MXPA00004638A - High throughput method for functionally classifying proteins identified using a genomics approach - Google Patents

Abstract

The present invention provides a method for functionally classifying a protein that is capable of unfolding due to a thermal change. The method comprises screening one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the protein, wherein a shift in the thermal unfolding curve indicates that the molecule binds to the protein or affects the stability in a measurable way;generating an activity spectrum for the protein wherein the activity spectrum reflects a set of molecules, from the multiplicity of molecules, that shift the thermal unfolding curve, of the protein and therefore are ligands that bind to the protein, comparing the activity spectrum for the protein to one or more functional reference spectrum lists;and classifying the protein according to the set of molecules in the multiplicity of different molecules that shift the thermal unfolding curve of the protein.

Description

High Processing Method to Classify Functionally Proteins Identified Using a Genomic Method Background of the Invention Field of the Invention The present invention relates generally to a method for classifying a protein based on the ability of one or more ligands to modify the stability, and particularly the thermal stability, of the protein, such as the modification of stability denotes an interaction between the ligand and the protein.

Related Art The pairs based on the ~ 3 x 109 nucleotides contained within the human genome code of approximately 60,000 to 100,000 essential proteins (Albert, et al., In: "Biological Model of the Cell", 3rd Ed., REF, 120111 Albert, B. D. et al., Eds. (1994); Rowen, L. et al. , Sci in ce 278: 605 (1997)). Human Genome Project researchers are rapidly identifying all genes in all 23 pairs of human chromosomes. The products of these genes are widely recognized as the future source of therapeutic targets for drug development in the coming decades. Although the sequence of the human genome will be fully completed within a few years, the elucidation of the function of these genes will be further delayed. Therefore, new technologies are required to understand the functional organization of the human genome and make the transition from "structural g_enomas", or sequence information, to "Functional genomes", or function of the gene, and the association with normal and pathological phenotypes (Hieter &Boguski, Sci in ce 278: 601 (1997)).

The difficulty of this comparison has been clearly illustrated by the recent discovery that the 4,288 genes in the elementary genome of E. col i, the function of approximately 40% of the proteins encoded by these genes are completely unknown (Blattner et al., Sci en ce 277: 1453 (1997)). In truth, of the 12 simple organisms from which the complete genomic information is available, with S. wax vi if ae is the largest with 12.1 megabases (6034 genes), only 44% to 69% of the genes have been identified using current computational sequence comparisons of the state of the art (Pennisi, E., Sci en ce 277: 1433 (1997)). However, the spirochete that causes syphilis has 1,014 genes, 45% of which have unknown function (Fraser et al., Sci in ce 281: 375-388 (1998)). As a result, there is a gap in functional information that represents a challenge for traditional methodologies, and at the same time an opportunity for the discovery of new targets for therapeutic intervention.

However, the classification of proteins with unknown function based on the homology of nucleotides or amino acids with proteins of known function is inaccurate and unreliable. Proteins that have structural homology may have dissimilar functions.

For example, lysozyme and a-lactalbumin have 40% homology in the sequence, but divergent functions. Lysozyme is a hydrolase and a-lactalbumin is a calcium-binding protein involved in the synthesis of lactose in the milk secretion of mammals that produce it (Qasba and Kumar, Cri t Rev. Bi och em. Mol. Ol. 32: 255-306 (1997)).

Some proteins have a similar function, although they do not have a homologous sequence. For example, trypsin serine protease and subtilisin exhibit a similar function, but do not exhibit homologous sequence or structural homology (Tong et al., Na ture S tru ctural Bi olgy 9: 819-826 (1998). cyclic-dependent AMP kinase from the bent quinaza family, and D-Ala: D-Ala ligase, from the family of "Grasp ATP" bent, do not have homologous sequences, although they share structural elements in common for the recognition of ATP and both are dependent enzymes-ATP (Denessiouk et al., Pro tei n Sci en ce 7: 1768-1771 (1998)). Some proteins that do not exhibit homologous sequences, exhibit some structural homology, even having dissimilar functions. Examples of these proteins are the bleomycin resistant protein, biphenyl 1,2-dioxygenase, and human glyoxalase (Bergdoll et al., Pro tei n Sci en ce 7: 1661-1670 (1998)).

Therefore there is a need for an accurate, reliable technology that facilitates the rapid, high-processing sorting of proteins of unknown function.

Brief Description of the Invention The present invention provides methods for functionally classifying a protein. The methods are related to the capacity of the molecules in the multiplicity of different molecules to modify the stability of a protein, and by means of this, to bind to a protein. Three of the methods do not involve a determination of whether the molecules that bind with proteins change the curve of the protein's thermal split. Three alternatives and different methods involve determining if the molecules that bind with a protein change the thermal splitting curve of the protein.

A. Methods that do not involve determining whether the binding molecules change the thermal splitting curve of the protein The present invention provides a method for functionally classifying a protein, the method comprising the separation of one or more of a multiplicity of different molecules by their ability to modify the stability of the protein, wherein the modification of the stability of the protein indicates that the molecule binds with the protein; generating a spectrum of activity for the sweeping protein, where the spectrum of activity reflects a subset of molecules, of the multiplicity of different molecules, that modify the stability of the protein and through this the ligands bind to the protein; comparing the spectrum of activity for the proteins with one or more lists of reference functional spectra; and classify the protein according to a group of molecules in the multiplicity of different molecules that modifies the stability of the protein.

The present invention also provides a method for functionally classifying a protein, the method of comparing the projection of one or more multiplicity of different molecules by its ability to modify the stability of the protein, wherein the modification of the stability of the protein indicates that the molecule binds to the protein; generating a spectrum of activity of the protein with -the projection, where the spectrum of activity reflects a subset of molecules, the multiplicity of different molecules, which modify the stability of the protein and therefore the ligand binds to the protein; comparing the spectrum of activity of the protein of one or more lists of reference functional spectra; and classify the protein according to the group of molecules in the multiplicity of different molecules that modify the stability of the protein.

The present invention also provides a method for functionally classifying a protein, the method comprising screening one or more multiplicities of different known molecules to bind to a particular class of proteins for their ability to modify the stability of the protein, wherein the modification of. the stability of the protein indicates that the molecule binds to a protein; generating a spectrum of activity for the projection protein, where the spectrum of activity reflects a subset of molecules, the multiplicity of different molecules, which modify the stability of the protein and through this the ligands bind to the protein; and classifying the protein according to a member of the class of proteins if one or more of the multiplicity of different molecules modify the stability of the protein.

