.25

ATOM 6001 N ASP 753 54.380 -7.346 32.601 1.00 44.35

ATOM 6003 CA ASP 753 53.099 -7.912 32.180 1.00 44.19

ATOM 6004 CB ASP 753 52.059 -6.814 31.985 1.00 46.22

ATOM 6005 CG ASP 753 51.512 -6.279 33.278 1.00 50.48

ATOM 6006 OD1 ASP 753 51.396 -7.062 34.248 1.00 52.15

ATOM 6007 OD2 ASP 753 51.170 -5.069 33.306 1.00 52.20

ATOM 6008 C ASP 753 53.244 -8.608 30.849 1.00 44.54

ATOM 6009 O ASP 753 52.770 -9.724 30.674 1.00 46.03

ATOM 6010 N LEU 754 53.880 -7.918 29.906 1.00 44.43

ATOM 6012 CA LEU 754 54.079 -8.438 28.563 1.00 43.70

ATOM 6013 CB LEU 754 54.570 -7.339 27.618 1.00 43.48

ATOM 6014 CG LEU 754 53.481 -6.350 27.201 1.00 44.67

ATOM 6015 CD1 LEU 754 54.095 -5.218 26.399 1.00 44.51

ATOM 6016 CD2 LEU 754 52.384 -7.069 26.408 1.00 42.07

ATOM 6017 C LEU 754 54.993 -9.642 28.512 1.00 43.14

ATOM 6018 O LEU 754 54.795 -10.536 27.697 1.00 41.32

ATOM 6019 N ASP 755 55.990 -9.671 29.383 1.00 44.74

ATOM 6021 CA ASP 755 56.897 -10.800 29.426 1.00 47.24

ATOM 6022 CB ASP 755 57.942 -10.575 30.517 1.00 51.26

ATOM 6023 CG ASP 755 59.121 -11.518 30.407 1.00 55.39

ATOM 6024 OD1 ASP 755 59.739 -11.793 31.455 1.00 60.61

ATOM 6025 OD2 ASP 755 59.443 ^• 11.970 29.283 1.00 57.16

ATOM 6026 C ASP 755 56.023 -12.005 29.771 1.00 47 67

ATOM 6027 O ASP 755 56.041 -13.032 29.081 1.00 45.99

ATOM 6028 N ARG 756 55.186 -11.816 30.789 1.00 46.72

ATOM 6030 CA ARG 756 54.272 -12.851 3 .256 1.00 46.25

ATOM 6031 CB ARG 756 53.519 -12.368 32 499 1.00 46.31

ATOM 6032 CG ARG 756 52.391 -1 .287 32.953 1.00 46.99

ATOM 6033 CD ARG 756 51.733 -12.776 34.227 1.00 48.10

ATOM 6034 NE ARG 756 51.320 -11.379 34.118 1.00 53.67

ATOM 6036 CZ ARG 756 50.294 -10.951 33.385 1.00 55.35

ATOM 6037 NH1 ARG 756 49.562 -11.812 32.684 1.00 54 . 10

ATOM 6040 NH2 ARG 756 50.008 -9.654 33.344 1.00 56 . 02

ATOM 6043 C ARG 756 53.282 -13.261 30.175 1.00 45 . 05

ATOM 6044 O ARG 756 53.213 -14.429 29.806 1.00 47 . 19

ATOM 6045 N ILE 757 52.550 -12.289 29.647 1.00 43 . 47

ATOM 6047 CA ILE 757 51 552 -12.553 28.617 1.00 43 . 80

ATOM 6048 CB ILE 757 50.842 -11.241 28.161 1.00 42 . 02

ATOM 6049 CG2 ILE 757 49.811 -11.536 27.086 1.00 39 . 63

ATOM 6050 CGI ILE 757 50.154 -10.578 29.361 1.00 40 . 00

ATOM 6051 CD1 ILE 757 49.600 -9.212 29.086 1.00 42 . 68

ATOM 6052 C ILE 757 52.148 -13.296 27.428 1.00 46 . 03

ATOM 6053 O ILE 757 51.549 -14.250 26.947 1.00 47 . 78

ATOM 6054 N VAL 758 53.359 -12.925 27.015 1.00 49 . 03

ATOM 6056 CA VAL 758 54.015 -13.584 25.884 1.00 51 . 51

ATOM 6057 CB VAL 758 55.412 -12.971 25.556 1.00 50 . 75

ATOM 6058 CGI VAL 758 56.105 -13.780 24.470 1.00 50 . 31

ATOM 6059 CG2 VAL 758 55.269 -11.541 25.081 1.00 52 . 52

ATOM 6060 C VAL 758 54.209 -15.050 26.212 1.00 54 . 30

ATOM 6061 O VAL 758 53.991 -15.915 25.369 1.00 54 . 80

ATOM 6062 N ALA 759 54.617 -15.311 27.450 1.00 57 . 65

ATOM 6064 CA ALA 759 54.858 -16.667 27.919 1.00 60 . 62

ATOM 6065 CB ALA 759 55.423 -16.637 29.327 1.00 60 . 32

SSSD/55145. v01

SSSD/55145. v()1

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01 TABLE 3

SSSD/55145 vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v()1

SSSD/55145. v01

SSSD/55145..01

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SSSD/55145. v()1

SSSD/55145. v01

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SSSD/55145. v01

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SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. .01

SSSD/55145. v01

SSSD/55145. v01 3.12

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01 ATOM 1844 OD1 ASP 1674 25.049 9.808 -7.172 1.00 56.20

