WO2005114198A2

WO2005114198A2 - Protein phosphorylation assay

Info

Publication number: WO2005114198A2
Application number: PCT/US2005/017711
Authority: WO
Inventors: Douglas S. Auld
Original assignee: Pharmacopeia, Inc.
Priority date: 2004-05-20
Filing date: 2005-05-19
Publication date: 2005-12-01
Also published as: WO2005114198A3

Abstract

The present invention provides methods for assaying the activity of protein kinases or phosphatases that involve reacting the enzymes with substrate, at least a portion of which is fluorescently labeled; allowing phosphorylated substrate to bind to a solid support coated with a phosphate-binding material; and measuring the fluorescence produced by bound, labeled substrate. The invention also provides methods for identifying substances that modulate the activity of protein kinases and phosphatases that comprise reacting the enzymes with a substrate, at least a portion of which is fluorescently labeled, in the presence and absence of test compounds, allowing phosphorylated substrate to bind to a solid support coated with a phosphate-binding material, measuring the fluorescence produced by bound, labeled substrate, and comparing the fluorescence from reactions carried out in the presence and absence of test compounds.

Description

PROTEIN PHOSPHORYLATION ASSAY

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Number 60/572,741, filed May 20, 2004, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION [0002] The present invention relates to methods for assaying the activity of protein kinases and phosphatases, and to methods for identifying substances that modulate the activity of protein kinases and phosphatases.

BACKGROUND OF THE INVENTION [0003] Signal transduction is largely regulated by the transfer and removal of single phosphate groups to and from protein targets, catalyzed by protein kinases and phosphatases. Protein kinases comprise approximately 2 % of the genes encoded by the human genome, and the two largest classes of kinases are the tyrosine and serine/theronine protein kinases. See, for example, Manning G, Whyte, DB, Martinez, R, Hunter, T and Sudarsanam, S: The Protein Kinase complement of the human genome. Science 2002; 298:1912-1934. Due to their role in signal transduction, protein kinases have become important drug targets in the pharmaceutical industry, second only to the class of proteins known as G-protein coupled receptors. Approximately 30 % of drug discovery research spending is focused on protein kinases. [0004] A wide variety of assay methodologies have been developed for both tyrosine and serine/threonine kinases, which can be classified into either antibody or non-antibody dependent techniques, depending upon whether a specific antibody is used to detect the phosphorylated product. Each of the assay methodologies can be further divided based upon whether they involve physical separation steps. In addition, the assays methodologies are classified as either homogenous or heterogeneous, depending upon whether one or more of the components are in the solid phase. [0005] Phosphospecific tyrosine antibodies were first used in tyrosine kinase assays ^v because such antibodies were easy to obtain. Tyrosine kinase assays that utilize phospho- tyrosine antibodies are described, for example, in Glenney, JR Jr, Zokas L: Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton: J. Cell Biology 1989;108:2401-2408. Phospho-specific serine/theronine antibodies were more difficult to develop, and a separate antibody typically had to be used for each phospho-peptide sequence, limiting the utility of antibody-dependent serine/threonine kinase assays. Kinase assays that did not require antibodies were therefore developed. [0006] A radioactive scintillation proximity assay (SPA) was developed in which a biotinylated or histidine tagged kinase substrate was phosphorylated using ³³P-ATP and the tagged substrate was detected after binding to streptavidin or nickel coated SPA beads, as described, for example, in Park, Y-W, Cummings, RT, Wu, L, Zheng, S, Cameron, PM, Woods, A, Zaller, DM, Marcy, Al, Hermes, ID: Homogeneous Proximity Tyrosine Kinase Assays: Scintillation Proximity Assay versus Homogeneous Time-Resolved Fluorescence. Anal. Biochem. 1999;269:94-104. The hazards of radioactive isotopes make SPAs unappealing, however. [0007] Other assay formats were developed and include assays that measure ATP depletion by employing luciferase-coupled reactions, generic scintillation proximity assays as described, for example, in Mallari R, Swearingen E, Liu W, Ow A, Young, SW, Huang, S-G: A generic high-throughput screening assay for kinases: Protein Kinase A as an example. J. Biomol. Screen. 2003; 8:198-204, and assays that utilize proteases to detect phosphorylation events as described, for example, in Rodems, SM, Hamman, BD, Christina, L, Zhao, J, Shah, S, Heidary, D, Makings, L, Stack, JH, Pollok, BA: A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay and Drug Development. Techn. 2002;1 :9- 19. More recently, metal ions have been used to trap phosphopeptide products in both kinase and phosphatase assays as described, for example, in Loomans EE, van Doornmalen AM, Wat JW, Zaman GJ: High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogenous assay for protein kinases. Assay Drug Dev. Technol. 2003 ;1 :445-453; Gaudet EA, Huang K-S, Zhang Y, Huang W, Mark D, Sportsman, JR: A homogenous fluorescence polarization assay adaptable for a range of protein/serine threonine and tyrosine kinases. J. Biomol. Screen. 2003;8:164-175; and Sportsman, JR, Daijo J, Gaudet, EA: Fluorescence polarization assays in signal transduction discovery. Comb. Chem. High Throughput Screen. 2003;6:195-200. [0008] Metal ions have been used in immobilized metal affinity chromatography (LMAC) for protein separation and analysis for more than 40 years. See, for example, Helfferich, FG: "Ligand exchange": A novel separation technique. Nature 1961;189:1001-1002. The principle underlying LMAC is rooted in Pearson's hard soft acid base theory (HSAB) in which metal ions act as Lewis acids and functional groups on proteins act as Lewis bases. For example, hard Lewis acids such as Fe³⁺ prefer to interact with hard bases such as the oxygen atoms present on PO₄ ^3" ions as described, for example, in Andersson, L, and Porath, J: Isolation of phosphoproteins by immobilized metal Fe affinity chromatography. Anal. Biochem.1986; 154:250-254. Such interactions are largely non-ionic in nature, have binding constants as high as 10 , which have been reported for phosphoserine binding to Fe as described, for example, in Andersson, L, and Porath, J: Isolation of phosphoproteins by immobilized metal Fe³⁺ affinity chromatography. Anal. Biochem.1986; 154:250-254, and are stable in denaturants. High salt concentrations are often used to limit the occurrence of nonspecific binding during metal ion/protein interactions as described, for example, in Porath, J, Olin, B: Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 1983;22:1621-1630; and Kastner, M: Immobilized metal ion affinity chromatography. J. Chromatogr. Libr. 2000;61:301-383. [0009] TMAP® (immobilized metal ion affinity-based fluorescence polarization) is a protein kinase and phosphatase assay format that utilizes the binding of metal coordination complexes to phosphate groups. For kinase assays, fluorescently labeled peptides are phosphorylated in a kinase reaction. Following the reaction, IMAP® nanoparticles derivatized with metal coordination complexes are added to the reaction mixture and bind the phosphorylated peptides, which causes a change in the motion of the peptides, resulting in an increase in the observed fluorescence polarization. The bulk fluorescence of the sample is measured in HvIAP®, resulting in susceptibility to interference from fluorescent compounds in the solution. In addition, due to the use of fluorescence polarization, MAP® assays cannot be easily miniaturized to volumes of less than 1 μL. Moreover, the substrates used in BVIAP® assays can be no more than 10 to 15 amino acids, and some kinases do not phosphorylate peptides this small, limiting the applicability of LMAP® to only certain kinases. [0010] A need therefore exists for protein kinase and phosphatase assays that can be used for all types of kinases, that do not utilize radioactivity, that can be readily mininaturized, that are not susceptible to fluorescent compound interference, and that do not place size limits on the substrate.