The present invention also provides a method for functionally classifying a protein, the method comprising classifying the protein according to the group of molecules in a multiplicity of different molecules that modify the stability of the protein.

B. Alternate and different methods that involve determining if the molecules that are bound change the thermal splitting curve of the protein The present invention provides a method for functionally classifying a protein that is capable of cleavage due to the thermal change, the method comprises the projection of one or more of a multiplicity of different molecules for its ability to change the thermal splitting curve of the protein , where a change in the thermal cleavage curve of the protein indicates that the molecule binds to the protein; generating a spectrum of activity for the projection protein, where the spectrum of activity reflects a subset of molecules, the multiplicity of different molecules, which change the thermal splitting curve of the protein and through this the ligands join to the protein; comparing the spectrum of activity for the proteins with one or more lists of reference functional spectra; and classifying the protein according to a group of molecules in the multiplicity of different molecules that changes the thermal splitting curve of the protein.

The present invention provides a method for functionally classifying a protein that is capable of unfolding due to thermal change, the method comprising screening one or more of a multiplicity of different known molecules to bind with a particular class of proteins for their abilities to change the curve of the thermal splitting of the protein, where a change in the thermal splitting curve of the protein indicates that the molecule binds to the protein; generating a spectrum of activity for the projection protein, where the spectrum of activity reflects a subset of molecules, the multiplicity of different molecules, which change the thermal splitting curve of the protein and through this the ligands join to the protein; and classify the protein as a member of the protein class if one or more of the multiplicity of different molecules change the thermal splitting curve of the protein.

The present invention also provides a method for functionally classifying a protein capable of unfolding due to the thermal change, the method comprises classifying the protein according to the group of molecules in a multiplicity of different molecules that change the curve of thermal splitting of the protein.

There are several advantages of the methods of the present invention in the drug discovery process, especially with respect to functional genomes. For example, the methods of the present invention provide the utility of extended cross-white because it is based on the thermodynamic properties common to all complex ligands / receptors. In addition, the methods of the present invention facilitate the direct evaluation of proteins that are white derived from genomic studies because knowledge of the function of the specific target is not necessary.

Another advantage provided by the methods of the present invention is that it can be universally applied to any receptor that is a drug that is white. It is not necessary to invent a new assay every time a new receiver becomes available for testing. Therefore, the projection of patterns that immediately overlap the preparation of the protein that is white. When the receptor under study is an enzyme, researchers can determine the order of the affinity range of a series of compounds more quickly and easily than those that can use conventional kinematic methods. In addition, researchers can detect ligand junctions with an enzyme, regardless of whether the binding occurs at the active site, at an allosteric cofactor binding site, or at an interface of the receptor subunit. The present invention also applies to receptors without enzymes.

Yet another advantage provided by the methods of the present invention is that the methods can be practiced using miniaturized assay volumes (e.g., 1-5 μL), which facilitate the use of 16 x high density microplate assay arrays. 24 (384 wells), 32 x 48 (1536 wells), or in addition, made-to-order arrangements. Only about 5 to 40 picomoles of protein are required (0.1 μg for a 25 kDa protein) per assay well, for a final protein concentration of approximately 1. to 4 μM. "So, 1.0 mg of the protein can be used to drive 103 to 104 trials in the miniaturized format.

Yet another advantage provided by the present invention is that the methods of the present invention facilitate the ultra high processing projection of the compound patterns (e.g., functional test patterns). Therefore, the methods of the present invention make possible a projection of 10,000 to 30,000 compounds per day per work station. With a speed, at least 2.5 to 6 target proteins can be screened per day, per workstation, against a functional test library of 4000 compounds. At least 500 a 1200 therapeutic targets can be screened per day, per workstation, against a functional probe library of 4000 compounds. In five years, one could sample 3 to 7.5% of the proteins encoded by the human genome per workstation.

Still another advantage provided by the methods of the present invention is that the wide dynamic range of the binding affinities that can be realized in the simple well test is extended by twenty orders of magnitude (for example, of femtomolar affinities (10 ~ 15 M) to millimolar (10 ~ 3 M)).

Yet another advantage provided by the methods of the present invention is that the milti-ligand binding interactions can be monitored by the close addition of free energy of the ligand bond of individual ligands.

In addition, the methods of the present invention provide information that is more accurate and reliable than the information provided by conventional methodologies of sequence homology, such as those reported in Tatusov, R. L. et al., Sci in ce 278: 631-637 (1997); and Heiter, P. and M. Boguski, Sci in ce 278: 601-602 (1997).

In addition, different classes of enzymes can be identified and differentiated based on the binding of different transition groups of analogous states. For example, benzeneboric acid (BBA) derivatives have found the reversible linkage with the various serine proteases such as subtilisins, from bacterial sources, and the a-quimot ripsin, from eukaryotic sources (Nakatani, H., et al. , J. Bi och em.

(Tokyo) 77: 905-8 (1975)). Similarly, analogs of the transition state of boroarginine, which have an arginine group in the Pl position for this mimic synthetic peptide, were found to be more specific inhibitors for the proteases of serine, thrombin, trypsin and plasmin (Tapparelli, et al. , J. Biol. Chem. 268: 4734-41 (1993)) with the observed specificity: Kd ~ 10 nM (thrombin), Kd -1,000 nm (trypsin), Kd ~ 10,000 nM (plasmin). This illustrates an important advantage that the methods of the present invention provide, with respect to the comparison of the sequences, a sounding to classify the proteins: the change of expected Tm of the binding of a transition analog state of the boronic acid must be much more characteristic of a serine protease (in spite of the bacterial or eukaryotic source) than the information provided by the single comparison of the sequence. Serine proteases from bacterial or eukaryotic sources are examples of the textbook of convergent evolution, and therefore have little homology in the sequence, despite the fact that they share the catalytic function.

Other features and advantages of the present invention are described in detail below with reference to the accompanying drawings.

Brief Description of the Drawings FIGURE 1A shows a flow diagram illustrating a method of the present invention. FIGURE IB shows another flow chart illustrating a method of the present invention.

FIGURE 2 is a schematic diagram illustrating an elevated view of a test apparatus that can be used to practice the microplate heat exchange test.

FIGURE 3 shows the results of the microplate heat exchange assays of single ligand binding interactions with three different classes of binding sites for human a-thrombin FIGURE 4 shows the results of microplate heat exchange assays of the multi-ligand binding interactions for human a-thrombin.

FIGURE 5 shows the cnt compounds in plate 1 of the probe functional library.

FIGURE 6 shows the spectrum of activity for Factor Xa that is generated using the compounds in plate 1 of the probe functional library.

