JP2000512737A - In vitro fluorescence polarization assay - Google Patents

Abstract

  (57) [Summary] The in vitro assay allows the identification of test substances that inhibit the mutual association of a pair of proteins. The method comprises the steps of providing a pair of proteins that are capable of associating, wherein one of said proteins has a covalently attached fluorophore, and said two proteins and at least one test substance. Preparing a mixed solution comprising: irradiating the mixed solution with polarized light of a suitable wavelength to cause excitation of the phosphor to be shown as emission of polarized light; and measuring the degree of emitted polarized light, Determining the effect of the presence or concentration of the test substance in reducing the emission polarization observed in a mixture of only two proteins. The inhibitory activity of the test substance is correlated with the reduced depolarization value.

Description

DETAILED DESCRIPTION OF THE INVENTION In vitro fluorescence polarization assayTechnical field of the invention   The present invention relates to protein-protein interactions, particularly for cell signal transduction. Materials and methods for identifying involved but inhibitory substances.Background of the Invention   Cell signal transduction, a series of events from extracellular to intracellular events Is an aspect of cellular function that occurs in both normal and disease states. Shi Numerous proteins that function as signal transduction molecules have been identified, Among them are, for example, receptor and non-receptor tyrosine kinases, phosphatases Receptor, ie anchored, or any other molecule with enzymatic or regulatory activity There are proteins and enzymes that reconvene. These molecules are generally , A signal that can specifically associate with another protein and alter cell activity Exhibit the ability to form a complex.   Signal proteins often contain domain-conserved sequences. However, this domain is a protein-protein interaction during signal transduction. Acts as a non-catalytic module that directs action. Such intramolecular domains include: In particular, SH2 domains, phosphotyrosine interaction ("PI") domains, W There are W domain and SH3 domain. One or two SH2 and PI domains Recognize proteins containing characteristic peptide sequences containing more than one phosphorylated tyrosine residue That is, it binds to this protein. WW and SH3 domains are also characteristic peptides Recognizes the protein containing the sequence, but this characteristic peptide sequence does not necessarily Hotyrosine residues are not included. These domains and the ones that contain them Protein, the production of proteins containing such domains (protein fragments And fusion proteins), the characteristic peptide sequences they recognize, and / or Are remarkable information on the clinical role of these protein interactions. The report has been described in the scientific literature.   Interactions between specific protein molecules are associated with causes or symptoms of disease or pathological condition If any, can interfere with the protein-protein interaction. Compounds prevent or treat the disease or condition in mammals, including human patients. It may be useful to treat.   To discover such inhibitors of protein-protein interactions The clinical tool for this is the binding assay. Song and Fields,Nature,340: 245-247 (1 989) using the well-known two-hybrid interaction / binding assay. Interactions between proteins and their partners have been studied [Fields et al. No. 5,283,173 issued Feb. 1, 1994. I want to.] In addition, such an approach would provide a predicted SH2-dependent interaction Is identified using yeast [Xing, Z et al.,Mol . Biol. Cell, 5: 413-421 (1994); And Osborne, M.A. et al.,Biotechnol., 13: 1474.1478 (1995)] Inhibition of a hybrid of two species [Chaudhurl, B .; et al.,FEBS Lett.,357: 221-226 ( 1995)]. In addition, it contains various SH3 domains. Design and preparation of proteins, peptide ligands for SH3 domains of interest On the creation of and the biological / clinical role of SH3-mediated interactions For background information on SH3 domains and their ligands, including International Patent Application No. PCT / US95 / 032, hereby incorporated by reference. See also No. 08. Design and creation of proteins containing various SH2 domains Generation of peptide ligands for SH2 domains of interest and mediation of SH2 For phosphotyrosine-containing ligands, including the biological / clinical role of interactions Information on receptor domains (eg, SH2 and PI domains) PCT / US97 / 02, which is incorporated herein by reference. See No. 635.   Proteins containing SH2 domains associate with their phosphotyrosine-containing ligands Competitive binding assays for detecting test substances that interfere with binding are described . See, for example, Pawson U.S. Patent No. 5,352,660. More recently The binding assay reported was surface plasmon resonance (Biacore) [eg, P anayotou et al,Mol . Cell. Biol., 13: 3567-3576 (1993)] or An assay based on radioligand is utilized. The former throughput is relatively high Low But the latter requires cumbersome filtration operations and is increasingly called radioactive waste Disposal problems result in large waste.   Quick and effective identification of inhibitors of protein-protein interactions With materials and methods designed to target a wide range of protein mediators, It will greatly contribute to the research for drug discovery. In addition, New protein or protein inhibitors associated with unfavorable or pathological conditions More efficient identification and development of pharmaceutical compositions with higher throughput. And will be possible.Summary of the Invention   The present invention provides a method for forming a binding complex by associating, that is, binding, Substances that inhibit or interfere with the binding of active protein pairs to each other By providing novel materials and methods for in vitro competition assays to identify To meet this need. The invention is particularly directed to intracellular proteins. Proteins or protein domains, especially those involved in cell signal transduction An assay for identifying compounds that can inhibit binding to a binding partner. this Such proteins include, for example, one or more, each having a protein ligand. Is the SH2 domain, PI domain, SH3 domain, or WW domain Includes proteins containing proteins.   In one aspect, the invention provides for inhibiting the association of a first protein with a second protein. It provides an in vitro assay for identifying harmful test substances. The method includes: At least one first protein and a second protein having a covalently attached phosphor; And forming a mixture containing the two test substances. Suitable for this mixture Irradiation with polarized light of a different wavelength causes excitation of the phosphor to be shown as polarized light emission. radiation The determined degree of polarization is measured to determine the effect of the presence or concentration of the test substance. Test substance The inhibitory activity is determined by the polarization of the first and second protein mixtures observed in the presence of the test substance. Values compared to those observed with the same protein mixture without the test substance It is shown by the fact that it is decreasing.   Inhibition of protein-to-protein association is the result of binding of the test substance to the first protein. Binding to a labeled ligand protein or peptide May be the cause. Therefore, this test method can compete with any of the binding partners. It can be viewed as a method of identifying test substances that bind together. As mentioned above, Once the degree of polarization of the emitted light is measured, it can be used to reduce the observed degree of emitted polarization. In the case of a mixture in which the effect of the presence or concentration of the test substance is observed and no test substance Is compared to Competitive binding of the test substance correlates with a decrease in the polarization value.   In yet another aspect, the present invention relates to a first protein, first identified by the method described above. It provides an inhibitor of the association of a protein with a second protein.   In yet another aspect, the invention relates to a component or component useful in the method of the invention. A reagent is provided, for example, a protein having a covalently attached fluorophore. This component or Reagents are further packaged in equipment with instructions for use in the described method. May be used.   Other aspects and advantages of the present invention are described in detail below in the detailed description of the preferred embodiments. You.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the structure of the fluorescent probe FMT1 described in Example 2.   FIG. 2A shows FMT1 of FMT1 tabulated for Src / total tracer bound versus total Src concentration. It is a saturation curve of the binding to SrcSH2. The nonlinear least squares method is a probe FMT Suitable for saturation experiments between 1 and the Src-SH2 domain. The calculated Kd is 0. 24, error 0.008 μM, chi-square 0.0027, and R 0.998 7   FIG. 2B tabulates the Rb / Rf pairing and shows the Scratchard component of the data of FIG. 2A. (Conversion). The calculated Kd is 0.26 μM, and the R value is 0 when linearly fitted. . 992.   FIG. 3 shows the following tetrapeptides, Ac-pYEEI (ring open), Ac-pYpY EEI (closed square), Ac-pYGGL (positive sign), Ac-pYEDL (open triangle Form), Ac-DGVpYTGL (closed triangle),% probe binding versus inhibitor 5 is a Src competition curve (2% DMSO) tabulating the concentration of a substance.   FIG. 4A depicts the experiment of Example 5, a long format 96 well plate. The arrow indicates the direction of dilution. White circles are sample wells, gray circles are Wells containing only lobes, and black circles contained probe and protein Well.   FIG. 4B depicts the experiment of Example 5, a 96-well plate in short format. Arrows indicate the direction of dilution for every four rows of wells. Circles are defined in Figure 4A As you did.   FIG. 5 shows a modified fluorescence probe used in the Src-SH2 assay described in Example 2. 1 shows the structure of a wing.   FIG. 6A shows the operation of the FP assay in the absence of an inhibitor. In this figure, A second protein or probe labeled with restain may bind to its binding partner (SH2 domain). (The first protein with a main). Light from a vertically polarized light source combines It remains polarized due to the slow rotation of the complex.   FIG. 6B shows the operation of the FP assay performed in the presence of an inhibitor. In this figure Inhibits the binding of the inhibitor to the first protein containing the SH2 domain, Prevents binding of the second protein (or probe) labeled with cein. small Unbound probes of the type rotate at a faster rate than the complex of FIG. 6A. Vertically polarized light source Is depolarized due to the fast rotation of the uncoupled probe.Detailed description of the invention   The present invention identifies test substances that disrupt or inhibit the association between a pair of proteins, To provide an assay based on fluorescence polarization (FP) to measure its ability It meets the needs of the industry. The new assay disclosed here and The material is robust and non-radioactive and adapts to various levels of automation. Has the advantage of I. Components of the test   In order to facilitate understanding of the present invention, the components of the assay are as follows. explain. A. Protein-to-protein interaction   The assay of the present invention is capable of inhibiting any protein-to-protein interaction. It is configured to be able to detect. "Protein-protein interaction" or The term "inter-association" of proteins refers to the spontaneous formation between two different proteins. Or a covalent or non-covalent complex or bond. Protein vs. protein An example of a protein association is the formation between a receptor and its naturally occurring ligand. Complex. A fragment of a protein, such as a peptide, Interactions that occur between proteins or peptides are also proteins-to-protein This is what the term quality interaction encompasses. Protein vs protein knots Examples of such cases are numerous in the art and include, for example, antibodies and protein antigens or epitopes. Binding to cell surface receptors, their binding to protein ligands, There are bindings between the gnal proteins and their protein binding pairs, and so on.   