SUMMARY OF THE INVENTION [0011] In certain embodiments, the present invention provides methods for assaying the activity of protein kinases that comprise preparing a reaction mixture comprising a kinase, at least one substrate, and adenosine triphosphate (ATP), wherein at least some of the substrate is fluorescently labeled; allowing the reaction to proceed for a time sufficient for phosphorylation of the substrate to occur; contacting the kinase reaction mixture with a suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support; and measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support, wherein the fluorescence is an indicator of kinase activity. [0012] Other aspects of the invention relate to methods for assaying the activity of protein phosphatases that comprise preparing a reaction mixture comprising a phosphatase and at least one phosphorylated substrate, wherein at least some of the substrate is fluorescently labeled; allowing the reaction to proceed for a time sufficient for dephosphorylation of the substrate to occur; contacting the phosphatase reaction mixture with a suspendable solid support coated with a phosphate-binding material for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support; and measuring the fluorescence produced by labeled, phosphorylated substrate bound to the solid support, wherein the fluorescence is an indicator of phosphatase activity. [0013] Still further embodiments of the invention are directed to methods for identifying substances that modulate the activity of protein kinases that comprise preparing a reaction mixture comprising a kinase, at least one substrate, and adenosine triphosphate (ATP), wherein at least some of the substrate is fluorescently labeled; preparing at least one additional reaction mixture comprising the kinase, at least one substrate, adenosine triphosphate (ATP), and a test compound, wherein at least some of the substrate is fluorescently labeled; allowing the reactions to proceed for a time sufficient for phosphorylation of the substrate to occur; contacting each kinase reaction mixture with a separate suspendable solid support coated with a phosphate- binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid supports; measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid supports; and comparing the fluorescence measured from the reactions carried out in the presence and absence of the test compound, wherein a difference in fluorescence indicates that the test compound is a modulator of kinase activity. [0014] Additional embodiments of the invention relate to methods for identifying substances that modulate the activity of protein phosphatases that comprise preparing a reaction mixture comprising a phosphatase and at least one phosphorylated substrate, wherein at least some of the substrate is fluorescently labeled; preparing at least one additional reaction mixture comprising the phosphatase, at least one phosphorylated substrate, and a test compound, wherein at least some of the substrate is fluorescently labeled; allowing the reactions to proceed for a time sufficient for dephosphorylation of the substrate to occur; contacting each phosphatase reaction mixture with a separate suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid supports; measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid supports; and comparing the fluorescence measured from the reactions carried out in the presence and absence of the test compound, wherein a difference in fluorescence indicates that the test compound is a modulator of phosphatase activity.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] Figure 1 depicts an example of a complex formed when a phosphorylated peptide is bound to a metal ion chelated to a suspendable solid support. [0016] Figure 2 illustrates a method for assaying the activity of a protein kinase in which a fluorescently-labeled substrate is phosphorylated in a kinase reaction and bound to a metal ion chelated to a suspendable solid support. Direct fluorescence produced by the bound, labeled peptide is measured using laser scanning imaging. [0017] Figure 3 A depicts the results of experiments in which various metal ions were tested for their ability to bind substrate and product peptides. [0018] Figure 3B depicts the laser scanning image of Zr coated polystyrene beads in the presence of either 10 nM Cy5-labeled substrate peptide or 10 nM Cy5-labeled product peptide. [0019] Figure 4 depicts the results of experiments in which substrate and product peptides were titrated against Zr™ coated polystyrene beads. The total Cy5-labeled peptide concentration was held constant at 5 nM, but the percentage of substrate and product peptide varied from 0 % to 100 %. [0020] Figure 5 depicts the results of experiments in which ATP was titrated against Zτ coated polystyrene beads in the presence of Cy5-labeled peptides. [0021] Figure 6 depicts the results of experiments in which MAPKAP-K2 was titrated in a MAPKAP-K2 assay while the ATP concentration was held constant. [0022] Figure 7 depicts the results of experiments in which ATP was titrated in a MAPKAP-K2 assay while the MAPKAP-K2 concentration was held constant. [0023] Figure 8 depicts the inhibition of MAPKAP-K2 activity by staurosporine. [0024] Figure 9 depicts the results of automation of a MAPKAP-K2 assay.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0025] As used herein, the term "substrate" refers to a peptide or polypeptide of any length that includes at least one serine, threonine, tyrosine, or histidine residue. The term "phosphorylated substrate" refers to a substrate to which a phosphate group is attached. [0026] As used herein, the term "suspendable solid support" refers to any solid support that is capable of being suspended in a liquid. "Solid support" refers to an inert, insoluble material to which a substrate can be bound or coupled, either directly or through one or more linking moieties. [0027] As used herein, the term "phosphate-binding metal" refers to a metal ion that has an affinity for, and binds to, phosphate groups found in peptides and polypeptides. [0028] As used herein, the term "coated" refers to a suspendable solid support to which at least one phosphate-binding metal ion is bound, either directly or indirectly, by any means. [0029] As used herein, the phrases "substances that modulate the activity of a protein kinase" and "modulator of kinase activity" refer to substances that either activate or inhibit the activity of a protein kinase. [0030] As used herein, the phrases "substances that modulate the activity of a protein phosphatase" and "modulator of phosphatase activity" refer to substances that either activate or inhibit the activity of a protein phosphatase. [0031] As used herein, the term "test compounds" refers to compounds that are potential activators or inhibitors of protein kinases or protein phosphatases. "Potential kinase and phosphatase inhibitors and activators" are compounds that may or may not reduce or increase, respectively, the activity of a kinase or phosphatase relative to the activity of the kinase or phosphatase in the absence of the compound. [0032] As used herein, the term "separate kinase reactions" refers to kinase reactions that are carried out in separate reaction vessels. [0033] As used herein, the terms "contacting," "contact," and all variations thereof, refer to any means that directly or indirectly cause placement together of moieties, such that the moieties come into physical contact with each other. Contacting thus includes physical acts such as placing the moieties together in a container, combining the moieties, or mixing the moieties. [0034] As used herein, the term "fluorescence" refers to light emitted from a fluorophore following absorption of radiation. A given fluorophore can absorb radiation of wavelengths corresponding to its absorption spectrum, and will emit unpolarized light of a different, and usually greater, wavelength. [0035] As used herein, the term "fluorophore" refers to a molecule that absorbs light at a characteristic wavelength and then re-emits the light most typically at a characteristic different wavelength. Fluorophores are well known to those of skill in the art and include, but are not limited to, cyanine dyes such as, for example, cyanine 5, cyanine 5.5, and cyanine 7, rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, texas red, coumarins, chelators within the lanfhanide ion series, phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC), and fluorescent dyes sold under the trademark Alexa Fluor™ dye. [0036] As used herein, the term "fluorescently labeled" refers to a substance to which a fluorophore is permanently or transiently attached. The term "unlabeled" refers to a substance to which a fluorophore is not attached. [0037] As used herein, the term "laser scanning imaging" refers to the detection of emitted light from laser excitation of a fluorophore attached to a substrate. In laser scanning imaging, light emitted from a fluorophore attached to a substrate that is bound to a suspendable solid support is optically discriminated from fluorophores attached to substrates that are free in the surrounding buffer through the optical separation of bound and free fluorescent molecules. [0038] As used herein, the term "hard Lewis metal ion" refers to a metal ion that is a hard Lewis acid. "Hard Lewis acids" are compounds that are capable of accepting an electron pair from a Lewis base, and, generally, are of small size, low polarizability, of high positive oxidation state, and do not have easily excited outer electrons, particularly d electrons. [0039] As used herein, the term "beads" refers to a solid support material in the shape of, for example, pellets, disks, fibers, gels, particles pads, slides, films, matrices, spherical particles, rotational elliptic particles, egg shaped particles. A bead may be, for example, solid, porous, rigid, semi-rigid, deformable, or hard and may be comprised of, for example, polystyrene, polyacrylamide, or a polyethylene glycol polystyrene co-polymer. [0040] One aspect of the present invention relates to methods for assaying the activity of protein kinases and phosphatases that involve reacting the enzymes with substrate, at least a portion of which is fluorescently labeled; allowing phosphorylated substrate to bind to a solid support coated with a phosphate-binding metal; and measuring the fluorescence produced by bound, labeled phosphorylated substrate. Advantageously, the methods of the invention do not require separation steps and also do not utilize antibodies, and thus can be used to assess the activity of tyrosine kinases as well as serine/threonine kinases and phosphatases. In addition, in certain embodiments of the invention, laser scanning imaging is used for detection of bound, labeled substrate and measurement of fluorescence therefore only occurs within the confocal volume. Bulk fluorescence is thus not measured, and interference from fluorescent compounds in solution is minimal. Furthermore, the present methods can be easily miniaturized to microvolumes. Finally, peptide and polypeptide substrates of any length can be utilized in the present methods. [0041] According to a preferred aspect of the invention, confocal microscopy is employed to determine the amount of labeled, phosphorylated substrate bound to a suspendable solid support. [0042] Confocal microscopy confines detection of an illuminated object, or sample, to a thin object plane. A view of a "slice" of the object, or sample, is obtained, which is achieved, for example, by placing a spacial filter, such as a pinhole, in the image plane located between the objective lens and a detector. Only light emitted from a narrow region near the object plane converges through the spacial filter. Light from other planes is blocked by the filter. Images are obtained of the object plane, for example, by scanning, in sequence, the points in the field of view, to obtain the "slice". [0043] Confocal microscopy using laser scanning is particularly preferred for use in certain embodiments of the invention. A suitable laser scanning microscope is sold as "LMAGN/2000" by Biometric Imaging Inc. (Mountain View, Calif.). Laser scanning microscopes are also described in U.S. Pat. Nos. 5,032,720; 5,120,953; 5,162,9465; 5,260,578; 5,283,684; 5,304,810; 5,351,152; 5,547,849; and 556,764, incorporated herein by reference in their entireties. Laser scanning imaging (LSI) offers an advantage over other separation-based techniques as bound and free ligand are separated optically using the confocal microscope. The confocal nature of the detection renders LSI relatively insensitive to fluorescent compound interference because the bulk fluorescence of the sample is optically separated from the particle- bound fluorescence. Fluorescence from the bulk sample is thus not detected. [0044] According to certain aspects of the invention, data obtained by confocal microscopy are analyzed to determine the