FIGURE 7 shows the spectrum of fibroblast growth factor receptor 1 (FGFR1) that is generated using the compounds in plate 1 of the probe functional library.

FIGURE 8 shows the result of a microplate heat exchange assay of the recombinant dimeric repressor l a c which binds a synthetic palindromic 21-mer operator sequence.

FIGURE 9 shows the result of a myosin microplate heat exchange test of the bovine muscle with adenosine triphosphate (ATP).

FIGURE 10 shows the result of a microplate heat exchange assay of the 3 ', 5' -dependent kinase protein-cAMP from bovine heart that binds to adenosine triphosphate-β-sulfate (ATP-γ-S).

FIGURE 11 shows the result of a microplate thermal change * test of bovine dihydrofolate reductase (DHFR) that binds to methotrexate.

FIGURE 12 shows the result of a microplate heat exchange test of bovine dihydrofolate reductase (DHFR) that binds to NADPH.

Detailed description of the invention In the following description, reference will be made to various terms and methodologies known to persons with experience in the arts of biochemistry and pharmacology. The publications and other materials that expose these known terms and methodologies are hereby incorporated as a reference in their entirety as they are thought to be set out in their entirety.

The present invention provides methods for functionally classifying a protein, which is capable of unfolding, according to the group of molecules in a multiplicity of different molecules that modify the stability of the protein. A protein can cause splitting by treatment with a denaturing agent (such as urea, guanidinium hydrochloride, guanidinium thiosuccinate, etc.), a detergent, to treat the protein with pressure, by heating the protein, etc.

The present invention provides methods for functionally classifying a protein that involves determining whether the thermal cleavage curve of the protein changes. Only molecules that change the thermal splitting curve are considered to be ligands that bind to the protein. Preferably, the microplate heat exchange test is used to determine if the curve of the thermal cleavage of the protein changes. The microplate heat exchange test involves determining whether the molecules tested for the joint change the thermal split curve. The heat transfer test in micro-flake is described in International Patent Application No. PCT / US 97/08154 (published on November 13, 1997 under No. WO97 / 42500); U.S. Patent Application No. 08 / 853,464, filed May 9, 1997; and U.S. Patent Application No. 08 / 853,459, filed May 9, 1997.

In a preferred embodiment, the present invention provides a method for classifying a target protein that is capable of unfolding due to a thermal change. In this embodiment, the target protein is contacted with a molecule of a multiplicity of different molecules in each of a multiplicity of vessels. The containers are then heated, at intervals, in a range of temperatures. Preferably, the multiplicity of containers is heated simultaneously. After each heating interval, a physical change associated with the thermal splitting of the target molecule is measured. In an alternate embodiment of this method, the containers are heated in a continuous manner. A graph of the curve of the thermal split is made as a function of the temperature of the target molecule in each of the vessels. Preferably, the midpoint of the temperature Tm, of each thermal splitting curve is identified and then compared with the Tm of the thermal splitting curve obtained by the target molecule in the absence of any of the molecules in the vessels. Alternatively, a total thermal splitting curve can be compared to other total thermal split curves using computational analytical tools.

The methods of the present invention which involve determining whether the molecules change the thermal cleavage curve of a protein are different from the methods that do not involve determining whether the molecules change the thermal splitting curve of a protein, such as the susceptibility to proteolysis, surface binding by the protein, the binding of the antibody by the protein, the protein molecular bond of the protein, the differential bond to immobilize the ligand, and the aggregation of the protein. These trials are well known to a person without experience in the art. For example, see U.S. Patent No. 5,585,277; and U.S. Patent No. 5,679,582. These soundings exposed in the. US Patents Nos. 5,585,277 and 5,679,582. They involve the comparison of the degree of folding and / or unfolding of the protein in the presence and absence of a molecule that is tested for its binding. These probes do not involve a determination of whether any of the molecules that bind to the protein change the thermal cleavage curve of the protein.

The term "functionally classifying proteins" refers to - classifying a protein according to a biological, biochemical, physical or chemical function, such as the ability to hydrolyze a phosphate radical (a phosphatase), to add a phosphate radical (a kinase), etc. The proteins can be classified as having one or more of the different functions, and the methods of the present invention are not limited to classifying proteins such as phosphatases, kinases, or other types of enzymes.

The terms "multiplicity of molecules", "multiplicity of compounds", or "multiplicity of containers" refer to at least two molecules, compounds, or containers.

The term "subgroup of molecules" in a multiplicity of different molecules refers to a group of molecules smaller than the multiplicity of different molecules. The term "mult i-variable" refers to more than one experimental variable.

The term "projection" refers to the test of a multiplicity of molecules or compounds by their ability to bind a white molecule that is capable of unfolding when heated. The projection process is a repetitive or iterative process, where the molecules are tested to bind a protein in a splitting test, and particularly in a heat exchange test. For example, if a subgroup of molecules within a functional probe library that projects its binding to a protein does not bind, when the projection is repeated with another subset of molecules. If the entire library fails to contain any molecule that binds to the protein, then the projection is repeated using molecules with another functional probe library.

As used herein, a "functional test projection" is an evaluation (eg, an assay) of its ability of a multiplicity of different molecules with a functional probe library to bind a target protein and modify the stability of the target protein. .

As used herein, a "functional probe library" refers to one or more different molecules that are tested for their ability to bind to a target protein and modify the stability, and particularly the thermal stability, of the protein in response to cleavage (for example, thermal splitting). In developing a stability test, and preferably the use of a microplate heat exchange assay technology, in the protein in the presence of each member of the functional probe library, the compounds can be incubated with the target protein individually and / or in groups to determine which ligands individually or in combination bind closely and specifically to the target protein.

A functional probe library can be any type of molecule library, including a protein library, a protein subunit library, a peptide library, a vitamin &; co-factors, an enzyme inhibitor library, a nucleic acid library, a carbohydrate library, a generic drug library, a library of natural products, or a combination of patterns. For the molecules in the functional probe library that binds to the target protein, the biological effect can be evaluated in vi vi and trials.

If the functional probe library is a combinatorial library, then a combinatorial library created using the DirectedDiversity® system is preferably created.

The DirectedDiversity® system is disclosed in U.S. Patent 5,463,564.

