JPH11510061A - Crystalline ZAP family proteins - Google Patents

Abstract

The present invention relates to human ZAP-70, in particular, the region of ZAP-70 containing the tandem Src homology-2 ("SH2") domain, its liganded or unliganded crystallinity. About the form. These crystalline forms are particularly useful for determining the three-dimensional structure of the protein. The conformation of the tandem SH2 region of ZAP is dependent on the biological function of ZAP and other members of the tandem SH2 domain-containing protein of the ZAP family, particularly biological interactions mediated by molecular interactions involving one or both SH2 domains. Provide useful information for designing a pharmaceutical composition that inhibits function.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Crystalline ZAP family proteins Copyright notice   Portions of the disclosure of this patent application contain material that is subject to copyright protection. Book The copyright owner may not file any patent application documents or Does not object to the copying of patent disclosures by some, but otherwise Holds all copyrights.Field of the invention   The present invention relates to human ZAP-70, particularly including the tandem Src homology-2 ("SH2") domain. Region of ZAP-70, its ligand bound (liganded) or unbound (unliganded) Related to crystalline forms. These crystalline forms represent the three-dimensional structure of the protein. It is particularly useful for building decisions. The three-dimensional structure of the tandem SH2 region of ZAP is ZAP and ZAP Biological function of other proteins in the family, particularly one or both SH2 domains Compositions that inhibit biological functions mediated by molecular interactions involving Provide useful information for product design.background   Patients who have been treated for an autoimmune disease or who have received organ or tissue transplants Require safe and effective immunosuppressants. For example, there are effective immunosuppressants Otherwise, transplant patients often reject the transplanted organ, To death. Immunosuppressants must block the immune response, but long-term The patient's body must be well-tolerated enough to be possible. An example For example, FK506, one of the compounds with immunosuppressive effects, can suppress liver transplant rejection. It has been used to prevent. However, patients receiving FK506 had severe renal disease. Toxicity has been observed, possibly requiring kidney transplantation after liver transplantation There is.   Research aimed at discovering new immunosuppressive drugs requires precise molecular mechanisms of the immune response Has been hindered by the lack of information on As a result, identification of new immunosuppressive drugs A significant portion of the research effort aimed at I have been. More recently, "structure-based" approaches to drug design have been attempted It has come to be. For example, FKBP protein, one of the cellular targets of FK506 Compounds designed to bind to were synthesized as candidates for immunosuppressants. this Their work provides an understanding of the actual molecular mechanisms of FK506-mediated immunosuppression. The result was disappointing because of the shortage. Now, FK506 and FKBP and Calcine Is known to bind as a complex with two proteins Have been. The immunosuppressive effect of FK506 is caused by the inhibition of calcineurin in T cells. Cause. However, calcineurin is also present in other cells, where it is heavy. As such, FK506 affects other cells and tissues, Cause a problem.   Meanwhile, another study found that protein is a critical mediator of the immune response. A tyrosine kinase, ZAP-70, was identified. Blocks the biological function of ZAP-70 This will lead to immunosuppression. Unfortunately, until now, the three-dimensional structure of ZAP-70 Details of the structure were completely unknown. I do not know the details of the three-dimensional structure of the protein If so, it would not have been possible to design inhibitors (inhibitors) based on their structure. We now show that tandem SH2 domain with and without various binding ligands The crystal of the critical region of ZAP-70 containing was obtained and its three-dimensional structure was determined. This information According to reports, inhibitors of ZAP-70 that can function as immunosuppressants, such as ZA Compounds that inhibit molecular interactions involving one or both of the P-70 SH2 domains Here, a rational design is possible for the first time. Some of the other proteins Although the three-dimensional structure of each SH2 domain is known, the three-dimensional structure of the tandem SH2 region has been determined. No one has ever reported a decision. And, as explained later, The known three-dimensional coordinates of the SH2 domain solve the structure of the tandem SH2 region of ZAP-70 Would have been insufficient.Summary of the Invention   The present invention relates to the region of human ZAP-70 spanning its two SH2 domains. So We refer to this area as the "ZAP tandem SH2 area" or simply "ZAP-NC" The region consists of the SH2 domain at the N-terminal side and the SH2 domain at the C-terminal side of human ZAP-70. This is because both of them are included (see FIG. 4). The present invention relates to a tan Complex with various ligands of sufficient quality to determine the tertiary (tertiary) structure of the protein We begin by obtaining crystals of human ZAP-NC, with or without the body.   When considering our research, obtaining protein crystals in any case It is a technique that is somewhat unpredictable. If you do not have an example of the traditional success in preparing and / or crystallizing Should be aware that Therefore, we first obtained the ZAP-NC crystal. And the results themselves were unexpected. Furthermore, our determination of the three-dimensional structure of ZAP-NC Means the first solution of the conformation of the tandem SH2 region from any protein Unexpected combination of surface features, including truly surprising structural features revealed . Our results are useful for many applications.   For example, using the knowledge gained about ZAP-NC, we can determine the tertiary structure of related proteins. Can be modeled. As an example, the structure of renin is the starting point of derivatization. Modeled using the tertiary structure of endothiapepsin There is. Cercarial elastase and tophozoite The model construction of the cysteine proteases has sequence identity (sequence identity) ty) was made from less than 35% of known serine and cysteine proteases. Get Using the model described, inhibitors in the low micromolar range were designed. (Proc. Natl . Acad. Sci.1933, 90, 3583). It does not rely on X-ray diffraction, and Another method for determining tertiary structure, such as NMR, which does not require quality crystallization, Structured for refinement using additional data collected by the decision method If a model is available, it will be simplified. Therefore, the tertiary structure of the ZAP tandem SH2 region Knowledge of the structure of other ZAP family proteins, including SYK, for example. It will be a meaningful window.   Knowledge of the tertiary structure of the tandem SH2 region, such as ZAP-NC, is It provides a tool for the study of ism and the identification of inhibitors of its function. For example In addition, the SH2 domain is an intramolecular and integral molecule essential for the biological activity of SH2-containing proteins. Known to be involved in child-to-child interactions, usually protein-protein interactions ing. Knowledge of the three-dimensional structure of the tandem SH2 region (Therefore, this may inhibit the interaction of the tandem SH2 region with its natural ligand). (Including molecules that can be used).   Thus, one object of the present invention is to provide a peptide sequence of the ZAP family of proteins. Having a peptide sequence derived from or selected from It is to provide a material containing protein. This protein has at least one , Preferably comprising two SH2 domains, eg, including the tandem SH2 region of ZAP-70 Consist of proteins containing SYK or other related tandem SH2 U. In the case of ZAP-70, this protein contains at least amino acid residues 3-279. It may be composed of a curable peptide sequence. Such crystalline materials may include one or more Heavy atom, for example, one or more lead, mercury, gold and / or It may contain a ren atom. Such heavy atom derivatives are, for example, one or two or more. Under conditions that allow for the incorporation of the heavy atom label above (eg, for the incorporation of selenomethionine So that the gene encoding the protein is expressed or Reacts with a reagent capable of binding a heavy atom (eg, trimethyl lead acetate) Or by sucking a substance containing a heavy atom into the crystal Can be.   The protein may be in the form of a complex with one or more ligand molecules. No. As used herein (hereinafter the same), the term “ligand” is used in the broadest sense. All substances capable of observable binding to protein are meant. Many SH2 The peptide sequence of the natural ligand for the domain ("ITAMs", see below) is known Information on consensus sequences for peptide ligands in the SH2 domain Have been debated. In the case of ZAP-70, as a natural ligand of ZAP-70 or other SH2 domains Peptide ligands of 15 to 19 residues from which the sequence is derived can be used. Those Ligands typically contain one or two phosphorylated tyrosine residues. Some Alternatively, one or both of the phosphorylated tyrosine moieties may be replaced with a phosphotyrosine mimetic reagent. (phosphotyrosine mimetic reagent) (Numerous examples are known in the art. Can be replaced by   Representative crystalline materials of the present invention having various physicochemical properties are disclosed below. ing. Preferred crystalline materials of the present invention have a resolution of about 3.5 ° better, more preferably Or 2.6Å or better resolution, even more preferably 2.2Å or better X-rays can be diffracted with better resolution, and the three-dimensional structure of the substance can be determined. Useful to do. (The smaller the value of Å, the better the resolution.)   The crystalline material of the present invention specifically has a crystal as shown in any of the attached annexes. Backbone of amino acids characterized by the structural coordinates given or listed in the Annex For the backbone atoms, the root mean square deviation (the mean squared deviation ZAP family proteins characterized by coordinates whose roots are less than 1.5Å Includes what is included.   The structural coordinates of the crystalline material of the present invention are displayed as a three-dimensional (three-dimensional) shape. ) Or other uses (the computer of the structural coordinates or the three-dimensional structure defined by the coordinates) (Including computer operations based on the coordinates or structure) A machine-readable storage medium, such as a computer hard drive, It may be stored in a machine-readable form on a floppy disk, DAT tape, etc. No. For example, a protein of the ZAP family, or a portion of such a protein Or machine-readable data that defines the three-dimensional structure of homologues with similar structures And display the protein structure as a three-dimensional depiction. can do. This typically involves reading data from this storage medium. Instructions for producing the depiction from such data. Computer is used. Therefore, the present invention relates to the structural structure of the crystalline material of the present invention. Data representing a marker, for example, the coordinates shown in the annex to this document, Has a memory containing along with any additional data and instructions for operating the Machines such as computers. Such data may be stored in other related structures. It can be used for a variety of purposes, such as elucidation and drug discovery.   For example, the first set of machine-readable data may be replaced with a second set of machine-readable data. Machine-readable using the first set of data and the second set of data together with the second set of data. Instructions for determining at least some of the coordinates corresponding to the two sets of data are programmed. Can be combined using a machine that has been programmed. As an example, the first set of data Is at least one of the coordinates of the ZAP or SYK protein shown in the annex to this document. The second set of data may include the Fourier transform of a part of the molecule or molecular complex. It may include X-ray diffraction data.   More specifically, one of the objects of the present invention is to associate ZAP family members with various resources. Conformational information on the novel complex with Gand, as well as other tandem SH2 regions, Unresolved individual SH2 domains, new ZAP family members and more Structural information about any mutein or other variant of To provide. For that purpose, the present inventors have proposed the crystalline material of the present invention or the same. With at least one SH2 domain, using some of the structural coordinates of Such proteins or protein: ligand complexes, including proteins To elucidate the three-dimensional structure of the crystalline form by, for example, a molecular replacement method. This To determine the three-dimensional structure of a protein or protein: Need to obtain X-ray diffraction data for crystals of Gand co-complex . The X-ray diffraction data is then used to identify existing structural coordinates using molecular replacement. Then, the three-dimensional structure of the protein or complex is determined. For example, as described in U.S. Pat. Model the structure of an unknown crystalline sample using known structure molecules as points . In this method, two molecules with similar unit cell structures, orientations and positions are similar. It is based on the principle that diffraction occurs. Molecular substitution is performed at the same position and And arranging known structures in unit cells in different orientations. After placing, in the unit cell Using atoms of known structure, we calculate the structural factors that would be obtained from a hypothetical diffraction experiment. Obtain by calculation. This is until the consistency between the known structure and the experimental data is obtained. In six dimensions (three angular dimensions and three spatial dimensions). Around here Similar structures can be refined using various refinement methods, often with higher resolution Can be fine-tuned to produce For example, as defined by experimental data Using the model obtained for the structure, this model produces a position shift of about 5% or less. It may be processed by a rigid approximation refinement method subject to limited additional rotation at 6 dimensions . The refined model is then further refined using another known refinement method. It may be densified.   For example, as indicated in the attached Annex I, Annex II or Annex III Using molecular replacement to take advantage of a set of coordinates, ZAP-NC, or a part of it, , The structure of a crystalline co-complex with a ligand other than the ζ1 peptide can be determined You. Similarly, using the same technique, SYK-NC, or a portion thereof, and its ligand The three-dimensional structure of the co-complex can be determined.   Another object of the invention is a protein comprising at least one SH2 domain, Describes the three-dimensional structure of the co-complex of this protein and its ligand by a homology model. It is an object of the present invention to provide a method for determining using a chemical method and the structural coordinates of the material of the present invention. E Morology modeling is a model of an unknown structure that has one or more related proteins. Using the structural coordinates of the quality, protein domain and / or subdomain Things. Homology modeling is used for proteins whose tertiary structure is to be elucidated. Or the homologous or homologous parts of the peptide to the conformation of the homologous structural element. It can be implemented by doing. Homology modeling uses amino acids (or By replacing the other components) with those of the relevant structure to be elucidated, Or reconstitute the whole. For example, complexing with the ζ1 peptide Utilizing homology modeling using the structural coordinates of ZAP-NC Can determine some of its three-dimensional structure. Obtained from evaluation of NMR data Determines the three-dimensional structure of SYK-C complexed with Thr-pTyr-Glu-The-Leu peptide A set of coordinates is shown in Annex IV. These coordinates are shown in Annex I-III Similar to ZAP-NC coordinates, can be used for storage, display, operation, and other uses Noh.   Thus, the crystalline material of the present invention can be used for other members of the ZAP-70 family of proteins. The structure of the bar, especially SYK, and at least one SH2 domain and binding A peptide (ie, a non-SH2 polypeptide), wherein the binding polypeptide is ZAP-NC or Or part of the inter-SH2 binding domain of SYK-NC (preferably at least 6 Amino acid), or at least about 25% peptide sequence similarity, or preferably Used to elucidate the three-dimensional structure of a newly discovered protein with identity It will be the starting material. Sequence similarity is determined using any conventional similarity matrix can do. For example, Dayhoff, M.O., Schwartz, R.M., Orcutt, B.C.,A tlas of Protein Sequence and Structure  1979, 5, Suppl. 3, 345; Greer, J. ,J . Mol. Biol. 1981153, 1027; and Gonnet, G.H., Cohen, M.A., Benner , S.A.,Science 1992, 256, 1443. At least one SH2 domain, Z Homologous to the AP or SYK interdomain linker, i.e., at least two Non-SH2 domain peptide sequence with 5% peptide sequence identity or similarity The protein they contain together is a ZAP family member for the purposes of this disclosure. Members.   To further illustrate, a structure defined by machine-readable data is The ability to associate with chemicals may be assessed by computer. Used here "Chemical substance" means a chemical compound, a complex of at least two chemical compounds. , As well as fragments of such compounds or complexes.   For example, a ZAP family protein, or part or co-complex A first set of machine-readable data defining a 3-D (three-dimensional, or three-dimensional) structure is A second set of machine readable defining the structure of a chemical or moiety of interest Along with the data, if the chemical or part is the ZAP family protein or The ability to associate with the moiety or complex and / or the location and And / or a machine programmed with instructions for evaluating orientation. Use and combine. Such methods involve the use of ZAP family proteins and such Provides insight into the location, orientation and energy of the association with the chemical.   Chemicals that can associate with ZAP family members Inhibits its interaction with natural ligands for May interfere with the mediated biological function. In the case of ZAP-70, such organisms The biological function involves the activation of T cells during the immune response. Such chemicals have potential It is a drug candidate.   A protein structure encoded by data can be used to visualize the structure And a graphic format that allows for a visual examination of the structure and its association with chemicals. Can be displayed in the same format. Alternatively, more quantitative or computer A data-like method may be used. For example, a chemical substance is described here (here). One of the present invention for assessing the ability to associate with any molecule or molecular complex The method includes the following steps: a) Using computer means, the chemical and its Fitting between binding pockets or other surface features of the molecule or molecular complex B) Analyze the results of this fitting operation and The association of the substance with the binding pocket is quantified.   The present invention further uses the structural coordinates of the crystalline material of the invention, or a portion thereof. Inside the three-dimensional structure, preferably inside the ligand binding site (site) or Identifies adjacent reactive amino acids such as cysteine residues; water accessible Such as surfaces that contain functional or space-filled van der Waals surfaces of all atoms Formation and visualization (imaging) of molecular surface; surface characteristics of the protein or complex Calculation and visualization of the size and shape of features such as ligand crystalline pockets A position inside the three-dimensional structure, preferably inside or adjacent to the ligand binding site; Localization of potential hydrogen bond donors and acceptors in the interior; Hydrophobic and hydrophilic, preferably inside or adjacent to the ligand binding site Region calculation; and selected functional groups of interest (eg, amino, hydroxyl, Carboxyl, methylene, alkyl, alkenyl, aromatic carbon, aromatic ring, hetero Aromatic ring, substituted and unsubstituted phosphoric acid group, substituted and unsubstituted phosphonic acid group, And unsubstituted fluoro and difluorophosphonic acid groups) Calculation and visualization of the area on or adjacent to the energy protein surface To provide Of proteins and their potential ligands Using the techniques described above to characterize interactions Of compounds that can specifically covalently bind to reactive amino acids (eg, cysteine) Alternatively, select and select the surface features of the protein (a set of (Eg, size, shape, charge, hydrophobic / hydrophilic, hydrogen bonding) Can be designed or selected. Construction Using the coordinates, the orientation and binding of a ligand to the protein in a complex state You can also predict or calculate numbers or relative affinities and use that information Can be used to design or select compounds with improved affinity.   In such cases, ZAP family proteins, or parts or complexes thereof The structural coordinates are machine readable, with instructions for performing the desired operation. And any necessary additional data (eg, potential ligand or Data that defines the structural and / or functional characteristics of the part, various amino acids (Such as data defining the characteristics of the acid molecule).   One method of the present invention provides a method for binding to ZAP family proteins. It provides for selecting compounds from a database of chemical structures. This method Structural coordinates of the crystalline material of the invention, for example, ZAP family proteins or one of them. Coordinates that define the three-dimensional structure of the part. One point that associates with the three-dimensional structure Or with respect to the advantage of interaction with two or more functional groups. Then, this feature One or more that have a tendency to favorably interact with the protein based on the Search chemical structure databases to find candidate compounds containing functional groups. The Compounds with structures that best match the point of favorable interaction with the steric structure are Identified.   Performing such a search with the aid of a computer is not required, but Are often preferred. In that case, ZAP family protein or a part thereof Or a first set of machines that define the 3D (steric) structure of a protein-ligand complex Define readable data as one or more moieties or functional groups of interest Together with a second set of machine-readable data, Instructions to identify preferred locations for favorable interactions are programmed Using a machine that is The third set of data, ie, the relationship between proteins and functional groups Data defining the location of the advantageous interaction is thus created. This third set of data Data is then combined with a fourth set of data defining the 3D structure of one or more chemicals. Together, each functional group is best located at its location of favorable interaction with the protein. Instructions to identify chemicals containing functional groups arranged in a Combine using a programmed machine.   