difference between signal associated with the suspendable solid support and background signal, to obtain a measure of the activity of a protein kinase or phosphatase. The methods of the invention are particularly advantageous for increasing the level of signal to background noise. By eliminating signal from labeled substrate contained in solution outside of the "slice" containing the measured solid support, the background noise is significantly reduced. [0045] The methods of the invention are especially effective for measuring fluorescence for samples containing suspendable solid supports that have been settled, for example, by gravity or by centrifugation. Confocal microscopy allows accurate measurement of a "slice" or "section" of liquid in a container. Thus, measurement can be taken of, for example, the bottom 10% of the sample where fluorescence bound to the suspendable solid support is concentrated. This is not possible in prior art assays using conventional optical detection since such assays do not eliminate signal from the volume above the solid particles. Elimination of this signal accounts, in part, for the very high signal to noise ratio achieved by the methods of the invention. [0046] Preferably, the suspended solid support is allowed to settle for about 10 minutes or more, or is centrifuged, so that more than about 75% of the solid support is contained in less than about 25% of the volume of the assay container, that is, a solid support layer forms on the bottom of the container. More preferably, more than 90% of the solid support is allowed to settle in less than about 10% of the volume of the container. In one preferred embodiment, the thickness of the layer of the solid support is about the same as the thickness of the confocal object plane. The time required for settling is a function of column height, and so is higher, for example, for samples in 96 well plates than for samples in 1536 well plates. [0047] The suspendable solid support should be small enough so that it does not block optical access to the rest of the solution upon settling to the bottom of the assay well. The support should be also be large enough, however, so that it does not remain in suspension for an extended period of time after the assay components are combined. The preferred supports are less than about 50 μm in diameter, most preferably less than about 20 μm in diameter. The diameter of the supports is preferably less than, although not significantly less than, the thickness of the confocal object plane. The supports are preferably greater than about 1 μm in diameter, so that the suspension does not require centrifugation to condense the supports to the bottom of the assay container. In certain embodiments of the invention, however, centrifugation is used to condense the suspendable solid support to the bottom of the assay container. [0048] A preferred suspendable solid support is a 6.2 μm bead made of polystyrene and commercially available from Spherotech (Libertyville, 111.). Such beads are avidin coated, typically containing 10⁶ binding sites per bead. Any suitable suspendable solid support, however, can be employed, including cellulose beads, controlled pore-glass beads, silica gels, and other types of polystyrene beads (optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxyl, or halo groups). Additional supports include grafted co-poly beads, poiy-acrylamide beads, latex beads, dimefhylacrylamide beads (optionally cross-linked with N-N^bis-acryloyl ethylene diamine), glass particles coated with hydrophobic polymers, etc., (i.e., having a rigid or semirigid surface). Divinylbenzene-crosslinked, polyethyleneglycol-grafted polystyrene type beads can be used, such as TentaGel S-NH₂ ^® beads (Rapp Polymere, Tubingen, Germany). [0049] If suspendable solid supports of the preferred size of about 1 μm to about 20 μm are used, the preferred density of the supports in the assay mixture will generally be about 100 to about 1000 suspendable solid support particles per microliter in a 1536 well plate and between about 30 to about 300 suspendable solid support particles per microliter in a conventional 96 well plate. The density of the suspendable solid support varies depending upon the size of the supports. [0050] In certain embodiments of the invention, a hard Lewis metal ion is bound to the suspendable solid support. The hard Lewis metal ion can be bound to the suspendable solid support by any desired means, including, for example, both covalent and non-covalent linkages. The hard Lewis metal ion may, for example, be bonded to a biotinylated chelate and then non- covalently linked to a streptavidin coated support. Covalent linkages are also known in the art. [0051] Some aspects of the invention involve assays to determine the activity of protein kinases or phosphatases. In protein kinase assays, a kinase is reacted with at least one substrate, at least some of which is fluorescently labeled, in the presence of adenosine triphosphate for a time sufficient for phosphorylation of the substrate to occur. In protein phosphatase assays, the phosphatase is reacted with a phosphorylated substrate, at least some of which is fluorescently labeled, for a time sufficient for dephosphorylation of the substrate to occur. The reaction mixture is contacted with a suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support. Increased or decreased binding of the fluorescently labeled, phosphorylated substrate to the suspendable solid support is determined by measuring the amount of fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support. For kinase assays, an increase in the signal produced by bound, phosphorylated substrate relative to that of controls is indicative of an active kinase. For phosphatase assays, a decrease in the signal produced by bound, phosphorylated substrate relative to that of controls is indicative of an active phosphatase. [0052] Some embodiments of the invention relate to methods for identifying substances that modulate the activity of protein kinases or phosphatases. In such methods, increased or decreased binding of a fluorescently labeled, phosphorylated substrate to a suspendable solid support coated with a phosphate-binding metal, which is indicative of enzyme activation or inhibition, is determined by measuring the amount of fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support. In this embodiment, substrate, at least some of which is fluorescently labeled, is reacted with a protein kinase or phosphatase in the presence or absence of test compounds to be screened for modulation of kinase or phosphatase activity for a time sufficient to allow phosphorylation or dephosphorylation, respectively, of the substrate to occur. For kinase assays, the reactions are conducted in the presence of adenosine triphosphate. For phosphatase assays, the substrate is phosphorylated. Following the reaction, the reaction mixture is contacted with a suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support. In preferred embodiments of the invention, the reaction mixture is examined by confocal microscopy, and substrate bound to the suspendable solid support appears as a region of increased fluorescence against a background of relatively constant fluorescence produced by unbound labeled substrate. [0053] The difference between the signal associated with the suspendable solid support and background signal is compared with that of control assays conducted in the absence of test compounds, to determine whether any of the test compounds modulate the activity of protein kinases or phosphatases. If a test compound is a kinase activator, the signal associated with the suspendable solid support will be greater than the signal generated by control assays conducted in the absence of test compounds. Conversely, if a test compound is a kinase inhibitor, the signal associated with the suspendable solid support will be less than the signal generated by control assays conducted in the absence of test compounds. Similarly, if a test compound is a phosphatase activator, the signal associated with the suspendable solid support will be less than the signal generated by control assays conducted in the absence of test compounds. Conversely, if a test compound is a phosphatase inhibitor, the signal associated with the suspendable solid support will be greater than the signal generated by control assays conducted in the absence of test compounds. [0054] The methods of the invention can be used to assay the activity of any protein kinase and any protein phosphatase. The methods of the invention can, for example, be used to assay both serine/threonine kinases and tyrosine kinases and any protein phosphatase. [0055] In certain preferred embodiments, the invention relates to methods for assaying the activity of protein kinases or phosphatases and methods for identifying substances that modulate the activity of protein kinases or phosphatases in which the reaction mixture contains both fluorescently labeled and unlabeled substrate, or fluorescently labeled and unlabeled phosphorylated substrate, respectively. Depending upon the particular instrument used to detect fluorescence, if the concentration of labeled substrate in the reactions is too high, bound, phosphorylated substrate cannot be differentiated from free substrate because the fluorescence produced by free substrate will obscure the fluouresence produced by bound, phosphorylated substrate. The particular instrument used to detect fluorescence thus sets an upper limit on the concentration of labeled substrate that can be present in the kinase and phosphatase reactions. For substrates with a high K_m, relatively high substrate concentrations are required for the enzymes to successfully catalyze the reactions, however. In such cases, to ensure that the substrate concentration is at or near the K_m of the substrate, and to also ensure that the concentration of labeled substrate is not greater than the maximum level dictated by the instrument used to detect fluorescence, both labeled and unlabeled substrate can be used in the reactions. In such embodiments, the concentration of the labeled substrate in the reaction mixture is, preferably, about 0.1 nM to about 100 nM, more preferably about 0.5 nM to about 50 nM, and even more preferably about 1 nM to about 10 nM, and the concentration of the unlabeled substrate in the reaction mixture is, preferably, about 0.1 nM to about 10 μM, more preferably about 0.5 nM to about 5 μM, and even more preferably about 1 nM to about 1 μM. In certain other embodiments of the invention, the protein kinase or phosphatase reaction mixture contains only labeled substrate. In such embodiments, the concentration of the labeled substrate in the reaction mixture is, preferably, about 0.1 nM to about 100 nM, more preferably about 0.5 nM to about 50 nM, and even more preferably about 1 nM to about 10 nM. [0056] In certain aspects, the invention relates to methods for assaying the activity of protein kinases and methods for identifying compounds that modulate the activity of protein kinases in which the concentration of adenosine triphosphate in the reaction mixture is from about 100 nM to about 5 μM, more preferably from about 500 nM to about 4 μM, and even more preferably about 2 μM. As is known to those skilled in the art, the preferred concentration of adenosine triphosphate in a kinase reaction depends upon the particular kinase whose activity is being determined. In general, the lower limit of the adenosine triphosphate concentration in a kinase reaction is dictated by the affinity of the particular kinase for adenosine triphosphate. [0057] The invention provides, in certain embodiments, methods for identifying substances that modulate the activity of protein kinases or phosphatases. Test compounds used in such methods can be potential kinase activators or inhibitors or potential phosphatase activators or inhibitors. In certain embodiments of the invention, separate kinase or phosphatase reactions are carried out in the presence of different test compounds in each reaction. Any number of separate reactions can be performed, and in prefened embodiments of the invention at least 10 separate kinase or phosphatase reactions are performed in the presence of different test compounds in each reaction, in more preferred embodiments, at least 384 separate kinase or phosphatase reactions are performed in the presence of different test compounds in each reaction, and in still more prefened embodiments, at least 1536 separate kinase or phosphatase reactions are performed in the presence of different test compounds in each reaction. [0058] Preferably, the methods of assaying the activity of protein kinases or phosphatases and methods for identifying compounds that modulate the activity of protein kinases or phosphatases are performed in microtiter plates having microvolume containers, such as the 1536 well plate described in U.S. patent application Ser. No. 60/037,636 filed Feb. 18,