As used herein, the term "activity spectrum" refers to the list of compounds (e.g., ligands) that bind to the protein and modify the stability (e.g., thermal stability) of the protein, and the respective affinities of the ligands with the target protein. The terms "functional test link profile" and "activity spectrum" are synonyms. A decrease in Tm suggests that the compound or molecule blocks the binding of another molecule that would stabilize the protein. For example, if a substance that forms metallic chelates decreases the Tm, this suggests that the protein binds with a metal (for example, an interaction between calcium and a-lactalbumin). If a reducing agent decreases the Tm, this suggests that the protein contains one or more disulfide bonds.

As used herein, the "functional reference spectrum list" refers to a list of target protein classes (including references to appropriate electronic databases), the associated ligands, and the corresponding constant links, which can be used to functionally classify a white protein. Alternatively, the list of the functional reference spectrum may be a group of one or more activity spectra for one or more known proteins. Thus, the activity spectrum for a given protein can serve as a "fingerprint" for this protein and for the functional class of proteins to which the protein belongs.

A "functional reference list" is a list of proteins that share one or more characteristics in common, such as a particular ligand, or exhibit a common activity.

As used herein, an "activity spectrum comparison agent" is a means of computation or a graph where one can compare the spectrum of activity, derived from observing the effects of the functional probe library on the target protein, with the list of the functional reference spectrum. For example, the agent comparing the spectrum of activity can be a programming device with a worksheet that is readily available to those without experience in the art. For example, Microsoft Excel (Microsoft Inc. Redmond, WA) can be used.

In several cases, a function of a gene can tentatively be assigned by means of homology for sequences of known functions (a "functional hypothesis" derived from sequence homology). The heat exchange test can be used to validate a functional hypothesis, or to identify the correct function of a list of possible functions involved by sequence homology. For example, there are proteins that hydrolyze ATP and convert hydrolysis energy into mechanical energy, known as "molecular motors". These proteins include DNA and RNA helicases, kinesins, caperonins to redouble proteins, and protein complexes at the base of bacterial flagella. These proteins all share the sequence homology in the hydrolysis-ATP domain, while their other functions are different. In an application of the methods of the present invention, the known sequence homology of a portion of a target protein (e.g., an ATPase domain) can be used to designate heat exchange assays using special functional test standards focused on different possible functions of the white protein (for example, the 'patterns containing molecules to test the special activities of caperonins, helicases, kinesins, and other molecular motors). Alternatively, a white jprotein can be identified by means of sequence homology as a tyrosine kinase, and the present invention could then be used to screen this target against a library of peptides containing different possible phosphorylation sites of the substrate. These examples illustrate that the present invention is highly complementary to the process of assigning function using sequence homology, because the present invention can be used to confirm, reject, or elaborate the hypothetical functions indicated by sequence homology.

Accordingly, the present invention also provides a method for functionally classifying a protein, the method comprising (a) screening one or more of a multiplicity of different known molecules to link a particular class of proteins for their ability to modify the stability of the protein , where the modification of the stability of the protein indicates that the molecule binds with the protein, (b) generate a spectrum of activity of the protein of the projection step, an where the spectrum of activity reflects a subset of molecules, the multiplicity of different molecules, which modifies the stability of the protein; and (c) classify the protein as a member of the protein class if one or more of the multiplicity of different molecules modify the stability of the protein.

It should be noted that the above process to elaborate or specify the function of the protein using a heat exchange assay can also be applied to the hypothesis generated using other methods of protein function assignment (eg, three-dimensional structures of the proteins). proteins and nucleic acids, cellular expression patterns of mRNA or a protein encoded by a target gene, and the phenotypic effects of altering a target gene to change its function at the level of the organism).

In addition, using the methods of the present invention, one can evaluate the binding of more than one ligand or more than one site in a protein, and classify the protein according to the subgroup that binds to the protein. For example, a protein of unknown function that is found to bind to DNA and Adenosinetriphosphate (ATP) can be classified as a protein that affects the structure of DNA. Thus, using the information concerning the linkage of the multiple ligands, the large number of the possible protein classification can be reduced to only a few similar classifications.

However, using the methods of the present invention, one can also screen a protein of known function from an additional function, previously unknown.

Preferably, the microplate heat exchange assay is used to screen the functional probe library of the molecules against the proteins.

The term "function" refers to the biological function of a protein, peptide or polypeptide. For example, a kinase is a protein wherein the function is to catalyze the covalent addition of one phosphate group to another protein.

The term "molecule" refers to the compound that is tested for binding affinity to the target molecule. This term encompasses the chemical compounds of any structure, including, but not limited to, nucleic acids, such as DNA and RNA, and peptides. More specifically, the term "molecule" encompasses the compounds in a compound or a combinatorial library. The terms "molecule" and "ligand" are synonymous.

The term "contacting a target protein" refers broadly to placing the target protein in solution with the molecule so that the link is projected. Less broadly, contacting refers to the change, turning, shaking or vibrating of a solution of the target protein and the molecule to screen the link. More specifically, contacting refers to the mixture of the target protein with the molecule to be tested for the link. Mixing can be achieved, for example, by repeated addition and discharge through the tip of a pipette. Preferably, contacting refers to the binding equilibrium between the target protein and the molecule that the link was tested. The contact may occur within a container or before the projected target protein and molecule are placed.

The term "container" refers to any container or chamber in which the receptor and the molecule to be tested the link can be placed. The term "container" covers the reaction tubes (for example, test tubes, microtubes, flasks, etc.). Preferably, the term "container" refers to a well in a microplate with multi-wells or microplate with microtitre.

The term "sample" refers to the contents of a container.

The term "spectral emission", "thermal change" and "physical change" encompass the release of energy in the form of light or heat, the absorption of energy in the form of light or heat, _ changes in turbidity and changes in properties polar of light. Specifically, the terms refer to fluorescent emission, fluorescent energy transfer, ultraviolet or visible light absorption, changes in the polarization properties of light, changes in the polarization properties of the fluorescent emission, changes in the rate of fluorescence change over time (eg, fluorescence lifetime), changes in fluorescence anisotropy, changes in fluorescence resonance energy transfer, changes in turbidity, and changes in the enzymatic activity. Preferably, the terms refer to fluorescence, and more preferably to fluorescence emission. The fluorescence emission may be intrinsic to a protein or it may be due to the fluorescence reporter molecule. The use of fluorescence techniques to monitor the unfolding of the protein is well known to people without experience in the art. For example, see Efink, M. R. Bi ophysi ca l J. 66: 482-501 (1994).

The term "unfolding" refers to the release of the structure, such as the crystalline arrangement of amino acid side chains, secondary, tertiary, or quaternary protein structures.

The term "double", "redouble" and "renaturalize" refers to the acquisition of the correct ordering of amino acid side chains, secondary, tertiary, or quaternary protein structures of a protein, which provides all the chemical and biological function of the biomolecule.