ATOM 1845 OD2 ASP 1674 24.786 7.640 -7.460 1.00 55.73

ATOM 1846 C ASP 1674 21.239 10.083 -5.321 1.00 45.94

ATOM 1847 O ASP 1674 20.402 10.200 -6.222 1.00 47.80

ATOM 1848 N ARG 1675 20.903 9.953 -4.040 1.00 45.98

ATOM 1850 CA ARG 1675 19.503 9.981 -3.608 1.00 43.76

ATOM 1851 CB ARG 1675 18.872 11.346 -3.887 1.00 48.61

ATOM 1852 CG ARG 1675 19.519 12.478 -3.142 1.00 58.37

ATOM 1853 CD ARG 1675 19.468 13.715 -3.992 1.00 70.39

ATOM 1854 NE ARG 1675 20.035 14.867 -3.306 1.00 79.14

ATOM 1856 CZ ARG 1675 19.612 16.116 3.472 1.00 82.95

ATOM 1857 NH1 ARG 1675 18.610 16.386 •4.308 1.00 82.00

ATOM 1860 NH2 ARG 1675 20.194 17.097 2 . 793 1 . 00 87 . 42

ATOM 1863 C ARG 1675 18.647 8.882 - 4 . 236 1 . 00 39 . 26

ATOM 1864 O ARG 1675 17.461 9.074 • 4 . 488 1 . 00 37 . 29

ATOM 1865 N ILE 1676 19.270 7.746 ^■ 4 . 526 1 . 00 35 . 86

ATOM 1867 CA ILE 1676 18.544 6.614 - 5 . 08 1 1 . 00 32 . 76

ATOM 1868 CB ILE 1676 19.324 5.927 - 6 . 192 1 . 00 3 1 . 73

ATOM 1869 CG2 ILE 1676 18.450 4.902 6 . 868 . 00 30 . 02

ATOM 1870 CGI ILE 1676 19.767 6.955 7.219 1.00 32.68

ATOM 1871 CD1 ILE 1676 20.658 6.371 -8.272 1.00 35.75

ATOM 1872 C ILE 1676 18.329 5.625 ^■3.946 1.00 31.08

ATOM 1873 O ILE 1676 19.264 4.962 ^■3.505 1 00 28.77

ATOM 1874 N TYR 1677 17.102 5.558 3.444 1.00 30.32

ATOM 1876 CA TYR 1677 16.779 4.653 -2.348 1 00 29.68

ATOM 1877 CB TYR 1677 15.846 5.329 -1 354 1.00 31.14

ATOM 1878 CG TYR 1677 16.523 6.395 -0 514 1 00 32.95

ATOM 1879 CD1 TYR 1677 16.616 7.721 -0.958 1.00 30.40

ATOM 1880 CE1 TYR 1677 17.208 8.707 ■0.171 1.00 27.57

ATOM 1881 CD2 TYR 1677 17.048 6.082 0 743 1.00 32.13

ATOM 1882 CE2 TYR 1677 17.642 7.059 1.543 1.00 31.50

ATOM 1883 CZ TYR 1677 17.711 8.366 1.081 1.00 31.12

ATOM 1884 OH TYR 1677 18.235 9.326 1.912 1.00 32.18

ATOM 1886 C TYR 1677 16.123 3.424 -2.933 1.00 28.88

ATOM 1887 O TYR 1677 15.268 3.537 3.811 1.00 32.20

ATOM 1888 N THR 1678 16.556 2.253 -2.481 1.00 26.34

ATOM 1890 CA THR 1678 16.023 0.988 -2.971 1.00 25.55

ATOM 1891 CB THR 1678 16.917 0.394 -4.043 1.00 28.81

ATOM 1892 OG1 THR 1678 18.221 0.179 -3.483 1.00 34.06

ATOM 1894 CG2 THR 1678 17.010 1.320 -5.267 1.00 27.25

ATOM 1895 C THR 1678 16.037 0.007 1.827 1.00 21.78

ATOM 1896 O THR 1678 16.505 0.312 -0.744 1.00 25.57

ATOM 1897 N HIS 1679 15.559 -1.198 -2.071 1.00 20.86

ATOM 1899 CA HIS 1679 15.580 -2.216 -1.030 1.00 20.30

ATOM 1900 CB HIS 1679 14.816 -3.453 -1.499 1.00 17.22

ATOM 1901 CG HIS 1679 13.367 -3.196 -1.797 1.00 19.02

ATOM 1902 CD2 HIS 1679 12.662 -3.275 -2.958 1.00 14.89

ATOM 1903 ND1 HIS 1679 12.459 -2.830 -0.826 1.00 18.98

ATOM 1905 CE1 HIS 1679 11.260 -2.697 -1.370 1.00 16.10

ATOM 1906 NE2 HIS 1679 11.359 -2.961 -2.663 1.00 15.18

ATOM 1908 C HIS 1679 17.050 -2.535 -0.761 1.00 20.44

ATOM 1909 O HIS 1679 17.428 -2.901 0.356 1.00 22.58

SSSD/55145. v01

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SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145 v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01 54.236 -1.504 36.569 1.00 51.86 53.036 -0.639 36.938 1.00 54.76

53.181 0.504 37.350 1.00 58.36 51.846 -1.179 36.769 1.00 59.25 55.006 -3.607 33.389 1.00 41.66 54.978 -4.841 33.355 1.00 40.25 54.759 -2.843 32.327 1.00 41.47 54.398 -3.387 31.018 1.00 40.00