1997, incorporated herein by reference in its entirety. In particular embodiments of the invention, a confocal scanning microscope sequentially scans the bottom of each well in the microtiter plate. The methods can also be carried out in conventional 96 well microtiter plates, or in any other container or on any surface capable of holding liquid samples and of being scanned by a confocal microscope. Examples include 12-well, 24-well, 384-well, 864-well plates, and microscope-slides. [0059] Any desired optically detectable fluorescent label can be used in the methods of the present invention. Fluorescent labels suitable for use in the invention are well known and include, for example, cyanine dyes such as Cy-5, Cy-5.5, and Cy7 (Amersham Corp.), fluorescein, rhodamine, Texas Red™, phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC), and fluorescent dyes sold under the trademark Alexa Fluor™. It is prefened that the fluorescent label fluoresce at a relatively high wavelength, such as, for example, higher than about 450 nm, to avoid interference from glass and plastic containers. The labels most preferably fluoresce above about 600 nm, and at less than about 800 mn. Labels that excite at about 400 nm can avoid photobleaching caused by near-UV light. [0060] The invention provides, in particular embodiments, methods for assaying the activity of protein kinases or phosphatases and methods for identifying compounds that modulate the activity of protein kinases or phosphatases in which phosphorylated substrate is bound to a suspendable solid support coated with a phosphate-binding metal. The suspendable solid support can be any material that is capable of being suspended in a liquid and to which a substrate can be bound, either directly or through one or more linking groups. Solid supports can be of any form or shape, including, for example, beads or particles. Solid supports can be comprised of many materials, including, for example, polymers, plastics, resins, polysaccharides, silicon or silica based materials, and membranes, hi preferred embodiments of the invention, the solid support is in the shape of spherical beads having a diameter of from about 1 μm to about 20 μm. In more preferred embodiments of the invention the beads have a diameter of from about 5 μm to about 15 μm, and in even more preferred embodiments of the invention the beads have a diameter of about 10 μm. [0061] In certain aspects, the invention provides methods for assaying the activity of protein kinases or phosphatases and methods for identifying compounds that modulate the activity of protein kinases or phosphatases in which a phosphorylated substrate is bound to a suspendable solid support to which a metal ion is bound. The metal ion has an affinity for, and binds, phosphopeptides, including phosphorylated substrates. In preferred embodiments of the invention, the metal ion is a hard Lewis acid such as, for example, Zr , Al^ιπ. Fe^m,Ga^m, La¹¹¹, or