A compound having a structure selected or designed by any of the aforementioned means, Binding ability to ZAP family proteins, ZAP family proteins Or the ability to inhibit binding to a non-natural ligand, and / or Tested for its ability to inhibit biological function mediated by family members Good.   The present invention also provides compounds capable of binding to ZAP family proteins. Also provided are pseudopeptide (peptidomimetic) methods for counting. One such The method is based on ZAP family proteins or their A three-dimensional description based on coordinates defining a three-dimensional structure is graphically displayed. Ligan The interaction of each part of the protein with the protein can be used to identify candidate parts for replacement. Characterize for One or more of the ligands interacting with the protein Replacement part selected based on knowledge of one or more candidate replacement parts And / or allow for additional interaction with the protein Another moiety may be added to the ligand to achieve   From the computational approaches and structural insights disclosed here, the ZAP family -Binds to proteinsDroppedAbility or substantialUnfortunateDesign of active molecules Or identification is also possible. To do this, the same applies to the data described here. Apply modeling and computer processing methods, but with the opposite of the goal, That is, a substantial binding affinity for one or more ZAP family membersChipped Compounds may be designed or identified. It is the ZAP family Research aimed at finding inhibitors of SH2-mediated interactions other than those mediated by members Can be useful. The goal of such research is that in this case undesirable side effects Lack of ZAP family-mediated activity, such as potential immunosuppression It is an inhibitor of other SH2-mediated interactions.   Compounds first identified by any of the methods described herein are also included in the invention. Is done.BRIEF DESCRIPTION OF THE FIGURES FIG. 1A   The N-terminal SH2 domain (ZAP-N) (P-C) to the right of the figure, and the inter-SH2 domain region in the middle toward the bottom of the figure. ZAP-NC and の₁The overall fold of the complex with the peptide (overall fo ld) Main chain ribbon display. Secondary structural elements are agreements in the SH2 domain²⁰Table with symbols according to It is shown. Includes the alpha carbon trace of the peptide. With ZAP-N Symbolizes all elements, and ZAP-C symbolizes only the center sheet and helix. Have been. The ends of proteins and peptides are indicated by N and C. The loop region is named by the secondary element to which it connects. That is, the BC loop and the B chain Connect to C chain. The N-terminus of the peptide is on the right side of the figure. This peptide is a T cell It consists of 19 residues starting from residue 48 of the mature ζ subunit of the receptor (TCR). Phospho Tyrosine is at relative positions 4 and 15. The main chain atom of each pYXXL motif Defining the plotted least squares plane yields a pair of planes at an angle of 120 °. Because the orientations of the two SH2 domains are staggered, these pYXXL motifs Separated by "S" shaped segments of peptides. This sequence contains ζAsp 8 residues and ζArg 1 It contains close to one complete turn of the α-helix between the two residues. Each pY And pockets for pY + 3 residues are visible. N terminalζ₁Residue surrounded by ζpTyr 4 Is noted. This figure was made with Sybyl (Tripos). Two Electron density map of the area near the interface between the SH2 domains of the same in 1.0s 2 | F for data between 15.0 and 1.9 mm_o|-| F_c| Calculate by coefficient be able to.FIG. 1B   The BC loop from βB5 to βC3 of ZAP-N is inserted into Lck SH2: middle T Coalescence²⁰And the two structures are fitted according to the secondary structural elements. You can check the relative position of the BC loop when you turn it on. The main of these loops The superposition of chain atoms results in an r.m.s. deviation (root mean square deviation) of 0.65 °. As well Results are also observed in the ZAP-C BC loop. Two isolated phosphopeptides In the complex with the SH2 domain, the BC loop directs nearly half of the hydrogen bond to a phosphate group. give. For both SH2 domains of ZAP-NC, the BC loop contains several waters. Has been extended to mediate the contact between the loop and the phosphate group. Extended Conformation is also observed for uncomplexed SH2 domains. And a complex with a peptide in which the phosphate group is replaced by a phosphonate group Has been¹⁴. This loop moves the hinge around βB5 and βC3 residues. Causes rearrangement (position change). The BC loop of ZAP-C has been extended, and ZAP- N hindered movement around BC due to its interaction with ZAP-C. receive.FIG.   Sequence alignment diagram of specific SH2 domain [SEQ ID NOS: 21 to 25]³³. Part surrounded by bold line The minutes indicate the segments used to measure the "complete" backbone r.m.s. Missing parts are excluded from calculation due to the presence of voids in one or more of the sequences Is done. The symbols below the alignment diagram indicate structurally conserved areas and have been reported so far. Nomenclature²⁰Is used. These areas are used to calculate the "core" r.m.s.deviation Was done. The part surrounded by a thin line in ZAP-N and ZAP-C represents the secondary structural element of ZAP-NC. Show. Src = avian Src; Lck = human p56-Lck; Syp-N = mouse Syp phosphatase (PT P1D) N-terminal SH2; ZAP-N (C) = N- or C-terminal SH2 of human ZAP-70.FIG.   Phosphotyrosine binding site. This phosphotyrosine residue facilitates direct comparison For the sake of simplicity, the same orientation is used in each figure. A. ΖpTyr 4 meeting with ZAP-C Residues selected for直接 Direct hydrogen bond to phosphate group of pTyr 4 Have been. Crystallographic water is indicated by ●. 551,555 And 556 water had a bridging contact between the phosphate group and the SH2 domain. The water of 622 is ζ₁To form a bridge. B. Close association with ζpTyr 15 Selected residue in the form. Like A, the dashed line indicates a direct hydrogen bond to the phosphate group. Tyr 238 and Lys 242 from ZAP-C complete hydrogen phosphate bond network I do. Water is indicated by ●, all of which are salts of phosphotyrosine into the SH2 domain. Involved in bridge formation. For clarity, some of the pockets Residues have been omitted. C. The interface between SH2 domains has a large hydrogen-bonded network. Contains. The interaction involving ζpTyr 15 has already been described elsewhere in this specification. Was. There are also some hydrophobic contacts, the most stimulating of which is Phe Side chains of 229 (ZAP-CβF), Tyr 238 (ZAP-CαB), and Thr 227 (ZAP-C EF loop) Arg 41 (ZAP-NBC loop) protrudes into the small hydrophobic recess formed by This is the part that is out. Arg 41 also has three hydrogen bond contacts to ZAP-C I have. This is an artificial parallel sheet with the F chain of ZAP-C. First as three residues in the BC loop of ZAP-N to form. These three hydrogen bonding contacts Only one of the contacts exclusively contains the main chain atom.FIG.   Activated T cell receptor ζ₁Explanatory drawing of ZAP-70 combined with the subunit. Left: T cell receptor (TCR) is a major histocompatibility complex (MHC) on antigen presenting cells The activation of T cells is initiated by associating with the peptide antigen bound to. TCR-MHC association leads to phosphorylation of the T cell receptor subunit on tyrosine in ITAM. Stimulate (most likely by Src family PTK, Lck or Fyn) . ZAP-70 binds to phosphorylated ITAM via its tandem SH2 domain (amino acids 1-259), The N-SH2 domain binds to the C-proximal pYXXL motif and the C-SH2 domain is N-proximal Binds in an orientation that binds to the pYXXL motif. Interdomain B (amino Acids 260-310) and the rest of ZAP-70, called the catalytic domain (amino acids 311-620). The main proposed location is shown. ζ The subunit is disulphide Two ZAP-70 molecules bind to the activated TCR complex because they exist as union dimers I could do that. Right: Doubly-phoshorylatedζ₁Combination with ITAM Explanatory drawing of a complex between two SH2 domains of ZAP-70 in a somatic state. SH2 of ZAP-70 Main is ζ₁Make multiple contacts with the peptide. Key determinants of binding are within ITAM Are the phosphotyrosine and leucine residues of the two pYXXL sequences. This structure has Unique to pY of the C-proximal pYXXL motif at the interface between two SH2 domains A perfect binding pocket appears. Furthermore, this crystal structure shows that interdomain A It also shows that a coiled coil helical structure is formed. This domain is Positions Two SH2 Domains for Meeting with ITAM and Interacts with Structural Changes Domain B and / or after engagement with the receptor May be involved in communicating to the main.FIG. Is a measure of the data measured by fluorescent polarization. Dual phosphorylation by associated Scatchard plot ζ-1 shows the binding curve of the ZAP-NC complex.FIG. Into a gridded box for receptor site mapping Shown is the contained ZAP-NC: ζ1 complex. Only the main chain and peptide of ZAP-NM are shown I have. The colors have the following meanings: oxygen and nitrogen are red and blue, respectively; Carbon atom yellow; N-terminal SH2 domain, inter-SH2 spacer and C-proximal SH2 domain The main carbon atoms are cyan, orange and green, respectively. The box is the space occupied by the peptide Note that it includes the space occupied by several interfacial regions of the SH2 domain. I want to be.FIG. Shows a representative part outline map of ZAP-NC. Figure 7A: Ring at -10 kcal / mol Receptor map of specific region for ZAP-NC + aminocation probe formed H. Pockets on the protein surface are indicated by solvent accessible surfaces. FIG. 7B: Individual amino acids very close to this contour map are clearly shown. ring The position of the segment is based on the hydrogen bond donor and the backbone chain of Arg192, Glu194 and Thr197. Strong interaction between the rubonyl oxygen atom and the side chains of Gln 195 and Tyr 198 Indicates that the utility will be available.FIG. Indicates a computer system.FIG. Indicates a storage medium of the present invention.Detailed description of the invention A. Introduction: ZAP-70 is immune by signaling through the associated T cell receptor complex Epidemiological response is mediated   T cell recognition of antigen presenting cells ultimately alters gene expression, secreted mediator Starting point for a series of intracellular processes that result in the production of Becomes^1,2. This recognition is mediated by the T cell receptor (TCR). TCR is antigen binding CD3 complex of subunits α and β, δ-ε and γ-ε heterodimer, It consists of a homodimer. Intracellular of each subunit, with the exception of α and β The part is a motif YXX (L / I) X_(7-8)1 to containing YXX (L / I) (X can vary) Has 3 peptide sequences^Three. Following receptor stimulation, these immune receptors Tyrosine activation motif (immunoreceptortyrosineactivationmotif, ie I (TMA) becomes a phosphorylated form of tyrosine residue, and this modified form Provides a binding site for the signaling protein.   TCR has no intrinsic protein tyrosine kinase (PTK) activity, but Src PTKs, both members of the SYK / ZAP-70 family, Related to the functioning of the body^Four. Current evidence suggests that Src family kinases Shows phosphorylation of TAM^Four. ZAP-70 then binds the ζ and CD3ε chains. Associates with dual-phosphorylated ITAM via its SH2 domain^Five, Itself is an early T Phosphorylated during cell activation⁶. ZAP-70 (ζ associatedprotein) is T-thin 70 kDa protein tyrosine expressed only in cells and NK cells (natural killer cells) Is a kinase⁷. ZAP-70 may play a critical role in T cell activation Are known. Genetic alteration of the ZAP-70 gene that results in reduced expression of human ZAP-70, CD4⁺Prevents T cell antigen activation, CD8⁺Inhibits T cell maturation and severely complex Causes immunodeficiency^8,9. Binding of ZAP-70 to the TCR blocks ZAP-70 association with the ζ chain. Since the blocking peptide also inhibits T cell signaling events, Is believed to be essential to us^Ten. For these reasons, ZAP-70 is a new It is an ideal target for the development of immunosuppressive therapies.   The first 259 residues of ZAP-70 are two SH2 domains connected by a 65 residue segment. Consists of the main, followed by the second connecting region and the catalytic domain⁷. SH2 Dome Inns consist of about 100 amino acids. Specificity of tyrosine phosphorylated protein Their role in recognition is essential for a variety of intracellular signaling events. (Recent^11,12Outlined). Several SH2 domains have been isolated (isolat ed) Short peptides containing phosphotyrosine (pY) when expressed as proteins Has been demonstrated to retain the ability to bind with high affinity. Isolated SH2 domain The selectivity for in is determined by the residues adjacent to the C-terminal side to phosphorylated tyrosine (pY + n). Depends on recognition. Several isolated SH2 domains (ligand bound and unbound) Three-dimensional structure)^13,14And one SH3-SH2 complex^FifteenAnd one SH3-S H2-SH3 protein¹⁶Was determined by X-ray crystallography or NMR. T To bind to CR, ZAP requires that both its SH2 domain be present and functional and that ITAM Need to be phosphorylated on both tyrosines^17-19.B. Structure determination   Despite the central role of ZAP-70 in the immune response, the tandem SH2 region of ZAP-70 Like conformation when ITAM interacts with ITAM in interactions required for its biological activity I didn't know anything about the child. In principle, X-ray crystallography I could handle the problem. However, other ZAP families like ZAP-70 and SYK Any tandem despite important biological functions mediated by Lee members Disclose some X-ray crystallographic data and other information about the three-dimensional structure of the SH2 domain Reports that appropriate crystals were or could be obtained There were no reports disclosing that. Even if a crystal is obtained, it will be used next The conformational data for each possible SH2 domain is required for structural refinement. Because it cannot be processed by the least squares minimization method Would not have been useful, at least in part, to elucidate its tandem structure. U.   Nevertheless, we found that the more N-terminal SH2 domain and the more C-terminal A region of human ZAP-70 that spans both the binding SH2 domain and the binding region ("ZAP-NC") Successfully produced a protein containing the peptide sequence of The crystals are crystallized in an unliganded form and in the form of a complex with various ligands. I got it. Using such a material, the present inventors have developed a ZAP- The three-dimensional structure of NC was elucidated. All disclosed here due to our success disclosed here Using materials and methods such as those described above, tandem SH2 domains, especially ZAP and SYK Proteins, including those of the various ZAP family members, Whether in form or not, it can be produced in a stable form and purified and crystallized. Now, we can say that we can determine their three-dimensional structure.material   As mentioned in either, ZAP-NC binds two src homology 2 (SH2) domains. One of the many proteins it contains. SH2 in a protein sequence The presence and boundaries of the main characterize amino acid sequence homology to known SH2 domains. Can be identified using a computer alignment program You. Generally, the SH2 domain of Src (between 140 and 255 amino acids) is used for this analysis. However, SH2 domains from other proteins can also be used. You. The alignment method typically used for such programs is Needleman-Wunch alignment. You. For example, “One applicable to similarity search of amino acid sequences of two proteins” General Methods "Needleman, S.B., Wunch, C.D.,J . Mol. Biol. 1970,48, 443-453 reference. The number of proteins identified as having an SH2 domain is growing significantly, Some contain multiple SH2 domains. For example, the column SH2 area ZAP-70 (strand spans amino acids 1-259), human SYK (str spans amino acids q-z), P Present in LCγ, P13K, rasGAP, SH-PTP1, and SH-PTP2.   We compared ZAP-NC with glutathione-S-transferase (GST) fusion protein. Expressed as quality. CDNA encoding residues 1-259 from human ZAP-70^Ten, PGEX2T Expression vector⁴¹Cloned intoE . coli BL21 orE . coli Traits in B834 Converted. The resulting construct contains a -LVPRGS- sequence with a thrombin cleavage site. GST-ZAP-NC fusion proteins linked by different polypeptide segments did.E . coli 834 auxotrophs⁴²Was replaced with selenomethionine in a defined medium. Used with sex methionine to produce selenomethionyl (SeMet) ZAP-NC. G The ST-ZAP-NC fusion protein is separated using glutathione agarose and then Cut with Rombin. Cleavage results in two polypeptides, GST and ZAP-NC. ZA Is the P-NC polypeptide a linker segment of the pGEX2T expression vector at the amino terminus? They contain two extra amino acids (Gly-Ser). These two extra amino Acid has no functional effect on ZAP-NC binding to peptide ligand It has been shown. ZAP-NC is bound to a phosphotyrosine agarose column and salt gradient ZAP-NC was separated from GST by eluting with (salt gradient). That Thereafter, ZAP-NC was further purified on a phenyl sepharose column. ZAP-NC: Peptide The ligand complex was prepared by mixing a two-fold excess of peptide with purified ZAP-NC and then mixing the mixture. -Chromatography using a Superdex 75 gel filtration column To generate the following. Purified ZAP-NC: Combined fractions containing peptide complex And used for subsequent crystallization experiments.   The N- and / or C-terminal truncations containing only within the SH2 homology boundary are truncated. Other ZAP-NC proteins, including the ZAP-NC protein taken, can also be used. Can be. In addition, the protein, up to the entire ZAP-70 protein (amino acids 1-620) Includes additional amino acids that extend to include additional domains (spacer B) It may be extended on the C-terminal side as described above. In addition, contains proteins such as human SYK Other tandem SH2s, especially from human and non-human ZAP family members Regions can also be prepared and used in a manner similar to that described herein. other It will be further understood that the expression system of can also be readily employed. For example, T7, Ma The encapsulated or cleaved epitope contains a lactose-binding protein fusion (MBP). Using the tandem SH2 protein together with labeling (His6, HA, myc, Flag)E . coli It may be produced in. For example, pVL1393 or a derivative can be epitope-tagged. Tandem SH2 protein fused (or not) to a fusion partner such as GST The baculovirus expression used for E. coli can also be employed. Mammals, yeast Conventional materials and methods for expression in mother or other cells can also be used. Wear.   Peptide ligands for co-crystallization with ZAP-NC or other tandem SH2 proteins Is, for example, the 細胞 1, ζ2, ζ3, ε, γ, or δ subunit of the T cell receptor. Or native, such as an ITAM sequence from the β or γ subunit of the IgE receptor Contains peptide sequences based on the ITAM sequence, which can be prepared by standard methods You. In most cases, such a ligand is a minimal ITAM sequence of 15 amino acids YXXLXXXXXXXYXXL [SEQ ID NO: 1], and one or both of the N and C termini Alternatively, it may further contain an amino acid. The N-terminus may be a free amino group, or It may be modified (eg, amidated). Similarly, the C-terminus is May also be amidated or otherwise modified. Tyrosine (each) It may be phosphorylated (pTyr). Alternatively, instead of pTyr, difluorophospho Tyr, phosphonomethylphenylalanine, hemi (semi) phosphorylated Tyr, or other A pseudo pTyr can also be used. The ligand is an amino acid relative to the native ITAM sequence. It may contain acid substitutions, insertions or deletions. Furthermore, the hybrid peptide Non-peptide ligands and non-peptide ligands can also be used. Of such ligands An example is shown in Table 1.Crystallization   Crystallization experiments were performed using the Crystal Screen 1 kit (Hampton Researc) for ZAP-NC crystals. h, Riverside, CA), starting with a sparse matrix screen This was done using the tanning technique. Crystals containing SYK-NC were obtained as described below. Was. In the case of ZAP-NC, it is stabilized in 0.5 N NaCl and subsequently dialyzed before crystallization experiments The best results were obtained using proteins which had been de-salted. That No special handling of the species was required for SYK-NC, but other tandem SH2-containing It may also be useful for quality.   For example, double phosphorylated 19-merζ₁Complex with ITAM peptide (ligand 5, Table 1) Crystals of ZAP-NC were grown from polyethylene glycol (PEG) 4000. So Has been elucidated by multiple isomorphous replacement with a resolution of 1.9 mm. Previously determined Cannot be solved only by molecular replacement using the coordinates for the SH2 domain Met. This is with other SH2 domains and between two ZAP domains. This is because sequence identity is low and there is an interdomain region of 65 residues. Crystallization Details of data collection, multiple isomorphous replacement (MIR), and refinement are provided below.   Specifically, double phosphorylation₁ZAP-NC complexed with peptide was added to 20 mM Tris PH 8.5, 30 mg / ml in 200 mM sodium chloride and 20 mM dithiothreitol Concentrated. This complex was treated with 4 mM lead trimethyl acetate (TML). 20% PEG 4 13.5 mg / ml on a precipitant solution of 000 and 20 mM dithiothreitol. Conjugate and 10% PEG 4000 in 50 mM sodium citrate, 100 mM ammonium acetate In 0.005% sodium azide and 20 mM dithiothreitol, pH 6.2 The vapor diffusion in the hanging drops contained Obtained. This crystal has a monoclinic system (P2₁, a = 50.11, b = 63.37, c = 54.00 °, β = 114.44 °).   In another of our experiments, similar conditions were used to include two molecules per asymmetric unit. Double phosphorylation was found₁Obtain crystals of ZAP-NC complexed with peptide I was able to do it. This crystal is also monoclinic (P2₁, a = 65.17, b = 62.00, c = 78.67Å, β = 111.32 °).   As detailed in Experimental Examples III (A) and (B), ligand bound and unbound Crystals of the combined ZAP-NC and SYK-NC proteins were obtained under similar conditions. Data collection   Diffraction data was obtained from graphite monochromated Cu Kα X-rays, using Rigaku R-AXIS II area scan. Collected using a dispenser. Diffraction data is collected as a 2 ° vibration image, DENZO⁴³so It was converted to integrated intensity. ROTAVA scaling parameters for each image TA⁴⁴Calculated by AGROVATA⁴⁴Applied in. Each set of data is based on a lead-containing ZAP-NC All SYK and ZAP except for proteins containing crystals were collected at room temperature. Crystals (with or without bound ligand) were collected at -160 ° C.MIR Analysis and refinement   SeMet ZAP-NC was crystallized by TML under the same conditions and data was collected. Lead and And the positions of the selenium atoms were determined from the difference Patterson function. Which set of data Also included was the measurement of anomalous variance. MLPHARE program⁴⁴With heavy atom parame When the data was refined, a phase was obtained at 2.8%. This MIR phase is referred to as DM program⁴⁴Solvent flattening / histogram matching g / histogram mapping) and a phase extension to 2.0 mm. Improved. Calculate the electron density map for each phase of MLPHARE and DM, and Ram⁴⁵Created a polypeptide chain model. Numbers using partial models and experimental phases SIGMAA for implementation of cycle phase combination⁴⁴It was used. X-PLOR⁴⁶using, Least square refinement by simulated annealing (SA method) . In this model, all the residues from Asp 3 to Asn 256 of the protein, zeta (ζ) -1 Of all 19 peptide residues and 113 water molecules, plus one lead and three Has a ren atom. TML binds to Cys 117. See Table 2.Structural coordinates, their storage and use   The structural coordinates of the crystalline material of the present invention can be obtained as detailed herein. You. By way of example, Annex I, Annex II, and Annex III include ZAP-NC: # 1 "monomer" (1 molecule complex per unit cell), ZAP-NC: $ 2, and ZAP-NC: ζ1 PDB for crystalline materials containing "dimers" (2 molecules of complex per unit cell) Indicates the structure coordinates in format.   