54.366 -2.279 29.966 1.00 40.55 53.316 -1.174 30.112 1.00 39.94 53.714 0.019 29.257 1.00 41.03 51.952 -1.696 29.722 1.00 37.80 55.383 -4.452 30.581 1.00 39.61 54.990 -5.470 30.027 1.00 42.08 56.670 -4.207 30.804 1.00 40.63 57.691 - 5.177 30.422 1.00 39.65 59.115 -4.639 30.677 1.0C 33.44 60.142 -5.694 30.351 1.00 31.57 59.372 - .433 29.825 1.00 25.19 57.458 -6 468 31.204 1.00 43.58 57.530 -7.563 30.646 1.00 44.81 57.116 - 6.339 32.481 1.00 46.24 56.869 -7.518 33.301 1.00 50.55 56.781 -7.137 34.783 1.00 53.70 58.090 -6.541 35.310 1.00 56.60 58.079 -6 243 36.792 1.00 56.20 53.387 -5.092 37.178 1.00 53.45 57.789 -7.170 37.573 1.00 60.28 55.622 -8.275 32.837 1.00 50.90 55.689 -9 474 32.555 1.00 51.03 54.501 -7.570 32.708 1.00 51.12 53.251 -8.184 32.265 1.00 48.76 52.122 -7.160 32.249 1.00 51.11 51.646 -6.805 33.636 1.00 54.97 51.592 -7 715 34.495 1.00 58.37 51.319 -5.618 33.864 1.00 56.38 53.381 -8.790 30.881 1.00 48.02 52.991 -9.935 30.672 1.00 48.32 53.925 -8.020 29.940 1.00 45.16 54.111 -8.490 28.571 00 44.82 54.696 -7.387 27.691 00 42.70 53.736 -6.263 27.298 00 42.92 54.500 -5.236 26.495 00 41.44 52.537 -6.822 26.502 00 42.86 55.001 -9.716 28.529 00 46.00 54.815 -10.606 27.708 00 45.88 55.975 -9.752 29.424 00 47.37

56.889 -10.873 29.516 00 48.88 57.898 -10.584 30.628 1.00 49.89 58.998 -11.616 30.717 1.00 51.73 59.640 -11.680 31.785 1.00 55.47

59.236 -12.354 29.738 1.00 50.98

SSSD/55145. vOI

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01 26.597 -10.647 -1.184 1.00 25.85

24.406 1.951 18.037 1.00 30.72

-1.809 12.914 3.754 1.00 43.57

59.590 13.738 33.131 1.00 26.96

4.442 -11.011 1.724 1.00 46.96

8.101 2.869 0.801 1.00 37.28

76.065 1.631 26.158 1.00 46.49

48.821 15.839 14.239 1.00 34.18

2.703 -11.324 8.959 1.00 39.16

82.922 26.478 12.953 1.00 43.77

8.998 -6.359 -3.309 1.00 39.51

-8.590 4.563 4.397 1.00 32.53

8.115 -13.800 8.351 1.00 41.64

51.643 6.187 10.821 1.00 31.70

20.737 3.915 15.522 1.00 17.40

73.254 3.698 20.947 1.00 27.49

5.343 -11.780 22.588 1.00 36.63

34.390 2.307 16.660 1.00 64.04

9.552 -11.846 6.934 00 28.23

8.463 4.098 -1.454 00 30.21

7.397 6.952 2.826 00 33.87

35.796 -1.428 0.072 00 30.27

45.044 10.052 11.102 00 28.75

45.209 11.756 21.279 1.00 31.80

-2.800 15.170 16.902 1.00 32.72

85.885 11.248 9.428 1.00 25.28

13 136 -2.420 1.867 1.00 20.56

75.900 3.542 20.641 1.00 39.79

13.075 7.580 -2.817 1.00 34.49

11.166 -10.189 0.573 1.00 36.71

13.814 -16.459 3.327 1.00 21 18 -6.419 -3.460 16.599 1.00 32.62

25.578 -12.834 3.624 1.00 43.32 -16.472 11.136 6.388 1.00 64.77 86.531 12.711 7.151 00 28.72 32.292 -4.665 1.511 00 30.98 45.116 7.369 11.774 00 30.59 81.035 12.317 16.907 00 41.72

2.905 -7.019 -2.101 00 26.20 31.895 -6.253 20.885 00 36.12 74.974 -2.640 12.464 00 58.90

7.514 6.734 -1.116 00 37.81 71.606 5.595 22.198 1.00 54.82 68.337 -5.037 8.955 00 40.80

0.191 -9.669 6.903 00 47.40 68.043 18.153 10.710 00 36.67

3.644 8.512 4.478 00 40.16 52.117 11.302 18.644 00 40.22 -10.220 6.750 4.981 00 25.00 76.944 1.425 -0.793 1.00 46.95 10.053 -11.958 17.014 1.00 38.99

34.348 14.128 18.169 1.00 42.98

SSSD/55145. v01

SSSD/55145. v01

SSSD/55145. v01

SSSD/55034. V01 TABLE 4

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034 V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01 ATOM 1243 OD1 ASP 1647 12.110 15.908 -1.009 1.00 61.21