Specific examples of protein-to-protein interactions demonstrate the method of the invention. We will use some of them here, but some of them One or more SH2 domains and / or SH3 domains (Syk, Zap , Src and Lck), and as the second protein, There are protein ligands. Zap and Syk proteins and their SH For more background information on the two domains, as well as peptide ligands, see here International Patent Application No. PCT / US96 / 1391, incorporated herein by reference See No. 8. B. First protein / peptide molecule of the binding pair   For the purposes of the present invention, and using the detection device now conventional, For example, the molecular weight is much larger than the second protein or peptide, It is preferred to use a first protein / peptide that interacts naturally. For the purposes of the present invention, proteins that participate in protein-protein interactions The larger one is called the first protein. Fireflies based on this assay What is labeled with the photophor is a second protein or peptide. In general, The first protein of the binding pair is the labeled second protein / peptide of the binding pair. Having a molecular weight at least twice as large as about 100 times the molecular weight of . In many cases, the molecular weight of the first protein of this binding pair will It is at least 25 to about 50 times the molecular weight of the biprotein / peptide.   The first protein is not always carefully purified during the production process, as described below. May not be required, but for specific quantitation purposes, e.g. for the affinity of two protein components To construct a saturation curve or to determine the Kd value, the concentration of the first protein It is important to know.   One of the "first proteins" specifically exemplified in Examples is It contains an SH2 domain that acts as a receptor for unphosphorylated peptides. This Receptors such as include a “phosphopeptide binding domain” (PBD), Proteins, fusion proteins, polypeptides, peptides or their fragments May be. PBD is a receptor present in a specific signal protein, for example. Domain that can bind to phosphorylated proteins or phosphopeptides Can direct protein-to-protein or protein-to-peptide association Things. For example, the “SH2” domain is one such receptor domain It is. The receptor comprises one or more SH2 or other phosphopeptides The polypeptide may include a binding domain. Receptors are larger proteins Or a peptide fragment thereof. The receptor is It is preferably derived from humans or other animals.   Including such receptors (eg SH2 domain, PI domain, etc.) Many proteins are known. See, for example, US Pat. No. 5,352,660. I want to be.   Another specifically exemplified "first protein" is the corresponding peptide sequence or Contains an SH3 domain that acts as a motif receptor. like this Receptors can be proteins, fusion proteins, polypeptides, peptides, Or fragments thereof containing SH3 or SH3-like domains. Good.   Another specifically exemplified "first protein" is the SH2 and SH3 domains It includes both. C. Second protein / peptide of the binding pair   The second protein is a ligand of the first protein or receptor (naturally occurring or its ligand). Other than that). The second protein is generally the two proteins of the binding pair. Smaller, and preferably labeled with a fluorescent component of the pair, and described herein. It may be a protein that forms a useful "probe" component in the method.   In one example, the second protein or "ligand" comprises one or more thyrones. Protein, fusion protein, peptide or fragment thereof containing a syn residue At least one of the tyrosine residues of the peptide ligand is phosphate Of specific and specific binding to phosphopeptide binding domains when It is possible. "SH2 ligand" is an example of such a ligand.   Receptors for peptide ligands, such as SH2, SH3, PI and WW domains A wealth of information on sequence specificity for, etc. is also well known. Departure When used as a probe in the light assay, this ligand is described in detail below. Labeled with a suitable fluorophore as described. Typically, the probe peptide Alternatively, the protein may have a Kd in the range of about 0.1 to about 1000 nM (typically). To the larger binding pair. More preferably, the two binding pairs are about 300 nM Better (ie numerically smaller) Kd, more preferably from about 5 to about 50 nm And Kd in the range of D. Method for producing first and second proteins / peptides of a binding pair   Enables recombinant production of desired proteins / peptides using various expression systems DNA sequence information and expression techniques are available. Sputum used in these tests To produce a protein, a protein containing at least one domain of interest May be expressed. The protein The protein portion, especially the larger of the two binding pair Alternatively, the protein may be expressed as a fusion protein by the usual method.   Any conventional method for producing proteins, including both prokaryotic and eukaryotic systems Materials and methods may also be utilized. For example, such a protein / peptide Depending on the baculovirus, bacterial, yeast or mammalian expression system, the full-length protein Protein, a fragment containing the receptor domain or a fusion protein. It may be expressed in the form. Such expression systems are within ordinary skill in the art. . For example, Molecular Cloning. A Laboratory Manual. , 2dedit., Cold Spring Harbor Laboratory, NY (1989). .   By using conventional protein / peptide expression technology, any size phase Interacting protein pairs can also be generated. Express protein component of the present invention Expression systems and their conventional components used to effect the expression are known to those skilled in the art and It does not limit the scope.   To illustrate, an expression vector for the protein or domain of interest. The desired protein, protein domain, or, in a normal expression vector, If known, encode the consensus homology domain of the domain of interest DNA, alone or preferably further ligated together with a flanking sequence. Can be built. If desired, such additional flanking arrangements Whether the amino acid increases stability, increases expression levels, Enhances its ability to interact with the protein, or for the following reasons: Routine experimentation to determine whether it is necessary or preferred to bind the fusion protein. It can be determined by a procedure. For example, in human Src, the SH3 consensus The suspension homology domain includes amino acids 91-140. We have amino acids 84-145 Was used to create the SH3 domain protein from the E. coli expression vector, Has an additional 7 amino acids on the N-terminal side of the homologous domain and 5 amino acids on the C-terminal side Is included.   The desired protein or protein domain may contain all of its natural connections, or Are two or more of the same or different domains As a tandem sequence containing in, or SH2-like domain, protein kinase Domain, glutathione S-transferase (GST), epitope labeling, Kinase recognition sequence, maltose binding protein, signal sequence, biotin-modified sequence Other unrelated domains, including, but not limited to, columns, etc. It may be expressed as a fusion protein having an in.   Modifying a protein or protein domain:   -Purification may be facilitated. For example, glutathione S transferase, Toose binding protein, metal chelating sequence (poly-histidine), protein A or expressed as a fusion to others.   -Identification or quantification may be facilitated. For example, biotin, phosphor, chromophore, Tyrone, spin label, radioactive or non-radioactive isotope label, magnetic particle, metal roller Modification by a covalent bond using an id, etc.   It may be attached to a defined solid carrier. For example, epitope labeling or Can be expressed as a fusion to other antigen domains, for example, N-terminal serine, In, lysine or other specific or specifically accessible protein characteristics. Operate to provide signs, etc.   -Unwanted features that complicate experiments may be removed. For example, unnatural Mutation of cysteine that participates in main dimerization, etc.   Increased stability under conditions of binding assays (eg, altering the natural coding sequence) And further encodes cysteine to form stabilized disulfides). E. FIG. "Test substance"   A "test or inhibitor" refers to a protein-protein interaction that participates. A compound that selectively binds to either the first protein or the second protein, or It is defined here as a composition. Alternatively, the test substance may be It selectively blocks or inhibits the interaction between these two proteins. You. For example, the inhibitor may have a higher binding activity than its naturally occurring binding protein. It may be one that can bind to the first protein by its binding activity, or It may be one that binds to a protein. Two proteins are tyrosine phosphorylated In the case of epta and its naturally occurring ligand, the inhibitor is a ligand. Is capable of binding to the receptor in competition with the One or more tyrosine phosphorylated peptides or It can selectively block or inhibit the interaction mediated by mein.   Ability to selectively bind to the first or second protein of interest; The source of the test substance or composition for which the Some include, for example, broths of microorganisms, cell extracts, conditioned media collected from cells, There are synthetic compounds and combinatorial libraries. The assay method of the present invention is applied to natural products and Screen compound libraries or structurally distorted diversity libraries It may also be used when trying to identify a desired inhibitor. The test substance is May be selected from a mixture of one or more test peptides, in which case the mixture The solution may be provided in the form of a library of synthetic peptides, or may represent a variety of peptides. It may be provided in the form of a phage library as indicated. F. "Fluorescent component"   "Phosphor" or "fluorescent component" means in a solution when excited by polarized light The fluorescent component that emits light back onto the fixed plane (ie, the light remains polarized) I am a child. Many fluorescent labeling components with a wide range of structures and characteristics are known. Suitable for use in practicing the invention. Similarly, they can be covalently linked to other molecules. Methods and materials for combining are also known [eg, Richard P. et al. Haugland, Molec ular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-19 94 (5^th edit, 1994, Molecular Probes, Inc. )]. Select phosphor When selecting, the lifetime of the excited state of the phosphor depends on the movement of the labeled probe or peptide. Be long enough to be able to measure the loss of polarization after emission Is preferred. Suitable phosphors include fluorescein, fluorescein isothiocyanate , Rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride Lido, phycoerythrin, and umbelliferone are included.   To avoid interference due to the fluorescence of the test substance, do not use the ultraviolet region of the spectrum. Utilizing phosphors with excitation and emission wavelengths in the visible range typically involves Is preferred. Preferably the phosphor is the smaller of the proteins to be labeled. That is, it may be covalently linked to a second protein, such as a peptide ligand, The linker used in this case is the improper movement of the phosphor, that is, the labeled peptide. Be short enough to avoid movements that are not correlated with the Is preferred.   