The present invention relates to the structural coordinates set forth in Annex I, II or III, or the columns therein. The root mean square deviation from the main chain atom of the listed amino acid residue is about 1.5 Coordinates that are less than Å, preferably less than about 1Å, and even more preferably less than about 0.5Å A crystalline material containing a ZAP family protein having a region characterized by Include all.   As will be appreciated by those skilled in the art, various computer solutions Analysis, a protein (or part or complex thereof) and ZAP-NC or other ZAP family proteins as described Or the complex, the degree of similarity between the three-dimensional structures it can. This type of analysis is based on the QUANTA Molecular Similarity app. Application (Molecular Simulations, Inc., Waltham, MA), version 3.3 Using a commercially available software application such as Id, Vol. 3, pages 134-135.   In the Molecular Similarity application above, the same structure between different structures Between different conformations of, and between different parts of the same structure. The procedure used in Molecular Similarity to compare structures involves the following four steps: Divide: (1) Load multiple structures to compare; (2) Atomic equivalence between these structures Provision of (atom equivalence); (3) execution of fitting work; and (4) solution of results Analysis.   Each structure is identified by a name. Target one structure (ie, fixed All the remaining structures are working structures (ie, moving structures). QUANTA The atomic equivalence in is defined by the input by the user. Therefore, we tamper equivalent atoms for all conserved residues between the two structures being compared. Are defined as carbon main chain atoms (N, Cα, C and O) Consider only the rigid fitting operation.   When using the rigid fitting method, the best fit with the target structure Translate and rotate the working structure to obtain. This fitting operation Fit root-mean-square difference of fit over a specific pair of equivalent atoms to an absolute minimum The least-squares fit to calculate the optimal translation and rotation to apply to the moving structure Use the singing algorithm. This number (Å) is reported by QUANTA ing.   For the purposes of the present invention, the relevant structural coordinates of the protein or complex of the present invention (Eg, coordinates listed in Annex I, Annex II or Annex III) Using chain atoms-When superimposed, main chain atoms of conserved residues (N, Cα, C, O) ZAP family proteins with a root mean square deviation of 1.5 の or less -The structural coordinates of all sets of a part of the protein or its molecular complex are the same. It is thought that. More preferably, the root mean square deviation is 1.0 ° or less. Most favorable More preferably, the root mean square deviation is less than 0.5 mm.   The term "root mean square deviation" is the arithmetic mean of the square of the deviation from the mean. Means square root. This is the deviation or variation from the trend or target Is an index that expresses For the purposes of the present invention, “root mean square deviation” Defined by the structural coordinates of Document I, Annex II or Annex III, and To As described, certain proteins from the backbone of the proteins of the invention, such as ZAP-NC Specifies the quality backbone variation.   The term "least squares" means that the best estimate of a value is the deviation of the observed value. This means a method based on the principle that the sum of squares is the value when the sum is the minimum.   Structural coordinates generated for the crystalline material of the present invention, for example, ZAP-NC, # 1, # 2 Or any of the various types indicated in Annex I, Annex II or Annex III. To use the structural coordinates of the complex, these coordinates must be displayed as a three-dimensional shape. Play, convert to 3D shapes, or manipulate them in other ways It is often necessary or desirable. This is typically a set of structural coordinates Like a program that can create a three-dimensional drawing of a molecule or part of it from This is performed using a commercially available software.   As an example, a computer for viewing protein structures or for other manipulations A non-restrictive list of data programs includes:   The storage and transfer of structural coordinates for the crystalline materials of the present invention and the Data storage material encoded with machine readable data for use in programming Provided, a machine-readable storage medium comprising: This data is The machine on which the instructions for use are programmed, for example, one of the types listed above. Or a computer loaded with two or more programs, It is possible to display a graphic 3D display of any molecule or molecular complex described here. Wear. Conventional computer readable storage media with data storage materials include Computer hard drives, floppy disks, DAT tapes, CD-ROMs, Other magnetic, magneto-optical, optical, floptical, and computer Other media that may be adapted for use with   Even more preferred are proteins of the ZAP family, such as ZAP-NC. Or SYK-NC or its structural coordinates, especially Annex I, Annex II or Generic Document III (or a derivative thereof such as zapNC-z1.pdb described elsewhere here) ) ZAP-NC: {1 or ZAP-NC: {2} Structural coordinates ± 1.5 mm or less A molecule defined by the root mean square deviation from the main chain atoms of the protein's amino acids Or machine readable, capable of displaying 3D graphics of molecular complexes Noh Data storage medium. A representative embodiment of this aspect of the invention is described in Annex I, Appendix Contains the coordinates of Generic Document II or Annex III, preferably in PDB format 3.5-inch diskette (floppy Disk), DAT tape or hard drive.   In another aspect, the machine-readable data storage medium comprises Annex I, Annex II Or the hull of the structural coordinates described in Annex III (also or a derivative thereof) Data storage material encoded with a first set of machine readable data including a Rier transform It has. This first set of data contains instructions for using this data. Machine that uses a rammed machine to include an X-ray diffraction pattern of a molecule or molecular complex Combined with the second set of readable data, the second set of machine readable Can be determined at least a part of the structural coordinates corresponding to the data.   FIG. 8 shows one version of these aspects. The illustrated system has a central processing unit. Unit ("CPU"), working memory (this is, for example, RAM <random access memory> Or “core” memory), mass storage memory (one or more Disk drive or CD-ROM drive), one or more cathode ray tubes (" CRT ") display terminal, one or more keyboards, one or more With two or more input lines (IP) and one or more output lines (OP) All of which are interconnected by a conventional bidirectional system bus.   Input hardware B, connected to computer A by input lines, It can be composed of devices. The machine readable data of the present invention is Or using one or more modems connected by a dedicated data line L Is also good. Alternatively or additionally, the input hardware may be a CD-ROM drive or May include a disk drive D. Using a CRT display terminal together with the keyboard Can also be used as input means.   Output hardware connected to computer A by an output line is also conventional. Device. As an example, the output hardware Using a program such as QUANTA described herein, the protein (or its Part of the CRT display terminal for displaying graphical depictions You. The output hardware is also programmable so that it can produce hardcopy output. Linter and a disk drive for storing system output for later use. They may have a live.   During operation, the CPU integrates the use of various input and output devices and Control access to data from and to working memory In addition, the order of the data processing steps is determined. Of the machine readable data of the present invention. Many programs can be used for processing. Examples of such programs are: As discussed herein, computer processing for drug discovery is being considered. For each component of the hardware system of FIG. It is specifically mentioned as appropriate in the description of the medium.   FIG. 9A illustrates a machine-readable version that may be performed by a system such as the system of FIG. 1 shows a cross section of a magnetic data storage medium 100 that can be encoded with useful data. Medium 100 can be a conventional floppy disk or hard disk, A suitable substrate 101 and a suitable coating 102 on one or both sides which may be conventional. You. Coatings have magnetic domains whose orientation or polarity can be changed by magnetism. Invisible). Media 100 is a disk drive or other data storage It may have a hole (not shown) for receiving the axis of rotation of device 24.   The magnetic domains of the coating 102 of the medium 100 are polarized or oriented, Machine-readable as described herein for execution by a system such as The retrievable data is conventionally or otherwise encoded.   FIG. 9B illustrates an example of a system that can be implemented by a system such as the system of FIG. Optical readout capable of encoding machine-readable data or sets of instructions, such as 2 shows a cross section of a possible data storage medium 110. The medium 110 is a conventional compact disc. Read-only memory (CD-ROM) or optical readable, magneto-optical writing possible A rewritable medium such as a simple magneto-optical disk may be used. The medium 110 is preferably A suitable substrate 111, which may be a conventional one, usually one side of the substrate 111. A suitable coating 112.   In the case of a CD-ROM, the coating 112 is reflective and encodes machine-readable data. Many pits 113 are carved. Laser light is reflected from the surface of the coating 112. By doing so, the arrangement of pits is read. Preferably a substantially transparent protective coating A 114 is provided on the coating 112.   In the case of a magneto-optical disk, the coating 112 does not have the pits 113, and the laser (see FIG. When heated above a certain temperature (not shown), its polarity or orientation is It has a number of magnetic domains that can be varied. The orientation of the magnetic domains is It can be read by measuring the polarization of the laser reflected light. Top with magnetic domain array The data as described above is encoded.C. ZAP-NC: Description of zeta ₁ tertiary structure   The first ITAM of the ζ subunit of the TCR (ζ₁) With a 19-mer peptide derived from the sequence The elucidation of the X-ray crystal structure of the tandem SH2 region (ZAP-NC) of human ZAP-70 We perform the first conformational characterization of this protein: ligand complex I was able to. This complex is an elaborate multiple between the peptide and both SH2 domains. Includes contact. The 65-residue inter-SH2 region is linked to the α-helix coiled coil. And help to form an interface between the two SH2 domains. Structure shown here IsBothSH2 domain recognizes phosphotyrosine in the second pYXXL motif It shows the surprising fact that it contributes. In this experiment, the SYK / ZAP family Demonstrates the first structural insights into the rotatory tyrosine kinase and details the TCR. This is the first time we have seen intracellular components.General topology   The first 259-residue segment of ZAP-70 has two SH2 domains in one helix Consists of those joined by regions. The entire fold is Y-shaped, with two SH2 domains Constitute one branch at the top of Y, with a 65 amino acid domain in between. Form the Y axis. The fragment of ZAP-70 used to determine the crystal structure Ends before the Nase domain. Therefore, we combine these two SH2 regions, ZAP-N and ZAP-C for ZAP N-terminal SH2 and ZAP C-terminal SH2 respectively Called. Each of these two SH2 domains, as previously reported, eg, v-Src¹³And p56-Lck²⁰There is a high structural similarity between them. Individual ZA Each P SH2 domain has a central antiparallel β sheet with two α helices Have. The region between SH2s begins with the β-strand, which is a continuation of the ZAP-N central sheet. This Following this are two interlaced to form a coiled coil motif An antiparallel α helix. Two SH2 domains are partially staggered And their central β-sheets are separated by about 29 ° at an angle of about 52 °. This distribution Position allows direct contact of the two SH2 domains essential for peptide binding. You. The central β-sheet of ZAP-C has several residues in ZAP-N, including some side-chain contacts. It is extended by an apparent hydrogen bond with the group.   ζ₁The peptide spans both sides of the SH2 domain and has both central β It straddles the sheet and makes many contacts with this protein surface. Join direction Is head-to-tail, ie, the N-terminal of the peptide is in contact with the C-terminal SH2 domain. peptide The N-terminal pYXXL segment of is a single library linked to an isolated SH2 domain. Found in phosphorylated peptides^20,21Binds to ZAP-C in a conformation similar to The peptide segment that separates the two pYXXL motifs is highly connected to ZAP-C. I'm touching. C-terminal phosphotyrosine is contributed by both SH2 domains It is joined in the formed pocket. The remainder of the second pYXXL motif is the first Motifs and other complexes^20,21As in.ZAP SH2 domain   Good electron density without breaks in all residues between Asp 3 and Asn 256 It is in. ζ₁The residues Gln 2 to Leu 18 of the peptide are in good electron density and The two terminal residues are weak but at an observable density. About the structural features of the SH2 domain And already defined nomenclature²⁰Was used here for clarity. N-terminal side SH2 Dome In (ZAP-N) the secondary structural elements are preserved, but A, E, F and G Somewhat long for chains. Due to the elongation of these chains, minor β-sheets (previously The sequence corresponding to the reported SH2 domain) is the central β-sequence of ZAP-N. It is an integral part of the More notably, helix A is only three residues long And the helix extends for nearly a full turn due to this part. The C-terminal SH2 domain of ZAP (ZAP-C) also has a small extension to some secondary structural elements. Having. The B, C, and E chains are one or two residues longer. Helicopter Box A also extends by one residue. F chain consists of only 2 residues and replaces FB loop doing. The BC loop of ZAP-C is also growing. Within each SH2 domain and all domains Very high electron densities were observed for a large number of structured water You. The presence of a very large number of observed waters in the phosphotyrosine binding pocket Is unique to ZAP-NC.Spiral interdomain A   The spacer between SH2s begins with a type II reverse turn and has a prominent contact with the A-chain of ZAP-N Followed by a long beta chain, thus forming an extension to the central beta sheet. Hydrogen bond Exists between the main chain atoms of Gln 111 and Tyr 12 and between Gln 111 and Ser 14. water Intervening contacts exist between the main chain atoms of Glu 109 and Tyr 12. Hydrophobic and hydrophobic Sex packing exists between Leu 108 and Tyr 12 and between Pro 110 and Phe 11.   Following this segment is five rotations that extend away from both SH2 domains Α helix (denoted as helix C), which forms the axis of the entire Y-shape of ZAP-NC Has formed. Following this helix is a single one consisting of Leu 133 and Glu134 It is a roll. A second α-helix (helix D) is formed by helix C Curved around the axis. Helix D is distorted and breaks into Pro 147 And a second break occurs at Ala 154 followed by Thr 155-Met 161 residues Short spanning 3_TenA helix follows. Helix C and D are both ZAP- Some hydrophobic contacts to C, most notably the p- of Phe 115 (αC) to Trp 233 Make a stacked arrangement. Some water-mediated hydrogen bonds form helix D and Z Exists between AP-C. These antiparallel helices form a coiled coil structure And has direct contacts between several hydrophobic residues that form its core.   For proteins containing multiple SH2 domains, separating the two domains The length of the region that is located varies greatly. This region may be as short as 10-15 residues (Eg, PLC-γ1, SHPTP-1 and -2), whereby the two SH2 domains are back- Will be back to back. SYK is between SH2 of length comparable to ZAP Domain and exhibits 68% sequence identity with the helical spacer described herein. It should retain the same overall conformation observed in ZAP-NC. Phosphatid The tandem SH2 domain of the p85 subunit of luinositol 3'-kinase (PI 3-K) Is also predicted to form a coiled coil of two antiparallel α-helices A much larger domain of 163 residues^{twenty two}Connected byBinding interaction on complexation ζ ₁   ZAP-NC: ζ₁The complex has both tyrosine residues phosphorylated and the sequence NQLpYNELNL First ITAM-containing segment of human TCR ζ chain having GRREEpYDVLD (SEQ ID NO: 14) (Ζ₁), And contains a 19 amino acid peptide. For clarity, The numbering of the tide residues starts with ΔAsn 1. This ζ₁The binding conformation of the peptide Although there is almost a complete α-helical turn between the bases ζAsn 8 and ζArg12, It's growing a lot. The main-chain conformation for each pYXXL motif is Observed Conformation of a High-Affinity Complex of the SH2 Domain with Peptides^20,21,23 Is similar to   ζExcluding Gly 10ζ₁All residues of the peptide are in contact with ZAP-NC. Peptide The area of the protein interface is 1300Å^TwoThat is all. This interface area is the protein-protein Those that are common in carbon interactions but are generally accepted for their nature of contact^{twenty four}What is Completely different. For example, antibody-antigen complexes or protease: protein inhibitor complexes The interface in the body is usually free of crosslinking water. ZAP-NC and ζ₁Interface with 2 Contains as much as one cross-linking water. The primary protein-protein contact is usually It is a property classified as hydrophobic (hydrophobic bond). In contrast, ZAP-NC and ζ₁ Half of the contact between is through direct hydrogen bonding. The total number of contacts allowed was , From those found for protein-protein structures with similar interfacial areas Quite a lot. The binding of phosphorylated peptides to individual SH2 domains is described Has been described as reminding of "lag"^{twenty one}, This general bonding arrangement It is also present in ZAP-C and ZAP-N. Each socket recognizes a phosphotyrosine residue A highly charged pocket and a second pocket that prefers hydrophobic residues at the pY + 3 position Become.Motif 1 (-pYNEL-) binding is limited to ZAP-C   The amino terminal pYXXL motif of ζ1 associates exclusively with ZAP-C. ζ₁ The first two residues of Asn 1 and ζGln 2 are mostly involved in intrapeptide interactions concern.主 Leu 3 main chain cal, only one contact between Leu 3 and ZAP-NC The hydrogen bond between bonyl and the NH1 of Arg 170 is typical of the pY-1 residue.   ポ ケ ッ ト Pockets for pTyr4 are helix A, B, C and D chains, BC loop Formed by residues from the Hydrophobic contacts include residues from βD, His 210, Tyr 211 and Leu 212 form one edge of the pTyr cavity. To achieve. Also, ζAsn 1 and ζGln 2 of the peptide itself make hydrophobic contact on the other side. Form. The side chain of Leu 212 is twisted away from the pTyr ring and is symmetrically related. (symmetry-related) Packed against Trp 131 from molecule. This Neighbor Trp Is any ζ₁Constitutes the only intermolecular crystal contact with the residue, which is also sparse to ζpTyr 4. In water contact. Direct hydrogen bond contacts to phosphate groups are made with only three residues It is. Arg 170 (αA) and Arg 190 (βB) interact through their terminal nitrogen You. Arg 192 is the only one in the BC loop long enough for a direct hydrogen bond to the phosphate group. And interact through its Nε. Because this BC loop is growing The pTyr binding region resembles a deep groove that follows the AA loop. pY-2 and pY-3 Residues are included as an integral part of the binding site, into which pTyr protrudes. This results in the formation of a channel. In this area, there are five very dense and low temperature factors Water exists and is part of a larger hydrogen bonding network.   As usual for complexes with the SH2 domain^20,21,23, PY + 1 and pY + 2 Residues extend along the surface of the protein. pY + 1 residue (ζAsn 5) Star intermediate T peptide (… pYEEI…) and Lck²⁰And v-Src^{twenty one}With the SH2 domain Make contacts similar to those found in the complex. pY + 2 residue (ζGlu 6) Away from the surface of the substrate.   ポ ケ ッ ト The pocket surrounding Leu 7 (pY + 3) is very deep, βD, EF loop, helix It is formed by residues from the B and BG loops. The size of this pocket Leu 7 contains only Tyr 211, Ile 223, Gly 226, Gly 245, and Leu 246. Or only 5 residues make direct contact. The depth of this pocket is dependent on many other SH2 domains Occupied by tyrosine in^{twenty three}Leucine in Helix B of ZAP-C Partly due to its existence. Even if tyrosine is not present, Very strong densities are seen for the two waters.主 The main chain of Leu 7 is that of Pro 224 Involved in water-mediated hydrogen bonding to carbonyl oxygen. To the overall shape of this pY + 3 pocket The second contribution of is given by the repositioning of the β-turn in the EF loop. It is. In an isolated SH2 domain complex, this loop is abrupt in this pocket. Participates in the formation of steep solvent-exposed walls. In ZAP-C, the EF loop moves toward the D chain. The rest of the peptide can continue on its way to ZAP-N to ride Wear.Intermotif (-NLGRREE-) binding   ζ₁The peptide segment separating the two pYXXL motifs into ZAP-C Consists of seven amino acids that make up most of their contacts. One complete rotation of the alpha helix Since close turns begin at ζAsn 8 and continue to ζArg 12, Most contacts are within the peptide. ζAsn 8 is the main ligand for the carbonyl oxygen of Gly 245 (BG). Arg 12 forms a direct chain hydrogen bond to the main chain carbonyl of Glu 225 (EF loop). It is involved in both contact and water-mediated hydrogen bonding. Another two water-mediated hydrogen bonds are ζAr g12 is linked to Gly226 and Lys228. Side chains of ζLeu 9 and ζArg 12 Closes the pY + 3 pocket of ZAP-C.   With two glutamic acid residues ζ₁This segment of the peptide is completed, Since these are the pY-1 and pY-2 residues of the second pYXXL motif, they are Make up the first contact. Glu 13 forms a main-chain hydrogen bond to the main-chain carbonyl of Asp 244 create. More interestingly, ΔGlu 13 is N-terminal due to its side chain carboxyl. Lys 242 (ZAP-C αB), an integral part of the phosphotyrosine pocket of the flanking SH2 domain Involved in direct hydrogen bonding to the side chain amino group. Glu 13 also has Lys 242 and To the guanidinium groups of Tyr 238 (ZAP-C αB) and Arg 17 (ZAP-N αA) It also retains Delwars contacts, which also contribute to the N-terminal pY pocket. ζGlu 14 Retains the characteristic pY-1 backbone carbonyl hydrogen bond to both terminal nitrogens of Arg 17, Arg 17 contacts the aromatic ring and the phosphate group of ζpTyr 15.Motif-2 (-pYDVL-) Binding Requires Both Domains   Probably ZAP-NC and ζ₁The most prominent feature of the complex with ζpTyr15 is the recognition pocket. Is composed of residues from both SH2 domains. This is This is the first report on a phosphotyrosine binding site with properties. ZA to ZAP-N Due to the P-C collision, ζpTyr 15 retracts into a deep tunnel.側 side chain of pTyr 15 To Arg 41 (BC loop), Val 47 (βC), His 58 (βD), and Pro 60 (βD). Make a van der Waals contact. The side chain of Arg 17 is located on the aromatic ring of ζpTyr 15 In addition to the carbonyl bridge of the pY-1 residue to the phosphate oxygen of ζpTyr 15, Forming an aromatic contact; This phosphate group is Tyr 238 (ZAP-C αB), Lys 242 (ZAP-C αB), closely associated with the side chains of Arg 17 (αA) and Arg 37 (αB), for a total of 6 direct Form hydrogen bonds. Six water-mediated hydrogen bonds form this phosphate group with Arg 17 (αA), C ys 39 (βC), Leu 40 (BC loop), Arg 41 (BC loop), and Lys 242 (ZAP-C αB). The relative importance of each residue at this interface is important for mutagenesis experiments. More can be determined. Four waters with strong electron density make this powerful network And the presence of these waters results in the interruption of ZAP-C on residues in the BC loop. (See below). In this configuration, each oxygen of the phosphate group is It has its full complement of hydrogen bonding partners.   