ATOM 1244 OD2 ASP 1647 10.337 17.173 -0.835 1.00 61.34

ATOM 1245 C ASP 1647 12.793 14.236 1.273 1.00 60.16

ATOM 1246 O ASP 1647 13.523 15.023 1.889 1.00 58.16

ATOM 1247 N ILE 1648 13.248 13.209 0.562 1.00 61.28

ATOM 1248 CA ILE 1648 14.658 12.878 0.439 1.00 62.12

ATOM 1249 CB ILE 1648 14.848 11.626 -0.444 1.00 59.97

ATOM 1250 CG2 ILE 1648 14.023 10.469 0.131 1.00 58.26

ATOM 1251 CGI ILE 1648 14.429 11.922 -1.883 1.00 55.69

ATOM 1252 CD1 ILE 1648 15.005 10.976 -2.890 1.00 54.38

ATOM 1253 C ILE 1648 15.470 14.047 -0.127 1.00 65.02

ATOM 1254 O ILE 1648 16.633 14.245 0.233 1.00 66.85

ATOM 1255 N HIS 1649 14.844 14.839 -0.995 1.00 65.85

ATOM 1256 CA HIS 1649 15.505 15.992 -1.589 1.00 66.73

ATOM 1257 CB HIS 1649 14.859 16.358 -2.934 1.00 65.67

ATOM 1258 CG HIS 1649 15.142 15.388 -4.038 1.00 66.47

ATOM 1259 CD2 HIS 1649 16.253 14.686 -4.355 1.00 67.11

ATOM 1260 ND1 HIS 1649 14.210 15.064 -4.999 1.00 65.21

ATOM 1261 CE1 HIS 1649 14.733 14.216 -5.867 1.00 66.52

ATOM 1262 NE2 HIS 1649 15.974 13.966 -5.494 1.00 66.25

ATOM 1263 C HIS 1649 15.505 17.200 -0.663 1.00 68.55

ATOM 1264 O HIS 1649 15.636 18.341 -1.116 1.00 69.35

ATOM 1265 N HIS 1650 15.273 16.963 0.629 1.00 71.25

ATOM 1266 CA HIS 1650 15.262 18.026 1.633 1.00 73.53

ATOM 1267 CB HIS 1650 13.849 18.551 1.860 1.00 76.79

ATOM 1268 CG HIS 1650 13.342 19.448 0.765 1.00 83.36

ATOM 1269 CD2 HIS 1650 13.509 20.772 0.537 1.00 86.47

ATOM 1270 ND1 HIS 1650 12.571 18.984 -0.270 1.00 87.02

ATOM 1271 CE1 HIS 1650 12.279 19.983 -1.076 1.00 88.66

ATOM 1272 NE2 HIS 1650 12.840 21.080 -0.609 1.00 88.34

ATOM 1273 C HIS 1650 15.872 17.580 2.965 1.00 73.11

ATOM 1274 O HIS 1650 15.686 18.241 3.977 1.00 73.23

ATOM 1275 N ILE 1651 16.599 16.464 2.949 1.00 72.64

ATOM 1276 CA ILE 1651 17.234 15.937 4.143 1.00 72.54

ATOM 1277 CB ILE 1651 17.660 14.472 3.942 1.00 74.59

ATOM 1278 CG2 ILE 1651 18.463 13.966 5.142 1.00 75.52

ATOM 1279 CGI ILE 1651 16.426 13.591 3.752 1.00 77.59

ATOM 1280 CD1 ILE 1651 16.747 12.141 3.472 1.00 80.12

ATOM 1281 C ILE 1651 18.463 16.769 4.523 1.00 71.47

ATOM 1282 O ILE 1651 19.326 17.022 3.688 1.00 72.40

ATOM 1283 N ASP 1652 18.529 17.197 5.784 1.00 70.34

ATOM 1284 CA ASP 1652 19.678 17.976 6.235 1.00 68.57

ATOM 1285 CB ASP 1652 19.272 18.878 7.411 1.00 72.80

ATOM 1286 CG ASP 1652 20.456 19.640 7.982 1.00 76.90

ATOM 1287 OD1 ASP 1652 21.463 19.888 7.287 1.00 79.62

ATOM 1288 OD2 ASP 1652 20.369 20.030 9.170 1.00 80.36

ATOM 1289 C ASP 1652 20.771 17.007 6.652 1.00 66.01

ATOM 1290 O ASP 1652 20.709 16.421 7.735 1.00 64.75

ATOM 1291 N TYR 1653 21.778 16.868 5.808 1.00 64.05

ATOM 1292 CA TYR 1653 22.906 15.978 6.074 1.00 63.55

ATOM 1293 CB TYR 1653 23.829 15.913 4.855 1.00 63.81

ATOM 1294 CG TYR 1653 23.316 14.993 3.771 1.00 65.65

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034 V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01 ATOM 2023 CB SER 1742 22.956 -5.808 -8.641 1.00 23.67