The term "denatured protein" refers to a protein that has been treated to remove the native ordering of side chains of amino acids, secondary, tertiary, or quaternary protein structures. The term "native protein" refers to a protein that processes the degree of ordering of side chains of amino acids, into structures of secondary, tertiary, or quaternary proteins that provide the protein with all the chemical and biological function. A native protein is a protein that has not been heated and has not been treated with cleaners or chemicals such as urea.

As used herein, the terms protein "and" polypeptide "are synonymous.

A "curve of unfolding" is to make a graph of the physical changes associated with the unfolding of a protein as a function of temperature, concentration of denaturing agent, pressure, etc. A "denaturation curve" is a diagram of the physical change associated with denaturation of a protein or nucleic acid as a function of temperature, concentration of the denaturing agent, pressure, etc.

A "thermal split curve" is a graph of the physical change associated with the unfolding of a protein or nucleic acid as a function of temperature. A "thermal denaturation curve" is a graph of the physical change associated with the denaturation of a protein or nucleic acid as a function of temperature. See, for example, Davison et al., Na ture S tru c t ure Bi olgy 2: 859 (1995); and Clegg, R. M. et al., Proc. Na ti. Aca d. Sci. U. S TO . 90: 2994-2998 (1993).

The term "change in the thermal splitting curve" refers to the change in the thermal splitting curve for a protein that binds to a ligand, relative to the thermal cleavage curve of the protein in the absence of the ligand.

The term "stability modification" refers to the change in the amount of pressure, the amount of heat, the concentration of the detergent, or the concentration of denaturing agent that is required to give a degree of physical change in a target protein that it is bound by one or more ligands. In relation to the amount of pressure, the amount of heat, the concentration of detergent, or the concentration of denaturing agent that is required to cause the same degree of physical change in a white protein in the absence of any ligand. Modification of stability can be exhibited as an increase or decrease in stability. Modification of the stability of a protein by a ligand indicates that the ligand binds with a protein. Modifying the stability of a protein by more than one ligand indicates that the ligands bind with the protein.

The term "thermal stability modification" refers to the change in the amount of thermal energy that is required to cause an established degree of physical change in the target protein that is bound by one or more ligands, relative to the amount of thermal energy which is required to originate the same degree of physical change in the white protein in the absence of any ligand. Modification of thermal stability can be exhibited as an increase or decrease in thermal stability. The modification of the thermal stability of a protein by a ligand indicates that the ligand binds to the protein. Modifying the thermal stability of a protein by more than one ligand indicates that the ligands bind to the protein.

The "average temperature Tm" is the midpoint of the temperature of a thermal split curve. The Tm can be easily determined using methods well known to persons skilled in the art. See, for example, Weber, P. C. et al., J. Am. Ch em. Soc. 116: 2717-2724 (1994); and Clegg, R. M. et al., Proc. Na ti. Aca d. Sci. . U. S TO . 90: 2994-2998 (1993).

As discussed above, it is preferred to determine the effect of one or more molecules on the thermal stability of a target protein according to the change in the Tm of the thermal cleavage curve of the protein. Alternatively, the effect of one or more molecules on the thermal stability of a target protein can be determined according to the change in the whole thermal splitting curve for the target protein.

The term "fluorescence test molecule" refers to an extrinsic fluorophore which is a molecule or a compound that is capable of associating with a unfolded or denatured receptor and then excites it with light of a defined wavelength, emitting fluorescent energy. The term fluorescence test molecule encompasses all fluorophores. More specifically, for proteins, the term encompasses fluorophores such as thioinosine, and N-ethenoadenosine, formicin, dansyl, dansyl derivatives, fluorescent derivatives, 6-propionyl-2- (dimethylamino) -naphthalene (PRODAN), 2-anilinonaphthalene, and N-arylamino-naphthalene sulfonate derivatives such as 1-anilinonaphthalene-8-sulfonate (1,8-ANS), 2-ani-lonaonaphthalene-6-sulfonate (2,6-ANS), 2-aminonaphthalene-8-sulfonate , N, -dimet il-2-aminonaphthalen-6-sulfonate, N-phenyl-2-aminonaphtal-ene, N-cyclohexyl-2-aminonaphthalene-6-sulfonate, N-phenyl-2-amino-naphthalene-6-sulfonate , N-phenyl-N-methyl-2-aminonaphthalene-6-sulphonate, N- (o-toluyl) -2-amino-naphthalene-6-sulfonate, N- (m-toluyl) AA-amino-naphthalene-6-sulfonate , N- (p-toluyl) -2-aminonaphthalene-6-sulfonate, 2- (p-toluidinyl) -naphthalene-6-sulfonic acid (2,6-TNS), 4- (dicyanovinyl) j ulolidine (DCVJ), 6-dodecanoi 1-2-dimethylaminonaphthalene (LAURDAN), 6-hexadecanoyl-2- (((2- (trimethylammonium) et yl) methyl) -amino) naphthalene chloride (PATMAN), red Nile, N-phenyl-1-naphthylamine, 1, l-dicyano-2- [6- (dimethylamino) naphthalen-2-yl] -propene (DDNP), 4,4'-dianyl-l, l-binaphthyl acid -5,5-disulfonic (bis-ANS), and dye derivatives of 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole, sold under the trademark of DAPOXYL ™ (Molecular Probes, Inc. , Eugene, OR), including the dyes of 5- (4"-dimet-ilaminophenyl) -2- (4'-phenyl) oxazole, provided by Di u, Z. et al., Photoch emi s try and Photobi ol ogy 66 (4): 424-431 (1997), and in Bi ol. Probes 25: pp. 8-9, Molecular Probes, Inc., Eugene, OR (1997).

Examples of dye derivatives of 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole, and the corresponding • catalog number of Molecular Tests, including 5- (4" -dimethylaminophenyl) -2- ( 4'-phenyl) oxazolbutyl-sulfonamide (D-12801), 5- ("-dimet-ylaminophenyl) -2- (4'-phenyl) oxazole- (2-aminoethyl) sulfonamide (D-10460), 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazolbut-1-sulfonamide (D-12801), 5- (4" -dimethanedylaminophenyl) -2- (4'-phenyl) oxazole-3-sulphonidophenylboronic acid ( D-10402), 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) -oxazolesulfonic acid (D-10430), 5- (4" -dimethylaminophenyl) -2- (4'-phenyl) oxazole- (2-bromoacetamidoethyl) sulfonamide (D-10300), 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole -2- (3- (2-pyridyldithio) propionamidoethyl) sulfonamide (D-10301), 5- ( 4"-dimet-ilaminophenyl) -2- (4'-phenyl) -oxazolesulfonyl (D-10160), 5- (4" -dimethyl-ylaminophenyl) -2- (4'-phenyl) -oxazole-3-sulfonamidopropionic acid, succinimidyl ester (D-10162), 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazolecarboxylic acid, succinimidyl ester (D-10161).