As described for the first pYXXL motif, the pY + 1 and pY + 2 residues (ζAsp 16 And ζVal 17) are unique to these positions in other SH2 complexes.^20,21,23Contact Make a touch. ζLeu 18 is a high affinity peptide complex with Src family SH2 domain Located in a hydrophobic pocket with similar dimensions to the pY + 3 pocket found in the body. 1 The main chain NH of ζLeu 18 to the carbonyl of Ala 72 on the EF loop Are combined. Contact to several hydrophobic residues is evident: βD binds Phe 59 Gives; interactions with EF loop include Ile 71, Ala 72, Gly 73, and Gly 74 Helix B gives Tyr 87; BG loop contacts via Gly 93 and Leu 94 make. ζ₁Asp 19 is at a weaker density, and Gly 93 (BG It appears that only one hydrogen bond to the carbonyl of step (p) is formed.Inter-domain contact   AP to ZAP-NC₁Unlike the vast contact area of ZAP-N and ZAP-C, two SH2 The total interaction area between the mains is small, only about 200 Å^TwoIs the size of ZAP-C The surface area of ZAP-N buried in the spacer between SH2 and SH2 is only about 400 Å^TwoAnd Z The corresponding buried area of AP-C is not much larger. ZAP-N of the whole complex Total burial of 620 Å^TwoWhich is about 1% of the total surface area of this domain. Equivalent to 3%. Conversely, the burial of ZAP-C is about 1000Å^TwoWhich is calculated as Occupies 20% of the total surface area. This difference is due to the ZAP-N BC loop, ZAP-C FB loop and And helix B and the convergence of the convex side of both helices of the spacer between SH2. This is due to the presence of the formed large channel that allows solvent access. this The irregularly shaped funnel has a diameter of about 5-7 mm and is completely surrounded by a length of about 12 mm After that, it is flared for a further 8 mm. ZAP-N and ZAP-C Each SH2 domain confers 9 residues on the interface only between the domains. contact Most are due to hydrogen bonding, and most of these are water-mediated. I However, there are also some Van der Waals contacts.   Total interface limited to two SH2 domains is absent in the absence of the peptide If so, the spacer between SH2 may be cut or swung If it is possible to stabilize in an appropriate direction for tandem coupling New Uncomplexed ZAP-NC in isoelectric focusing gels as multiple bands Exist, these bands are ζ₁Addition of peptide breaks down into one band . The microheterogeneity seen in this uncomplexed ZAP-NC is due to conformational variability. Matches.Comparison with other SH2: phosphopeptide complexes   Folding despite low sequence identity (33%) between ZAP-N and ZAP-C The overall similarity is noteworthy. Side chain position is significantly better between the two domains Is stored in The root mean square (r.m.s) deviation of the entire main chain is 1.07Å. Src The same measurement for ZAP-N or ZAP-C for family SH2 domains (individual) The fixed value is typically 1.50Å, despite similar% identity values. You. As reported for the traditional structure of individual SH2 domains^14,20,21,Roux Loop region shows largest positional variation, loops AA, BC, CD, and EF are most prominent . The CD loops of both ZAP-N and ZAP-C bind to the Src family SH2 domain. With a large truncation (truncation) Is also evident from the sequence of many SH2 domains^{twenty three}.   ζ₁PYXXL motifs are in a similar main-chain conformation in the complex, but The direction (orientation) of rosin varies between ZAP-N ZAP-C.芳香 The aromatic ring of pTyr 4 is p5 6-Lck²⁰And v-Src^{twenty one}Very well overlaps with both pTyr. But the other For ζpTyr 15, the aromatic ring is only 0.7 に toward the guanidinium of Arg 17 The position moves and shifts 0.8 ° away from the D-chain. This is ζ₁Is the ZAP-C domain Direction to move in, as well as phosphate groups and Tyr 238 and Lys on ZAP-C 242 and strong hydrogen bonding interactions. Both pTyr ports of ZAP-NC The ket is large enough to hold some water and thus other SH2 The reason why the pocket is larger than the main is that the position of the BC loop fluctuates. BC (phosphotyrosine binding) loop found in both SH2 domains of ZAP The extended position of is observed for the uncomplexed SH2 domain, Explained as a hinge in the binding of tyrosine phosphorylated and phosphonated peptides Have been^14,21. This loop is reoriented, but BC The internal conformation of the loop is strongly retained.   The pY + 3 pocket of ZAP-C is significantly larger than this site in other SH2 domains. No. This is due, in part, to the ZAP-C EF residues Pro 224 and Glu 225 position change. Except for the absence of Tyr in αB, this pocket The positions of the other side chains forming the target are very similar to the corresponding sites in Lck and Src. I have.   In addition, compared to the crystal structures of all other SH2 domain complexes, ZAP-NC There are a number of waters. Some are involved in cross-linking of phosphotyrosine to this protein I do. These intervening water molecules bind individual ZAP SH2 domains to phosphorylated ITAM ligand. May contribute to the weak affinity of the^17-19,25. Also, multiple burials or traps Water is present at all of the various interfaces.Biological significance   ZAP-70 tandem SH2 domain of the invention in complex with a component of the T cell receptor ζ chain The structure of the inn is the first to see the intracellular mechanism of TCR (summarized in Fig. 4) Give the opportunity. Some unique features of this structure are that each SH2 domain is independent Instead of functioning as a modular module, the interaction between these domains Suggests that ZAP-70 plays a critical role in recognition of TCR phosphorylated ITAM sequence doing. This structural information can also be used to interpret genetic and biochemical data. Important and provides a basic framework for exploring the mechanism of action of ZAP-70.   The tandem SH2 domain of ZAP-70 binds to the phosphorylated ζ and ε subunits of TCR. Show high selectivity, while isolated SH2 domains from other proteins Is indiscriminately bound by many tyrosine phosphorylated proteins in whole cell extracts Do^Five. Ligand binding and selectivity for the isolated SH2 domain is Some residues C-terminal to tyrosine and phosphotyrosine, especially at the pY + 3 position Mediated by recognition of hydrophobic residues¹². High ZAP-70 to double phosphorylated ITAM sequence Degree selectivity appears to be the result of a number of structural features. Two ζ or ε chains The distance between the pYXXL motifs is tightly associated by coiled coils between SH2s. It becomes a partner with proper spacing for a pair of separated SH2 domains. This pair of The association of the SH2 domain with phosphotyrosine and other ITAM residues Interaction at the domain interface, thus concealing a single phosphotyrosine at the domain interface Stabilizes the conformation that allows for the formation of pockets.   ZAP-NC has high affinity for dual phosphorylated ITAMs and selection for ζ and ε chains Despite its selectivity, the individual SH2 domains of ZAP-70 Was not found to bind to^Five. ZAP-NC is a monophosphorylated ITAM-based The parent has a 100- to 1000-fold lower parent to the corresponding double phosphorylation Unite with union^17-19,25. Therefore, both SH2 domains for high affinity binding Must cooperate and the two phosphotyrosine residues are present and properly positioned Must be. This structural expression of cooperativity and selectivity, ie, both Convergence of residues from the SH2 domain captures pTyr15, indicating that ZAP-NC and ζ₁With It is also the most prominent feature of the complex.   In this regard, the SH2 domains are subject to their natural molecular context. Unfold their natural folds when extracted from their phosphorylated proteins Previously presumed to have full capacity for quality recognition and binding^12,14. we Indicates that the N-terminal SH2 domain of ZAP-70 was expressed in an isolated state. Provide structural evidence that they are incomplete. Only the pY pocket of ZAP-C Domain also requires contributions from neighboring domains or proteins Suggest that you might.   From the crystal structure, the orientation that ZAP-70 assumes after associating with the TCR, as shown in FIG. C₁N-proximal pYXXL motif, and ZAP-N as its C-proximal motif, We are convinced that each will be aligned. We believe that this orientation is the catalytic domain of ZAP-70. Activates and activates downstream substrates in the signal transduction cascade. We also believe it may be important for positioning for phosphorylation.   ζ_TwoIn all ITAMs of the TCR except for the above, there is a 7 residue interval between the two pYXXL motifs. Ζ_TwoIs an interval of 8 residues. In vitro binding of ZAP-NC to synthetic phosphopeptides In the experiment, the binding hierarchy was ζ₁≧ ζ_Two> Ε ≧ ζ_ThreeShow that^{twenty five}. This result The results suggest that one more residue is allowed between the pYXXL motifs. Conversely, ε Deletion of 2 amino acids in the corresponding region of Zap rapidly reduced ZAP-70 binding and produced IL-2 Vanish¹⁷. Thus, the distance between the two pYXXL motifs depends on the association and signalin Important for To determine the relative contribution of each residue in ITAM, CD8-ζ systematically replaced with alanine₁Experiments with chimeras were performed. So Results indicate that only the substitution of pY and pY + 3 residues was To completely eliminate the bug²⁶. With the exception of these residues, the specific ITAM The exact sequence is less important for selectivity than the distance between pYXXL motifs. IT To assess the contribution of the AM sequence to selectivity, more Many residue changes are required.   As mentioned earlier, the inter-SH2 region allows two SH2 domains to associate Constrain within distance. However, a significant portion of the antiparallel helix is in the SH2 domain This region is also facing away from the Regulation of protease activity and / or clustering of receptors Can be given. Evidence that this domain forms a coiled coil of an alpha helix Indicate that these structural units are normally involved in protein-protein interactions ing²⁷So very interesting. It is already predicted that a coiled coil will be formed. The inter-SH2 region of the p85 subunit of PI3-K is the PI110 catalytic subunit of PI3-K. Necessary and sufficient for the interaction of p85 with^{twenty two}. One that is clear from our structure The intriguing possibility is that this ZAP interdomain regulates its kinase activity. To be involved in the clause. This interdomain directly or indirectly enhances catalytic activity May inhibit, this inhibition is reduced by binding of SH2 domain to ITAM there is a possibility. PI 3-K, a PTK homologous to ZAP-70^{twenty five}And SYK²⁸The experiment by We support such a model. Known PI-3K SH2 binding site derived from PDGFβ receptor Addition of a phosphotyrosine-containing peptide corresponding to the Tyr751 region Causes activation of PI-3K²⁹. Similarly, a resource derived from the γ subunit of the IgE receptor Phosphorylated ITAM peptide increases SYK kinase activity 5-10 fold^30,31. Between SH2 Another function of the region is to regulate ZAP-70 activity, such as Lck and / or Fyn. May bind to proteins or proteins that are substrates for ZAP-70 That is. Tyrosine 126 in the inter-SH2 region is phosphorylated by Lck in vitro.³² And may be involved in interactions with other SH2 domain-containing proteins. Most Later, the inter-SH2 domain is a potential ZAP-70 minute in the activated TCR complex. It may also be important for intermolecular association between offspring.   SYK also considers its functional similarity to ZAP and 57% sequence identity. , Are expected to exhibit the features of the above structure. SYK is expressed in several types of hematopoietic cells Causes binding to the ITAM sequence in the cytoplasmic domains of IgE and B cell receptors Function in mast cells and B cells respectively²⁸. ZAP-70 and SYK When comparing arrays³³Most of the residues in ZAP-NC that contact pTyr 15 Stored in the corresponding location. The N-terminal SH2 domain of SYK is phosphotyrosine Does not bind to ligand or phosphotyrosine affinity column³⁴. this is As in ZAP-NC, this phosphotyrosine site also forms a complete pocket. This suggests that the C-terminal SH2 domain is required for synthesis. IgE receiving Double-phosphorylated peptide derived from γITAM induces SYK activation³⁰. I Various ZAP-NC and ζ₁And thus the SH2 domain taken by this activated kinase Can show the main conformation.   ZAP-70 emerges as an attractive target for the development of safe and powerful immunosuppressive drugs Have been. ZAP-70 has been shown to be required for T cell-mediated immune responses in humans, Defects do not affect other tissues⁸. Therefore, ZAP antagonists (antagonists) Specifically inhibits T cells, FK506 and cyclosporin^35,36More non- Targets calcineurin, a protein that is specifically expressed^37,38Currently The toxicity of the immunosuppressive drugs used could be avoided. This kind of protein Phosphatase is required for the T cell immune response while at the same time in some other tissues. But it works. As a result, cyclosporine and FK506 are found in the kidney and central nervous system. Can cause serious side effects in patients with organ transplant rejection. Giving is severely restricted³⁶. To extend the routine use of such treatments to autoimmune diseases In addition, a new immunosuppressive drug having low toxicity is required.   One means of inhibiting T cell activation is to bind to ZAP and prevent its association with the TCR. Small (i.e., preferably having a molecular weight of about 1200 or less, more preferably about 750 or less). Below, even more preferably about 500 or less), preferably non-peptide, membrane permeable To develop molecules. Such compounds are available in any of the SH2 domains of ZAP-70. Or bind to the SH2 interdomain interaction, preferably with high affinity. Can be. Our crystal structures reveal the molecular details of the ZAP conformation and Provides a means to consider the interaction of TCR with TCR. Each SH2 ligand binding site and unexpected The unique structural feature of the association between the SH2s of the two For structural and structurally biased compound libraries. Can be used here.D. Structure elucidation of other tandem SH2 proteins using ZAP-NC three-dimensional structure   Now that the structure of ZAP-NC has been elucidated, we have identified two SH2 domains, especially the ZAP file. Other proteins, including Millie members, are also formed by association between domains. It has a unique binding pocket that has been Think about what can be used to design things. Most relevant to ZAP The protein currently believed to be SYK (see sequence diagram). ZAP-NC Using the structure of, it is possible to obtain a 3D model of Syk-NC by homology modeling. Wear. Before elucidating the structure of ZAP-NC, the SH2 domain of Syk and the SH2 domain of known structure were The sequence identity to the main is low, and none of the SH2 domains elucidated so far This method is difficult, if not impossible, because it does not contain two SH2 domains. there were. Another known protein with a tandem SH2 domain is PL Cγ, P13K, rasGAP, SH-PTP1, and SH-PTP2. Also two SH2 domains Proteins will continue to be used for genomic sequencing or other cloning methods It is expected to be discovered by law.E. FIG. Use of structure in drug discovery Use of the tandem SH2 domain structure of ZAP-70 in computer-based drug design (CADD)   The conformation of the tandem SH2 domain (ZAP-NC) of the protein tyrosine kinase ZAP-70 The availability makes possible a structure-based drug discovery approach. To the structure Based on a new (de Novo) Molecular design, computer-assisted lead ( lead) molecule optimization and computerized candidate drug structure based on structural criteria Including sorting. From the structure of the peptide ligand, a new pseudo peptide (peptido mimetic) module into a conformationally-restricted page. It may be developed directly by peptide substitution design or database search. Or, Using the target protein structure with the ligand removed, the structure-based read You can also make discoveries. Multiple uncomplexed state of ZAP-NC state) by several methods to give additional target conformation Can also. The coordinates of the experiment and the resulting uncomplexed model are Structure and potential ligand or chemical moiety ("fragment" or "seed") Receptor site mapping to identify sites of favorable interaction energy between Can be processed by such techniques as Following such an assessment, the fragment Operations such as toseed ligation and growth may be performed. Fragment seed concatenation Are structures that include “connected” and “seed”, that is, each having an advantageous interaction A chemical structure containing two or more mapped parts at appropriate intervals to reach the site Construction, means how to design. Growth is based on receptor site mapping or Sets up structures that extend certain molecules or moieties to fill available space. Means to measure. Based on the receptor site mapping data, Potential ligands can also be selected from the database. Whatever source, potential Target or suboptimal ligands use their receptor site maps Multiple conformations and orientations according to energy priority. It can be refined by filtering. Finally, p72^SykColumn of Due to the high degree of sequence similarity to the SH2 domain, knowledge based on the structure of ZAP-NC Either homology template method or minimization following repeated site mutations This will create a high quality Syk-NC model. Sy created The structure of k-NC is then processed as another protein target by the methods outlined above. May be. These methods and their application to ZAP-NC are described below.   ζ ₁ peptide pseudo peptideIs, from the binding conformation of a peptide ligand, Depending on the design, one or more peptide segments can be By searching for existing sources or enhancing existing ligand-protein interactions (That is, the components of a ligand can be transferred through accessible protein contacts, Greater interactions with target proteins due to overhanging or otherwise hidden water By substituting alternative parts that can be Knowledge of the binding conformation of a peptide may be relevant to conformational constraints and peptide bond displacement. Can be suggested. The less biased approach is Substitution to one or more moieties of a peptide ligand, preferably a non-peptide A computer to search a three-dimensional database to determine substitutions. Computer algorithm. In this way, the conformational assignment of its biological activity is Compounds, ie, particularly biologically relevant conformations of the peptide ligand A compound based on The algorithm for this purpose is Cast-3D (Chemical Abstracts Service), 3DB Unity (Tripos, Inc.), Quest-3D ( Cambridge Crystallographic Data Center) and MACCS / ISIS-3D (Molecular De sign Limited). These geometric sir Dimensions that are prohibited using the size and shape requirements of the binding site Can be reinforced by a three-dimensional search that excludes Services applicable to ZAP-NC Program that can be used to synchronize geometric and three-dimensional requirements in CAVEAT (University of California, Berkeley), HOOK (MSI) and A There is LADDIN (Daylight Software). Each of these search protocols , An existing common database, the Cambridge Structural Database (the Cambridge Str uctural Database) or other available chemical manufacturers' chemical databases Can be used in combination.   For retention of potential pharmacophoric elements clearly present in peptides In addition, hydrogen bond donating or accepting that can replace ordered water molecules Incorporation of a group into the ligand structure results in significant entrainment that produces a favorable binding free energy. Usually gives tropy gain. Such ordered water is identifiable from its structure Yes, other well-ordered waters were also fully solvated, as explained in more detail below. It may be located during computer simulation of the structure.   Generation of an alternative binding site conformation of the target proteinBut phosphotyros Of the binding region, the nature of the interface between the two SH2 domains, and Overall, the induced fit for both ligand and protein after binding, That may be desirable due to the possibility of conformational changes. For example, with β-chain B The loop connecting C (BC loop or phosphotyrosine binding loop) is a Src family It was reported to act as a functional hinge in the Lee SH2 domain. Also, Charged residues in the schotyrosine binding pocket can change the orientation of the side chains. Wear. Metropolis Monte Carlo or molecular dynamics simulation (MCPro < Yale University>, AMBER <UCSF>, CHARMm <Harvard University> and GROMOS <ET H / Groningen>). Used locally to create an uncomplexed Boltzmann distribution, and thus for molecular design An effective set of additional conformations can also be provided. Another side chain lioness (End change of orientation), Dead End Elimination And A^*Algorithm (University of Southhampton) for each side chain iteration Systematic conformational search, or each residue type has the same backbone twist Can also be tested by comparing with one member of the Protein Data Bank. You. Convert the effective conformation of the BC loop (or loop EF or BG) to a similar anchor Brookhav on loops with anchoring geometry en) Search for protein databank or run within selected loop Apply the dam backbone conformation and filter the results to fit the anchor residues Can be created. Throat based on this knowledge We also have a force-field minimization to create possible geometries Yields the first structure that can be used.   In addition to the inherent flexibility of the peptide binding site, two SH2 domains The interface between them provides an opportunity to explore additional conformational states. Two SH2 Interfaces limited to domains only 200 Å^TwoGives the total buried surface area of Consist of hydrogen bond contacts, many of which are mediated by water. Isoelectric focusing gel According to the experiment by uncomplexed ZAP-NC,₁Two that weaken after binding to a peptide It suggests a conformational mobility between the SH2 domains of S. cerevisiae. ZAP function is 2 Requires that two SH2 domains associate simultaneously with the dual phosphorylated ITAM of the TCR Thus, gross displacements between the two SH2 domains are regulatory. May be worth it. That is, the conformation in which the SH2 domain is separated is inactive. And an inhibitor that stabilizes this alignment state would be attractive. sufficient Dynamics simulations of ZAP-NC solvated in The emergence of dissociated structures and further target conformations of uncomplexed proteins Can give insight.   