Sc^πι. In certain embodiments, the metal ion is bound to the suspendable solid support through a metal chelate such as, for example, nitriolacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxylmethylethylenediamine) (TED), or glycine hydroxamic acid (GH). Metal ions and metal chelates are familiar to those skilled in the art, and additional metal ions and chelates can be used in the methods of the invention. Any means can be used to attach a metal chelate to the suspendable solid support. Such means are familiar to those skilled in the art. [0062] Figure 1 depicts a complex formed when a phosphorylated peptide is bound to a metal ion chelated to a suspendable solid support. Figure 2 illustrates a method for assaying the activity of a kinase in which a Cy5-labeled substrate is phosphorylated in a kinase reaction and is bound to a metal ion chelated to a suspendable solid support. Direct fluorescence produced by the bound, labeled peptide is measured using laser scanning imaging. [0063] In certain preferred embodiments of the invention, the suspendable solid support is comprised of sepharose or polystyrene. Various means familiar to those skilled in the art can be used to attach metal chelates to sepharose and polystyrene. For example, sepharose and polystyrene can both be epoxy activated. Any means can be used to epoxy-activate sepharose and polystyrene including, for example, treating the sepharose or polystyrene with butane- 1,4- dioldiglycidyl ether. Other means for epoxy activation are familiar to those skilled in the art. Epoxy-activated sepharose or polystyrene can then be treated directly with the metal chelate iminodiacetic acid ( DA) to attach the chelate to the sepharose or polystyrene. To attach the metal chelate nitriolacetic acid (NTA) to epoxy activated sepharose or polystyrene, N-(tert- butoxycarbonyl)-L-serme (N-Boc-L-Ser) can be added to epoxy activated sepharose or polystyrene at pH 10.0 for 24 hours at room temperature. The Boc group can then be removed by treatment with 2 M HC1 for fours hours at room temperature. The free alpha-amino group can then be treated with bromoacetic acid to form L-serine, N,N-bis(carboxymethyl) that contains the NTA chelate. Other means for attaching metal chelates to epoxy activated sepharose and polystyrene are familiar to those skilled in the art. [0064] Alternatively, in other embodiments of the invention, the suspendable solid support is comprised of sepharose or polystyrene that is amine activated. Any means can be used to amine-activate the sepharose or polystyrene. To attach the metal chelate iminodiacetic acid (LDA) to amine activated sepharose or polystyrene, the amine-activated sepharose or polystyrene can be treated with 2 M bromoacetic acid in 2 M NaOH for 48 hrs at room temperature. [0065] In addition, in other embodiments of the invention, avidin or streptavidin can be attached to the suspendable solid support, and a biotinylated metal chelate can be bound to the solid support through interaction of the avidin or streptavidin with the biotin. In preferred embodiments of the invention, the suspendable solid support is comprised of streptavidin-coated polystyrene, and a biotinylated metal chelate is bound to the polystyrene through interaction of the streptavidin with the biotin. In other embodiments of the invention, the suspendable solid support is comprised of sepharose, and streptavidin or avidin is attached to the sepharose. The streptavidin or avidin can be attached to the sepharose by any means, including, for example, through one or more linking moieties. A biotinylated metal chelate is bound to the sepharose through interaction of the streptavidin or avidin with the biotin. [0066] Certain aspects of the invention relate to methods for assaying the activity of protein kinases or phosphatases and methods for identifying compounds that modulate the activity of protein kinases or phosphatases in which the kinase or phosphatase reaction mixture is contacted with a suspendable solid support coated with a phosphate-binding metal by mixing the reaction mixture with a solid support that is suspended in a buffer having a pH from about 1 to about 9, more preferably from about 3 to about 7, and most preferably about 5. In preferred embodiments, the mixture formed by mixing the kinase or phosphatase reaction mixture with the suspendable solid support is allowed to settle for at least 30 minutes, more preferably at least two hours, and most preferably at least three hours, prior to measuring the direct fluorescence produced by labeled substrate bound to the suspendable solid support. Alternatively, the mixture formed by mixing the kinase or phosphatase reaction mixture with the suspendable solid support is settled by centrifugation prior to measuring the direct fluorescence produced by labeled substrate bound to the suspendable solid support.