ATOM 2024 OG SER 1742 24.324 -5.891 -9.023 1.00 26.64

ATOM 2025 C SER 1742 23.524 -6.984 -6.545 1.00 23.09

ATOM 2026 O SER 1742 22.993 -8.104 -6.603 1.00 21.90

ATOM 2027 N GLN 1743 24.719 -6.782 -5.997 1.00 23.62

ATOM 2028 CA GLN 1743 25.466 -7.895 -5.416 1.00 23.26

ATOM 2029 CB GLN 1743 26.953 -7.754 -5.702 1.00 24.32

ATOM 2030 CG GLN 1743 27.255 -7.828 -7.170 1.00 23.04

ATOM 2031 CD GLN 1743 26.684 -9.076 -7.810 1.00 24.83

ATOM 2032 OE1 GLN 1743 27.176 -10.178 -7.584 1.00 21.07

ATOM 2033 NE2 GLN 1743 25.647 -8.907 -8.625 1.00 22.66

ATOM 2034 C GLN 1743 25.227 -8.121 -3.927 1.00 23.85

ATOM 2035 O GLN 1743 25.744 -9.083 -3.366 1.00 25.36

ATOM 2036 N ARG 1744 24.458 -7.240 -3.290 1.00 22.69

ATOM 2037 CA ARG 1744 24.155 -7.395 -1.868 1.00 21.65

ATOM 2038 CB ARG 1744 23.635 -6.087 -1.277 1.00 21.22

ATOM 2039 CG ARG 1744 24.623 -4.962 -1.342 1.00 21.63

ATOM 2040 CD ARG 1744 24.013 -3.656 -0.863 1.00 19.06

ATOM 2041 NE ARG 1744 24.869 -2.563 -1.318 1.00 24.44

ATOM 2042 CZ ARG 1744 24.461 -1.322 -1.564 1.00 22.49

ATOM 2043 NH1 ARG 1744 23.184 -0.972 -1.378 1.00 18.95

ATOM 2044 NH2 ARG 1744 25.337 -0.438 -2.034 1.00 22.19

ATOM 2045 C ARG 1744 23.095 -8.470 -1.712 1.00 22.45

ATOM 2046 O ARG 1744 22.363 -8.772 -2.654 1.00 25.62

ATOM 2047 N PRO 1745 23.065 -9.139 -0.559 1.00 21.78

ATOM 2048 CD PRO 1745 24.025 -9.114 0.563 1.00 21.02

ATOM 2049 CA PRO 1745 22.057 -10.175 -0.362 1.00 20.99

ATOM 2050 CB PRO 1745 22.532 -10.879 0.919 1.00 21.12

ATOM 2051 CG PRO 1745 23.240 -9.777 1.676 1.00 19.86

ATOM 2052 C PRO 1745 20.726 -9.485 -0.146 1.00 22.18

ATOM 2053 O PRO 1745 20.680 -8.281 0.128 1.00 23.04

ATOM 2054 N THR 1746 19.646 -10.236 -0.297 1.00 19.31

ATOM 2055 CA THR 1746 18.335 -9.689 -0.085 1.00 19.12

ATOM 2056 CB THR 1746 17.307 -10.334 -1.045 1.00 19.86

ATOM 2057 OG1 THR 1746 17.299 -11.763 -0.886 1.00 22.54

ATOM 2058 CG2 THR 1746 17.668 -10.002 -2.479 1.00 22.97

ATOM 2059 C THR 1746 17.961 -9.975 1.367 1.00 19.91

ATOM 2060 O THR 1746 18.676 -10.711 2.058 1.00 19.93

ATOM 2061 N PHE 1747 16.884 -9.381 1.855 1.00 21.80

ATOM 2062 CA PHE 1747 16.456 -9.678 3.224 1.00 23.46

ATOM 2063 CB PHE 1747 15.353 -8.720 3.686 1.00 21.84

ATOM 2064 CG PHE 1747 15.872 -7.368 4.082 1.00 24.84

ATOM 2065 CD1 PHE 1747 16.627 -7.207 5.237 1.00 22.23

ATOM 2066 CD2 PHE 1747 15.611 -6.248 3.293 1.00 22.97

ATOM 2067 CE1 PHE 1747 17.124 -5.944 5.598 1.00 19.42

ATOM 2068 CE2 PHE 1747 16.111 -4.991 3.646 1.00 17.14

ATOM 2069 CZ PHE 1747 16.862 -4.846 4.801 1.00 18.02

ATOM 2070 C PHE 1747 15.992 -11.133 3.295 1.00 22.28

ATOM 2071 O PHE 1747 16.189 -11.796 4.304 1.00 23.76

ATOM 2072 N LYS 1748 15.430 -11.632 2.199 1.00 23.46

ATOM 2073 CA LYS 1748 14.971 -.13.014 2.140 1.00 25.84

ATOM 2074 CB LYS 1748 14.344 -13.327 0.782 1.00 26.89

SSSD/55034. V01

SSSD/55034 V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. V01

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSΞD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034 VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034 VOl

SSSD/55034. VOl 54.102 -8.489 28.523 1.00 46.37

54.664 -7.385 27.625 1 00 44.16 53.621 -6.366 27.152 1 00 46.35 54.296 -5.272 26.343 1, 00 45.11 52.514 -7.070 26.349 1.00 42.89 55.004 -9.703 28.481 1.00 47.08

54.818 -10.590 27.659 1.00 45.02

55.969 -9.755 29.385 1.00 49.68

56.890 -10.876 29.487 1.00 51.62

57.883 -10.586 30.615 1.00 54.90

59.009 -11.589 30.702 1.00 59.00

59.694 -11.608 31.746 1.00 63.70

59.223 -12.346 29.728 1.00 60.31

56.059 -12.117 29.817 1.00 51.50

56.119 -13.150 29.138 1.00 47.11

55.237 -11.958 30.844 1.00 51.81

54.362 -13.009 31.328 1.00 51.44

53.635 -12.519 32.582 1.00 54.52

52.45.9 -13.358 33.027 1.00 55.00

51.815 -12.727 34.255 1.00 59.54

51.417 -11.335 34.026 1.00 64.01 50.366 -10.960 33.301 1.00 65.76 49.598 11.866 32.721 1.00 63.56 50.061 -9.676 33.183 1.00 66.59 53.361 -13.440 30.260 1.00 50.03

53.267 -14.622 29.960 1 , 00 49.98

52.645 -12.483 29.673 1.00 46.87

51.656 -12.789 28.644 1.00 44.28

50.919 -11.532 28.125 1.00 40.46

49.923 -11.923 27.062 1.00 38.44 50.202 -10.830 29.277 1.00 39.74 49.481 -9.551 28.920 1.00 40.68 52.251 -13.528 27.454 1.00 44.20