More specifically, the methods used by the inventors will be described in the following examples. Among the fluorescent components, the covalent bond to the second protein molecule (peptide ligand) Chemically attached. In Example 2, phospho-Tyr containing peptides were added in vitro. Labeled with fluorescein. Those skilled in the art will appreciate such phosphopepti It will be appreciated that any method can be used to create the code. G. FIG. "Fluorescence polarization"   PerrinJ . Phys. Rad., 1: 390-401 (1926), the FP first described as a phosphor Emission can be depolarized by a number of factors, the most of which The dominant factor is rotational diffusion, in other words, the rate at which molecules rotate in solution. It is based on seeing. "Degree of polarization" occurs between absorption and the subsequent emission of a photon. Is the measured value of the average angular displacement of the phosphor. The angular displacement of this phosphor was originally Depends on the rate and extent of rotational diffusion during the lifetime of the excited state. Is dependent on the viscosity of the solution and the size and shape of the diffused fluorescent species. It depends. If the viscosity and temperature are kept constant, the degree of polarization is the molecular volume of the phosphor. Or directly related to size. In addition, polarization values are dimensionless (vertical and horizontal) Fluorescent brightness), and is not affected by the brightness of the phosphor. F. Additional information on signal transduction domains specifically illustrated as targets (I) SH2 or SH-2-like domain   The term "SH2 domain" generally refers to the Src homology region 2 (SH2 region). Say the same sequence. Src homology region 2 is a non-catalytic domain of ~ 100 amino acids Originally, its effects on both catalytic activity and substrate phosphorylation Virus Frc and viral Src identified in cytoplasmic tyrosine kinase (T. Pawson, Oncogene, 3, 491 (1988) and I. Sadowskl et al., Mol. Cell. Biol. 6, 4396 (1986)). The SH2 domain has been found in a variety of eukaryotic proteins. Some of them work by transducing intracellular signals. Many in the industry It is known. Examples of proteins containing SH2 domains (including equivalents from various species) And (1) a member of the protein tyrosine kinases of the src family (Sr c, Lyn, Fyn, Lck, Hck, Fgr, Yes), (2) Shc, (3) ) Tsk, (4) Btk, (5) VAV, (6) Grb2, (7) Crk, and (8) Signal transduction and transcription (STAT) proteins are included. in addition, For example, ZAP-70, p85 phosphatidylinositol 3 'kinase (PI3 K), Syk, GTPase activating protein (GAP), and phospholipase Many proteins, such as C gamma, have two SH2 domains. SH2 Dome Proteins containing humans include humans, rodents, sheep, cows, C elegance, and shows. Identified in Drosophila, Xenopus, Plasmodium, Freshwater Sponge, and Hydra I have.   Novel SH2 or SH2-like domains from unknown DNA, RNA or protein sequences One method of identifying sites is to use a number of computer alignment programs. Is to use one of the grams. One example is the World Wide Web (W Run on the WW) site htt: //ulrec3.unil.ch/software/profilescan.html Pfscan. To use this program, Quality sequence, see "Profile" describing its SH2 domain motif And examine. Based on this program information, the specific benefits of profiles are: The ability to explain protein motifs that are significantly branched using these It is. These profiles are usually multiple alignments of the first set of sequences. It is obtained from: One profile, in addition to the array itself, Type of residue is assigned to which position in the domain, which amino acid Is conserved, which amino acids are not conserved and can be inserted at any position or region And which regions are essential. pfsc For further information on an and PROSITE, see ISREC (Switzerland Institute for Experimental Cancer Research) Io Informatics Group website http: // u Obtained at lrec3.unil.ch/index.html.   As an example, we have used the pfscan program to distribute the peptide of human Src. The columns were analyzed. The results are shown below. This program uses this Src's SH2 domain. In encompasses the region from amino acids 150-247 of the Src peptide sequence. Was clearly identified. In addition, the SH3 and kinase domains are also pfscan Identified. NScore raw from-to Profile | Description 26.9695 1792pos. 150-247 PS50001 | SH2 Src homology 2 (SH2) domain 20.2947 1182pos. 83-144 PS50002 | SH3 Src homology 3 (SH3) domain 43.4246 2912pos. 269-522 PS50011 | Protein_Kinase_DOM Protein Protein kinase   NScore for a match is stored in a random database of any size. Negative decimal logarithm of the expected number of good (or better) matches. N If Score is << 1, this converges to the probability of finding a match in the database . The expected number of matches depends on the size of the database. Decimal logarithms of magnitude must be subtracted before calculation.                     -log (NExp) = NScore-log (DBsize) However, in this formula, (NExp = predicted number of expected matches) and (DBsize = Database size by number).   The following table shows how NScore works in the SwissProt database. To probabilities in non-redundant (nr) protein databases Here are some examples showing how to do it. The calculation is 18,531,385 residues for SwissProt (log = 7.27) 58,154,119 residues (log = 7.76) for nr database It is based on the size of the database. The expected number of potential matches is: And so on.   The segments of the test sequence have an NScore value of SH2 profile> 7.5 , Preferably> 8, more preferably> 9, more preferably> 10. It is good to include main.   As a second example, the N-terminal 160 amino acid sequence of human ZAP-70 is pfsca n. The results show that one SH2 domain contains amino acids 10-102 It turned out that it was binding. NScore raw from-to Profile | Description 16.4402 1082pos. 10-102 PS50001 | SH2 Src homology 2 (SH2) domain   SH2 domains are other computer alignment programs, eg For example, DNAstar Computer Package (Madison, WI) It is also possible to identify by using MegAlign in the above. To do this, One or more known SH2 domains and a test sequence are combined by the cluster method. Align. For known SH2 domains^Three25%, in some cases 30- A sequence having an amino acid sequence that is 50%, otherwise> 50% identical, is SH2 homologous Identified as a domain. Identical between test sequence and different known SH2 domains The position at which the amino acid sequence of SEQ ID NO: 1 may vary except for one particular position. until today All of the SH2 domains identified in FIG. An arginine residue is conserved at residues 0-45. In human src, this Le Ginine is Phe for the abbreviations F, Leu for L, Val for V, Arg for R, G for E It is found in the sequence FLVRES when the Ser amino acid residue is represented by lu, S.   Another way to identify SH2 or SH2-like domains is to use the SH2 domain. A nucleotide or protein database that summarizes the characteristics of the Is to search for. In the SWISS-PROT database, this is either FT or Is listed in the "Features" heading. SWISS-PROT database Is accessible on the WWW at EBI http://www.ebi.ac.uk. For example, In the file listed for Src (P12931), SH2 The area including the main is assumed to be 150-247.   Yet another way to identify SH2 or SH2-like domains is to use cDNA generation. The current library is screened for the known SH2 domain with a phosphorylated peptide ligand. Leaching and isolating the cDNA for the SH2 protein. May be. Low stringency screening using PCR or SH2-specific probes May be able to do this. SH2 domain or including SH2 domain Protein can be isolated from naturally occurring sources (eg, cells, tissues, organs, etc.) Often produced recombinantly in bacteria, yeast or eukaryotic cells, It can be created in a test tube using a translation system, or created by synthesis (for example, ).   Some SH2 or SH2-like domains are co-ordinated by the pfscan program. And may exhibit significant homology to known SH2 domain sequences. May not be detected by the computer alignment program I don't know. However, these sequences are identical or different from known SH2 domains. It may have a similar three-dimensional structure, work as an SH2-like domain, May act to bind to phosphotyrosine-containing peptides or proteins Absent. Some of the known three-dimensional structures of SH2 domains have already been elucidated. SH2 domains feature two antiparallel beta sheets consisting of 5 or 6 beta chains. It is a sign. The region that forms the alpha helix is or does not exist in this domain. Or not. SH2 domain or SH2-like domain is an x-ray crystallographic Has an SH2-like domain structure when dissolved by Will be recognized. Or, depending on the selection, the structure predicted by homology / modeling A particular protein sequence may be identified as an SH2-like domain using No.   http://ulrec3.unil.ch/pr_details/alignments/SH2.msf (profile The trick is available at http.//ulrec3.unil.ch/cgi-bin/get_pstprf?SH2) The obtained SH2 used to create the profile of SH2 for pfscan The alignment of the domains was approximately 390 of proteins from various species. Based on the alignment of SH2 domains. Swiss-Prot Of the protein containing the SH2 domain used in the database alignment The list includes the following (P #### is Swiss-Prot data Base receiving Accession number).  Summary of Methods for Identifying SH2 Domains in Test Peptides or Nucleotide Sequences Is as follows. 1. The cDNA or RNA is translated into a one letter symbol protein sequence. this Is a component such as EditSeq of DNAstrider or DNAstar package. It could be done with a pewter program. 2. Go to http://ulrec3.unil.ch/software/profilescan.html on the WWW site Good. 3. Copy the test sequence into the appropriate box in the pfscan form. 4. Submit the form to the pfscan server. 5. The result is sent back via web browser or email.   As explained in the above paragraph, the SH2 domain and SH2-like domain It may be used for implementation. Using the information provided here and from the examples provided below By analogy, any SH2 domain, SH2-like domain, PID or PID-like Domains, and their peptide ligands can be identified as, for example, ZAP, Syk, Src or Could be used in place of the SH2 domain of Fyn to practice the invention . (Ii) PID or PID-like domain   The phosphotyrosine-binding domain replacing the SH2 domain is a so-called phosphotyrosine phase Interaction domain (PID). This domain has about 160 amino acid residues on average. The first identified, though containing a group, was in the Shc protein. Consensus pTyr-Xaa-Xaa-Xaa-Xaa (after phosphotyrosine Domain that is followed by three or more amino acids) In contrast, the PID domain is a consensus Asn-Xaa-Pro-pTy Recognize an array with r (also called NPXY in one letter symbols). In this application The invention described also relates to PID domains and PID-like domains. This In the case of the SH2 domain, the sequence encoding the PID domain The ligand that replaces the coding sequence and recognizes the PID domain is the SH2 domain Replaces ligand. Phosphorylation of the PID ligand is determined by v-Sr as described herein. would be achievable with c. Alternatively, protein kinase can be used for PID May be able to phosphorylate the ligand. In addition, the dandelion in the cell Protein kinases may also catalyze the phosphorylation of PID ligands.   Much information about these domains is known in the art. PID domain A detailed description of the property can also be found on the WWW site http: //www.bork.embl-heidelb You can also see it at erg.de/Modules/pid-gif.html. The following information is on this site It is obtained from Documentation-PROSITE   Excluding SH2, the phosphotyrosine interaction domain (PI domain or PID ) [3] is the second phosphotyrosine found in the converted protein Shc [1, 2]. Binding domain. Shc converts activated growth factor receptors into mammalian cells. Connects to a signaling pathway that regulates growth, but converts tumoral tyrosine kinases They may also participate in sex. PI domain contains a number of growth factor receptors Asn-Pro-Xaa- found in many tyrosine-phosphorylated proteins It specifically binds to the Tyr (p) motif. PID is also related to Shc. The protein Sck [1] and some unrelated regulators listed in the following list It is also found in protein [3].         Mammalian Shc (46 kD and 52 kD isoforms) has one N-terminal         It contains a PID, a collagen-like domain and a C-terminal SH2 domain.         ・ Human Shc-related protein Sck is composed of one PI domain and SH2 domain.         Including the main.         -Mammalian X11 is significantly expressed in the nervous system. This is about 100 of PID         It contains two disc homology regions (DHR) downstream of AA.         ・ The Drosophila nuclear Numb protein is the Drosophila embryo.         Necessary to determine cell fate during sensory organ formation. This is one         It has a PID.         ・ Sea Elegance hypothesis protein F56D2.1 is N-terminal metalloprotein         Includes an Nase domain followed by one PID.         -Rat FE65. WW domain and two PIDs in the sequence of FE65         Found that this protein was probably signal transduced.         You can see that they are involved in         Drosophila impaired proteins are cells of CNS axons and body wall muscle         It is a tyrosine phosphorylated protein found in the quality. Neural development of the embryo         Are linked. Contains one N-terminal PI domain.         ・ Cell division-induced reactive phosphoproteins generated by alternate splicing in mice         The isoforms P96, P93 and P67 contain one N-terminal PID.         This is also true for the differentially expressed human ortholog Doc-2.         You.         ・ The human EST05045 protein fragment has one PID.         