Receptor site mappingIdentifies energetically favorable binding sites on macromolecules Encompasses a variety of computer operations. The most straightforward approach is electrostatic or parental Empirically determined oily potential, curvature, and hydrogen bonding characteristics According to the physical properties of the macromolecule target, accessible surfaces (and Includes "painting" other generated features. Characterize the surface of macromolecules in this way Such methods for doing this are described in Grasp (Columbia University), DelPhi (Biosym Techno logies), MOLCAD (Tripos, Inc.) and Hint (Virginia Commonwealth Universit) y). The next molecular design is the identified surface Includes the identification or design of ligands that have characteristics complementary to the property. A more advanced Algorithms determine the interaction between the target and potential ligands or fragments. Includes the actual calculation of enthalpy. In practice, the protein or protein of interest From the coordinates of the denature fragment (which may be rotated or otherwise transformed) Are all undesired ligands (or parts thereof) and / or all undesired ligands. Remove unwanted solvent molecules. This coordinate is then assigned to the atomic location By attaching and processing parameters, processed targets for mapping (Target). The target may be split into discrete binding sites. target Alternatively, the divided site can be transferred to the MCSS program (Molecular Simulations, Inc.) As in the case of the above, a predetermined functional group flag that can be relaxed into a desired place next Fragment or a single fragment probe on top Are placed inside a regular grid of site points, which are placed one after the other. this Examples of programs using the site-lattice algorithm include Glin / Grid (Molecula r Discovery, Ltd.), Ludi (Biosym Technologies), Leapfrog (Tripos, Inc.) And Legend (University of Tokyo). Either technique requires an Talpy contributions are estimated in the molecular mechanical force field and the appropriate position of the selected functional group is determined. Determined systematically.   In the site-grid approach, a desired portion of the target is surrounded by a defined grid. One box is defined. The grid resolution, ie the spacing between grid points, is May be determined, or may be set by a computer program. In addition, hydrophobic Other parameters of each point in the grid, such as character or other properties, should be specified as well. Can be. One or more selected moieties, functional groups, molecules or molecular flags A probe (that is, a computer model) of the -Determine the interaction energy of the target pair for each such grid point . Collect data of selected parts, functional groups, etc. Capture, visualize on computer monitor, or various texts or figures It may be printed out in a table format.   As another approach to placing parts at each of a set of grid points, As described above, using multiple copies of selected fragments, parts, molecules, etc. Multiple copies of the protein near the protein target (defined by coordinates as described above) By overlapping, the target may overflow. This model is then Loop minimization (ie, molecular dynamics minimization) calculations to reach the point of favorable interaction. Or identify the area. The data can be handled in the same way as the grid method.   One application of this method to the structure of ZAP-NC is to use peptide ligands (ζ₁)When Includes crystal structure coordinates with both experimentally observed water molecules removed. Thus The binding site is a van der Waal at any position previously occupied by the peptide. Includes all protein residues that are within the Ruth distance. This "old" joint Position is expanded to include most of its proximal protein surface, and Additional crevices not occupied by known ligands to any SH2 domains (crevi ces) and depressions are potential "auxiliary" binding regions Occupancy can significantly contribute to the inhibition of ZAP association with the T cell receptor. ZAP- Similar provisions for the NC coupling surface can be obtained empirically or obtained by the modeling method described above. The same applies to any other coordinates. Receptor site mapping, as described here Other methods include the SH2 domain in ZAP-NC (or ZAP family-NC) structures or other ZAP family to design or select ligands that can bind to It can be applied to Lee's 3-D structure.   The receptor site map is described above.Database searchGeneration of ligand by And new designs for new chemicalsGrowth / consolidation methodSeeds for give. Ligand growth program places appropriate functional groups at each site point First access the extensible fragment dictionary. You. Then a genetic algorithm or subgraph isomorphism Depending on the protocol, attach a small aliphatic chain or ring to the fragment. Internal freedom Correction of the degree and translation or rotation of the candidate model in the coupling cavity , A probability improvement may be introduced. The resulting multiple sets of molecules can be Considers constraints, complementarity of electrostatic and hydrophobic interactions, and solvation estimates Score and filter by function. Establishment of new ligand for ZAP-NC Ludi (Biosym Techno Corp.) logies), Leapfrog (Tripos, Inc.) and Legend (University of Tokyo), Grow (Upjohn), B uilder / Delegate (University of California, San Francisco) and Sprout (University of California) versity of Leeds). Clique detection method is site mapping And another strategy for ligand growth. DOCK (University of Califor nia, San Francisco) and similar programs to reach specific binding sites. Spheres with the smallest set of atomic dimensions. Next, a sphere with atoms filling the binding site To orient the ligand so that it overlaps the center (ie, “nucleus”) of the Try database search. The complementarity of the shapes, including the steric requirements of the cavity, Augmented by point scoring functions and potential energy functions .   Ligand optimization(From any source) using ZAP-NC conformation Can be made. ZAP-NC receptor site map or hydropathy (hydrophobic) Use the profile to identify preferred positions for the functional group components of the ligand And use this to minimize the otherwise minimal standing of the ligand structure itself. Will normally be suppressed by minimal steric considerations Filter or restrict the search for conformation of the ligand structure You. Simulated annealing (simulated annealing) becomes available as apparent binding sites become available.  annealing, SA), distance geometry, metropolis Monte Carlo, or coupling By using the force field directly in the stochastic search of the style, It is possible to determine the mode of ligand binding. Ligand binding to ZAP-NC Autodock (Scripps Clinic), DG EOM (QCPE # 590), Sculpt (Interactive Simulations, Inc.) or the molecules mentioned above Any of the kinetic programs. A set of possible combinations that are easy to process Change the binding affinity of each model considered in a predictable manner Modifications of test ligands that are considered to easily identify the appropriate binding mode be able to. For example, one ligand can be matched to one binding model and May be modified so as to contain a functional group at a position that also matches the model. Next Then, the "disproved" model can be removed using the combined data. Wake up If you create one appropriate model by repeating the removal of the unlikely connection style The potential for improving this lead (the appropriate model) is its local protein ring It becomes easily apparent from the border.   Another protocol for ligand optimization is 3D data combining knowledge of binding sites. Includes database search. Modeling involves the biologically active Multiple candidates for the conformation can be indicated. A probe for precise conformation Identified several constrained mimics of each possible conformer 3D search to include. Unconstrained ligand structure-activity The relationship will indicate which functional groups are retained in the constrained mimetic. Finally, the steric and electrostatic requirements of the binding site prioritize the possibilities obtained. It may be a filter to give.   Due to the structure of ZAP-NC according to the present invention, accurate homologous proteinModel buildingAnd that They can then be used in drug design. This protein tyrosine Kinase p72^SykSH2 domain and interdomain coiled-coil region of ZAP-7 Sharing a high degree of sequence similarity with 0. ZAP-NC structure is a knowledge-based template construction method, Or local molecular mechanical minimization after repeated point mutations Makes it easy to use for developing reliable Syk-NC models. Syk-NC model Examples of programs applicable to development include Composer (Birbeck College), Model er (MSI) and Homology (Biosym Technologies). The obtained model Can then be processed with any of the CADD techniques described above.F. Compound characterization   Compounds designed, selected and / or optimized by the methods described above Binding activity to proteins containing one or more SH2 domains May be evaluated. A variety of techniques can be used, many of which are in the art. It is well known. For example, a compound may be conjugated to a phosphorylating ligand for that of the SH2 domain. The activity as a competitive binding inhibitor can be evaluated. For example, Pawson See U.S. Patent No. 5,352,660 (October 4, 1994). 1 or 2 or more Surface plasmon to evaluate the binding properties of compounds to the SH2 domain on A ringing (SPR) method may be used (see, for example, Panayotou et al, 1993, Molecular and Cellular Biology13: 3567-3576), fluorescence proximity The same applies to methods and other methods known in the art.   The SPR method uses real-time two or (Pharmacia Biosensor, Piscatawy, NJ) developed an SPR method that uses gold (Provided as a disposable biosensor “chip”) and adjustable by the user Focus the polychromatic light beam on the interface between the buffer compartment Te This gold film has a 100 nm thick “hydro `` Gel '' is attached, which is the covalent immobilization of the analyte of interest (covalent immobili zation). Irradiated light interacts with free electron cloud in gold film When used, plasmon resonance increases. The resulting reflected light produces optimal resonance. The spectrum is worn at the shifted wavelength. Separates the reflected polychromatic light into its component wavelengths (prism BIAcore shows the generated surface plasmon Create an optical interface that accurately reports resonance behavior. When designed as above, Plasmon resonance-and hence, the wear spectrum-is the evanescent field (This corresponds approximately to the thickness of the hydrogel) and is sensitive to mass. Mutual One component of a pair that acts is immobilized on the hydrogel, and the component of the other When supplied through a buffer chamber, the interaction between these two components is Mass accumulation and its corresponding plasmon resonance effect (measured by wear spectrum). ) Can be measured in real time. This method can be Fast, extremely sensitive real-time molecular interactions without the need for component labels System measurement is possible.   Fluorescence polarization (FP) is the IC of the association reaction between two molecules.₅₀value And K_dProtein-protein and protein-ligand interactions to obtain values It is a measuring method that can be easily applied to the action. In this method, one of the molecules of interest is Must be conjugated to a fluorophore. This is generally The smaller molecule of the system (in the case of SH2, a phosphotyrosine-containing peptide) is there. Sample containing both ligand-probe complex and protein receptor The mixture is excited with vertically polarized light. Light is absorbed by the fluorophore of the probe for a very short time Re-emission occurs. The degree of polarization of the emitted light is measured. Polarization of emitted lightYes Depending on several factors, the most important factors are the viscosity of the solution and the apparent molecule of the fluorophore. Quantity.   With proper control, the degree of polarization of the emitted light is limited to changes in the apparent molecular weight of the fluorophore. Make it dependent. This apparent molecular weight indicates that the probe-ligand complex is in solution. Free of charge or bound to a protein receptor. To FP Based join assays have a number of important advantages. The main one among them is true Under highly homogeneous equilibrium conditions₅₀Value and K_dValue measurement, speed of testing and convenience for automation Convenience and ability to screen in cloudy suspensions and colored solutions It is.   Automation of such an FP assay is performed using a 96-well fluorescence polarization plate reader. Done. This reader has a sensitivity level of 1 nM for fluorescein-labeled molecules. The reading of the polarization value can be carried out with a microscope, and each plate can be read in 3 minutes.   The fluorescence polarization equilibrium binding assay was applied to ZAP, Syk, and Src domains. N, C-ZAP The binding curve of the double-phosphorylated へ -1 sequence to Also shown in FIG.   A compound may be a specific SH2-containing protein and its natural ligand (or its (Or some analogs based on it) to identify any other SH2-ligand Preferentially over the inhibition of interaction, for example, at least an order of magnitude, more preferably at least It is often desirable to inhibit also by two orders of magnitude (on any scale).   Such compounds are of interest using appropriate cell-based assays or animal models An activity that inhibits cells or other biological events mediated by pathways involving interactions Sexability may be further evaluated. Diverse cells of a compound or other biological Cell-based assays and animal models suitable for assessing inhibitory activity on events are available in the art. Known in the art. New assays and models are being developed one after another, and the scientific literature Has been reported to.   For example, compounds that bind to ZAP-70 can be prepared using any of the conventional assay methods and materials. Can be assessed for biological activity in inhibiting T cell activation. An example For example, compounds that bind to ZAP can inhibit CD4 + and CD8 + T lymphocytes in vitro. And cytotoxic T cells within the dose range used to demonstrate in vitro activity Can be assayed for lack of in vitro toxicity. A set of in vivo models To determine the breadth profile of the compound's immunosuppressive activity, Profiles of positive controls such as crossporin and FK506 It may be compared with. The comparison is based on other currently accepted immunosuppressive compounds, i.e. It may be done for pamycin, cyclophosphamide, and leflunomide. The first in vivo screening models include delayed-type hypersensitivity testing and allogeneic skin Transplantation, and popliteal lymph node hyperplasia. Optimal profile in the above model Compounds that have demonstrated efficacy in rheumatoid arthritis, transplantation, graft-versus-host disease, Designed to confirm immunosuppressive activity in certain therapeutic areas, including asthma Advance to a more accurate model.   Compounds that bind to SYK are blocked in mast cell or basophil granule loss assays. The harmful activity can be evaluated. Histamine, leukotriene, hormone Specific mediators, such as sex mediators and / or cytokines Inhibitory activity of compounds of the invention on cell release, and phosphatidylinositol Its biological activity with respect to the level of protein hydrolysis or tyrosine phosphorylation. As an indicator of physical activity, it can be characterized by conventional in vitro assays. [Example For example, "IgE-induced histamine release from a rat basophil leukemia cell line: release And Non-Releasable Clones ", Edward L. Barsumian, Chaviva Isersky, Mari anne G. Petrino, Reuben P. Siraganian,Eur . J. Immunol. 11: 317-323; Forr est, MJ, 1991,Biochemical Pharmacology 42: 1221-1228 (activated netrofil < netrophils> measurement of N-acetyl-β-glucosaminidase); and Stephan, V .M. et al.,J . Biol. Chem. 267: 5434-5441 (1992). ] For example, Hista Min release is available from AMAC Inc. Using kits available from (Westbrook, ME) It can be measured by an immunoassay. Bind to SYK in this way Assess the biological activity of the compounds and determine between them or between leflunomide (and And its active metabolites, A771726), vanadate, staurosporine, genistay Or other chemicals containing clinically relevant compounds that can be used as positive controls It can be compared to known active compounds such as compounds.   Generally speaking, in such a test, the IC₅₀Interesting at values of 150-300 μM, 50- Good at 150 μM and very interesting below about 50 μM.   The compounds that bind to SYK are the sensitized guinea pig tracheal strips (strips). Tissues can also be tested in an ex vivo assay for their ability to block antigen-induced contraction You. Activity in this assay is useful for predicting the efficacy of potential anti-asthmatic drugs It has been shown. Numerous animal models of asthma have been developed so far, They can be used (for review, Larson "Experimental model of reversible airway obstruction")THE LUNG Naka, Scientific Foundations, Crystal, West et al. (Ed), Raven Pre ss, New York, pp. 953-965 (1991); Waner et al., 1990,Am . Rev. Respir. Di s.  141: 253-257). Species used in animal models of asthma include mice, Rats, guinea pigs, rabbits, dogs, sheep, and primates are included. Use Other possible in vivo models are Cross et al.,Lab . Invest. 63: 162-170 (1990) And Koh et al.,Science, 256: 1210-1213 (1992).   As another example, signals related to the initiation, retention or spread of cancer growth (proliferation) Compounds that bind to SH2-containing proteins involved in transduction of It can be assessed by in vitro or in vivo assays. For example, Ishii,J . Antibiot.  XLII: 1877-1878 (1989) (in vitro evaluation of cytotoxic / antitumor activity);  Sun et al., U.S. Patent No. 5,206,249 (issued April 27, 1993) In vitro evaluation of growth inhibitory activity); and Sun et al., Supra (xenografted in mice). Xenograft models using various human tumor cell lines and various animal models See Dell).   One or more (eg, 5 to 7 days) Investigating toxicology in doses is generally performed using the intended route of administration for efficacy studies. Will be applied. Such investigational toxicological studies should be performed at the maximum tolerated dose, intraperitoneal or oral. Individual bioavailability from the route of administration and early This is done to determine an estimate of the integrity limit. Appropriateness of the compound in animal models Initial bioavailability and pharmacokinetics of the compound to help determine appropriate dosage criteria The (blood purification value) may be determined by a standard cold or radioactive assay.Illustrative drug design   An example of one approach to using the structure of ZAP family members in drug design For this, we use the structural coordinates of the ZAP-NC: ζ1 complex (see, for example, Annex I). Potential amino acid residues to interact with their ligand molecules Was characterized. For example, using the program Syby1, N-terminal SH2 domain that is within 10 mm of the We identified amino acid residues from the in and C-terminal SH2 domains. Ligan Residues from that region that can participate in hydrophobic interactions with moieties on the molecule Are listed in Table A. Hydrogen bonding interaction or ion with a moiety on the ligand molecule Table B lists the acidic residues from that region that can participate in sexual (salt bridge) interactions Raise it. Hydrogen bonding interaction or ionicity with a moiety on the ligand molecule (salt bridge) The basic residues from that region that can participate in the interaction are listed in Table C. That region that can participate in hydrogen bonding interactions with moieties on the ligand molecule Are listed in Table D. Hydrogen bond accepting phase with a moiety on the ligand molecule It has a main chain amide carbonyl at an appropriate position that can participate in interactions. Residues from the region are listed in Table E. Hydrogen bond donating ability with a moiety on the ligand molecule A region having a main chain amide nitrogen at an appropriate position that can participate in the interaction. The residues from the region are listed in Table F.   Similarly, we consider the amino acid residues from the C-terminal and N-terminal domains. Of these groups are present at the interface between these two domains, and Disrupts juxtapositioning of domains required to bind to vesicular receptors To identify amino acid residues that could interact with the ligand molecule Was. That region that can participate in hydrophobic interactions with moieties on the ligand molecule Are listed in Table G. Hydrogen bonding interactions with moieties on the ligand molecule Or acidic residues from that region that can participate in ionic (salt bridge) interactions Are listed in Table H. Hydrogen bonding interaction or ion with a moiety on the ligand molecule Table I lists the basic residues from that region that can participate in sexual (salt bridge) interactions List. Can participate in hydrogen bonding interactions with moieties on the ligand molecule Neutral residues from that region are listed in Table J.   Similarly, we have identified amino acids from the interdomain (also called spacer A) region. Of the acid residues, they exist at the interface between the two α-helical coils in this region, and To disrupt the observed folding of the -ZAP domain Amino acid residues that could interact with the gand molecule were identified. Ligan Residues from that region that can participate in hydrophobic interactions with moieties on the molecule Are listed in Table K. Hydrogen bonding interaction or ion with a moiety on the ligand molecule Table L lists the acidic residues from that region that can participate in sexual (salt bridge) interactions Raise it. Hydrogen bonding interaction or ionicity with a moiety on the ligand molecule (salt bridge) Table M lists the basic residues from that region that can participate in the interaction. That region that can participate in hydrogen bonding interactions with moieties on the ligand molecule Table N lists the neutral residues from   Using the amino acid residues listed in Tables A-N, The binding site on the ZAP-70 protein can be determined. The binding sites are approximately Includes a subset consisting of the aforementioned residues that are within 10Å. For example, residue CYS39 , ARG41, SER42, PRO60, GLU62, LYS220 and ASP230 contain such binding sites .   We have identified new types of ZAP family members based on the aforementioned types of assessments. Come up with a class of ligands. Specifically, this type is one or more of Is one or more residues defined by two or more residues, preferably the residues described above. Compound containing one or more moieties capable of interacting with a binding site Become. The set of subsets of such compounds comprises at least two parts, At least one of which is a substituted or unsubstituted phosphate or phosphate mimetic (eg, (Substituted or unsubstituted phosphonic acid moieties). Those parts are replaced In certain embodiments, the substituents can be alkyl, aryl, or arylalkyl .   The term alkyl refers to both saturated and unsaturated, straight-chain, branched, cyclic or Polycyclic aliphatic hydrocarbons are meant to encompass one or more carbon sources. May contain oxygen, sulfur, or nitrogen in place of the child, and hydroxy, C₁~ C₈Alkoxy, acyloxy, carbamoyl, amino, N-acylamino, Selected from the group consisting of ton, halogen, cyano, carboxyl, and aryl It may be optionally substituted with one or more functional groups. Alkyl groups are preferred Or a lower alkyl group, that is, one having 1 to 8 carbon atoms.   The term aryl is used to describe stable cyclic, heterocyclic, polycyclic and heteropolycyclic unsaturated. Sum C_Three~ C₁₄Part, meaning, by way of example and not limitation, Phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoi , Bilimidinyl, and oxazoyl. These further include hydroxy, C₁~ C₈Alkoxy, C₁~ C₈Branched or straight chain alkyl, acyloxy, carb Moyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, Substituted with 1 to 5 substituents selected from the group consisting of ano and carboxyl (For example, Katritzky, Handbook of Heterocylic Chemistry reference).   The ligand may contain one or more amide bonds, but preferably Non-peptide. Preferably, the molecular weight of the ligand is 1200 or less, more preferably It is 750 or less, more preferably 500 or less. Peptides and peptidic molecules Two or more natural α-amino acids are peptide bonds (where the amino acid is proline Except in certain cases).   