51.643 -14.469 26.959 00 40.28 53.440 -13.111 27.014 00 47.56 54.102 -13.745 25.874 00 48.90

55.543 -13.177 25.609 00 47.01 56.198 -13.920 24.456 00 44.38

55.493 -11.714 25.262 00 47.85 54.249 -15.232 26.149 00 51.79

54.043 -16.055 25.258 00 49.80 54.622 -15.550 27.386 00 54.80

54.825 -16.925 27.814 00 57.15

55.406 -16.948 29.212 00 56.77

53.524 -17.717 27.777 00 60.83

53.487 -18.849 27.296 00 63.59

52.452 -17.112 28.271 00 61.74

51.151 -17.760 28.295 00 61.29

50.280 -17.149 29.388 1.00 60.41

50.808 -17.323 30.812 1.00 58.68

49.917 -16.603 31.815 1.00 59.64

50.899 -18.799 31.138 1.00 57.84

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

SSSD/55034. VOl

Claims

CLAIMSWhat is claimed is :

1. A crystalline form of a polypeptide corresponding to the catalytic domain of a protein tyrosine kinase.

2. The crystalline form of claim 1, wherein said protein tyrosine kinase is a receptor protein tyrosine kinase .

3. The crystalline form of claim 2, wherein said receptor protein tyrosine kinase is selected from the group consisting of PDGF-R, FLK, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , and MUSK.

4. The crystalline form of claim 1, wherein said protein tyrosine kinase is a non-receptor protein tyrosine kinase.

5. The crystalline form of claim 4, wherein said non-receptor protein tyrosine kinase is selected from a group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

6. The crystalline form of claim 1, comprising one or more heavy metal atoms.

The crystalline form of claim 1, wherein said protein tyrosine kinase is FGFR.

8. The crystalline form of claim 7, wherein said FGFR is FGFRl.

9. The crystalline form of claim 8, defined by atomic structural coordinates set forth m Table 1.

10. The crystalline form of claim 7, comprising at least one compound.

11. The crystalline form of claim 10, wherein said compound is a nucleotide analog.

12. The crystalline form of claim 11, wherein said nucleotide analog is AMP-PCP.

13. The crystalline form of claim 12, defined by atomic structural coordinates set forth m Table 2.

14. The crystalline form of claim 10, wherein said compound is an indolinone compound.

15. The crystalline form of claim 14, wherein said indolinone compound has a structure set forth m formula

I or II:

or a pharmaceutically acceptable salt, isomer, metabolite, ester, amide, or prodrug thereof, wherein

(a) A,, A₂, A₃, and A₄ are independently carbon cr nitrogen;

(b) R_j is hydrdgen or alkyl;

(c) R₂ is cxygen in the case of an oxindolinone or sulfur in the case of a thiolindolinone ;

(d) R₃ is hydrogen;

(e) R₄ , R₅, R₆ , and R₇ are optionally present and are either (i) independently selected frcm the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, SO,NRR\ SO_jR, SR, N0 , NRR¹, OH, CN, C(0)R, 0C(0)R, NHC(0)R, (CH₂)_nC0₂R, and CONRR' or (ii) any two adjacent R₄ , R₅ , R₆, and R, taken together form a fused ring with the aryl portion of the oxindole-based portion of the indolinone ;

(f) R₂ ' , Rj', R₄ ' , R₅', and R₆ ' are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0_?NRR ' , SO.R, SR, N0_?, NRR', OH, CN, C(0)R, OC(0)R, NHC(0)R, (CH₂)_nCO_?R, and CONRR ^• ; (g) n is θ, 1, 2, or 3 ;

(h) R is hydrogen, alkyl or aryl; (i) R¹ is hydrogen, alkyl or aryl; and (j) A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1 , 2 , 3 -triazole , 1 , 2 , 4 -triazole , oxazole, isoxazole, thiazole, isothiazole, furan, 1,2,3- oxadiazole, 1 , 2 , 4-oxadiazole, 1 , 2 , 5 -oxadiazole , 1,3,4- oxadiazole, 1 , 2 , 3 , 4 -oxatriazole, 1, 2 , 3 , 5-oxatriazole, 1, 2 , 3-thiadiazole, 1 , 2 , -thiadiazole, 1 , 2 , 5-thiadiazole, 1, 3 , 4-thiadiazole, 1 , 2 , 3 , 4 - thiatriazole , 1,2,3,5- thiatriazdle, and tetrazdle, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0₂NRR\ SO₃R, SR, NO,, NRR', OH, CN, C(0)R, 0C(O)R, NHC(0)R, (CH₂)_nC0₂R or CONRR'.

16. The crystalline form of claim 15, wherein said indolinone compound is 3- [ (3 - (2 -carboxyethyl ) -4 - methylpyrrol-5-yl) ethylene] -2 -indolinone .

17. The crystalline form of claim 15, wherein said indolinone compound is 3 - [4- (4-formylpiperazine-1- yl) benzylidenyl] -2 - indolinone .

18. The crystalline form of claim 16, defined by the atomic structural ccdrdinates of Table 3.

19. The crystalline form of claim 17, defined by the atomic structural coordinates of Table 4.

20. The crystalline form of claim 1, having monoclinic unit cells.

21. The crystalline form of claim 20, wherein said monoclinic unit cells have dimensions of about a=208.3 A, b=57.8 Λ, c=65.5 A and 0=107.2°.

22. The crystalline form of claim 20, wherein said monoclinic unit cells have dimensions of abdut a=211.6 A, b=51.3 A, c=66.1 A and 3=107.7°.