I do. References [1] Kavanagh WM, Williams LT, Science 266: 1862-1865 (1994 ) [2] Braiki, P. et al. Biol. Chem. 269,32031-32034 (1994) [3] Bork, P., Margolis, B. Cell 80,693 (1995)   Alignment of PI domains based on numerous PI domains collected from various species Is a WWW site http://ulrec3.unil.ch/prf_details/alignments/PID It is described in .msf. Other such alignments are web sites. Http://www.bork.embl-heidelberg.de/Modules/piali.html. (Iii) SH3 domain and SH3-like domain   The term "SH3-like domain" generally refers to the Src homology region 3 (SH3 region). Refers to homologous sequences. The Src homology 3 region is originally a virus Fps and a virus. In Src cytoplasmic tyrosine kinase, both catalytic activity and substrate phosphorylation A non-catalytic domain consisting of ~ 60 amino acids identified due to its action (T. Pawson, Oncogene, 3491 (1988) and I. Sadowski et al., Mol. CellBio). l. 6,4396 (1986)). SH3 domains have been found in various eukaryotic proteins. However, some function during intracellular signal transduction. SH3 do Maine Examples of proteins containing (including equivalents from various species) include (1) sr Members of the c family protein tyrosine kinases (Src, Lyn, Fyn, Lc k, Hck, Fgr, Yes), (2) Grb- having two SH3 domains 2, (3) Sprk, threonine / serine protein kinase, (4) Tsk, (5) Btk, (6) Vav, (7) GTPase-activating protein (GAP) (8) neutrophil oxidase complex p40, p47, and p67 proteins; and 9) phosphatidylinositol 3 'kinase, (10) Crk, (11) phos Follipase C gamma, (12) Abl. Dandelion containing SH3 domain Bodies have been identified in humans, rodents, cows, C. elegans, and yeast. So Other SH3 domains selected from the scientific literature or identified by sequence analysis, Alternatively, the clone may be formed by the method described above. SH3 domains and their resources For a wealth of information on Gand, see eg PCT / US95 / 03208 Please refer to.   Some SH3 or SH3-like domains contain 18 conserved amino acids. No acid acid or any computer alignment professional Exhibit significant homology to known SH3 domain sequences as detected in gram Maybe not. However, these sequences are known from the SH3 domain. May have the same or similar three-dimensional structure as the It might work. Some of the known three-dimensional structures of SH3 domains Is already known. The SH3 domain consists of two inverted 5 or 6 beta chains It features a parallel beta sheet. The domain that forms the alpha helix is this domain May or may not exist. SH3 domain or SH3-like domain Is SH3-like when dissolved by x-ray crystallography or NMR spectroscopy It will be recognized that it has a domain structure. Alternatively, depending on the selection, Using the structure predicted by Deling, a specific protein sequence was identified as an SH3-like domain. May be identified. II. Assay protocol   The in vitro assays of the present invention may be capable of competitively binding to a first Identification of a test substance that inhibits the association between one protein molecule and a second protein molecule Use FP to do this. Fluorescence polarization is a method of determining ligand vs. protein and protein. It is a very useful way to study protein-protein interactions. The present invention relates to a firefly When the molecular weight changes due to cleavage or binding of a photo molecule to another molecule Based on the observation that the polarization also changes. Low molecular weight and / or high flexibility Phosphors have low polarization values, while high molecular weight and / or hard phosphors have polarization values. Is high.   The assay of the present invention utilizes such intrinsic properties of the fluorescent component. This way Then, a mixed solution containing the following components is prepared.   (A) capable of binding to or associating with smaller second protein molecules, for example A predetermined amount of a first protein molecule such as a tyrosine phosphorylated receptor,   (B) a smaller, predetermined amount covalently bound to or labeled with a phosphor; Second protein. In the following example, a fluorescent label is covalently attached to the peptide ligand. To form a low-molecular-weight fluorescent-labeled probe.   (C) a predetermined test substance which is considered to have a competitive inhibitory effect.   This mixture is the first and second protein when mixed in the absence of the test substance. It is achieved under suitable conditions that allow complex formation between them. Thus, this way If the test substance is actually an inhibitor of protein-protein complexation, Quality, those conditions may cause it to competitively bind to the first or second protein. It is a suitable condition that also allows   A plane having a wavelength sufficient to excite the phosphor mixture of (a), (b) and (c). Irradiate with polarized light. Thus, the light emitted from the fluorescent second protein is the fluorescent second protein. Polarized to varying degrees depending on the molecular volume of the protein. In the unbound state in solution The polarization readings are low because the low molecular weight peptide is rotating at high speed.   In the presence of a binding / interacting partner, the first protein, eg a receptor Fluorescent second protein with lower molecular weight is the first protein with higher molecular weight Binds to proteins such as tyrosine phosphorylated proteins or peptide receptors. Sign The second protein binds to its target, the first protein, and is irradiated with plane-polarized light. The rotation of the large first protein: second protein complex is slower, That The polarization reading will increase. Thus, the method of the present invention Track the change in polarization ratio in the horizontal and vertical planes of. This is conventionally used with fluorescent labels Tracking of changes in the absorbance intensity at a particular wavelength range, This is in sharp contrast to. The change measured in the present invention is a labeled second protein. Is a direct measure of binding to the first protein.   Free labeled second protein vs. bound second protein: first protein This difference in polarization values of the protein complex is determined by measuring the binding to release ratio of the second protein, To analyze its binding to the first protein in the presence of a test substance Used. Such measurements may be performed in either saturation or competition experiments. . The FP assay of the present invention can thus be used in many solutions, including the cytoplasm of cells. Can be.   It is not necessary to separate the components in the mixture to determine the degree of polarization of the emission. last The effect of the presence or concentration of the test substance depends on the ratio of the polarization levels of the mixture, Comparing the polarization levels of the same amount of the first and second proteins / peptides in the absence It is determined by   Competitive binding between the first or second protein and the test substance rather than between the two proteins If a protein-to-protein complex does not form between The protein will remain free in solution and a low degree of polarization will be measured. Inspection If the substance is not an inhibitor or a good inhibitor, a complex is formed and mixed The degree of polarization of the combined liquid will increase. Thus, in the absence of a test substance, Protein: Observed emission polarization from the known polarization level of the second protein complex Decreasing light depolarization value is noticeable in the presence of inhibitory test substances Is done.   The method of the present invention is not based on a change in the intensity of the absorbance in a specific wavelength range, but on the emission wavelength. In order to track the change in polarization ratio in the horizontal and vertical planes of the long range, the method uses detection in solution. Even when the absorbance of the test compound is high, the compound is not easily affected by the interference.   The method of the invention is amenable to automation. For example, the steps outlined above All or some of the steps may have two or more steps for any given test substance. Step or one or more test substances or test substance concentrations The steps may be performed by a device programmed to perform the steps automatically. With one example Thus, a standard fluorometer equipped for polarization experiments or measurements can be used in the present invention. May be used both when irradiating the mixed solution and when measuring polarized light. Energize phosphor Suitable wavelengths to occur depend on the nature of the phosphor, as described above. Typically In order to define the wavelength range determined from the excitation and emission wavelengths of the phosphor, A filter is used. Fluorescein carboxamide peptide has 4 It will be common to use an 85 nM excitation cutoff filter. Furthermore, the optical path The instrument, including the test chamber in which the sample is to be evaluated, will be placed in the Non-polarizing materials should be used for either part. For such applications, Sticks and optical fibers are generally avoided; optical glass, quartz, etc. It is used favorably.   In addition to using standard fluorometers, Jolly FPM1 (for individual samples) or Jolly FPM2 (for high-throughput assays in 96-well format) A fluorimeter for use can also be used. Such fluorimeters are most suitable for polarimetry. Optimized and have significantly higher sensitivity than standard fluorometers .   Other automated equipment also has mixing steps combined with other steps, and And / or control mixture without test substance and test mixture with test substance Could be used for both polarization comparisons. A person skilled in automation A variety of devices could then be used to generally automate the assays of the present invention.   In yet another aspect, the present invention relates to a method, for example, It provides components or reagents such as proteins possessed by covalently binding an optical body. You. This component or reagent may be used in a single device with instructions for use in the manner described further. It may be packed in. III. Inhibitor   As long as a compound is identified as a single inhibitor, it can e.g. Can be prepared using known methods, such as recombinant methods or chemical synthesis. it can. In addition, it can be obtained from the source that was originally identified. A. Control screening   Proteins using the assays of the present invention: , One or more other proteins: Control screening of a set of ligands To identify non-specific inhibitors or to confirm the specificity of the inhibitors Can be. By using various methods, many of which are known in the art, The test compound identified as an inhibitor by the method described in Further binding activity to the target protein or further containing its domain May be evaluated for their binding activity to proteins. Control screening It can be performed using the methods and materials of the invention or, for example, cell-based assays. Set Alternative methods for detecting binding may be used [eg Panayotou et al,M ol . Cell. Biol. , 13: 3567-3576 (1993)].   The inhibitors identified in the assay system of the present invention are further evaluated for toxicity and pharmacological activity. Can be evaluated by conventional methods. For example, identified as an inhibitor The test compound can then be further analyzed using appropriate cell-based assays or animal models. Proteins: cytological or mediated by pathways involved in ligand interactions May be evaluated for activity in inhibiting other biological events. Wide range The inhibitory activity of test compounds on cytological and other biological events in humans Cell-based assays and animal models suitable for are known in the art. new New assays and models are constantly being developed and reported in the scientific literature.   Non-limiting examples include the shape of the signal leading to the appearance of asthma or allergic symptoms A compound that binds to the SH2 domain involved in transduction is You may evaluate by a sphere degranulation assay. Identified as SH2 inhibitor by the method of the present invention Histamine, leukotrienes, hormonal mediators and / or Or inhibitory activity on the release of specific mediators such as cytokines into cells, Fatty inositol hydrolysis or its production on the level of tyrosine phosphorylation Characterize physical activity as a measure of biological activity using conventional in vitro assays [Eg Edward L. Barsumian et al,Eur . J. Immunol.,11: 317-323 ( 1981); J. Forrest,Biochem . Pharmacol.,42: 1221-1228 (1991) (Activated N-acetyl-beta-glucosaminidase was measured from neutrophils) and V.A. M. St ephan et al.,J . Biol. Chem.,267: 5434-5441 (1992)].   