EXPERIMENTAL EXAMPLES [0067] The following examples are illustrative of certain embodiments of the invention and should not be considered to limit the scope of the invention.

Materials [0068] The metal chloride salts A1C1₃, FeCl₃, ZrC , LuCl₃, ScCl₃ and GaCl₃ (> 99% pure) were purchased from Sigma-Aldrich. Staurosporine, MES, Tris, and NaCl were also from Sigma-Aldrich. Biotin-X-nitriloacetic acid was purchased from Molecular Probes. Streptavidin- coated polystyrene beads (6 - 8 μm in diameter) were purchased from Spherotech (Libertyville, Illinois). The serine/theronine kinase MAPKAPK2 was purchased from Upstate (Waltham, MA). [0069] Peptides containing the following sequences: 1. Cy5-KKLNRTLSVA (substrate peptide) 2. Cy5-KKLNRTLS(PO4)VA (product peptide) 3. KKLNRTLSVA (unlabeled peptide) were synthesized at SynPep (Dublin, CA). All peptides were > 95% pure as judged by HPLC and showed the expected mass spectrum (SynPep analysis).

Example 1: Metal-ion loading of a suspendable solid support [0070] A 500 μL suspension of streptavidin-coated polystyrene beads (40 mg/mL supplied in aqueous 0.02% sodium azide) was placed in an eppendorf tube and centrifuged for 2 minutes at 4000 rpm in a table top micro-centrifuge. The sodium azide solution was decanted and discarded, 500 μL of 0.1 M MES pH 5.5 buffer was added, and the process of centrifugation was repeated. The supernatant was discarded, 500 μL of 0.1 M ZrCl in 0.1 M MES pH 5.0 was added, followed by the addition of 5 μL of 2.8 rαM biotin-X-nitriloacetic acid (prepared in water). Nitriloacetic acid is a tridentate chelate capable of binding a variety of metal ions. Zr coating of the beads was then allowed to proceed for 1 hour at room temperature. The process of centrifugation was repeated and the supernatant was discarded. The beads were then washed three times with 500 μL of 0.1 M MES pH 5.5 by repeating the centrifugation process. Finally, the beads were stored in 500 μL of 0.1 M MES pH 5.5 buffer and placed at 4 °C until ready for use. Some aggregation of Zr™ coated beads was noted during preparation. To prevent this, 0.03% CHAPS was added to the bead stock and the stock tube was briefly sonicated.