23. The crystalline form df claim 10, ccmprising one or more heavy metal atdms .

24. A pdlypeptide ccrresponding to the catalytic domain of a protein tyrosine kinase, containing at least about 20 amino acid residues upstream of the first glycine in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues downstream of the conserved arginine located at the C-terminal boundary of the catalytic domain.

25. The polypeptide of claim 24, wherein said protein tyrosine kinase is a receptcr protein tyrosine kinase .

26. The pdlypeptide df claim 24, wherein said prdtein tyrdsine kinase is a non-receptor protein tyrosine kinase.

27. The polypeptide of claim 25, wherein said receptor tyrosine kinase is selected from the group consisting of FGF-R, PDGF-R, KDR, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, R0R1, and MUSK.

28. The polypeptide of claim 26, wherein said non- receptor kinase is selected from the group consisting of

SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

29. The polypeptide df claim 24 having the amine acid sequence shown in SEQ ID NO : 4.

30. A method of using the polypeptide of claim 24 to form a crystal, comprising the steps of:

(a) mixing a volume of polypeptide solution with a reservoir solution; and (b) incubating the mixture obtained in step

(a) over the reservoir solution in a closed container, under conditions suitable for crystallization.

31. A method of obtaining an FGF receptor tyrosine kinase domain polypeptide in crystalline form, comprising the steps of:

(a) mixing a volume of polypeptide solution with an equal volume of reservoir solution, wherein said polypeptide solution comprises 1 mg/mL to 60 mg/mL FGF- type tyrosine kinase domain protein, 10 mM to 200 mM buffering agent, 0 mM to 20 M dithiothreitol and has a pH of about 5.5 to about 7.5, and wherein said reservoir solution comprises 10% to 30% (w/v) polyethylene glycol, 0.1 M to 0.5 M ammonium sulfate, 0% to 20% (w/v) ethylene glycol or glycerol, 10 mM to 200 mM buffering agent and has a pH of about 5.5 to about 7.5; and

(b) incubating the mixture obtained in step (a) over said reservoir solutidn in a clcsed container at a temperature between 0° and 25° °C until crystals fcrm.

32. The methdd of claim 31, wherein said pdlypeptide sdlution comprises about 10 mg/mL FGF receptor tyrosine kinase domain, about 10 mM sodium chlcride, abdut 2 mM dithiothreitol, about 10 mM Tris- HCl and has a pH of about 8; the reservoir buffer comprises about 16% (w/v) polyethylene glycol (MW 10000), about 0.3 M ammonium sulfate, abdut 5% ethylene glyccl or glycerol, about 100 mM bis-Tris and has a pH of abdut 6.5; and the temperature is abdut 4°C.

33. The method of claim 31, wherein said polypeptide solution comprises a compound.

34. A cDNA encoding an FGF receptor tyrosine kinase domain protein, wherein a coding strand of the cDNA has the nucleotide sequence cf SEQ ID NO : 5.

35. A method of determining three dimensional structures of protein tyrosine kinases with unknown structure comprising the step of applying structural atomic ccordinates set forth in Table 1, Table 2 , Table 3, or Table 4.

36. The method of claim 35, comprising the following steps: (a) aligning a first computer representation of an amino acid sequence of a prctein tyrosine kinase of unknown structure with a second computer representation of a protein tyrosine kinase of known structure by matching homologous regions of amino acid sequences of said first computer representation and said second computer representation;

(b) transferring computer representations cf amino acid structures in said prctein tyrdsine kinase of known structure to computer representations of corresponding amino acid structures in said protein tyrosine kinase with unknown structure; and

(c) determining a low energy ccnfdrmation of the protein tyrosine kinase structure resulting from step (b) .

37. The method of claim 35, comprising the following steps:

(a) aligning the positions of atoms in the unit cell by matching electron diffraction data from two crystals; and (b) determining a lew energy conformation of the resulting protein tyrosine kinase structure.

38. The method of claim 35, comprising the fcllowing steps: (a) determining the secondary structure of a protein tyrosine kinase structure using NMR data; and

(b) simplifying the assignment of through- space interactions of amino acids.

39. The method of any one of claims 35, 36, 37, or

38, wherein said protein tyrosine kinase with or without known structure is a receptor protein tyrdsine kinase.

40. The method of claim 39, wherein said receptor protein tyrosine kinase with or without known structure is selected from the group consisting of FGF-R, PDGF-R, FLK, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, R0R1, and MUSK.

41. The method of anyone df claims 35, 36, 37, or

38, wherein said protein tyrosine kinase with or without known structure is a non-receptor protein tyrcsine kinase .

42. The methdd df claim 41, wherein said prctein tyrosine kinase with or without known structure is selected from the group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

43. A method of identifying a potential modulator of protein tyrosine kinase function by docking a computer representation of a structure of a compound with a computer representation of a structure of a cavity formed by the active- site of a protein tyrosine kinase, wherein said structure of said protein tyrosine kinase is defined by atomic structural coordinates set forth in Table 1, Table 2, Table 3, or Table 4.

44. The method of claim 43, comprising the following steps: (a) removing a computer representation of a compound complexed with a protein tyrosine kinase and docking a computer representation of a compound from a computer data base with a computer representation of the active-site of the protein tyrosine kinase; (b) determining a conformation of the complex resulting from step (a) with a favorable geometric fit and favorable complementary interactions; and

(c) identifying compounds that best fit said active-site as potential modulators of protein tyrdsine kinase functicn.