One particular residue or plurality at the binding site of the ligand or a moiety on the ligand The ability to interact with a set of residues depends on the proximity of the ligand moiety to the residue of interest. It can be determined by paying attention. Proximity and protein Determined by physical methods such as X-ray crystallography or NMR evaluation of the ligand co-complex. Or the structure of the ligand using a program as described above. The determination may be made through a study of the model of docking with the structure of the protein. Numerous commercial programs evaluate modeled or experimentally determined structures And conveniently identify atoms involved in hydrogen bonding or hydrophobic interactions Can be. In general, hydrogen bonding (this includes salt bridges and other ionic interactions) Traverses a distance of about 2.8-3.5 よ り, more usually about 3.2 約, and about 180 ± 60 ° At a donor-H-acceptor angle of Hydrophobic interactions depend on the nature of the atoms involved. Accordingly, up to about 5 °, more preferably up to about 4.5 °, more usually up to about 4 ° It happens across distances. Again, one of many commercial computer programs Using the hydrogen bonding and hydrophobicity between the ligand moiety and the protein atom Interactions can be identified.G. FIG. Pharmaceutical compositions and uses of inhibitors of ZAP family members   Ligand-specific compounds that bind to one or more ZAP family members Based on the nature of the function of SH2-containing proteins, especially newly discovered proteins For classification, use as a biological reagent in the binding assays described here. Can be.   In addition, one or more SH2 domains may be identified as described above. From molecular interactions mediated by ZAP family proteins containing Can be used to inhibit the appearance of certain biological events. The present invention Thus, such proteins and their natural ligands [ie, the ZAP family Natural proteins (typically) or parts thereof that bind intracellularly to proteins Or similar) or is mediated by such an interaction Provide methods and materials for inhibiting (completely or partially) biological activity You. In this method, a compound identified or obtained as described herein can be used as an example. For example, to introduce this compound into cells whose molecular interactions should be inhibited. Mixing or contacting with ZAP family protein by the method. After compound introduction Inhibits the interaction of ZAP family proteins with their natural ligands, Can be easily detected. Inhibition of such interactions is a consequence of the biology of SH2-mediated events. Can be used for research aimed at better understanding.   Generally, inhibitors of SH2-mediated interactions include, for example, SH2-containing proteins and Symptoms arising from cellular processes mediated by interaction with natural ligands Or it may be useful for diagnosis, prevention or treatment of disease. For example, the development of osteoporosis Patients who need it to prevent or reverse the process or reverse the process By administering an SH2 binding or blocking agent that selectively binds to Src SH2 The patient can be treated. SH2 binding or blocking agents may be therapeutically useful Pathological conditions involve the SH2 domain-containing proteins Src, PLCg, and Grb7. There were many others, including breast cancer that had been. Prostate as another related medical condition Cancer, in which case Grb2, PLCg, and P13K (all of these SH2 domains Targeting) may be useful in treating or preventing this disease. No. Inhibition of Grb2 or Abl SH2 domain interaction with Bcr-abl It may also be useful in the treatment of leukemia (CML) and acute myeloid leukemia (AML). SH2 inhibition Yet another related use of the agent is to target the SH2 domain of the STAT protein. Diseases mediated by interferons, growth factors, or cytokines (eg, , Inflammatory disease).   Of particular interest is the interaction of ZAP family members with their natural ligands. A drug that blocks action. For example, it is believed to be involved in T cell activation. Inhibitors of ZAP-70-related interactions are useful for treating and preventing autoimmune diseases It may be useful as an immunosuppressant in the prevention of skin and organ transplant rejection. SYK Inhibitors of the interaction of steroids with natural ligands cure asthma and uncontrollable allergic reactions Would be useful for treatment and prevention.   Inhibitors selected or identified according to the present invention can be prepared using conventional materials and means. In the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier and / or other excipients Can be prescribed. Such a composition would provide a ZAP family protein Cure of a disease or condition resulting from a cellular event involving more mediated molecular interactions For therapy, it can be administered to humans or non-human animals. Such a pair The administration of the composition is optional using appropriate formulation compositions, as is well known in the art. By parenteral, oral, inhalation and the like. Inhibition of the present invention The agent may be a conventional excipient, that is, a pharmaceutically acceptable organic or non-toxic agent suitable for parenteral administration. It can be used as a mixture with organic carrier substances.Pharmaceutical applications   Compounds identified as described here are involved in pharmacologically important cellular events. Due to its ability to inhibit the required protein-protein interaction, For the treatment or prevention of various diseases and disorders in mammals in need In pharmaceutical compositions and methods.   Mammals include rodents such as mice, rats and guinea pigs, and dogs. , Cats, horses, cows, sheep, non-human primates, and humans.   Preferred methods of such treatment and prevention are to prevent, alleviate the disease or disorder. Administering to the mammal an effective amount of the compound for summation or healing. this Effective amounts may be determined by conventional assays well known in the art, including those described herein. It can be easily determined by evaluating the compounds of the invention.Therapeutic / prophylactic administration and pharmaceutical compositions   The present invention addresses the symptoms and / or severity of the diseases or disorders mentioned above. Treating, preventing and / or alleviating an elephant individual by administering an effective amount therefor Provide a way to Target individuals include, but are not limited to, cattle, pigs, chickens And the like, preferably mammals, and particularly preferably humans.   Various drug supply systems, such as liposome encapsulation, microparticles, microcapsules, etc. Are known and can be used for the administration of the inhibitors of the present invention. One interesting supply The formula is for administration via the lung, as described in more detail below. Other deployment methods Including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal Internal, epidural and oral routes are included. Inhibitor administration can be, for example, (bolus) injection, epithelial or mucosal skin lining (eg, oral mucosa, rectal and intestinal mucosa) Absorption through membranes, etc.), and can be carried out by any convenient route. It may be administered together with a physically active agent. Administration can be systemic or local. nose Preferred routes of administration for the treatment or prevention of tracheal or pulmonary conditions are oral, nasal, Or a tracheal aerosol (aerosol) or nebulizer.   Thus, in specific embodiments, the inhibitors of the present invention may be administered to the body in need of treatment. It may be desirable to administer locally to the site. This means, for example, that Although not illustrated, local infusion during operation, topical application, injection, catheter means, suppository means, Or it may be achieved by a skin patch or implant means. This buried material Porous, non-porous, including membranes and fibers, such as sialastic membranes Or of a gelatinous material.   The present invention also provides a pharmaceutical composition. Such compositions are therapeutically (or prophylactically) effective amounts. And a pharmaceutically acceptable carrier or excipient. Such a charge The body can be, but is not limited to, saline, buffered saline, dextrose Water, glycerol, ethanol, and combinations thereof. Carrier and And the compositions can be sterilized. The formulation should be in a form suitable for the mode of administration. It is.   The pharmaceutical compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents, as desired. Can have. The composition can be used in liquid solutions, suspensions, emulsions, tablets, capsules, Tablets, sustained release formulations, or powders (powder). The composition also comprises Formulation as a suppository, with traditional binders and carriers such as glyceride Can also. Oral formulation compositions include pharmaceutical mannitol, lactose, starch, stear Magnesium phosphate, sodium saccharin, cellulose, magnesium carbonate, etc. And standard carriers.   In certain embodiments, the pharmaceutical compositions of the present invention are suitable for intravenous administration to humans. And formulated according to the usual methods. Typically for intravenous administration Is a solution in sterile isotonic aqueous buffer solution. If necessary, this composition Also contains a solubilizing agent and a local anesthetic to relieve pain at the injection site. sell. Generally, the components are mixed separately or together in unit dosage form For example, like an ampoule or sachet with the amount of active ingredient It is supplied as a lyophilized powder or anhydrous concentrate in a hermetically sealed container. This composition If the product is to be administered by infusion, add sterile medicinal water or saline. It can be supplied using an infusion bottle. When administering the composition by injection Prepare an ampoule of sterile water for injection or saline so that the ingredients can be mixed prior to administration. You may make it.   Administration of an effective amount of an inhibitor to an individual can involve administering the compound directly to a lesion on the individual's skin. It can also be achieved locally by giving. For this purpose, the inhibitor is Pharmaceutically acceptable topical carriers such as tablets, ointments, lotions or creams. Is administered or applied as a composition having Topical carriers include, but are not limited to, water , Glycerol, alcohol, propylene glycol, fatty alcohol, trig Includes lyserides, fatty acid esters, or mineral oil.   Other topical carriers include liquid paraffin, isopropyl palmitate, poly Ethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5% ) Aqueous solution or sodium lauryl sulfate (5%) aqueous solution. Antioxidant Even if other materials such as additives such as humectants, viscosity stabilizers, etc. are added as necessary Good.   In some cases, the inhibitor may be placed on, inside, or below the skin It is expected to be placed inside. Such equipment receives the compound. Patches, implants, and implants that release to the skin by a dynamic or active release mechanism There is.   Materials and methods for making various formulation compositions are well known in the art. [For example, US Pat. Nos. 5,182,293 and 4,837,311 (tablets, capsules And other oral and intravenous formulations)].   Effective doses of the inhibitors of the present invention range from about 0.01 to 50 mg / kg of body weight of the mammal. It is preferably about 0.1 to 10 mg / kg, and is administered once or in divided doses. General In addition, this inhibitor can be used in patients in need of such treatment in a daily dosage range of about 1-2000 mg / person. It can be administered in a box.   The amount of the inhibitor that is effective in treating or preventing a particular disorder or condition will depend on the disorder or condition. Or may vary depending on the nature of the condition and can be determined by standard clinical techniques. You. In addition, in vitro or in vivo assays may be used to help determine optimal dosage ranges. It may be adopted in some cases. Effective doses should be determined in vitro or in animal model test systems. May be extrapolated from dose-response curves obtained from the system. Inhibitors as active ingredients The exact dosage level should be determined by the attending physician or other The health care provider should determine the route of administration and the age of the individual or patient , Weight, gender, and general health; nature, severity and clinical stage of the disease; And at the same time, depending on known factors, including the use of other treatments.   The present invention also relates to a pharmaceutical composition of the invention comprising one or more components comprising one or more components. Drug packs or kits comprising two or more containers are also provided. In such a container, Directed by a government agency that oversees the manufacture, use, or sale of drugs or biological products. A prescribed form of precautionary statement can optionally be attached, which is intended for human administration. Reflects the approval of the regulatory authority for manufacture, use or sale of the product.Lung administration   In one aspect of the invention, the inhibitor is administered to the lung, for example by aerosolization. Administration. This route of administration is intended for the treatment of bronchial or pulmonary infections or tumors. It may be particularly useful for treatment or prevention.   Lung administration can be performed, for example, by various delivery devices known in the art [eg, Newman, S.P., 1 984, Aerosols and the Lung, Clarke and Davia (eds.), Butterworths, Lo ndon, England, pp.197-224; PCT publication number WO92 / 16192 (October 1, 1992); PCT public Publication No. WO91 / 08760 (June 27, 1991); NTOS patent application by Roosdorp and Crystal  7-504-047 (filed on April 3, 1990)]). You. The supply device is not limited to this, but includes a nebulizer (nebulizer), And inhalers, and powder inhalers. Various supply devices are commercially available. They can also be used. Examples include the Ultravent nebulizer (Mallinckrodt, Inc., St. Lou). is, Missouri); Acorn II nebulizer (Marquest Medical Products, Englewood, Colo) rado); Ventolin metering sprayer (Glaxo Incs., Research Triangle Park, North Ca rolina); Spinhaler powder inhalers (Fisons Corp., Bedford, Massachusetts). Or Turbohaler (Astra). Such instruments are usually cast from such instruments. It is necessary to use a formulation suitable for the drug, in which the propellant is present In some cases.   Ultrasonic nebulizers are used to produce aerosols of respirable size from liquids. Sprays tend to be more efficient (Smith and Spino in cystic fibrosis Pharmacokinetics of Drugs "Consensus Conference, Clinical Outcomes for Evaluation  of New CF Therapies, Rockville, Maryland, 10-11 December 1992, U.S. cyst Sexual fibrosis foundation).   Aerosol particles may be formed using a nebulizer (nebulizer). Any of a variety of chemically acceptable inert gases can be used as aerosolizing agents. You. Physiologically acceptable surfactants (eg, glycerides), excipients (eg, lactose) Other components such as carriers, diluents and the like can also be included.   The invention is not limited in scope by the specific embodiments described herein. Not. Indeed, in addition to those described herein, various modifications of the present invention The description will be clear to the skilled person. Such modifications are also within the scope of the appended claims. Is included.   Although various patents, patent applications, and publications are referenced herein, the disclosures of The entire contents of the indication are incorporated herein.Experimental example I. Preparation of protein A. ZAP-NC: expression, purification and complex formation Cloning   ZAP-NC was developed as a glutathione S-transferase (GST) fusion protein. It was revealed. DNA sequence encoding residues 1-259 from human ZAP-70 [A.C. Chan, M . Iwashima, C.W. Turck, A .; Weiss,Cell 71 649-662 (1992)]. [D.B. Smith, K.S. JohnsonGene 67, 31-40 (1988)].E . coli  BL21 orE . coli Transformed into B834. The resulting construct is At the bin cleavage site and two extra residues (G and S) at the N-terminus of ZAP-NC Coded against.Expression   A typical preparation involves growing and inducing a 2 liter culture (BL21) in BHI medium. ZAP-NC was produced. The culture is grown at 25 ° C until the OD 595 nm reaches 0.8, Induction was performed with 1 mM IPTG for 5 hours.   Selenomethionyl (SeMet) ZAP-NC,E . coli 834 auxotrophs [D.J. Leah y, H.P. Erickson, I .; Aukhil, P.Joshi. W. HendricksonProteins 19 48-54 (1 994)] to replace methionine with selenomethionine in a defined medium, I let you. The SeMet ZAP-NC was treated with 50% D / L-selenomethionine at 0.5% titration. 10 L of a defined medium [J.O. Boles, W.H. Tolleson, J.C. Schmi dt, R.B. Dunlap, J.D. Odom,J . Biiol. Chem. 267, 22217-22223 (1992)] Propagated. The culture is grown at 30 ° C until the OD 595 nm reaches 0.8 and left for 10-15 hours. Induced.Purification   After isolating the GST fusion protein using glutathione agarose, And cut it. ZAP-NC uses tandem SH2 domains on a phosphotyrosine agarose column. And separated from GST by elution with a salt (concentration) gradient . Then, ZAP-NC was subjected to hydrophobic interaction chromatography on a phenyl sepharose column. Further purification by luffy. 500 mM NaCl and 10 mM at 4 ° C. Stored under argon with dithiothreitol. Typical purifications are listed below You. Both ZAP-NC and SeMet ZAP-NC are purified by N-terminal analysis and SDS gel electrophoresis. The degree was determined to be> 95%. Mass Spectrometry for ZAP-NC and SeMet ZAP-NC Nomethionine contamination rate> 95%.Preparation of the complex   The complex of ZAP-NC plus ζ1 peptide (NQLpYNELNLGRREEpYDVLD) After adding a 2-fold excess of peptide to the gel, the sample is run through a gel filtration column. Prepared. The peptide was dissolved in 2000 μl of 100 mM Tris, pH 8.0, and 6 mg Added to Parkin. Incubate the sample at room temperature for approximately 30 minutes, then filter with a 0.2 micron filter. Filtered. Superdex 75 16/60 column with 20 mM NaCl and 5 mM DDT  Equilibrated in Tris, injected sample, and collected fractions in 3 mL. The complex is 65.6mL Eluted and pooled peak fractions based on protein A (280). Complex analysis Was performed using a homogeneous 20% native gel.Typical ZAP-NC purification Cell lysis:   1) About 20 g of cells were dissolved in 40 mL of buffer A using a French press cell.   2) Diluted 1: 1 with buffer B.   3) The mixture was centrifuged at about 30,000 × g for 30 minutes, and the supernatant was taken out.Glutathione column 2.6 × 10:   1) The supernatant was injected onto a glutathione column equilibrated in buffer B.   2) About 100 mL of buffer B was washed with buffer C up to the baseline.   3) The fraction was eluted with buffer D, and fractions were collected in 5 mL portions and pooled.Thrombin cleavage   1) After adding 200 mM NaCl to the pool, human thrombin was added at 1 μg / mg protein. added.   2) The cells were cultured at room temperature in a nutator. Take samples while continuing the culture , Run on an SDS gel. The reaction was completed in 40 minutes. (Stop the reaction by adding PMSF be able to. )   3) After cleavage was completed, the pool was diluted 5-fold with 20 mM Tris pH 8/5 mM DTT.Phosphotyrosine column: 26 x 7.5 cm:   1) The pool was injected onto a column equilibrated in buffer E.   2) Washed and eluted with 5 CV (column volume) gradient against buffer F. 5m L fractions were collected.   3) The peaks were pooled.Phenyl Sepharose column: 26 × 17.5cm:   1) Pool 3M (NH_Four)_TwoSO_Four1: 1 dilution.   2) The column was equilibrated with buffer G.   3) Inject protein, wash to baseline, elute with 3 CV gradient against buffer H Was. The peak fractions were pooled.   4) 500 mM NaCl and 5 mM DTT were added to the peak pool. Stored at 4 ° C. under argon .Buffer used B. Syk-NCCloning and expression   The DNA sequence encoding residues 6-265 of human Syk was cloned into the pET expression vector. ,E . coli BL21 (DE3) [Shiue, L. et al. et al.Molecular and Cellular Biology  Fifteen, 272-281 (1995)]. In a typical preparation, 200 μg / mL Syk-NC by growing and inducing 2 liters of culture in BHI medium supplemented with picillin Was produced. The culture was grown at 25 ° C until the OD 595 nm reached 1-2, and 1 mM IP After induction with TG, the cells were collected 4 hours later.Purification   All operations were performed in a cold room at 4 ° C. Cells are twice as large as a French press cell Lysis buffer (20 mM Tris pH 8, 500 mM NaCl, 5 mM DTT and 1 mM pefabloc) And dissolved. The supernatant was collected by a high-speed centrifuge, and buffer A (20 mM Tris pH8) was added twice. , 5 mM DTT) and equilibrated with the same buffer. Applied to anion exchange columns. The effluent (flowthrough) was added to buffer B (20 mM Tris (pH 7.4, 5 mM DTT, 50 mM NaCl) after overnight dialysis and 50 mL phosphotyrosine Injected into a Garose column. Syk-NC protein bound to the column was converted to 4 CV of 50 mM Eluted with a 22M NaCl salt gradient. Collect Syk-NC protein and add buffer C (50 mM Mes pH 6.2, 5mM CaCl_Two, 5 mM DTT). After dialysis, protein sample Centrifuged, then applied to Source 15S column (16 × 10 cm) equilibrated in buffer C did. After washing the column with buffer C, a salt gradient of 5 CV from 0 to 750 mM NaCl was applied. And eluted. The peak fractions were pooled. SDS gel electrophoresis shows that this protein Showed> 95% purity. Predicted by N-terminal sequencing and mass spectrometry Confirmed sequence. The protein concentration was determined by measuring the absorbance at 280 nm. Purified protein was stored at 4 ° C. with 10 mM DTT.Syk-NC And peptide complex   Complexes with a number of different γ and ζ peptides of different lengths were made. Typical In the experiments, a two-fold excess of peptide was dissolved in 100 mM Tris buffer and added to 10 mg Syk-NC. I got it. After incubating this sample at room temperature for 30 minutes, 20 mM Tris pH 8, 100 mM NaCl, 10 mM The solution was applied to a Superdex 75 column (16 × 60 cm) equilibrated in MDTT. 3mL fractions And the peak fractions were pooled.C. Syk-C experiment   The C-terminal SH2 domain of human Syk encoding residues 163-265 was cloned into pGEX2TK. Cloned into the current vector,E . coli Transformed into BL21 (DE3) [Shiue, L .; et al.Molecular and Cellular Biology Fifteen, 272-281 (1995); Law, C.L. et al .J . Biol. Chem. 269, 12310-12319 (1994)]. 1g / L^FifteenNH_FourCl and / or 3g / L¹³C Growth and induction of 2 liter culture in M9 medium supplemented with glucose To produce isotopically labeled glutathione S-transferase (GST) -Syk-C The labeled Syk-C SH2 was obtained. Immediately prior to induction, unlabelled glucose in M9 medium:¹³ Add 4.15 g / L of a mixture containing C in a 9: 1 ratio and label separately. Was done (about 10%)¹³A C Syk-C sample was prepared. In a typical preparation, the culture is And grow to an optical density (OD) of 595 nm at 1.0 with 1 mM isopropyl-b-D-thiol. The cells were induced with galactopyranoside (IPTG) and collected after 5 hours. Cells until use- Stored at 80 ° C.Purification   Lyse the cells and affinity purify the protein with glutathione agarose. Was. GST fusion protein is digested with thrombin, and phosphotyrosine agarose and And 98% pure SH2 domain by SDS gel electrophoresis. Got in. Typical purifications are summarized below. N-terminal sequencing and mass spectrometry Sequence analysis confirmed the predicted sequence. Purified protein is excess dithiotray Stored under argon with toll (DTT).Preparation of the complex   0.5 mL of NMR buffer (50 mM Tris-d₁₁, 0.15N NaCl, 10mM DTT-d₈, 0.025% NaN_Three , PH = 7.0), a two-fold molar excess of pTyr76 peptide was added to S buffer dissolved in the same buffer. Trial of Syk-C SH2 protein: pTyr76 peptide complex in addition to yk-C protein A preparation was prepared. The mixture was cultured overnight at 8 ° C., concentrated, and centrifuged. Equilibrated at 14 ° C. using a 10 microconcentrator. 