Preferably the term "fluorescence test molecule" refers to dyeing derivatives of 1,8-ANS or 2,6-TNS and 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole, sold under the registered trademark of DAPOXYL ™, such as those provided by Di u, Z. et al., Pho tochemi s try and Ph o tobi olgy 66 (4): 424 -431 (1997). Even more preferably, the term refers to 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole dye derivatives, sold under the trademark of DAPOXYL ™, such as those provided by Diwu, Z. et al., Pho tochemi s try and Pho tobi olgy 66 (4): 424-431 (1997). Even more preferably, the term refers to sodium salts of 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazolesulfonic acid (D-12800).

The term "conveyor" encompasses a platform of another object, in any form, which is capable of supporting at least two containers by itself. The conveyor can be made of any material, including, but not limited to glass, plastic, or metal. Preferably, the conveyor is a millipore microplate. The terms microplate microtiter plate are synonymous. The conveyor can be removed from the heating element. In the present invention, a plurality of conveyors are used. Each conveyor holds a plurality of containers.

The terms "spectral measurement" and "spectrophotometric measurement" are synonyms and refer to the measurement of changes in light absorption, turbidity measurements, measurements of visible light absorption and measurement of ultraviolet light absorption are examples of Spectral measurements The measurements of the intrinsic fluorescence of a target protein, and the fluorescence of an extrinsic fluorophore that is complex with or bound to a target protein are also examples of spectral measurements and spectrophotometric measurements.

The term "polarimetric measurements" is related to measurements of changes in the properties of light polarization and fluorescence emissions. Circular dichroism and optical rotation are examples of polarization properties of light that can be measured polarimetrically. Measurements of circular dichroism and optical rotation are taken using a spectropolarimeter. "Non-polarimetric" measurements are those that are not obtained using a spectropolarimeter.

Recognition of the cellular and / or biological function of proteins can be a valuable advantage in drug discovery, where this can be useful in developing a detailed understanding of the therapeutic hypotheses of drug function, when designing specific strategies for the drug. drug design, and to reveal the potential side effects of the drug.

There are tens of thousands of different enzymes and receptors that are targets of potential drugs, and are more constantly discovered through genome sequence studies. These proteins and cellular receptors have specific functions in the biological system, which are defined practically by the molecular ligands with which specific interactions are formed. Typical interactions that have functional significance include interactions of enzymes with molecular ligands similar to analog substrates, cofactors, adapter domains, nucleic acids, etc., and receptor interactions with specific ligands, other receptors, structural surface compounds cellular, nucleic acids, polysaccharides, etc.

Over time it will be possible to isolate, or clone and label proteins that are targets of drugs, in several cases these will have no basis of functional recognition around the protein that can assist in successor stages of the drug discovery process. However, a substantial fraction of known protein molecules fall within the classes of mechanisms that share important characteristics, including their ability to bind specific types of ligands of molecules, including enzyme cofactors, enzyme substrates or analogous substrates, etc. Consequently, it is possible to classify different proteins that from another inpdo are unknown their function by their ability to specifically bind several types of ligands, alone or in combination.

When a protein binds to a biological ligand in a functionally significant medium, there is an effect on the physical state of the protein that is reflected in its relative stability to its unbound state. Consequently, one can functionally classify a protein with previously unknown function by incubating it with a test panel of ligands and cofactors (functional sounding library), and measuring which ligand has effects on the stability of the protein. Alternatively, one can determine a previously unknown function of a protein of previously unknown function by incubating it with a sounding board of biological ligands and cofactors (biological probe library), and measuring which ligands have an effect on the stability of the protein.

As has been established from the thermodynamic studies of the protein-ligand interactions, when two associated molecules form a favorable and specific complex interaction, the binding interactions are associated with a reduction in the total free energy of the complex and a net stabilization of the protein-ligand complex relative to the unlinked protein. In practical terms, this means that when an enzyme or receptor interacts with its cofactors or analogs of specific cofactors, the enzyme or receptor will be stabilized by the interactions. However, it is possible that there may be special situations where the ligand that binds can destabilize the target protein. For example, some proteins contain more than one domain or allosteric sites where one or more ligands can be linked.

SUMMARY OF THE METHODS OF THE PRESENT INVENTION The methods of the present invention, as well as the other information, are shown in Figures IA and IB.

Identification of a Putative White Gen White proteins are proteins that bind to a drug that may have therapeutic potential and whose characterization may be useful in the drug discovery process. Several genes that are potential targets for therapeutic intervention are identified through the phenomenological correlation that is related to a genetic defect with a disease state (for example, when an inherent disease correlates with a genetic defect in a specific enzyme or receptor). ) or through differences in protein expression patterns in diseased tissues vs. normal.

In several cases it is possible to determine some "function" of the product of a gene through the homology of the sequence with a homologous protein with known functional or structural data. However, in a substantial fraction of cases, the homology of the sequence may not be sufficient to establish the functional relationship, and an alternate means is needed to establish the function in a way that can directly facilitate the drug discovery process.

B. Cloning and Expressing the Protein In order to practice the methods of the present invention, it is necessary to obtain the target protein in sufficient quantities for the biological assay. Proteins that are new potential therapeutic targets and / or require functional characterization can be isolated directly from the natural source using a variety of established biochemical isolation procedures.

The availability of the complete gene sequences of the genome sequence data facilitates the cloning and expression of the target protein identified by means of genomic methods. For example, the known white DNA sequences can be used to design the oligonucleotide probes to select the full length of the cDNA clones containing all the cDNA coding of the gene of interest of a library representative of any cDNA clone. In another example, the known sequence of blank DNA can be used to design the first PCRs behind the selective amplification and clone the gene of interest of the total genomic DNA. These and other high processing and expression cloning methods are well known to those skilled in the art. Thus, all the data length of the gene sequence automatically provides the direct means for the high processing, the parallel production of the target proteins, a first stage necessary in any strategy of functional screening based on molecules, of high processing.