45. The methdd of claim 43, comprising the following steps:

(a) modifying a computer representation of compound complexed with a protein tyrosine kinase by the deletion of a chemical group or groups or by the addition of a chemical group or groups;

(b) determining a conformation of the complex resulting from step (a) with a favorable geometric fit and favorable complementary interactions; and (c) identifying compounds that best fit the protein tyrosine kinase active-site as potential modulators of protein tyrosine kinase function.

46. The method of claim 43, wherein said method comprises the following steps:

(a) removing a computer representation of a compound complexed with a protein tyrdsine kinase; and

(b) searching a data base for data base compounds similar to said compounds using a compound searching computer program dr replacing portions of said compound with similar chemical structures from a data base using a compound construction computer program.

47. The method of any one of claims 43, 44, 45, or 46, wherein said protein tyrosine kinase is a receptor protein tyrosine kinase.

48. The methdd of claim 47, wherein said receptcr protein tyrdsine kinase is selected frcm the grdup ccnsisting df FGF-R, PDGF-R, FLK, CCK4 , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , and MUSK.

49. The methdd of anydne df claims 43, 44, 45, or 46, wherein said protein tyrosine kinase is a non- receptor protein tyrosine kinase.

50. The method of claim 49, wherein said protein tyrosine kinase is selected from the group consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.

51. a potential modulator of protein tyrosine kinase function identified by the method of any one df claims 43, 44, 45, cr 46.

52. The potential mddulatdr df claim 51, wherein said odulatcr is selected from a ccmputer data base.

53. The pdtential modulatdr of claim 51, wherein said mcdulator is constructed from chemical groups selected from a computer data base.

54. The potential modulator of protein tyrosine kinase function of claim 51, wherein said modulator is an indolinone compound of formula I or II:

(I)

or a pharmaceutically acceptable salt, isomer, metabolite, ester, amide, or prodrug thereof, wherein

(a) A_lr A_2/ A₃, and A₄ are independently carbon dr nitrogen;

(b) R₂ is hydrogen dr alkyl;

(c) R₂ is oxygen in the case cf an dxindolinone or sulfur in the case of a thiolindolinone;

(d) R₃ is hydrogen;

(e) R₄, R₅, R₆, and R₇ are optionally present and are either (i) independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, S0₂NRR\ S0₃R, SR, N0₂, NRR', OH, CN, C(0)R, OC(0)R, NHC(0)R, (CH₂)_nC0₂R, and CONRR' or (ii) any two adjacent R₄, R₅, R₆, and R₇ taken together form a fused ring with the aryl portion of the oxindole-based portion of the indolinone ;

(f) R₂', R₃', R₄ ' , R₅', and R₆ ' are each independently selected frcm the group ccnsisting df hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, SO_?NRR', S0₃R, SR, N0₂, NRR', OH, CN, C(0)R, OC(0)R, NHC(0)R, (CH,)_nC0₂R, and CONRR' ;

(g) n is 0, 1, 2, or 3;

(h) R is hydrogen, alkyl or aryl; (i) R' is hydrogen, alkyl or aryl; and

(j) A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1 , 2 , 3-triazole, 1 , 2 , 4-triazole, oxazole, isoxazole, thiazole, isothiazole, furan, 1,2,3- oxadiazole, 1 , 2 , 4 -oxadiazole , 1 , 2 , 5-oxadiazole , 1,3,4- oxadiazole, 1 , 2 , 3 , 4-oxatriazole , 1 , 2 , 3 , 5-oxatriazole, 1 , 2 , 3 -thiadiazole, 1, 2 , 4-thiadiazole , 1 , 2 , 5- thiadiazole , 1, 3 , 4 -thiadiazole, 1 , 2 , 3 , 4-thiatriazole , 1,2,3,5- thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(0)R, SO_?NRR', S0₃R, SR, N0₂, NRR', OH, CN, C(0)R, OC (O) R, NHC(0)R, (CH₂)_nCO_?R or CONRR¹.

55. A method of identifying a potential modulator df protein tyrosine kinase function as a modulator of protein tyrosine kinase function, comprising the following steps:

(a) administering said potential modulator td cells;

(b) ccmparing the level of protein tyrosine kinase phosphorylation between cells not administered the potential modulator and cells administered said potential modulator; and (c) identifying said potential mddulator as a modulator of protein tyrosine kinase function based on the difference in the level of protein tyrosine kinase phosphorylation .

56. A method of identifying a potential modulator of protein tyrosine kinase function as a modulator of protein tyrosine kinase function, wherein said method comprises the following steps:

(a) administering a preparation of said potential modulator to cells; (b) comparing the rate of cell growth between cells not administered the modulator and cells administered the modulator; and

(c) identifying said potential modulator as a modulator of protein tyrosine kinase function based on the difference in the rate of cell growth.

57. A method of treating a disease associated with a protein tyrosine kinase with inappropriate activity in a cellular organism, wherein said method comprises the steps of:

(a) administering a modulatdr df prdtein tyrdsine kinase functidn td the drganism, wherein said modulator is in an acceptable pharmaceutical preparation; and (b) activating or inhibiting the protein tyrosine kinase function to treat the disease.

58. The method df any one of claims 55, 56, or 57, wherein said prdtein tyrdsine kinase is a receptcr protein tyrosine kinase.

59. The method df claim 58, wherein said receptcr prdtein tyrdsine kinase is selected from the group containing FGF-R, PDGF-R, FLK, CCK , MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1 , and MUSK.

60. The method of any one of claims 55, 56, or 57, wherein said protein tyrosine kinase is a non- eceptor protein tyrdsine kinase.

61. The methdd df claim 60, wherein said non- receptcr protein tyrosine kinase is selected frc a grdup consisting of SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, and ACK.