For example, histamine release obtained from AMAC (Westbrook, Maine) It can be measured by radioimmunoassay using a possible instrument. This Thus, the biological activities of the inhibitors identified by the method of the present invention are evaluated, and Known active compounds and clinical compounds that can be used with each other and as a positive control Can be compared.   Generally, such assays have an IC50 score of 150-300 μM. Is considered a target, a score of 50-150 μM is considered good, and the score is About 50 μM is of most interest. Before the in vivo model or In addition to the in vivo model, the inhibitors identified according to the present invention may be further ex vivo, Ability to block antigen-stimulated contraction of guinea pig sensitized tracheal strip tissue. You may inspect. Activity in this assay predicts efficacy of potential anti-asthmatic drug Has been found to be useful in doing so.   Numerous animal models of asthma have been developed and are available [ See Larson, “Experimental Models of Reversible Airway Obstruction” , In THE LUNG, Scientific Foundations, Crystal, West et al. (Eds) Raven Pr ess, New York, pp. 953-965 (1991); Warner et al.,Am . Rev. Respir. Dis.,14 1: 253-257 (1990)]. Species used in animal models of asthma include Mouse, rat, guinea pig, rabbit, dog, sheep and primates are included. Other in vivo models available are Cross et al.,Lab Invest.,63: 162-170 ( 1990); and Koh, et al.,Science,256: 1210-1213 (1992).   Further examples include the traits of signals involved in the initiation, continuation or spread of cancer growth. An inhibitor identified by the method of the present invention, which binds to a protein involved in transduction, Related conventional in vitro and in vivo assays may be used. For example, Ishii et al. ,J.Antibiot., XLII: 1877-1878 (1989); and U.S. Patent No. 5,206,249 ( For publication, see April 27, 1993). B. Use of inhibitors identified according to the invention   Inhibitors identified according to the present invention can be used to identify specific proteins, especially newly discovered inhibitors. To classify proteins functionally, tests such as those used here are required. May be used as a biological reagent. The protein family or protein Lass may be functionally defined based on ligand specificity. Further, according to the present invention, Using the identified inhibitor, the target protein or protein-to-ligand Can inhibit the occurrence of biological events caused by tuple-mediated molecular interactions Wear. Inhibition of such interactions may impair the regulation and biological significance of such events. It can be useful for research aimed at better understanding.   Such inhibitors include, for example, cellular processes mediated by the interaction of interest. May be useful in the diagnosis, prevention or treatment of symptoms or diseases caused by For example That require an SH2 binding or blocking agent that selectively binds to SrcSH2 It can be used to prevent or suppress the development of osteoporosis, Can be performed on the patient.   There are many other conditions where phosphopeptide binding or blocking agents may be useful therapeutically Among them are SH2 domain containing proteins Src, PLC gamma and There are breast cancers that have been implicated in Grb7. Before any other relevant condition There is a gonad carcinoma, in which case the targeted Grb2 containing all SH2 domains , PLCg, and PI3K may be useful in treating or preventing this disease. G Inhibition of the interaction of rb2 or Abl SH2 domains with Bcr-abl May be useful in the treatment of myeloid leukemia (CML) or acute myeloid leukemia (AML) Not.   Yet another related use of PBP inhibitors is in the use of STAT protein PBD. Aiming at interferon-mediated diseases, growth factor-mediated diseases, or cytokines It may be the prevention of a mediated disease (eg an inflammatory disease). Involved in T cell activation Agonists that block the SH2 domain of ZAP-70 are useful for treating autoimmune diseases Maybe. Inhibitors that block one or both SH2 domains of ZAP-70 Quality may also be useful as an immunosuppressant in preventing rejection of transplanted skin or organs. No.   Similarly, as a further example, SH3 inhibitors may have SH3-based interactions. In the diagnosis, prevention or treatment of conditions or diseases caused by mediating cellular processes May be useful. For example, selectively bind to SrcSH3, or SrcSH SH3 inhibitors that inhibit the interaction with In order to prevent or prevent the progression or progression of osteoporosis, Can be applied. There are many other conditions where SH3 inhibitors may be useful therapeutically Among them, neutrophil oxidase p47 and SH3 of p67 complex are related. Alleged restenosis, rheumatoid arthritis, gout, asthma, emphysema, immune vasculature There are inflammation, ulcerative colitis, psoriasis and acute respiratory failure syndrome. Other relevant conditions Has chronic myeloid leukemia targeted at the SH3 domain of GrbB-2 There is. Recently, the BCR-abl oncogene in CML has been shown to interact with GrbB-2. It has been shown to participate in the ras pathway for growth stimulation through action. In these cells, the SH3 domain of GrbB-2 interacts with the SOS oncogene. Inhibiting its use would block its ability to stimulate cell growth. Still other related conditions include cancers such as breast cancer, glioblastoma, head and neck Head and ovarian tumors, and in these conditions the SH3 domain of GrbB-2 Will be aimed at. For example, amplification of EGF and PDGF receptors The accompanying tumor inhibits the interaction of GrbB-2 (SH3) with Ras, Blocking the activation of the Ras pathway might be able to inhibit it. In addition, Src file SH3 domain of Milli's kinase is T cell, B cell, mast cell, and NK cell It is thought to be involved in the activation of tyrosine kinases Tsk and Btk. The SH3 domain is responsible for the function of T cells (SH3 of Tsk) and B cells (SH3 of Btk). SH3 inhibitors identified according to the present invention, If administered to a patient in need thereof, immune function may be suppressed.   A substance that inhibits a protein-ligand interaction identified by the method of the present invention The quality can be a pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or other additives, It can be made using conventional materials and means. Such a composition is human Or cells involved in non-human animal-protein-ligand interactions It can be administered to treat a disease or condition caused by the event. This Administration of such compositions may involve the use of suitable formulations, as is known in the art. Or by any conventional route (parenteral, oral, inhalation, etc.). Such inhibition The substance is converted into a conventional excipient, i.e., a pharmaceutically acceptable organic or suitable for parenteral administration. It is also possible to use a mixture with an inorganic carrier substance. C. Pharmaceutical compositions and methods   i. composition   Inhibitors identified according to the present invention can be converted to drugs using conventional materials and means. A physiologically acceptable carrier and / or other excipient (ie, a drug suitable for parenteral administration) Therapeutic (or prophylactic) in admixture with a chemically acceptable organic or inorganic carrier substance) Above) can be made into pharmaceutical compositions containing an effective amount of the inhibitor. Such carriers include saline, buffered saline, dextrose, water, glycerol, and the like. Rolls, ethanol, and combinations thereof, including but not limited to Not. Carriers and compositions may be sterile. The formulation is suitable for the mode of administration There must be.   The composition may optionally contain small amounts of wetting or emulsifying agents or pH buffering agents. You may. The compositions can be liquid solutions, suspensions, emulsions, tablets, pills, capsules, It may be a depot preparation or a powder. The composition is a traditional combination, such as triglycerides A suppository can also be prepared by using an agent and a carrier. Oral preparations include, for example, pharmaceuticals Grade mannitol, lactose, starch, magnesium stearate, sacchar Includes standard carriers such as sodium phosphate, cellulose, magnesium carbonate, etc. May be.   In one specific example, the composition is a pharmaceutical composition adapted for intravenous administration to humans. The composition is prepared according to a normal procedure. Typically for intravenous administration The composition is a solution in sterile isotonic aqueous buffer. If necessary, the composition Soluble solubilizers and a local anesthetic to ease pain upon injection may also be included. In general, the ingredients may be provided separately or indicate, for example, the amount of active ingredient. Lyophilized powder in unit dose in an airtight container such as an ampoule or sachet It is provided as a mixture as an anhydrous concentrate. Attempt to administer the composition by infusion If necessary, be prepared in an infusion bottle containing sterile pharmaceutical grade water or saline. Can be. When administering the composition by injection, sterile water for injection or physiological diet The components may be mixed prior to administration using a saline ampoule.   Topical compositions include, for example, drugs such as gels, ointments, lotions, or creams. These include physically acceptable topical carriers, such as water, glycero , Alcohol, propylene glycol, fatty alcohol, triglyceride , Fatty acid esters, or mineral oils, but are not limited thereto. No. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene Len glycol, ethanol (95%), polyoxyethylene monolaurate water Solution (5%) or aqueous sodium lauryl sulfate (5%). As needed Add other materials such as antioxidants, wetting agents, viscosity stabilizers, and similar substances. You may get.   Materials and methods for making various formulations are known in the art [eg, See U.S. Patent Nos. 5,182,293 and 4,837,311]. ii. Method   The present invention provides a method for treating, preventing, and / or treating the symptoms of the above diseases or abnormalities. By administering to the subject an effective amount of an inhibitor to reduce the severity It provides a way to achieve this. Subjects were cattle, pigs, chickens, Animals, including but not limited to animals, Preferably it is a mammal, most preferably a human. "Mammals" , Mice, rats, rodents such as guinea pigs, dogs, cats, horses, cattle , Sheep, non-human primates, and humans. Such an effective amount Inhibitors identified according to the present invention can be used in the art, including the assays described herein, It can be easily determined by evaluating with a known ordinary test.   Administration of such compositions can be accomplished by any suitable formulation, as is well known in the art. It may be done by any conventional route using agents. Various delivery systems are public Is known, and it can be used to administer inhibitors, For example, there is encapsulation in liposomes, microparticles, microcapsules and the like. Special attention One form of delivery is pulmonary artery administration. Other delivery methods include intradermal, muscle This includes intra-, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, nasal and oral routes. It is not limited to these. Inhibitors can be administered by epithelium Or by absorption through mucocutaneous layers (eg, oral mucosa, rectal and intestinal mucosa, etc.) ) And may be administered with other biologically active agents.   Administration can be systemic or local. Treatment of nasal, bronchial or pulmonary conditions Alternatively, the preferred route of administration is oral, nasal, or bronchial aerosol or nebula. It is the. Thus, in certain embodiments, the inhibitor may be localized to the area in need of treatment. It may be preferable to administer the quality. This can be done, for example, by a surgical Intraoperative local infusions, local administration, injections, catheters, suppositories, skin patches or Plant use, including but not limited to Implants, whether porous or non-porous, can be made with sialastick. tic) It may be a gel-like material including a membrane such as a membrane, or a fiber.   In addition, administration of an effective amount of an inhibitor to an individual will This can be achieved by topical administration directly to the affected area of the skin of the individual. Several In some cases, placing the inhibitor on the skin, in the skin, or in a device placed under the skin Can be considered. Such devices may be provided with a passive or active release mechanism. Patches, implants and injections that release the compound to the skin are included.   