C. Thermal Stability Screening In order to develop a microplate heat exchange assay of a target protein, it is necessary to determine the assay conditions that are optimal for performing the assay. Proteins are linear polymers of amino acids that spontaneously bend into stable, highly organized 3-dimensional structures. The biological activity and functions of a target protein, include virtually all the specific bonds and catalytic properties that characterize the protein, depends on its three-dimensional structure.

Virtually all domains of the active bent protein behaves thermally as organic crystals that fuse with a first-order, cooperative, well-defined transition pseudophase: for example, they melt in a state of partial disorder, similar to organic liquids, with a well-defined melting temperature (Tm) that reflects the free energy stabilization of the protein with three dimensional structure under the experimental conditions of the solvents. The microplate thermal exchange technology uses fluorescent dyes sensitive to the medium to detect sensibly the process of thermal splitting and to directly monitor the effects on the stability of the protein that increase from the perturbations of the medium in the solvent or through the binding of ligands to the protein.

The stability of the folded state in three dimensions of a protein can potentially be disturbed in several ways. One way is to alter the environment of the aqueous solvent where the protein molecules initially bent from a disorganized polymer in the organized 3-dimensional state. By changing the properties of the entire solvent around the protein, the stability of the bent state can be altered relative to the stability of the unfolded state. This can provide a useful strategy to find optimal conditions for measuring ligand binding and is the principle behind stability screening.

D. Optimization Screening of the Microplate Thermal Change Test The screening of the optimization test is a group of conditions and fluorescent dyes that are used with the white protein to determine the optimal conditions for developing the microplate heat exchange test. The protein is subjected to a variety of solution conditions and or fluorescent dyes in order to evaluate the behavior of the protein and / or the labeling.

Examples of variations in conditions could include the addition of organic solvents, variations in pH, salts, etc. which have the potential to alter the relative stability of the doubled and unfolded states of the protein. Examples in variations in dyes could include those whose differences in change, polarity, excitation, wavelength, wavelength emissions, antecedent signal strength, or other properties that offer advantages in accuracy of measurements, miniaturization or optimization of the signal with noise under specific test conditions. The optimization of the conditions that facilitate the stability of screening is an empirical process and can be easily practiced by a person without experience in the art.

E. Functional Sounding Library A substantial fraction of the protein molecules that can serve as targets for potential drugs fall into the mechanical classes that share important characteristics. For example, several enzymes use ATP as an energy cofactor, others use pyridine nucleotides as cofactors, some use both as cofactors, etc.

By examining the scientific literature or through experimental means, it is possible to compile a substrate group of enzymes, substrates analogs, cofactors, protein adapter domains, nucleic acid analogs, polysaccharides, fatty acids, nucleic acids, transmission peptides, or other molecules that have been determined to specifically bind to the defined class of protein molecules, or where the functional significance has been attached with a link to a functionally known class of molecules.

As used herein, a "functional probe library" refers to one or more different molecules that are tested for their ability to bind to a target protein and modify the thermal stability of the protein in response to thermal cleavage. When developing a thermal stability test (preferably using a microplate heat exchange assay technology) in the protein in the presence of each member of the functional probe library, the compounds can be incubated individually with the target protein and / or in groups to determine which ligands individually or in link tightly and specifically to the white protein.

Examples of molecules that may comprise a functional sounding library include, but are not limited to the following. 1. Vitamins and Coenzymes NADH / NAD, NADPH / NADP, ATP / ADP, ATP -? - S, Acetyl-CoA, biotin, S-adenosyl-methionine, thiamine pyrophosphate (TPP), sulphated oligosaccharides, heparin-like oligosaccharides, GTP, GTP -? - S, gamma-S, pyroxidal-5-phosphate, flavin nucleotide (FMN), falvinadenine dinucleotide (FAD), folic acid, tetrahydrofolic acid, methotrexate, vitamin Kx, vitamin E succinate salt, vitamin D3, vitamin D3-25-hydroxy, vitamin D3-la-25-dihydroxy, vitamin Bi2, vitamin C, vitamin B, coenzyme A, coenzyme An-butylrilo, transretinoic acid, and hematin. 2. Functional groups of amino acid residues and their mimics Block construction: Guanidino groups Imidazole groups Phenyl groups Phenolic groups Indole groups Aliphatic chains Derivatives of simple and blocked amino acids __ High order structures: Peptide hormones Vasopressin Insulin TRH Corticotropin Glucagon Domains of SH2, SH3 domains, plextrin domains, etc.

Bioactive Peptides Lectins 3. Chelates of Metals Calcium chelates (Calbioquem, San Diego, CA) Iron chelates . Metallic Ions Transitional Metals Calcium, magnesium . Carbohydrates Block construction Glucose Galactose ___ Xylose High order biomolecules Cellulose Starch Fructose Mañosa Sucrose Lactose Bioactive carbohydrates (available from Sigma Chemical Co., St. Louis, MO) 6. Nucleic acid Block construction Uracil Thymidine Cytosine Adenine Guanine High order structures : Oligonucleotides Deoxyribonuoleic acid (DNA) Ribonucleic acid (RNA) The methods of the present invention can also use screening proteins, against nucleic acid libraries that appear synthetically and naturally (eg, oligonucleotides) to test the different classes of proteins that bind nucleic acids. For example, there are several proteins that bind DNA that can be identified by its ability to bind to the DNA sequences of particular classes. Large libraries that contain different nucleic acid sequences (for example, the 4096 different possible synthetic hexamers) can be purchased or synthesized.At all concentrations, all or part of the cognate binding site of proteins that bind nucleic acids at the site can be detected In the event that the protein appears to bind several sequences, it links the sites that can be reconstructed by synthesizing various combinations of nucleic acid sequences, and then the microplate heat exchange assay, or another assay, can be used to measure the affinities of link.