5 from 2mL to 200mL Complete equilibration was ensured by multiple buffer changes. Of the filtrate and the final complex solution An aliquot was collected and analyzed by HPLC. All NMR samples contain 2-4 mM protein Was.Typical Syk-C purification Cell lysis:   1) Approximately 7 g of the frozen pelleted cells were diluted with 2 volumes of PBS, 0.5% Triton X-100, and 500 mM NaCl. , 5 mM DTT, 2 mM EDTA, 1 mM PMSF.   2) Transfer the homogenate at 16,000 psi using a 20K manual injection French press cell. And dissolved in two treatments.   3) The lysed cells were again diluted with twice the amount of the new lysis buffer and stirred for 10 minutes Thereafter, cell debris was sedimented by centrifugation at 30,600 × g for 40 minutes.   4) The supernatant was filtered through a 0.8 μm Supor membrane filter before injection into the column. Full operation The crop was performed at 4 ° C.Glutathione agarose chromatography   1) A 26 × 100 mm glutathione agarose column was equilibrated with PBS and 2 mM DTT.   2) After injecting the filtered bacterial lysate at 2 ml / min, the column was washed with PBS, 0.5% Triton X Washed with -100, 500 mM NaCl, 2 mM DTT, then with PBS, 2 mM DTT.   3) GST fusion protein was converted to 100mM Tris, 100mM NaCl, 20mM reduced glutathione, 2m Eluted with M DTT, pH 8.0.GST Enzymatic cleavage of fusion proteins   1) One unit of human protein per mg of total protein in glutathione agarose eluate Thrombin was added.   2) Thrombin was reacted at 4 ° C. for about 16 hours with slow magnetic stirring.   3) After confirming the completion of cleavage with 20% SDS gel, the reaction was stopped with 1 mM PMSF.   4) Before injecting the cleaved fusion protein into a phosphotyrosine agarose column The solution was filtered through a 0.2 μm Supor membrane.Phosphotyrosine agarose chromatography   1) A 26 × 100 mm phosphotyrosine agarose column was buffered with buffer A (20 mM Tris, 100 mM M NaCl, 5 mM DTT, pH 7.4).   2) After injecting the cleaved fusion protein at 2 ml / min, backwash to the baseline with buffer A Was cleaned.   3) Syk-C was eluted with 4 column volumes (CV) of 0-100% buffer B gradient. Buffer B = A + 1.9M additional NaCl.PEI Chromatography   1) Equilibrate a 16 × 53 ml polyethyleneimine column with phosphotyrosine buffer A Was.   2) Inject the pooled phosphotyrosine peak at 5 ml / min into PEI and add buffer A Washed. Syk-C was contained in the effluent.D. ZAP-C experiment Cloning and expression   The C-terminal SH2 domain of human ZAP-70 is replaced with glutathione S-transferase (G ST) produced as a fusion protein. Residues 155 to 258 of ZAP-70 as pGEX expression vector -E . coli Transformed into BL21. In a typical fermentation, 200m g / mL ampicillin in LB medium ZAP-C was produced. The culture is grown at 37 ° C. until the OD at 595 nm is 1, After induction with 1 mM IPTG, the cells were collected 4 hours later. Immediately before induction, the temperature was reduced to 25 ° C. 1 g / L^FifteenNH_FourGrowth and induction of culture in M9 medium supplemented with Cl^FifteenN-labeled ZAP-C Produced. Cells were stored at -80 ° C.Purification   Lyse ZAP-C cells and affinity the protein with glutathione agarose Purified. GST fusion protein is digested with thrombin and phosphotyrosine agarose > 95% purity by sodium dodecyl sulfate (SDS) gel electrophoresis SH2 domain was obtained. Purified protein is excess dithiothreitol (DTT) and stored under argon.Typical ZAP-C purification Cell lysis:   1) 50 g of frozen ZAP-C cells were added to 2 volumes of PBS, 0.5% Triton, 500 mM NaCl, 5 mM DTT , 2 mM EDTA, and 1 mM PMSF.   2) The resulting homogenate was subjected to 3 min at 16,000 psi using a Minnie Rannie cell disruptor. Dissolved by repeated treatments.   3) The cell lysate was centrifuged at 30,000 × g for 40 minutes.   4) The supernatant was collected and filtered through a 0.8 μm membrane before injection into the column. All operations at 4 ° C went.Glutathione agarose chromatography   1) A 26 × 100 mm glutathione agarose column was equilibrated with PBS and 2 mM DTT.   2) After injecting the filtered bacterial lysate at 2 ml / min, the column was washed with PBS, 0.5% Triton, Washed with 500 mM NaCl, 2 mM DTT, then with PBS, 2 mM DTT.   3) GST fusion protein was converted to 100 mM Tris, 100 mM NaCl, 2 mM DTT, 20 mM reduced gluta. Eluted with thione, pH 8.GST Enzymatic cleavage of fusion proteins   1) 1 unit of human toro per 1 mg of protein in eluate of glutathione agarose Was added.   2) Thrombin was reacted at 4 ° C. overnight with slow stirring.   3) The next morning, add additional thrombin (1 unit / mg protein) to the protein solution and cut The cleavage reaction was monitored on an SDS gel. After completion, the reaction was stopped with 1 mM PMSF.Phosphotyrosine agarose chromatography   1) Using a 26 × 80 mm phosphotyrosine agarose column with buffer A (20 mM Tris, 100 mM NaCl, 2 mM DTT, 2 mM EDTA, pH 7.4).   2) After injecting half of the cleaved fusion protein into this column at 2 ml / min, buffer The solution A was washed to the baseline. Elution of ZAP-C with 4 column volumes of 0-100% buffer B gradient (Buffer B = A + 1.9 M NaCl).   3) This phosphotyrosine chromatography is then performed on the other half of the cleaved fusion tag. Applied to protein. ZAP-C fractions were collected and analyzed by UV / visible spectroscopy.II . Preparation of ligand   All peptides are TFA-labile side-chain protected N-fluorenylmethoxy Applied Biosystems 431A or 433A synthesizer using bonyl (Fmoc) amino acid Synthesized by automated solid-phase synthesis on an Iser. The synthesis is Rink resin (Rink, H.  Tetrahedron Lett. 1987, 28, 3787-3790) on a 0.25-0.50 mmol scale. gave. Amino acid (1.0 mmol) uses HBTU / HOBt / DIEA (1: 1: 2) as activator And joined. The coupling reaction was for 30-50 minutes. Acetyl assembled peptide (Ac in DMA_TwoO / pyridine) before phosphorylation (one reaction for 5 minutes). Or after phosphorylation (2 × 10 minutes reaction).Phosphorylation ^39,40   Mix resin-bound peptide (0.25 mmol) and tetrazole (25-40 equivalents / OH) And dried under vacuum overnight in the presence of NaOH pellets in a desiccator. In the flask, Then N_TwoAnd add DMA (6 mL). (t-BuO)_TwoPNEt_Two(10 equivalents / OH) and mix. Sonicate the compound for 60-90 minutes. Filter the resin, DMA (3 x 5 mL) and CH_TwoCl_Two(Four × 5 mL). CH_TwoCl_Two(5 mL) and mCPBA (2-5 eq / OH) Sonicate the mixture for an additional 20-50 minutes. Filter the resin, CH_TwoCl_Two(6 × 5 mL ) And suction dry.Resolution and deprotection groups   Phosphorylated peptide-resin was converted to TFA: phenol: water (90: 5: 5) or TFA: water : Ethandithiol: Anisole: Phenol (95: 5: 5: 5: 2) 90 ~ Treat for 120 minutes. The resin is filtered, washed with TFA and the filtrate is concentrated by rotary evaporation You. Diethyl ether was added to precipitate the crude peptide, which was filtered off, Et._TwoIn O Wash and dry. Combine peptides with gel filtration, preparative HPLC, and gel desalting And purify.   Gel filtration: 0.1M NH for crude peptide_FourHCO_Three(10-20 mL) Apply to a G-25 column (2.6 x 100 cm) and elute at about 0.5 mL / min. 254 eluents Or monitor at 278 nm and identify product-containing fractions by analytical HPLC, pool and freeze-dry. Dry.   Preparative HPLC: Reverse-phase Kromasil C8 column with UV monitoring of peptides at 220 nm (Particle size 10 microns, pore size 100 mm, 20 × 250 mm). The product is treated with 0.1% TFA 60/40 MeCN / H in aqueous solution_TwoO (0.1% TFA) or 25mM Et_Three60/40 Me in N phosphate CN / 25mM Et_ThreeElute with either gradient of N phosphate pH 7. The latter buffer Requires desalting of the isolated pure peptide, which results in a lyophilized product. Apply to Sephadex G-10 or G-15 column (2.6 × 30 cm), 0.1M NH_FourHCO_ThreeBy 1 Performed by eluting at ２2 mL / min.Ligand (9)   Nα-Fmoc- (O, O-diethyl-α, α-difluorophosphonomethyl) phenylalanine (FmocF_TwoPmp (OEt)_TwoOH) and solid-phase synthesis of ligand (9) according to the method described above. (Burke, T.R., Smyth, M.S., Otaka, A, Nomizu, M., Roller, P.P., Wo lf, G., Case, R., Shoelson, S.E., phosphatase resistant SH2 domain inhibitors Non-hydrolyzable phosphotyrosyl mimics for preparation,Biochem. 1994, 33, 6490-64 94). This peptide is converted to TFA: phenol: H_TwoO: ethanedithiol: anisole (18: 1: 1: 1: 1) to separate the crude bis-O, O-diethyldifluoro A lophosphonate-containing product was obtained. The last deprotecting group converts the crude product to TMS-I at room temperature: This was performed by treating with MeCN (1: 1) for 20 minutes. Evaporate the solvent and elute the residue with 0.2M It was dissolved in sodium phosphate (pH 7.0: 50 mL). This solution was added to diethyl ether (6 × 15 mL) and lyophilized. The product was purified as described above.III . Crystallization and structure determination A.ZAP-NCIs crystallized in a complex with various ligands (see Table 1) as follows: Was:   1. Zap / ζ1: Binary complex of ZAP-NC and ζ1 19-mer (ligand 5, Table 1) 20 mM Tris, 200 mM sodium chloride, and 20 mM dithiothreitol at pH 8.5 Was concentrated to 30 mg / ml in a buffer containing Complex with ZAP-NC ζ1 peptide Further treatment with 4 mM trimethyl lead acetate. The crystals are 20% polyethylene glycol 13.5 mg / ml over a pool of 4000 and 20 mM dithiothreitol precipitant solution. Protein complex, 10% polyethylene glycol 4000, 50 mM quencher at pH 6.2 Sodium acid, 100 mM ammonium acetate, 0.005% sodium azide and 20  3 weeks with multiple hanging drops containing mM dithiothreitol Within a while they grew spontaneously. Large crystals overnight due to microseeding was gotten. Each of the obtained crystals has a monoclinic system (P2₁, A = 50.11, b = 63.37, c = 54.00 °, and β = 114.44 °). Ligand 8 (Table Crystals of ZAP-NC complexed with 1) were also obtained under the same conditions.   2. Zap / ζ2: formation of a binary complex between ZAP-NC and ζ2 19-mer (ligand 6, Table 1) The crystal underwent some changes below under conditions similar to those used for Zap / ζ1. Obtained. Concentration of the protein was performed using 10 mM Tris at pH 8.5, 0.5 M sodium chloride, Performed in 20 mM DTT. 20-26 mg / ml protein before preparing for crystallization And 2 mM trimethyl lead acetate for 1 hour. Each drop corresponds to a Zap / ζ1 complex. In addition to the conditions described above, 20 mM sodium acetate and 0.2 M sodium chloride Was contained. The final pH during the drop is between 6.4 and 6.5. Crystallization of Zap / ζ1 Performed by micronucleus inoculation (no spontaneous crystallization was obtained). The crystal is an asymmetric single One molecule per position monoclinic (P2₁, A = 50.00, b = 63.19, c = 54.22 Å, and β = 114.6 °), and the X-ray was diffracted to a resolution of 2.2 °.   3. Zap / ζ3: formation of a binary complex of ZAP-NC and ζ3 19-mer (ligand 7, Table 1) Crystals were prepared for ZAP-NC / つ い 2, except that the drops contained sodium acetate. Obtained as described. Crystallization was obtained after micronucleus inoculation with Zap / ζ1 crystals. Was. The crystals are monoclinic (P2₁, A = 49.85, b = 63.38, c = 54.01 °, and β = 114.43 °), and the X-ray was diffracted to a resolution of 2.6 °.   4. Zap / difluorophosphono-ζ1: ZAP-NC and ζ1 19-mer difluorophospho The crystals of the binary complex with the analogs (ligand 9, Table 1) are based on ZAP-NC / ζ1 As described, obtained after micronucleus inoculation with Zap / ap1 crystals. Crystals are unpaired Monoclinic system (P2₁, A = 49.77, b = 60.87, c = 53.58 mm, and And β = 117.09 °), and the X-ray was diffracted to a resolution of 2.2 °.   5. Zap / IgE γTAM 19-mers: ZAP-NC and IgE γTAM 19-mers (ligand 1, Table 1) Crystals of the binary complex with) were prepared as described for ZAP-NC / ζ1, except that pH 6. 6, obtained after micronucleus inoculation with Zap / ζ3 crystals.   6. Zap / IgE γTAM 16-mer analog: ZAP-NC and IgE γTAM 16-mer analog (Riga The crystal of the binary complex with the compound 10 and Table 1), as described for ZAP-NC / ζ1, However, by micronucleus inoculation of Zap / γ crystals at pH 7.0 in the presence of 2% glycerol. Was obtained.   7. Zap / ligand 11: Mix ZAP-NC with 3 mM ligand 11 (Table 1) and mix Treated with methyl lead. The crystals were made up of 10% PEG 4K, 50 mM Tris, pH 8.23 and 10 mM DT. 12.5 mg / ml ZAP-NC protein, 1.5 mM ligand 11 and 1.5 mM vinegar in a solution of T Obtained from a mixture of trimethyl lead acid. B. SYK-NC was crystallized with or without ligand (see Table 1) as described below. Was:   1. SYK-NC (no ligand) is 20 mM Tris, 0.2 M sodium chloride, pH 8.0 , Concentrated in 40 mM DTT. Crystals are 10% PEG 4k, 0.2 M sodium chloride, 50 mM Obtained at 12.5 mg / ml protein in phosphate buffer, pH 7.3, 30 mM DTT.   2. syk / ζ1: formation of a binary complex of SYK-NC and ζ1 19-mer (ligand 6, Table 1) The crystals were 50 mM Hepes, pH 7.2, 9% PEG4k, 4% 2-propanol, 0.25 M NaCl. And 11 mg / ml syk / ζ1 complex in 30 mM DTT.   3. syk / γ19: Binary complex of SYK-NC and IgEγ19 mer (ligand 1, Table 1) The crystals of 50 mM Tris pH 7.68 or 50 mM imidazole pH 7.36, 11% PEG 4k, 3.5% 2-propanol, 0.3 M sodium chloride, 11 mg / Obtained with the ml syk / ζ1 complex.   4. syk / γ15: Binary complex of SYK-NC and IgE γ15 mer (ligand 2, Table 1) Body crystals were obtained under several different combinations of conditions described below.     a. 10% PEG 4K, 50 mM citrate / phosphate buffer, pH 5.6, 0.1 M ammonium chloride 18 mg / ml complex in monium, 0.01% sodium azide, 30 mM DTT.     b. 10% PEG 4K, 50 mM sodium citrate buffer, pH 5.6, 0.1 M Ammonium, 0.5% methylpentanediol, 0.01% sodium azide, 30 mM 18 mg / ml complex in DTT.     c. 10% PEG 6K, 50 mM phosphate buffer, pH 6.2, 0.2 M sodium chloride, 50  18 mg / ml complex in mM ammonium acetate, 0.01% sodium azide, 30 mM DTT body.   5. syk / γ25: Binary complex of SYK-NC and IgE γ25-mer (ligand 3, Table 1) Body crystals were obtained under several different combinations of conditions described below.     a. 16% PEG 2K, 50 mM sodium citrate, 5% glycerol, 20 mM DTT , 12.5 mg / ml SYK-NC / ligand complex in pH 6.46.     b. 10% PEG 4K, 50 mM sodium citrate, 0.1 M ammonium acetate, 20 12.5 mg / ml SYK-NC / ligand complex in mM DTT, pH 6.3.   6. syk / γTam 27-mer: SYK-NC and IgE γ27-mer (ligand 4, Table 1) The crystals of the primary complex are just above for the case of IgE γ25-mer (ligand 3, Table 1) Was obtained in the same manner as described above. C. ZAP-NC: ζ1 3D structure   ZAP-NC: X-ray diffraction obtained using crystals of ζ-1 complex (one complex per unit cell) The data was analyzed as described elsewhere (see also Table 2) and the crystalline The coordinates defining the three-dimensional structure of the coalescence were obtained. ZAP-NC: ζ2 The structure of the complex is ZAP-NC: ζ2 XAP diffraction data of the complex and ZAP-NC as indicated by the coordinates in Annex I : Determined by the molecular replacement method using the structure of the ζ-1 complex. ZAP-NC: $ 1 model The rigid body approximation was refined by using The obtained model was compared with ζ1 peptide for ζ2 After substitution with the peptide, it was reconstructed by conventional refinement. ZAP-NC: ζ-1 "dimer" The X-ray data of the complex (2 complexes per unit cell) is also ZAP-NC: ζ-1 ("monomer") structure It was analyzed by the molecular replacement method using the structure. Complete ZAP-NC: ζ-1 "monomer" model Rigid body refinement using individual SH2 domains And reconstructed the helical domain region. Their structural coordinates are given in the annex below. Document I (ZAP-NC: ζ-1 complex, "monomer"), Annex II (ZAP-NC: ζ2 complex) and And Annex III (ZAP-NC: ζ-1 complex, "dimer") contain a protein databank file. It is shown in the format. Such data is tailored to the worker's computer. May be transferred to any desired medium and formatted as desired.   The present invention preserves those coordinates, as well as their internal coordinates (ie, their unique Which holds the internal relationship of You. Those skilled in the art will appreciate that these coordinates are, for example, dynamic simulations.  other transformations, including molecular mechanics calculations such as simulation), minimization, etc. Understand that it can be done. The present invention further provides for ZAP-NC or other ZAP files. The coordinates of the corresponding area of the family member, in particular Annex I, Annex II or Appendix Such transformations (or the formation of another conformation, for example) And the products of this transformation (ie, the derivation of the coordinates). Conductor). IV. Modeling   Silicon running Irix 5.2 to illustrate receptor site mapping techniques, Silicon On the Graphics Onyx workstation, set up a Molecular Discovery program (Molecular Discovery Ltd; Goodford, P.J. “Biologically important macromolecules A Computer Method for Determining Energetic Advantageous Binding Sites "J . Med. Chem.  1985, 28, 849-857), we evaluated ZAP-NC as follows. (1) From the ZAP-NC + ζ1 protein data bank (PDB) coordinate file, Pide and all crystallographically observed water molecules were removed (Sybil's Remove Atom With features). (2) Convert the obtained PDB file into a set of molecular mechanics parameters suitable for protein studies. (Such suitable parameters include the computer User extension data files that are commonly associated with the program). (3) The entire peptide binding surface of each SH2 domain and the interface region between the two Created a three-dimensional box to surround. The dimensions of this box are 60Å × 40Å × 37Å, 0.50.5 Regular grid points arranged at intervals were specified to fill the box. Figure 6 shows the position of the box Shown in (4) At each lattice point, 46 atoms (atomic) and polyatomic probes Placed. These probes provide parameters that represent a wide variety of chemical moieties. Include. The energy of the interaction between each probe and the protein is determined by Lennard-Jo Empirical, including multiple well-defined terms for nes, electrostatic and hydrogen bonding potentials According to the potential energy function, computer calculation was performed at each point of the lattice. each The degree of burial of the probe is calculated by computer Calculated. (5) A secondary contour map was created. This means that some potential energy Can be used to visualize the site of advantageous interaction of the lobe. One example is shown in FIG. You.   The data from this receptor site mapping experiment was used by Silicon running Irix 5.2. On a Graphics Onyx workstation, write the DAT text in tar format. Moved to the soup. This map data is file extension for each secondary contour map Uses child .cnt and .lont and ASCII output. The orientation of the map is ZapNC-zl.pdb, which is a file from Brookhaven Protein Databank. It is a format. This contour file is Sybyl 6.1 (Tripos, Inc., St. Louis, MO). File name is Molecular Discover program System, version 12 probe nomenclature.   As described in the section on computer processing, the above or other programs Using the information provided by site mapping, potential drug support sites (pharmacological Group, pharmacophore) to determine the spatial arrangement, thus searching the 3-D database Seed points or growth from or selection from a selected point A new program can be provided that attempts to conclude. Used by computer method For an example of the basic structure of a drug-supporting site for See Fesik (1993) cited above.   For example, data defining a secondary contour map is stored in a software program such as Sybyl. 3D structure to identify preferred positions for selected functional groups using May be displayed on the screen. Review those screen displays visually and show them on the map. Preferred position (ie characterized by favorable interaction energy with target protein) Positions identified for each selected functional group). It is possible to select the corresponding compound that contains the moieties appropriately arranged for Wear. Using a computer program such as LeafFrog, one compound can be From one or more selected parts located in the It can also be "grown" mathematically. Or for two or more parts Starting from the position of interest mapped and defining the spatial relationship between those parts. Define the "vector" to be determined, and then use those Selecting or designing a compound that embodies the selected moiety in a spatial relationship Can also. A database containing the 3D structures of potential ligands is determined experimentally Or computer-generated structures. Choice or design professional Seth, ALADDIN (Van Drie et al, 1989, J. Comput-Aided Mol Des 3, 225-251). ), MACCS-3D (Moock et al, 1990, Chemical Information Systems, Bawden & Mi tchell, Chichester, pp 42-49), 3D SEARCH, ChemDBS-3D, SYBYL / 3D and CA Can be done with the help of a computer using a program like VEAT . See, for example, Fesik, 1993, J Biomolecular NMR 3, 261-269.V. Test method (1) Binding test (a)Competitive binding assay: Measurement of binding is performed by surface plasmon resonance (SPR) and applied techniques. [Malmqvist, M.,Current Opinions in Immunology Five, 282-286 (1993); Malmq vist, M., Nature 361: 186-187 (1993); Jonsson, U., Malmqvist, M.,Advances in Biosensors , JAI Press Ltd. London, 1992, pp. 291-336; Jonsson, U. et  al,Bio Techniques 11 (5): 620-627 (1991)]. Wear. The SH2 domain is typically pre-incubated with various concentrations of the test compound. SH2 binding to phosphopeptide ligands with immobilized test compounds The ability to competitively inhibit is first measured. Results are measured in the absence of competitors Percentage inhibition is calculated by comparison with the binding. I c₅₀Value reduces binding by 50% Represents the concentration of inhibitor required. Details of each measurement are described below. All measurements , 10 mM HEPES (pH 7.4) / 150 mM NaCl / 3.4 mM EDTA / 0.05% Tween 20 ± 10 m Performed at 25 ° C. in HEPES buffered saline (HBS) consisting of MDTT.Primary screening  -The test compound (e.g., 50 μM) is added to the target SH in HBS ± 10 mM DTT. Preincubate with 2 at 4 ° C for a minimum of 1 hour. The compound is The ability to inhibit binding of the SH2 domain to the phosphopeptide is measured using SPR. Compounds that inhibit SH2 / phosphopeptide association by a predetermined percentage (eg, ≧ 50%) The product is subjected to the following secondary screening.Secondary screening  -Log dilutions of test compound (10^-Four,Ten^-Five,Ten^-6, ...) Pre-incubate with target SH2 in HBS ± 10 mM DTT. The compound The ability of a product to inhibit binding of the SH2 domain to a target phosphopeptide using SPR Measure. % Inhibition relative to compound concentration (compared to SH2 domain in the absence of inhibitor) From the plot), IC₅₀Find the value of ・Details of the column ZAP test: Peptide corresponding to human T cell receptor ζ chain ITAM-1 [NQ LY (PO_Four) NELNLGRREEY (PO_Four) DVLD] [SEQ ID NO: 15] to a larger peptide [Ac-KGG NQLY (PO_Four) NELNLGRREEY- (PO_Four) DVLD-NH_Two] Synthesized as part of [SEQ ID NO: 16] Used to form sensitive biosensor surfaces. Specifically, Activate surface chip CM5 with 200 mM EDC / 50 mM NHS to react with primary amine. The ITAM peptide was immobilized via the N-terminal lysine. Not yet The reaction site was blocked with ethanolamine (1M in water) and the chip was Gin hydrochloride was used to clean up non-covalently bound peptides. The test is 10 nM pp70^ZAPPerformed in HBS + 10 mM DTT with (1-259) +/- test inhibitors. ・N-ZAP Test details: Peptide corresponding to human T cell receptor ζ chain ITAM-1 [NQLYN ELNLGRREEY (PO_Four) DVLD] [SEQ ID NO: 17] to a larger peptide [Ac-KGGNQLYNELNLG RREEY- (PO_Four) DVLD-NH_Two] Synthesized as a part of [SEQ ID NO: 18] Used to form sensitive biosensor surfaces. Test is pp70^ZAP(1-259) R195K (Arginine 195 is replaced with lysine to inactivate the C-terminal SH2 domain. Performed in HBS + 10 mM DTT using the substituted mutant) +/- test inhibitor. ・C-ZAP Test details: Peptide corresponding to human T cell receptor ζ chain ITAM-1 [NQLYN ELNLGRREEY (PO_Four) DVLD] [SEQ ID NO: 17] to a larger peptide [Ac-KGGNQLYNELNLG RREEY- (PO_Four) DVLD-NH_Two] Synthesized as a part of [SEQ ID NO: 18] Used to form sensitive biosensor surfaces. Test is pp70^ZAP(1-259) R37K (place arginine 37 with lysine to inactivate the N-terminal SH2 domain) Exchanged mutant) or pp70^ZAP(161-259) +/- HBS + 10 mM D using test inhibitors I went inside the TT. ・Column Syk Test Details: Pp72 corresponding to γ-chain ITAM of human FcεRI^sykPeptide Riga DGVY (PO_Four) TGLSTRNQETY (PO_Four) ETLK] [SEQ ID NO: 19] to a larger peptide Do [Ac-CGGDGVY (PO_Four) TGLSTRNQETY- (PO_Four) ETLK-NH_Two] Synthesized as part of [SEQ ID NO: 20] And used to form a Syk-sensitive biosensor surface. Specifically, Iosensor chip CM5 was replaced with 200 mM ethyl-3- (3-dimethylaminopropyl) -calcium. After activation with bodimide hydrochloride (EDC) / 50 mM N-hydroxysuccinimide (NHS) Develop a surface reactivity to the primary amine, treat with ethylenediamine, Form an amine-rich surface, m-maleimidobenzoyl-N-hydroxysuccin Imidoester (sulfo-MBS; 25 mM NaHCO_ThreeThiol To produce surface reactivity against Immobilized via terminal cysteine. Unreacted sites are blocked with β-mercaptoethanol. Lock the chip and release the non-covalent peptide using 6 M guanidine hydrochloride. It was cleaned to become. Assay is 20 nM pp72^syk(1-265) +/- Performed in HBS. ・C-Syk Test details: Pp72 corresponding to human phosphorylated γ chain ITAM of human FcεRI^sykPep Tide ligand [DGVY (PO_Four) TGLSTRNQETYETLK] to a larger peptide [Ac-CGGDG VY (PO_Four) TGLSTRNQETYETLK-NH_TwoSynthesized as part of] and described above for column syk Was used to form a C-Syk sensitive biosensor surface as described above. The test is 270 nM pp72^syk(163-265) +/− Test inhibitors were performed in HBS.