Amount of inhibitor that will be effective in treating or preventing a particular disease or condition Will depend on the nature of the disease or condition, but will depend on standard clinical techniques. Can be determined. In addition, in vitro or in vivo assays are available depending on selection Thus, the data may be used to clarify the optimal dose range. Effective amount of test tube It may be extrapolated from dose-response curves obtained in internal or animal model test systems. For example In general, a typical effective dose of an inhibitor, when administered in a single or multiple doses, is approximately Within the range of about 0.01 to about 50 mg / kg, preferably about . It is in the range of 1 to about 10 mg / kg. Generally, inhibitors are such For patients in need of treatment, a daily dose of about 1 to about 2000 mg per patient May be administered.   The precise dose level of the inhibitor as an active compound will be Other health professionals should make a decision and identify the phosphopeptide bond interaction in question. Effect, route of administration, age, weight, gender and health of the individual, nature of the disease, Known, including severity and clinical stage, and with or without concomitant therapeutics It will depend on the factors. C. Utensil   The present invention may further comprise one or more prescription ingredients of the pharmaceutical composition of the present invention. A pharmaceutical pack or device comprising one or more containers . Depending on the choice, such containers may contain the manufacture, use, or use of pharmaceutical or biological products. You may put a note in the form of a government agency that regulates sales, but this is not recommended. Such notices have been approved by the agency for manufacture, use or sale for human administration. It shall reflect that.   Pharmacologically acceptable surfactants (eg, glycerides), additives (eg, Other components such as (toose), carrier, and diluent may be further included.   The following examples illustrate various aspects of the present invention. These examples are attached Does not limit the scope of the invention as defined in ("background'' B) References, pending patent applications, published patent applications cited throughout this application , Issued patents, and information contained on the website are all references and Here, it is clearly stated that it is incorporated here. IV. An example   The following examples illustrate various aspects of the present invention. These examples are attached Does not limit the scope of the invention as defined in   Fluorescein and fluorescein (in the low nanomolar probe concentration range) 96-well plate reader (FPM2, Jolly Y.) Instruments) to develop an FP assay based on 96 wells Became possible. In these examples, the FP assay and the Src-SH2 The components required to measure binding to the main are discussed.Example 1-Peptide Synthesis   Fmoc-Rink Amidole with a substitution level of 0.3-0.6 mmol / g Zin [4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) fe Nonoxyresin (Advanced Chemtech)] I went in motion. The standard FMOC synthesis method was used. Washing and deprotection solvents used Is dimethylacetamide (DMA), the coupling used (original: coupli ng) The solvent was N-methylpyrrolidone (NMP). Coupling of amino acids (Original: coupling) contains four equivalents of amino acids and four equivalents of coupling ( Original: coupling) Reagent 2- (1H-benzotriazol-1-yl) -1,1,1 3,3-tetrafluoroboron fluoride tetramethyluronium (TBTU), and eight equivalents Of N-methylmorpholine (NMM) was used per equivalent of resin amine. use The amino acids thus obtained were Fmoc-Gly, Fmoc-Glu (Tbu), Fmoc-I le, Fmoc-Thr (Tbu), and Fmoc-Tyr (Tbu). . Deprotection of Fmoc was performed using a 20% piperidine DMA solution.   Standard methods [eg Merrifleld,J.Am Chem.Soc., 85: 2149-2154 (1963) Was used to phosphorylate the solid phase. Resin and 1-1, H- Tetrozole to P_TwoO_FiveAnd dried under vacuum overnight. 50 equivalents of 1-1, H-tet 1 ml / 0.1 g of dry weight (0.1-0.5 g) Dry DMA was added. The resin was agitated for 20 minutes to swell. 10 equivalent dibenge N, N-diethylphosphoramidite (manufactured by Toronto Research Chemicals) ) Is then added and the resin mixture is stirred for 15 minutes, sonicated for 30 minutes and Stir for 5 minutes. The resin was washed 5 times with DMA. 3 equivalents of chlorobenzyl per Add Oxide DMA solution, stir for 30 minutes, sonicate for 30 minutes to oxidize I let you. Finally, the peptide was washed 5 times with DMA, CH_TwoCl_TwoThree times and three times with MeOH After washing twice, vacuum drying was performed overnight.   Final cleavage from the resin and deprotection of the side chains was achieved in a 90: 10: 10: 5 ratio. Trifluoroacetic acid (TFA): H_TwoO: ethanedithiol (EDT): tri-i This was performed using isopropyl silane (TIPS). After adding the cleaning agent to the resin, TFA was added with stirring for 2.5 hours. The resin was filtered. N_TwoLeeward For a few hours to remove TFA. Resuspend the crude peptide slurry in water Let Extracted three times with diethyl ether at the ice temperature for one minute. N_TwoExtra ether downwind Was removed. The crude peptide mixture was lyophilized overnight.   All peptides were purified by the following method. Lyophilized crude peptide was added to DM It was redissolved in SO to a concentration of 100-300 mg / ml. Pepti Was purified using a semi-preparative reverse-phase HPLC column (manufactured by Baydac). . A series of 15-30 ul injections of 100% crude peptide in DMSO were used. Was. Purity was determined by reverse-phase HPLC for analysis using a diode-array spectrophotometer. The examination was performed using a column. One attempt is sufficient to achieve a purity of more than 90%. Was.Example 2-Probe synthesis   A fluorescent component, 5-carboxyfluorescein, was obtained from the core middleT antigen. Pentapeptide based on the known Src-SH2 high affinity tetrapeptide sequence The combined fluorescent probe (FIG. 1 or FIG. 5) was designed as an example. This pep The sequence of the tide ligand is N-terminal to facilitate coupling. Core middle T antigen high affinity Src-SH2 sequence with glycine (pYEE GpYEEI including I).   There are several reasons for choosing 5-carboxyfluorescein. this is, It is one of the few defined isomers fluorescein. This is This results in a conjugate that is less flexible than available fluorescein, which Is important to minimize the “propeller effect” that can interfere with FP-based measurements. It is important to note that an activated version is commercially available (Molecular Pro Beeves).   The probe sequence was prepared as follows. Peptide GpYEEI The resin component was directly bound to the resin component. This peptide resin is Rink Assembled with resin and phosphorylated as described above. The resulting array is It was Fmoc-GpYEEI-LINK. 20% pipette for peptide / resin Release protection with lysine DMA solution, remove FMOC-protecting group, The free amino end has been left available for use in rings. Fill with DMA Minute And then washed with 1.1 equivalents of 5-carboxyfluorescein succinimidyl Steal (Molecular Probes) was added to 6 equivalents of diisopropylethyl alcohol. Added with min. After 1.5 hours of coupling (coupling) After washing once with NMP and three times with DMA, the coupling described above (original: coupling) ) Was repeated. The completed probe is cleaved and treated as described above, A probe named MT1 was created.   Two exemplary markers for the SH2 domain of Src, created as described above. The identification probe is:   Fluorescein-GpYEEI-NH_Twoas well as   Fluorescein-pYpYpYIE-NH_TwoIt is.Example 3-Protein Creation-Src   Residues 145-251 encoding human Src [Tanaka, A. and Fujita, D.J.,Mol .Cell.Biol .6: 3900-3909 (1986)] in the pT7 expression vector Bacteria BL21 (DE3) were transformed. Growth of 27 liter culture in minimal medium Protein was made from length and induction. In a typical fabrication, optics at 595 nm Cultures were grown at 37 ° C. until the density (OD) was 1.0. 1 m of culture M with isopropyl-BD-thiogalactopyranoside (IPTG). The temperature was reduced to 25 ° C. Cultures were harvested after 21 hours.   Using a French Press cell at 16,000 psi, cells were washed with 50 mM phosphoric acid. Potassium acid, 250 mM NaCl, 5 mM DTT, 2 mM EDTA, 1 m m of PMSF, pH 7.0, was dissolved in. Protein is weak-strong cation exchange The purified product was carboxy-sulfone (JT Baker). Column 50 mM potassium phosphate, 5 mM DTT, 0.02% NaN_ThreePH 7 . 0, equilibrated and filtered bacterial lysate was added at 2 ml / min. . The Src protein was eluted with a 1 M NaCl gradient. The eluate is Centriple Use a Top 10 Concentrator (Amicon's 10,000 MW cutoff) And concentrated and centrifuged at 3000 × g. Next, this protein is Potassium phosphate, 50 mM NaCl, 5 mM DTT, 1 mM EDTA, 0 mM . 0 2% NaN_ThreeSephacryl S-100 (fur) equilibrated at pH 7.4. The mixture was purified by gel filtration using a column (manufactured by Macia). SDS gel electrophoresis and RP- Purity as determined by HPLC is> 95%. The purified protein is 50 mM potassium phosphate, 500 mM NaCl, 10% glycerol, 5 mM DTT, 5 mM EDTA, 0.02% NaN_Three, PH 7.4, frozen in Keep in state.Example 4-Src-SH2 binding assay based on fluorescence polarization A. FP-Overview   All polarization methods use standard cut-off filters (excitation = 485 nm; emission = 530 nm) with a FPM2 96-well plate reader (Jolly) Was. Use saturation experiments to maximize protein and assay stability, and K for pair-probe interaction_dFor the purpose of judging The conditions were checked. B. Saturation experiment   For saturation experiments, the probe was used at a fixed concentration, and Src-SH2 increased in concentration. And added. This is usually the case with such assays using radioligands. This is the reverse of the method.   A saturation experiment was performed, in which the concentration of the Src-SH2 domain was higher, 40 Variations were performed from μM to the lower 39 nM. The fluorescent probe FMT1 (FIG. 1) The fixed concentration was kept at 20 nM. Protein concentration and protein concentration using amino acid analysis And both concentrations were determined. The results are shown in FIGS. 2A and 2A, respectively, obtained from this experiment. B shows the saturation curve and the scratchard analysis.   From the data shown in FIGS. 2A and 2B, the parent of the probe to the receptor domain That compatibility is appropriate for performing a competitive binding assay, and that saturation to a single site Possible binding was observed, with the assay of the invention and with competitive reversible binding to a single site. It turns out that it fits. C. Competitive experiment   Probes and proteins were used at fixed concentrations and peptides were Added in increasing degrees.   The designed probe FMT-1 has a .about.0.2 value with respect to Src-SH2 under standard buffer conditions. It has a Kd of 3 μM. Buffer conditions change for observed Kd values Some differences will occur.   As shown in FIG. 3, the Src competition assay (2% DMSO buffer) was Peptide, Ac-pYEEI (ring open), Ac-pYpYEEI (closed square), A c-pYGGL (plus sign), Ac-pYEDL (open triangle), Ac-DGV Performed on pYTGL (closed triangle). FIG. 3 shows the professional results obtained in one set of experiments. Figure 3 shows a competition curve plotting the percentage of active binding versus inhibitor concentration.                                   Table 1                       Representative peptide competitive IC50   This type of data can be obtained using the materials and methods of the present invention.₅₀Has a range of values It demonstrates that competitive binding assays can be performed with inhibitors.   Exemplary protocols according to the present invention are both manual and automatic, as follows: Done. D. Manual test   Two different plates can be used in each experiment. 2% DMSO play And 20% DMSO plate.Buffer   All buffer components were of low fluorescence grade (Panbella Corporation). Standard (STD) buffer is 20 mM phosphoric acid (pH 7.4), 100 mM Na Cl, 2 mM DTT, 1 mM EDTA, and 100 μg / ml BGG. No. Standard buffer is Millipore water or NaCl, EDTA and phosphate stock Create with clean water as clean as possible. The buffer is made up to volume with the same tap water.   The five buffers required for these experiments were standard (STD) buffer, STD buffer + 4% DMSO, 100% DMSO, STD buffer with only labeled probe Solution, and a buffer to which Src protein and a labeled probe are added.   