Example 2: Detection with the laser scanning imager [0071] Fluorescence was detected using the FMAT 8200 (PE Biosystems, Foster City, CA U.S.A). The instrument possesses a helium-neon red-laser focused through an objective lens with a 100 μm depth of field. The laser raster scans a 1 mm² area to produce the image of fluorescent particles or cells. For detection in 384-well plates (FMAT, PE Biosystems), the photomultiplier was set at 700 mV for detection of Cy5 fluorescence. The analysis parameters used Wand-Goldman processing with a minimum event count cut-offset to ten. Example 3: Discrimination of substrate from product peptide [0072] Al^πι, Fe^m, Lu^πI, Sc^m, Ga^ιπ, and Zr metal ions were testing for binding discrimination between substrate and product peptides. Group III metal ions such as Lu^ra and Sc¹¹¹, as well as the metals Zr and Ga^πι, have been reported to bind to phosphorylated species. See, for example, Posewitz, MC, Tempst, P: Immobilized gallium(ILT) affinity chromatography of phosphopeptides. Anal. ChemΛ999;ll:2S83-2S92; and Liu, JJ, Hartman, DS, Bostwick, J: An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor- stimulated phosphoinositide hydrolysis. Ana. I Biochem. 2003;318:91-9, incorporated herein by reference in their entireties. Using the procedure described in Example 1, the metal ions were each coated onto streptavidin-coated polystyrene beads. Binding of the metal ions to substrate peptide (Cy5-KKLNRTLSVA) and product peptide (Cy5-KKLNRTLS(PO₄)VA) was separately measured after adding 5 nM of the substrate or product peptides to the wells of 384- well plates containing beads coated with the different metal ions. The beads were allowed to settle for 1 hour followed by detection with the laser scanning imager as described in Example 2. [0073] As shown in Figure 3 A, none of the metal-coated beads exhibited any detectable binding to the substrate peptide. However, different responses were obtained to the product peptide. The largest signal was obtained with the Zτ^w coated beads, although both the Ga^m and Lu^πι coated beads exhibited a good signal. The lower performance of the Fe¹¹¹ coated beads may have been due in part to the ability of iron to quench the fluorescence of the Cy5-labeled peptide. An image of the Zr^ coated beads derived from the FMAT in the presence of the substrate or product peptides is shown in Figure 3B.

Example 4: Titration of phosphopeptide [0074] The sensitivity of detecting product peptide (Cy5-KKLNRTLS(PO4)VA) with the FMAT described in Example 2 was tested. Zr¹^ coated polystyrene beads were added to the wells of a 384-well plate using a 1 :300 dilution of the 40 mg/mL bead stock diluted in 0.1 M MES pH 5.5, 50 nvM NaCl, 0.03% CHAPS buffer. The total concentration of Cy5-peptide was held constant at 5 nM but the percentage of substrate and product peptide was varied from 0 % to 100 %. The beads were allowed to settle for 1 hr followed before detection. [0075] Figure 4 shows that a linear response to the product peptide was observed with the signal increasing as the amount of product peptide increased to 100 % of the total peptide in the well. Example 5: Titration of ATP [0076] An ATP titration was performed to test the sensitivity of the assay signal to the phosphate groups present on ATP. Sensitivity to ATP was tested using Zr coated polystyrene beads, which were added to the wells of a 384-well plate using a 1:300 dilution of the 40 mg/mL bead stock diluted in 0.1 M MES pH 5.5, 50 mM NaCl, 0.03% CHAPS buffer. Cy5-ρeρtide was held constant at 2 nM product peptide and 8 nM substrate peptide. The beads were allowed to settle for 1 hour before detection. [0077] As shown in Figure 5, half the signal was lost at approximately 5 μM ATP, with complete loss of signal occurring at 100 μM ATP.

Example 6: MAPKAP-K2 assay [0078] A kinase reaction was performed as follows. 20 μL of 1.25 μM ATP and 6.25 nM Cy5-substrate peptide in kinase buffer (10 mM Tris-Cl pH 7.4, 10 mM MgCl_2> 1 mM DTT ) were added to the wells of a black clear bottom 384-well plate (Falcon, #3962). 5 L of MAPKAPK2 enzyme (6.25 ng/ L, Upstate, Charlottesville, VA, U.S.A) in kinase buffer was added to the wells. The final concentrations of the assay components were 1 M ATP, 5 nM peptide, and 18 nM MAPKAP-K2 kinase (approximately 0.1 Units). The reaction was incubated at room temperature for 1 hour, and 25 L of 20 ng/ L Zr^-coated polystyrene beads were then added to each well. The beads were allowed to settle in the plate for at least 1 hour at room temperature, and the plate was then read on the FMAT as described in Example 2. [0079] Staurosporine dose-response curves were collected by serial dilution of a staurosporine stock prepared in DMSO. The concentration ranged from 100 M down to 10 nM and was chosen based upon the reported IC_5ϋ of approximately 1 M for staurosporine-mediated inhibition of MAPKAP-K2. The final DMSO concentration was 1% in the wells. DMSO concentrations up to 10% had no effect on metal-peptide binding (data not shown).

Example 7: MAPKAP-K2 titration [0080] As shown in Figure 6, MAPKAP-K2 was titrated in a MAPKAP-K2 assay while the ATP concentration was held constant at 1 μM. Maximal signal was achieved at between 10 nM and 20 nM MAPKAP-K2. Example 8: ATP titration in the presence of MAPKAP-K2 [0081] As shown in Figure 7, ATP was titrated in a MAPKAP-K2 assay while the MAPKAP-K2 concentration was held constant. A bell shaped curve was obtained. The MAPKAP-K2 activity first increased with increasing ATP concentration. However, as the ATP concentration increased further, the ATP competed^' with the peptide and the signal decreased.

Example 9: Inhibition of MAPKAP-K2 activity by staurosporine [0082] The inhibitor staurosporine was used to demonstrate that the signal generated in the MAPKAP-K2 assay depends upon MAPKAP-K2 activity. As shown in Figure 8, an IC₅₀ of approximately 1.0 μM was obtained for staurosporine-mediated inhibition of MAPKAP-K2.

Example 10: Assay automation [0083] As shown in Figure 9, a 384-well MAPKAP-K2 assay was automated using a PlateMate® (Cybio, Jena, Germany) equipped with a 384-pipett head. Twenty μL of 2.5 μM ATP and 12.5 nM substrate peptide prepared in enzyme buffer were added to the wells of a black clear bottom 384-well (Falcon, #3962). Five μL of 1.6 ng/μL MAPKAP-K2 enzyme was then added to each well and the phosphorylation reaction was allowed to proceed for 1 hour at room temperature. Finally, 25 μL of 20 ng/μL Zr^-coated polystyrene beads in MES pH 5.5 buffer were added and the beads were allowed to settle for 1 hour before reading on the FMAT as described in Example 2. [0084] The signal to background was 5 -fold. The Z' for this assay was determined to be 0.6, which indicates sufficient quality to perform high-throughput screening (Zhang JH, Chung TDY, Oldenburg, KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 1999;4:137-142).