(2) Cell-based assays (a) T cell assay (T cell receptor dependent transcription)Purpose and description of the test : This test is based on the human Jurkat of a compound. It measures the ability of the T cell line to inhibit TCR activation of the IL-2 transcription pathway. You. This Jurkat cell is used to control the upstream promoter, which normally regulates IL-2 production Element, NF-AT binding site¹⁸Construct containing the β-galactosidase gene under the control of Transfected. Activation of cells produces β-galactosidase Live.Cell drug treatment and stimulation : The compound to be assayed is serially diluted in an assay buffer. The diluent is added to the Jurkat cells for a one hour preincubation. After pre-incubation, the plate is coated with an antibody against the CD3 component of the TCR. Transfer cells. This antibody cross-links the TCR and does not activate the receptor signaling pathway You. After incubating the cells for 4 hours, the amount of β-gal produced is measured.Measurement of β-galactosidase : Using this measurement, β-gallon produced by the MUG assay Quantify the amount of custosidase. MUG (4-methylumbelliferone galactose) Is cleaved with β-galactosidase to give the fluorescent derivative 7-hydroxy-4-methyl bear. Produces phosphorus. The observed fluorescence is dependent on the amount of cleavage product and thus on β-galactosidase. Correlates with the amount of dase. For each well, β-gal with no compound added Is determined as 100%. Convert the raw data to the inhibition rate (%) And then the data are processed as a percentage of the controlled release relative to the concentration of the test compound. Cut.(b) Cytotoxic T lymphocyte killing Purpose and description of the test : This assay is based on human cytotoxic lymphocyte cells of a compound It measures the ability of the system to inhibit the cytotoxic function. Mitogen stimulus removal Blood lymphocytes from human CD8⁺ CTL was prepared. Mitogen-activated cells are in IL2 And re-stimulated every 3 weeks with an antibody against the T cell receptor complex, CD8 + Cells were sorted and cloned. Clone T9 was selected for use in the CLT assay. Was. The T cells were non-specifically selected, ie, did not use specific antigens for induction. Therefore, the target chosen for this assay was the B cell hybridoma OKT3. This cell line has on its surface an anti-CD3 (one of the subunits of the T cell receptor) Antibody). Recognition of the T cell receptor by this antibody is Induce Roses.Cell drug treatment and stimulation : The compound to be assayed is serially diluted in an assay buffer. The diluent is added to the CTL for a one hour preincubation. Spare incubator After the incubation,⁵¹Cr-labeled OKT3 cells and 3: 1 (effector: target) And incubate for 3 hours. This effector: target ratio The rate produces a specific release of about 30%. ⁵¹ Cr Measuring emissions : Centrifuge the cell mixture and remove the supernatant. Released into medium Was done⁵¹The amount of Cr is measured with a gamma counter, and the value of specific release is determined by detergent dissolution. The value for the target cells is determined as 100%. This data is plotted against the test compound concentration. Plot as specific release (%).(3) Animal model: (a) Delayed type hypersensitivity   The first screening model is delayed type hypersensitivity. Dinitro on the abdomen of mice Apply sensitizing chemicals such as fluorobenzene or oxazarone (sensitization) I do. Seven days later, a low concentration of the same compound is applied (attacked) to the ear of the sensitized mouse. You. Antigen processing and presentation, T lymphocyte activation, leukocyte infiltration, body fluids Mediator release, increased microvascular permeability, and plasma effusion all sensitized This is a phenomenon caused by the attack of a mouse, which results in the formation of edema. Edema at 24 o'clock Appears as a 2-3 times increase in ear thickness within the interval.   Test compounds or standards at various times before and after the sensitization or challenge phase It can be administered (topically or parenterally). Increased ear thickness may be due to immunosuppressants and And some compounds, including steroids. This model is One primary model for dermatitis.(b) Allogeneic skin transplant   Allogeneic skin transplantation is used to determine the immunosuppressive activity of a test compound. this In the model, the thoracic skin of a donor mouse (Balb / c) was transferred to a recipient mouse (C57bl / 6). Surgically implanted over the rib cage. The host rejection of this skin graft provides skin contraction, dryness Proven by dryness and erythema. The average graft survival is 10-11 days, 80% of the pieces are rejected by 12 days. Active novel immunosuppressive compounds Like the compound, try to prolong the survival of the graft.(c) Popliteal lymph node hyperplasia   This model directly evaluates T lymphocyte proliferation in vivo. From Balb / c mice Spleen cells are isolated and administered into C3H mouse foot pads. 4 Within days, popliteal lymph nodes may be removed from recipient mice and weighed. it can. Other hematological evaluations, including FACS scans on T lymphocyte subpopulations The law can also be implemented. Active compounds, like existing immunosuppressive compounds, Let's stop the increase.(d) Rheumatoid arthritis   Evaluation of anti-arthritic effects includes adjuvant induction in rats and / or mice, Available in several models, including carrageenan-induced and collagen-induced arthritis It is possible. Measurements are taken after 20-30 days as the volume of the legs increases. Leg The ability of the test compound to prevent the induction of swelling is determined daily for 12 consecutive days after injection of the inducer. Test by treatment. Ability of test compound to reverse the development of leg swelling To administer the test compound for 12 consecutive days starting on day 12 after injection of the inducer. To be tested. Water displacement plethysmography (volume variation) Recording method). Histology is also a good endpoint for these studies. Above MRL / lpr mouse model is required to display rheumatoid arthritis. This model Is a circulating rheumatoid factor, pannus formation, and Develops rheumatoid arthritis that mimics human symptoms, including the presence of bone and cartilage erosion This is a spontaneous autoimmunity model.(e) Systemic lupus erythematosus   Systemic lupus erythematosus (SLE) is another autoimmune disease with several animal models I am sick. Several mouse strains express SLE spontaneously. One of such systems One is the MRL / lpr mouse. These mice develop nuclear over time (20-30 weeks) It expresses autoantibodies against dsDNA of antigen and renal basement membrane. This is the fixation of complement And the formation of an immune complex. Renal damage revealed by onset of proteinuria Become. The remaining physiological, hematological and immunological aspects described below for the CGVHD model Many anomalies also exist. Cyclosporine, cyclophosphamide and leflunomie Immunosuppressive compounds, such as drugs, prevent and reverse the disease process in this model. Can be. Interestingly, these mice resembled rheumatoid arthritis A pathological phenomenon also appears.   The mouse chronic graft-versus-host disease (CGVHD, described below) model has many of the clinical features of SLE. It is a model of SLE including. Activity in this model is more clinically relevant It was shown to be predictive of activity in the high SLE model.(f) Transplant   The allograft (skin graft) assay is often used as an initial test for immunosuppressive activity . This model is useful as a screen, but is useful for assessing graft survival. Using “clinically acceptable” physiological endpoints, internal organs (heart, liver, kidney, It can also be supplemented by assays based on animal transplant models, including bone marrow) transplants. Very limited Only a limited number of rodent models require test compound efficacy. One rodent model After observing the activity in an animal model (eg, a dog, pig, or It is common to test test compounds in non-human primates). The active compound is Reduces acute and chronic rejection and prolongs graft survival.(g) Graft-versus-host disease (GVHD)   Chronic GVHD (CGVHD) is CD4⁺Can be used to model dependent humoral immunity. this is BDF₁In mice (this is the offspring of a DBA / 2 male x C57BL / 6 female mating), Spleen isolated from DBA / 2 mice: induced by administration of lymph node cells It is. This is due to a) CD4⁺T lymphocytes (Lyl⁺;mouse Markers) disregulation and stimulation of activity, and b) abnormal TB Produces lymphocyte cooperation. The resulting pathological condition is often a systemic area. Similar to Tematodes (SLE). Some measurable endpoints appear within 14 days This includes circulating anti-host IgG and IgE antibodies, altered T measured in vitro and B cell proliferative activity, complement utilization, hemagglutination, slow progressive wasting, skin abnormalities , Splenomegaly, lymphocyte hyperplasia, and proteinuria. A small number of these endpoints Only measurements are needed for the measurement. Active compounds limit T lymphocyte dysregulation, It is to eliminate the change of these factors. Many steroids (eg, prednisolo Yes), cyclosporine, FK506, cyclophosphamide, and leflunomide Yes Deviation is also active in this model and can be used as a positive control.   Acute GVHD model (AGVHD) is also BDF₁Made with mouse. In this case, the C57BL / 6 Spleen isolated from mouse: lymph node cells are administered. This is due to the lack of MHC I molecules. CD8 due to conformity⁺This results in dysregulation and irritation of T lymphocytes. Finally, de Donor's cytotoxic T lymphocytes and NK cells rapidly reject host tissues, Causes a relatively rapid death of the event. Progression of AGVHD in this model is (Including the number and type of T cells), cytokines (TNF, IL-1, IL-2 and / or IL-4), low body weight, hypogammaglobulinemia, circulatory system as an indicator of aplastic anemia Hematological signs (granulocytopenia, thrombocytopenia), ex vivo NK or CTL activity, and And the viability of the host. Active compounds can alter the above factors. Comb prolongs survival over 4-6 weeks or more.(h) asthma   Asthma offers another opportunity for safe immunosuppressive treatment. Atopic asthma patients Have antibody-mediated hypersensitivity and often have late-occurring late phase reaction) is likened to a DTH response. Asthma most recently defined as an inflammatory disease (1992). Since then, several publications by prominent asthma scholars have Activated CD4 in bronchoalveolar lavage fluid and blood of patients with primary asthma⁺And CD8⁺T Demonstrates the presence of lymphocytes. The proportion of these cells changes during asthmatic conditions . In addition, some of the T cell binding cytokines (IL-1, IL-2, IL-4, IL-5, and And TNF) are all involved in clinical and experimental asthma. Inflammatory events in asthma Is now thought to be driven by T lymphocytes. Inhalation of cyclosporine Early clinical trials showed that local immunosuppression was a defect underlying the asthma It suggests that hypersensitivity can be alleviated.   A guinea pig model of antigen-induced lung abnormalities is used as a model for asthma. This dynamic The product is a seroconverter for the IgE class, as is the case in atopic asthmatics. To generate high circulating titers of anti-ovalbumin antibodies Sensitive to albumin activity. Aerosol attack on sensitized guinea pigs was measured Possible eosinophil-rich pulmonary infiltrates (eosinophils increase about 16-fold), pulmonary edema, and small airway This results in mucosal obstruction, all of which are exacerbated by airway overload, a defect underlying A rapid response (approximately 3- to 4-fold increase in reactivity) occurs. Acute bronchoconstriction clearly present It sharpens the presence of the pathophysiological sequelae described above. Active compounds are It significantly reduces or eliminates the weather.   The above description is illustrative rather than limiting of the scope of the present invention. Less than Given the above description, numerous materials and methods used in the practice of the present invention Changes will be apparent to those skilled in the art. All such obvious modifications are within the scope of the present invention. Should be considered within the enclosure.References

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), UA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM , AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, G B, GE, HU, IS, JP, KE, KG, KP, KR , KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, TJ , TM, TR, TT, UA, UG, UZ, VN (72) Lou, Shaod             Massachusetts, United States             02151, Libya, Lantern Road 242,             Apartment 14 (72) Inventor Laird, Ellen Earl             Massachusetts, United States             02166, Newton, 2-2, Commonweed             Luss Avenue 2340 (72) Inventors Callas, Jennifer L             Massachusetts, United States             02173, Lexington, Haze Avenue             ー 27 (72) Inventors Zoller, Mark Jay             Massachusetts, United States             02193, Weston, Sutton Place             7 (72) Inventor Holt, Dennis A             Massachusetts, United States             01775, Stowe, Brookmill Road 7

Claims (1)

[Claims] 1. Peptides containing the tandem SH2 region of ZAP family proteins or parts thereof A material comprising a crystalline form of the protein containing the sequence. 2. The material of claim 1, further comprising one or more heavy metal atoms. 3. The protein is Annex I, Annex II, Annex III, or Annex I For the coordinates of V or the backbone atoms of the amino acids listed there, Includes an area characterized by coordinates whose mean squared deviation is less than or equal to 1.5 mm. 2. The material according to claim 1, wherein 4. Claims: The protein is in a complex with one or more ligand molecules. 2. The material according to claim 1, wherein 5. The protein is the tandem SH2 region of human ZAP-70 (at least amino acids 3-279). Or the tandem SH2 region of human SYK (containing at least amino acid residues 6 to 265). 2. The material according to claim 1, wherein the material comprises: 6. 6. The method of claim 1, wherein the X-ray is diffracted with a resolution better than about 3.5 °. The material according to any of the above. 7. A protein containing an SH2 domain or the A method for determining a three-dimensional structure of a complex,   (a) taking X-ray diffraction data of the protein or co-complex crystal,   (b) calculating the three-dimensional structure coordinates of the material according to any one of claims 1 to 6; Prepare, and   (c) analyzing the X-ray diffraction data for the existing structural coordinates using molecular replacement; Determine the three-dimensional structure of the SH2 domain-containing protein or co-complex. Do A method that includes: 8. The protein binds ZAP-NC or a portion thereof with a ligand other than the ζ1 peptide. 8. The method according to claim 7, which is a co-complex. 9. The protein is a co-complex of SYK-NC or a portion thereof and its ligand. The method of claim 7, wherein Ten. A protein containing an SH2 domain or the A method for determining a three-dimensional structure of a complex,   (a) using the structural coordinates of the material according to any one of claims 1 to 6; And   (b) The homology modeling of the existing structural coordinates allows the SH2 domain Determine the three-dimensional structure of the containing protein or co-complex, A method that includes: 11． By selecting compounds that can bind to ZAP family proteins So,   (a) Use coordinates that define the three-dimensional structure of the ZAP family protein or a portion thereof. I mean,   (b) establishing the advantage of interaction with one or more selected functional groups; Characterize (characterize) points related to body structure,   (c) providing a database of one or more candidate compounds; and   (d) From this database, it is best to consider the points of favorable interaction with the conformation. Identify compounds with a matching structure, A method that includes: 12． The compound thus identified is   (a) binding ability to ZAP family proteins,   (b) binding of ZAP family proteins to their natural or unnatural ligands Ability to inhibit, and / or   (c) the ability to inhibit biological functions mediated by ZAP family members, 12. The method of claim 11, further comprising testing for. 13. Machine readable data, and an instruction to use this data is programmed. The method according to any one of claims 1 to 6, when using a rammed machine. 3D drawing of molecules or molecular complexes containing proteins, or parts thereof A data storage material on which the data that can be displayed is encoded, A machine-readable data storage medium. 14. Machine readable data, and an instruction to use this data is programmed. Annex I, Annex II, or Annex II when using a rammed machine Root mean based on I coordinates or from conserved protein backbone atoms ZAP family proteins or based on coordinates with a squared deviation of 1.5Å or less ZAP family proteins: 3D drawing of ligand complexes or their parts Data that can be displayed, comprising a data storage material that is encoded, Machine readable data storage medium. 15． A data storage material on which a first set of machine-readable data is encoded A machine readable data storage medium, wherein the first set of data is machine readable. To use the second set of data that can be issued and the first and second sets of data Machine readable when combined with the programmed instructions using a programmed machine At least some of the coordinates corresponding to the two sets of data can be determined, where: The first set of data may be Annex I, Annex II, Annex III or Annex IV And at least a portion of the coordinates according to Or a data storage medium comprising an X-ray diffraction pattern of a molecular complex. 16． A method for displaying a three-dimensional figure of the material according to any one of claims 1 to 6,   (a) reading data stored in the machine-readable storage medium according to claim 13; Using this data, the protein or protein defined by this data can be Is a three-dimensional depiction of the protein: ligand complexes or parts thereof 14. The machine-readable storage medium according to claim 13, wherein the instructions are programmed. Prepare the machine that is loaded, and   (b) reading the data with this machine and displaying a stereoscopic depiction; A method that includes: 17． By designing compounds that can bind to ZAP family proteins So,   (a) Based on coordinates defining the three-dimensional structure of the ZAP family protein or a portion thereof The 3D description is displayed graphically,   (b) between multiple parts of the ligand known to bind to the protein Characterize the interaction to identify candidate parts for replacement,   (c) providing a knowledge base of one or more candidate replacement portions;   (d) from this knowledge base, retrieve one or more selected portions of the ligand At least the binding affinity of the ligand to the protein Identifying one or more replacement moieties, some of which retain; A method that includes: 18． By designing compounds that can bind to ZAP family proteins So,   (a) Use coordinates that define the three-dimensional structure of the ZAP family protein or a portion thereof. I mean,   (b) favors the interaction of one or more selected functional groups with the protein Characterize points related to their conformation to identify favorable points in terms of gender ,   (c) is known to bind to the protein near its characterized point Characterizing one or more portions of the ligand;   (d) providing a knowledge base of one or more molecular fragments or molecules;   (e) From this knowledge base, the preferred points identified in (b) and the portion of the ligand Identifying one or more fragments or molecules capable of connecting   (f) The modified ligand can bind to the protein with the modified ligand. One or more thus identified in the orientation and position chosen to be Modifying by the covalent attachment of such a fragment or molecule to it, A method that includes: 19. How to determine the orientation of a ligand bound to a ZAP family protein Law,   (a) Use coordinates that define the three-dimensional structure of the ZAP family protein or a portion thereof. I mean,   (b) the advantage of one or more selected functional group interactions with the protein; Characterize points related to their conformation to identify favorable points in terms of gender ,   (c) It is known to bind to the protein, but the binding configuration of the ligand The one or more functional groups of the ligand whose locus is unknown are replaced with the functional group phase identified in (b). Immobilized at a selected site consistent with a favorable point of interaction, and   (d) a series of alternative conformations for the immobilized ligand and / or Perform modeling calculations to generate orientation, A method that includes: 20. By designing compounds that can bind to ZAP family proteins So,   (a) a protein identified by the method of claim 19: a ligand; The conformation and / or orientation of   (b) characterize the interaction between the protein and its ligand moiety for replacement Identifying candidate portions of the ligands of   (c) providing a knowledge base of one or more candidate replacement portions;   (d) From this knowledge base, locate the selected part of the ligand (s). At least one of the binding affinities of the ligand for the protein. Part identifies one or more replacement moieties retained; A method that includes: twenty one. By designing compounds that can bind to ZAP family proteins So,   (a) a protein identified by the method of claim 19: a ligand; The conformation and / or orientation of   (b) the advantage of one or more selected functional group interactions with the protein; Points related to the three-dimensional structure of the protein to identify favorable points in terms of sex Characterize,   (c) is known to bind to the protein near its characterized point Characterizing one or more portions of the ligand;   (d) providing a knowledge base of one or more molecular fragments or molecules;   (e) From this knowledge base, the preferred points identified in (b) and the portion of the ligand Identifying one or more fragments or molecules capable of connecting   (f) The modified ligand can bind to the protein with the modified ligand. One or more of the thus identified in the orientation and position selected Modify by the covalent attachment of such a fragment or molecule to it, A method that includes: twenty two. Including any combination of the steps according to any one of claims 17 to 21 To select or design compounds that can bind to ZAP family proteins Law.