One liter is 100 ml of 1 M NaCl, 1 M phosphoric acid (pH 7.4). 20 ml, 500 mM EDTA 2 ml, 5 mg / ml BGG 20 ml , And tap water consist of 858 ml.   Make a standard buffer and store at 4 ° C. Prior to each use, 2 mM DTT is added. This standard buffer is also used to make a standard buffer + 4% DMSO stock. this May be stored at 4 ° C. and DTT may be added prior to use.Protein and control peptide   The SRC protein is used at a final concentration of 0.75 μM. One of SRC stock solution An example is a 416.6 μM solution in STD buffer. The peptide Ac-pYEEI is Use at a final concentration of 100 μM. It is preferable to make a 2x stock of this Newprobe   Fluor-GpYEEI is used as a probe at a final concentration of 20 nM. Supply The probe stock used is a 10 μM solution in STD buffer.   As an example, make only 25 ml of protein and only 2 ml of probe. To do so, follow the steps below.   (A) 27 ml of standard buffer (add 2 mM DTT)   (B) Add 2 × or 40 nM probe-1515 = 108 μL   (C) Remove 2 ml for probe only   (D) Add 2 × or 1.50 μM SRC to the remaining 25 ml = 90 μL   This gives a protein and probe solution.Second stock   Each compound / peptide and control peptide are placed in separate tubes. These are 2 × stock.   A first stock of 50 mM in 100% DMSO has been made and then the desired fruit Make a second stock of STD buffer + 4% DMSO at appropriate concentrations for testing Is desirable.   An example of a second stock is an example of an assay (long format, 2% DMS For O), combine 2 mM stock in STD buffer + 4% DMSO I do. The other is 10 for long format assays using 20% DMSO. 5 mM stock in 0% DMSO. Another second stock solution shows STD buffer + 4% D for G format assay # 1 and # 2-2% DMSO 400 μM stock in MSO. Short format # 1 and # 2- 20% DMSO is a 1 mM stock in 100% DMSO.Long format certification   A 1: 2 dilution is used for the first well in a final volume of 1 mM. Plate 1-2% DM For SO, the assay protocol is as follows.   (A) In columns 2-12, 50 μl of standard buffer + 4% DMSO.   (B) 100 μl of 8 different compounds in each well of column AH in column 1 (2 mM stock / SB + 4% DMSO).   (C) 50 μl (1: 2) system down to plate 10 in horizontal direction up to column 10 Perform column dilution.   (D) Add only 50 μl of probe to column 12.   (E) Add 50 μl of protein and probe to column 1-11.   (F)-100 μl final volume per well.   (G) Read the plate with FPM2.   For plates 1-20% DMSO, the assay protocol is as follows: .   (A) In columns 2-12, 20 μl of 100% DMSO.   (B) 40 μl of 8 different compounds in each well of column AH in column 1 5 mM stock / 100% DMSO).   (C) 20 μl (1: 2) system down to plate 10 in horizontal direction up to column 10 Column dilution.   (D) Add only 50 μl of probe to column 12.   (E) Add 50 μl of protein and probe to column 1-11.   (F) Add 50 μl of STD buffer to the whole plate.   (G) —100 μl final volume per well.   (H) Read the plate with FPM2.Short format # 1 certification   1: 3 dilution in a final volume of 200 μM to the first well. Compound / peptide replicates Used. These examples are for a 1: 3 dilution but the amount may be any desired May be changed for dilution.   For plates 1-2% DMSO, the assay protocol is as follows.   (A) 50 μl of standard buffer + 4% DMSO for rows BD and FG on the side.   (B) For each well of the horizontal row A of the vertical row 1-10 and the horizontal row E of the vertical row 1-12 , 75 μl of duplicated 11 different compounds (400 μM / SB + 4% DMSO ).   (C) From the AD and from the EH, 2 vertically down the plate Make 5 μl (1: 3) serial dilutions.   (D) Add only 50 μl of probe to column 12.   (E) 50 μl of protein in AD in column 1-11 and EH in 1-12 And probe.   (F) —final volume per well is 100 μl.   (G) Read the plate with FPM2.   For plate 2-20% DMSO, the assay protocol is as follows: .   (A) 20 μl of 100% DMSO for rows BD and FG.   (B) For each well of the horizontal row A of the vertical row 1-10 and the horizontal row E of the vertical row 1-12 Are 30 μl replicates of 11 different compounds (1 mM / 100% DMSO).   (C) 10 μm from AD and then from EH to the lower part of the plate in the vertical direction. l (1: 3) Serial dilution.   (D) Add only 50 μl of probe to column 12.   (E) 50 μl of protein in AD in column 1-11 and EH in 1-12 And probe.   (F) —final volume per well is 100 μl.   (G) Add 30 μl STD buffer to the entire plate.   (H) Read the plate with FPM2.Short format # 2 certification   1: 3 dilution in 200 μM final volume to first well. Single compound / peptide Used in These examples are for a 1: 3 diluent, but the amount may vary depending on the degree of dilution desired. It may be changed as needed.   For plates 1-2% DMSO, the assay protocol is as follows.   (A) In columns 2-4 and 6-8, 50 μl of standard buffer +4 in 10-12 % DMSO.   (B) 75 μl of each of the 16 different compounds (40 0 μM / SB + 4% DMSO).   (C) 75 μl of 6 different (400 μM / SB) only for AF in column 9 + 4% DMSO) compound.   (D) From 1-4, then from 5-8, then from 9-12, horizontally below the plate 25 μl (1: 3) serial dilutions toward the top.   (E) Add only 50 μl of probe to column H of column 9-12.   (F) 50 μl in horizontal row AH, vertical row 1-8, and AG vertical row 9-12 Add protein and probe.   (G)-Final volume must be 100 μl per well.   (H) Read the plate with FPM2.   The following protocol is followed for the plate 2-20% DMSO assay.   (A) 20 μl of 100% DM in columns 2-4 and 6-8 and 10-12 SO.   (B) 30 μl of 16 different compounds (1 m) in each well of columns 1 and 5 M / 100% DMSO).   (C) 30 μl of 6 different compounds (1 mM / 1 00% DMSO). Total 22 compounds.   (D) From 1-4, then from 5-8, then from 9-12, horizontally below the plate Serially dilute 10 μl (1: 3) toward the top.   (E) Add only 50 μl of probe to column H next to column 9-12.   (F) 50 μl per row AH in vertical columns 1-8 and AG in vertical columns 9-12 Add protein and probe.   (H) Add 50 μl STD buffer to the entire plate.   (I)-Final volume must be 100 μl per well.   (J) Read the plate with FPM2. E. FIG. Automated test   (1)Short dilution   This assay uses 200 μM for the first well and 4 wells down from there. 2 compounds / plate, plate against Genesis Deck "Landscape (Original language: landscape) direction, performed in 1/3 dilution steps .   (I) 20% DMSO.   (Ii) 2% DMSO.   (2)Long dilution   This assay uses 1 mM in the first well and up to 10 wells below, using 8 compounds. Objects / plates, plates against the Genesis Deck are "landscape" : Landscape) direction in half dilution step.   (I) 20% DMSO.   (Ii) 2% DMSO.   Diluted solution   Five buffers:   -Buffer + 4% DMSO   -100% DMSO   -Buffer containing only probe   -Buffer containing protein and probe   -Buffer   (3)Short format   Each compound is in a separate well of a 96-well plate.   Plate 1-2% DMSO   50 μl of 4% DM in columns 2, 3, 4, 6, 7, 8, 10, 11 and 12 SO.   -In all the horizontal rows of columns 1 and 5 and in each well of columns A to F of column 9 75 μl of 22 different compounds (400 μM stock, 4% DMSO).   -75 μl of buffer + 4% DMSO in wells G9 and H9.   -While discarding the last 25 μl, plate 1 to 4 and 5 to 8 , And serially dilute 25 μl (1: 3) from 9 to 12. Now the liquid / well 50 μl remain.   50 μl of protein in the whole plate except wells H9, 10, 11 and 12 Add quality and probe.   Add only 50 μl of probe to wells 9, 10, 11 and 12   -Final volume per well is 100 [mu] l.   -Read the plate.Plate 2-20% DMSO   20 μl of 100 in columns 2, 3, 4, 6, 7, 8, 10, 11 and 12 % DMSO.   -In all the horizontal rows of columns 1 and 5 and in each well of columns A to F of column 9 Is 30 μl of 22 different compounds (1 mM stock, 100% DMSO).   -30 [mu] l of 100% DMSO in wells G9 and H9.   -While discarding the last 25 μl, plate 1 to 4 and 5 to 8 , And serially dilute 10 μl (1: 3) from 9 to 12. Now the liquid / well 50 μl remain.   50 μl of protein in the whole plate except wells H9, 10, 11 and 12 Add quality and probe.   Add only 50 μl of probe to wells H9, 10, 11 and 12.   -Add 30μ STD buffer to the whole plate.   -Final volume per well is 100 [mu] l.   -Read the plate.   (4)Long format   Each component is placed in a separate well of a 96-well plate.   Plate 1-2% DMSO   -50 µl of 4% DMSO in column 2-12.   -100 μl of 8 different compounds in each well of column AH in column 1 vertical.   -50 μl of std. Add 3   -Plate up to column 10 while discarding the last 50 μl (all horizontal columns) Make a serial dilution of 50 μl (1: 2).   -50 μl of wells A11 to H11 while discarding the last 50 μl Perform column dilution.   50 μl of dandelion on the whole plate except A12, B12, C12 and D12 Add quality and probe.   Add only 50 μl of probe to A12, B12, C12 and D12.   -Final volume per well is 100 [mu] l.   -Read the plate.   Plate 2-20% DMSO   20 μl of 100% DMSO in row 2-12.   40 μl of 8 different compounds in each well of column AH of column 1   20 μl of std. Add 4.   Plate up to column 10 with the last 20 μl being discarded (all horizontal columns) To 20 μl (1: 2).   20 μl system in wells A11 to H11 while discarding the last 20 μl Perform column dilution.   50 μl of dandelion on the whole plate except A12, B12, C12 and D12 Add quality and probe.   Add only 50 μl of probe to A12, B12, C12 and D12.   -Add 30 [mu] l STD buffer to the whole plate.   -Final volume per well is 100 [mu] l.   -Read the plate. * If diluted, plates can be read between 5 and 30 minutes. * The assay plate is transferred to the FPM2 instrument for a 3 minute reading.Example 5-Assay of ZAP, SYK and LCK based on FP   The SH2 domain of Src, exemplified in Examples 1-4 above, was bound to a phosphor. In addition to its examples with phosphoTyr-containing peptide ligands, the assay also included protein Z It was also used for ap, Syk and Lck. These three proteins are based on salt concentration, Minor changes to production parameters such as DTT concentration, protein concentration, temperature, etc. It is made in a manner similar to the creation of Src in E. coli as described above. S rc has only one SH2 domain, while ZAP and Syk have two SH2 domains. Domains, and the Lck protein contains one SH2 domain and one SH3 Domain and is included.   The "first protein" created for these assays was the Srca145- 251 are created in substantially the same manner as described above. These proteins Are shown in the table below. The term "NC" refers to the N- and C-termini of the first protein. Mean that both contain the SH2 domain. Zap, Syk and L The sequence of ck is known in the art. PCT / US96 / 13918 See also                                   Table I         First proteinAmino acid residues of naturally occurring proteins         NC-ZAP aa1-259         NC-Syk aa1-260         Lck aa 109-266   Probes for each assay are designed and used as described above. For example, N, C- ZAP protein, ie, two SH2 domains of human ZAP are included in the series Probes for proteins include, for example, fluorescein-GpYNELN LGRRGEEpYEVL-NH_TwoIt is. Another example, N, CSyk A protein that contains two SH2 domains of human Syk Probes for proteins include, for example, fluorescein-ApYTGLSTRN QETpYETL-NH_TwoIt is. This SRC probe is also used for Lck Was done.   The Lck protein is labeled with a fluorophore-labeled phosphopeptide for its SH2 domain. Ligand, a fluorophore-labeled peptide ligand toward its SH3 domain, To the SH2 domain and SH3 domain Assays were performed on double ligands.   The assay format shown in Example 4 was reproduced for these binding pairs and results were obtained. .   From the data shown in FIGS. 2A and 2B, this program for this receptor domain Lobe affinity is adequate for performing competitive binding assays, and to a single site Saturable bond was observed, which was compatible with the assay of the present invention, It can be seen that it is compatible with competitive reversible binding. The data obtained from the saturation test is used in the present invention. To assess the availability of a particular probe when used within the scope of this method. Can be For example, if the probe is working at a certain level, If (eg Kd <10 μM and mP difference value> 50 (in the presence and absence of protein) Polarization differences observed in the presence)), suitable for use in the competition assay of the invention It is shown that.Example 6-Human GRB2-SH2 binding assay based on fluorescence polarization A. Test-Overview   All assays include SrcS described in the previous example, including buffer and instrument usage. The H2 assay was performed in substantially the same manner as the H2 assay, except that human G (instead of SrcSH2) RB2 protein and SH2 of GRB2 (instead of Src-specific probe) Replaced with domain specific probe. Human GRB2 protein tested by the assay For the quality, amino acid residues 55-152 of huGRB2 [see Cell 70: 431-442 (1992)] Using a longer sequence encompassing the SH2 domain. Is also good. The production and purification of the protein is similar to that described above, but in particular GRB2 Is the same as using a phosphotyrosine column to purify the SH2 domain of is there. B. Saturation experiment   As before, fix probe concentration and increase Grb-2-SH2 concentration A saturation experiment was performed.   