Claims

What is Claimed:

1. A method for assaying the activity of a protein kinase comprising: preparing a reaction mixture comprising the kinase, at least one substrate, and adenosine triphosphate (ATP), wherein at least some of the substrate is fluorescently labeled; allowing the reaction to proceed for a time sufficient for phosphorylation of the substrate to occur; contacting the kinase reaction mixture with a suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support; and measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support, wherein the fluorescence is an indicator of kinase activity.

2. The method of claim 1 wherein the protein kinase is a serine/threonine or tyrosine protein kinase.

3. The method of claim 1 wherein a hard Lewis metal ion is bound to the suspendable solid support.

4. The method of claim 3 wherein the hard Lewis metal ion is Zr , Al¹¹¹, Fe¹¹¹, Ga^m, La^m, or Sc^m.

5. The method of claim 3 wherein the Hard Lewis metal ion is bound to the suspendable solid support through a metal chelate.

6. The method of claim 5 wherein the metal chelate is nitriolacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxylmethylethylenediamine) (TED), or glycine hydroxamic acid (GH).

7. The method of claim 1 wherein the suspendable solid support is comprised of sepharose.

8. The method of claim 7 wherein streptavidin or avidin is attached to the sepharose, or the sepharose is amine or epoxy activated.

9. The method of claim 1 wherein the suspendable solid support is comprised of polystyrene.

10. The method of claim 9 wherein the polystyrene is streptavidin or avidin coated or amine or epoxy-activated.

11. The method of claim 10 wherein a hard Lewis metal ion is bound to the suspendable solid support through a biotinylated metal chelate.

12. The method of claim 1 wherein all the substrate is fluorescently labeled.

13. The method of claim 1 wherein the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support is measured by laser scanning imaging.

14. A method for assaying the activity of a protein phosphatase comprising: preparing a reaction mixture comprising the phosphatase and at least one phosphorylated substrate, wherein at least some of the substrate is fluorescently labeled; allowing the reaction to proceed for a time sufficient for dephosphorylation of the substrate to occur; contacting the phosphatase reaction mixture with a suspendable solid support coated with a phosphate-binding material for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid support; and measuring the fluorescence produced by labeled, phosphorylated substrate bound to the solid support, wherein the fluorescence is an indicator of phosphatase activity.

15. The method of claim 14 wherein a hard Lewis metal ion is bound to the suspendable solid support.

16. The method of claim 15 wherein the hard Lewis metal ion is Zr™, Al¹¹¹, Fe^m, Ga^m, La¹¹¹, or Sc¹¹¹.

17. The method of claim 15 wherein the Hard Lewis metal ion is bound to the suspendable solid support through a metal chelate.

18. The method of claim 17 wherein the metal chelate is nitriolacetic acid (NTA), iminodiacetic acid (LDA), tris(carboxylmethylethylenediamine) (TED), or glycine hydroxamic acid (GH).

19. The method of claim 14 wherein the suspendable solid support is comprised of sepharose.

20. The method of claim 19 wherein streptavidin or avidin is attached to the sepharose, or the sepharose is amine or epoxy activated.

21. The method of claim 14 wherein the suspendable solid support is comprised of polystyrene.

22. The method of claim 21 wherein the polystyrene is streptavidin or avidin coated or amine or epoxy-activated.

23. The method of claim 22 wherein a hard Lewis metal ion is bound to the suspendable solid support through a biotinylated metal chelate.

24. The method of claim 14 wherein all the substrate is fluorescently labeled.

25. The method of claim 14 wherein the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support is measured by laser scanning imaging.

26. A method for identifying substances that modulate the activity of a protein kinase comprising: preparing a reaction mixture comprising the kinase, at least one substrate, and adenosine triphosphate (ATP), wherein at least some of the substrate is fluorescently labeled; preparing at least one additional reaction mixture comprising the kinase, at least one substrate, adenosine triphosphate (ATP), and a test compound, wherein at least some of the substrate is fluorescently labeled; allowing the reactions to proceed for a time sufficient for phosphorylation of the substrate to occur; contacting each kinase reaction mixture with a separate suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid supports; measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid supports; and comparing the fluorescence measured from the reactions carried out in the presence and absence of the test compound, wherein a difference in fluorescence indicates that the test compound is a modulator of kinase activity.

27. The method of claim 26 wherein at least 384 separate kinase reactions are carried out in the presence of different test compounds in each reaction.

28. The method of claim 26 wherein a hard Lewis metal ion is bound to the suspendable solid support.

29. The method of claim 26 wherein all the substrate is fluorescently labeled.

30. The method of claim 26 wherein the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support is measured by laser scanning imaging.

31. A method for identifying substances that modulate the activity of a protein phosphatase comprising: preparing a reaction mixture comprising the phosphatase and at least one phosphorylated substrate, wherein at least some of the substrate is fluorescently labeled; preparing at least one additional reaction mixture comprising the phosphatase, at least one phosphorylated substrate, and a test compound, wherein at least some of the substrate is fluorescently labeled; allowing the reactions to proceed for a time sufficient for dephosphorylation of the substrate to occur; contacting each phosphatase reaction mixture with a separate suspendable solid support coated with a phosphate-binding metal for a time sufficient to allow phosphorylated substrate to bind to the suspendable solid supports; measuring the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid supports; and comparing the fluorescence measured from the reactions carried out in the presence and absence of the test compound, wherein a difference in fluorescence indicates that the test compound is a modulator of phosphatase activity.

32. The method of claim 31 wherein at least 384 separate phosphatase reactions are carried out in the presence of different test compounds in each reaction.

33. The method of claim 31 wherein a hard Lewis metal ion is bound to the suspendable solid support.

34. The method of claim 31 wherein all the substrate is fluorescently labeled.

35. The method of claim 31 wherein the fluorescence produced by labeled, phosphorylated substrate bound to the suspendable solid support is measured by laser scanning imaging.