The following Grb-2-specific SH2 probe ("FPgb2") is described in Example 1. It was synthesized using the technique described above. FPgb2, Fluor-GpYVNV-NH2   However, in the above formula, “Fluor” means a peptide containing the sequence GpYVNV. Represents 5-carboxyfluorescein attached to the tide via an amino bond. this The sequence of the tetrapeptide is specific for binding to the SH2 domain of Grb-2 . This probe has features suitable for acting as probes for competition assays, and G Suitable saturation curves for RB2 SH2 proteins (eg, Kd and total mP difference).Example 7-SrcSH3 binding assay based on fluorescence polarization A. Test-Overview   All assays include SrcS described in the previous example, including buffer and instrument usage. The H2 assay was performed in substantially the same manner as the H2 assay, except that the SH2 domain of Src and SH were used. A protein containing a peptide sequence that fills in the three domains (amino acid residues 84-2 51) (see [Mol. Cel Biol. 7 (5): 1978-1983 (1987)]), and (Sr A probe specific to the SH3 domain of Src (instead of the c-specific SH2 probe) Was replaced. If it contains the SH3 domain of Src (SH2 domain of Src Larger or smaller Src protein (with or without) Note that it may be used. Protein production and purification are basically As described above, see, for example, Example 3. Provide the following details: Purification of Src-SH2-SH3 domain: 1) French press dissolution, 50 mM potassium phosphate, 5 mM DTT, 2 m M EDTA, 1 mM PMSF, pH 7.0. 2) 40 uM WP carboxy-sulfone column-> 633 mg 3) Column of phosphotyrosine agarose-> 512mg 4) 50 mM potassium phosphate, 10% glycerol, 500 mM NaCl Dialysis against 5 mM EDTA, 0.02% NaN3, pH 7.0.   B. Saturation experiment   As in the previous example, the probe concentration was fixed and Src-SH2-SH3 protein was used. Saturation experiments were performed with increasing concentrations of the material.   The following SrcSH3-specific probe ("FPSH3") technology described in Example 1 Was synthesized using Fluor-PLARRPPLPPLP-NH2   However, “Fluor” in the above formula refers to the binding to the SrcSH3 domain. A specific peptide containing the sequence PLARRPPLPPLP via an amino bond Represents the combined 5-carboxyfluorescein. This probe can be used for competition assays. Features suitable to act as lobes and suitable saturation curves with Src protein (E.g., suitable Kd and total mP differences for protein binding).Example 8-SrcSH2-SH3 Double Bond Assay Based on Fluorescence Polarization   A. Test-Overview   Proteins such as Src contain both SH2 and SH3 domains. A few The protein is not isolated via either the SH2 or SH3 domains. Can bind to such SH2-SH3 proteins via both at any one time . One example is the protein p130CAS, which is an SH2 and SH3 domain. It is believed that it binds to Src through both of the proteins. SH130 of p130CAS And SH2 binding sequences have been identified by conservative and site-directed mutagenesis ( Nakamoto et al. JBC 271, 8959-8965, 1996). Residues 733-738 (RRL PSP) remains (possibly phosphorylated and involved in SrcSH2 binding). In the same way as the group Tyr-762, Is shown. Fluorescent probes for FP assays are based on these sequences. It was designed based on. If such a probe is used in the assay, an SH2-specific inhibitor And SH3 specific inhibitors can be screened simultaneously. One such probe ("FPC130") is: Fluor-RPLPPSPPKFTSQDSPDGQYENSEGGWMEDp YDYVHL   This is the native sequence obtained from p130CAS (733-767). Affinity Alter the protein specificity and, in particular, process the protein for the desired protein of interest. In order to increase the overall affinity of the probe, based on the above, but different components SH2 And / or a derivative probe using the SH3 sequence may be used.   All assays included the SrcSH described in Example 4, including buffer and instrument usage. The two tests were performed in substantially the same manner, but the Src (instead of SrcSH2) Protein containing peptide sequence filling between SH2 domain and SH3 domain And the SH2-SH3 probe described above (instead of the Src-specific SH2 probe) Was replaced.   B. Saturation experiment   As in the previous example, the probe concentration was fixed and the protein (in this case Src- A saturation experiment was performed with increasing concentrations of SH2-SH3).   The FPC130, which is a SrcSH2-SH3 specific probe, is a technique described in Example 1. Synthesized using the technique. Fluor-RPLPPSPPKFTSQDSPDGQYENSEGGWMEDp YDYVHL However, “Fluor” in the above formula is specific to binding to the SrcSH3 domain. Binding via an amino bond to a peptide containing the sequence PLARRPPLPPLP Represents 5-carboxyfluorescein (can it be replaced with another fluorescent probe? Maybe.) This probe is suitable for acting as a probe for competition assays. And a suitable saturation curve for the Src protein (eg, for protein binding). Suitable Kd and total mP difference).Example 9-Human Src-SH2 Binding Based on Fluorescence Polarization Using the Modified Probe Test A. Test-Overview   Using a phosphor that is not fluorescein, Src-SH2 (SH2 domain Applicable to any fragment of Src, including developed. This phosphor is a family of fluorescent molecules, including core phosphors, 4,4-difluoro-4bora-3a, 4a-diaza-s-indacene (original: indacene) (commercially available from Molecular Probes). This fluorescence-pepti The structure of the probe is:This phosphor is BODIPY-TRX, a Texas Red family phosphor. It has spectral properties very similar to optical bodies. The use of a red phosphor allows Inspection that may be used in a wider range and its fluorescence properties may resemble fluorescein itself Compounds can be screened.   All assays included the SrcSH described in Example 4, including buffer and instrument usage. The procedure was performed in a manner similar to that of the two assays, except that the new probe shown above was used. Also, the fluorometer is different to match the spectral characteristics of the probe of this modification A filter was used. Specifically, the excitation / emission filters used above were 591/635, as opposed to 485/530 for use.   The synthesis of the peptide portion of the probe is as described in Example 1, except that the peptide NH2- pYEEI was created. The probe further comprises the released amine peptide NH2-p YEEI in 66% DMSO buffer (34% 0.1 M sodium bicarbonate) By binding to the succiimidyl ester of the BODIPY-TRX probe Synthesized. The reaction was completed by silica gel TLC (4: 1: 1, butanol, H2O, Acetic acid solvent system) and was performed in 24 hours. The resulting peptide is very hydrophobic And purified on the same TLC system described immediately above. B. Saturation experiment   As in the previous example, the probe concentration was fixed and the concentration of Src-SH2 protein Saturation experiments were performed at increasing degrees. The only difference is that, as explained in A above, An alternative set of filters for the new probe.   This probe has sufficient Kd and difference in total mP as a probe for competition assay. Presented at the time of protein binding.   The present invention is not limited in scope by the specific embodiments described herein. No. Indeed, various modifications of the invention in addition to those described herein will become apparent from the foregoing description. It will be clear to the traders. Such modifications are intended to cover the appended claims. As intended.   The disclosures of patents, patent applications, and publications cited herein are all references and references. It has been incorporated.

────────────────────────────────────────────────── ─── 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), AU, CA, JP, U S (72) Inventor Zoller Mark Jay.             United States 02193 Massachusetts             Sutton Place 7 Weston, Tutu

Claims (1)

[Claims] 1. A test for identifying test substances that inhibit the association of two protein molecules. An in vitro test method,   (A) providing a first protein molecule and a second protein molecule capable of being associated with each other; Wherein the second protein molecule has a covalently bonded phosphor. Is a step,   (B) containing the first and second protein molecules and at least one test substance; Creating a liquid mixture;   (C) irradiating the mixture with polarized light having a suitable wavelength to excite the phosphor to emit polarized light. A step to indicate as   (D) measuring the degree of emitted polarized light;   (E) a mixture of the first and second protein molecules in the absence of the test substance The effect of the presence or concentration of the test substance on reducing the emission polarization observed in Determining the inhibitory activity of the test substance with the reduced depolarization value. Steps that are related A method that includes 2. One of the protein molecules is an SH2 domain, a PI domain, an SH3 domain. At least one domain selected from the main and WW domains 2. The method of claim 1, wherein the other of the protein molecules is a ligand thereof. . 3. Wherein one of the protein molecules is an SH2 domain or a PI domain; And at least one domain selected from the group consisting of: Comprises the phosphotyrosine-containing peptide sequence. 4. Said one or more steps comprises two or more steps For a test substance, or for one or more steps, multiple test substances or tests 2. Performed on a device programmed to perform automatically on substance concentration. The method described in. 5. The method of claim 1, wherein the phosphor is fluorescein. 6. A method for identifying a substance that inhibits the mutual association of a pair of proteins, comprising: The method comprises the steps of: testing the protein of the pair and one or more tests involving candidate inhibitors. Mixing the substance with the substance, and the presence of Determining if the association has been inhibited,   (A) a step of using a pair of proteins, wherein one of the proteins is A step covalently bonded to the phosphor,   (B) irradiating the mixture of the protein and the test substance with polarized light having a suitable wavelength; Showing the excitation of the phosphor as polarized light emission;   (C) measuring the degree of polarized light emitted;   (D) polarized light observed in a mixture of the proteins in the absence of the test substance From the luminescence, determine the effect of the presence or concentration of the test substance on reducing it Step, wherein the competitive binding of the test substance is correlated with the observed depolarization value. Steps and A method comprising: 7. One of the proteins is an SH2 domain, a PI domain, an SH3 domain. Including at least one domain selected from the group consisting of 7. The method of claim 6, wherein the other of said proteins is its ligand. 8. One of the proteins is one of an SH2 domain and a PI domain And at least one domain selected from the group consisting of 8. The method of claim 7, comprising a tyrosine containing peptide sequence. 9. Said one or more steps comprises two or more steps For a test substance, or for one or more steps, multiple test substances or tests 7. Performed on a device programmed to perform automatically on substance concentration. The method described in. 10. 7. The method of claim 6, wherein said phosphor is fluorescein. 11. A pair of proteins identified by the method of claim 1 or claim 6. Inhibitors. 12. Any of receptor tyrosine-phosphorylated peptides and / or their ligands An in vitro assay method for identifying a test substance that competitively binds to crab,   (A) providing a receptor for a tyrosine phosphorylated peptide;   (B) providing the ligand for the receptor, wherein: The ligand having a covalently attached fluorescent moiety, a step,   (C) illuminating a mixture containing (a) and (b) and the test substance with polarized light of a suitable wavelength; Illuminating to show the excitation of the phosphor as polarized light emission;   (D) measuring the degree of emitted polarized light;   (E) In reducing the polarized light emission observed with a mixture of only (a) and (b) Determining the effect of the presence or concentration of said test substance on said test substance, Competitive binding is a function of reduced depolarization values, steps and A method that includes