There are several proteins that link DNA that can be identified by their ability to bind to particular classes of DNA sequences with lower efficiency. For example, it is well known that some transcription factors link a variety of sequences rich in A / T, preferably G / C rich sequences. It is known that telomerases recognize sequences rich in G / C. It is known that helicases bind small fragments of single-stranded DNA with low specificity. A smaller, more generic library could contain the following compounds to detect these and other proteins that bind DNA: tracts rich in AT: d (T) 32 / d (A) 32 d (ATAT) 8 / d (TATA) 8 d (AAAT) 8 / d (TTTA) 8 d (AAATT) 6 / d (TTTAA) 6 d (AAATTT) 6 / d (TTTAA) 6 d (AAAATTTT) 4 / d (TTTTAAAA) 4 - GC-rich tracts: d (C) 32 / d (G) 32 d (GCGC) ß / d (CGCG) 8 d (GGGCCC) 6 / (CCCGGG) 6 d (GGGGCCCC) 4 / d (CCCCGGGG) 4 others d (CA) 32 / d (GT) 32 d (CT) 32 / d (GA) 32 d (GA) 2 / d (TC) 32 Single stranded compound of the above double sequences. d (T) 4o / d (A) 2o (an example of a fragment containing both single and double stranded DNA) chromosomal DNA of human cut "all genomic amplification" applied to different chromosomes of human sperm DNA of cut salmon Microbial DNA cut from supercooled plasmid DNA PCR amplification products from specific chromosomal regions (eg, telomeres and centromeres) Other known recognition sites for transcription, processing RNA, transposition 7. Lipids Block construction: Hill Phosphoric Acid Glycerol Palmitic Acid Oic Acid Cholesterol High Order Structures Phosphatidylcholine 8. Enzyme Inhibitors Protease inhibitors (Sigma Chemical Co. St. Louis, MO) PMSF Luepept ina Pepstatin-A Bestat ina Cristatin aldehyde peptide (cysteine protease inhibitors) Protein tyrosine kinase inhibitors (Calbiochem, San Diego, CA) Inhibitors of protein phosphatase (Calbiochem, San Diego, CA) __. Kinase Protein Inhibitors (Calbiochem, San Diego, CA) Kinase Protein Activators (Calbiochem, San Diego, CA) Phosphodiesterase Inhibitors (Calbiochem, San Diego, CA) Phospholipase Inhibitors Transition State Analogs Similarly, zinc metalloproteases, such as the enzyme converted to angiotensin, and carboxypept idase, would be identifiable (a) by de-stabilization with EDTA or orthophenanthroline (Zn2 + chelation) and (b) by stabilization in the presence of hydroxamates and phosphoramidates that mimic the transition state of hydrolysis with peptide bond catalyzed with Zn 2 + The functional sound library may also include compounds of amine hormones, and alkaloid compounds.

The functional sound library can be a library of generic drugs. Alternatively, the functional sound library can be a library of natural products. For example, see In cycl opedi a of Common Na tural Ingredi on ts Used in Foods, Drugs a nd Cosme ti cs, 2nd Edition, Leung and Foster, Eds., Wiley Interscience (1996).

Functional Sounding Screening In addition to optimizing conditions that modify the stability of the protein, another means to affect the stability of a bent protein is specifically binding molecules with an unfolded or doubled state of the protein. Since virtually all actively biological proteins are doubled with organized structures of three dimensions, the greatest interest concerns ligand molecules that bind and stabilize the doubled state of the protein.

As discussed above, a functional probe screening is an assay of the ability of a multiplicity of different molecules in a functional probe library to bind to the target protein and modify the stability of the target protein in response to thermal cleavage. Using the technology, one can directly measure the binding affinity of a small or large molecule of ligand with a target protein through its effect on the average splitting temperature Tm (or thermal splitting profile) of the protein. For molecules that bind to the doubled state of the protein, which includes most of the biological ligands of interest, there is a quantitative relationship between the binding affinity of the ligand and the fact that the Tm of the protein is the bound state. in relation to the Tm of the protein in the unfolded state.

Most proteins have functions that are reflected by their ability to bind molecules of large or small ligands with high specificity and high affinity. Several proteins correspond to functional classes (for example, kinase, phospotasse, pyridine-dependent nucleotide of oxidoreductases, etc.) that bind specific cofactors or catalyze specific reactions using a limited group of catalytic mechanisms. Consequently, molecules in a functional class such as kinases, which use ATP as a cofactor, will generally link a non-hydrolysable ATP cofactor analogous to AMPPNP, a property that would be detected using the methods of the present invention.

However, several proteins will bind a combination of ligands or make multiple interaction groups with the domains of the biological adapter. To the extent that these interactions are independent, they will generally produce additive perturbations in the stability of the unlinked form of the protein.

When a protein has been tentatively assigned to a particular protein class, one can re-screen the protein using a library of known compounds or molecules to bind to this class of protein.

G. Activity Spectrum After developing the thermal stability test (preferably using the microplate heat exchange assay technology) in the protein in the presence of each member of the functional sounding library, one can determine which ligands bind closely and specifically with the protein white and modify the thermal stability of the target protein. The list of compounds (e.g., ligands) that bind to the target protein and modify the thermal stability of the target protein, and the respective affinities of the ligands for the target protein comprise the spectrum of activity of the target protein.

H. List of the Functional Reference Spectrum As discussed above, a "functional reference spectrum list" is a list of target protein classes (including references for an appropriate electronic database), the associated ligands, and the corresponding binding constants, which can be used to functionally classify a white protein. Alternatively, the list of the functional reference spectrum may be a group of one or more activity spectra of one or more known proteins.

As discussed above, a "functional reference list" is a list of proteins that share one or more characteristics in common, such as binding to a particular ligand, or exhibiting a common activity. An example of a functional reference list is given in Table 1. The characteristics shared by the proteins listed in Table 1 is that they link NAD and exhibit hydrogenase activities. The list of proteins in the Table 1 illustrates how a functionally related class of proteins can be discriminated according to their ability to bind different groups of ligands. For example, a protein that binds nicotinamide adenine dinucleotide (NAD), NADPH, or NADH, and malate, as shown by its ability of these compounds to modify the thermal stability of the protein, could be classified as a hydrogenated maleate . As another example, a protein wherein the thermal stability is modified by ethanol and NAD could be classified as a dehydrogenase alcohol.

Table 1 Functional Reference List Aldehyde Dehydrogenase Class 3 d Alcohol Human Dehydrogenase a-hydroxysteroid dehydrogenase Malate Dehydrogenase Horse Liver Dehydrogenase Alcohol Dehydrogenase Alcohol Glyceraldehyde-3-Phosphate Dehydrogenase β-Alcohol Human Dehydrogenase Dihydropteridine Reductase D-2-Hydroxyisocaproate Dehydrogenase Brassica Napus Enoyl Acp Reductase 7-Hydroxysteroid Dehydrogenase 'Holo-D-Glyceraldehyde-3-phosphate Dehydrogenase Glutathione Reductase D-Glyceraldehyde-3-phosphate Dehydrogenase Glutathione Reductase 3-Isopropylamate Dehydrogenase β-3-Alcohol Human Dehydrogenase Isolate Dehydrogenase Horse Liver Dehydrogenase Alcohol Table 1 Functional Reference List M4 Lactate Dehydrogenase Dihydrolipoamide Dehydrogenase Udp-Gal 4-Epimerase D-3-phosphoglycerate Dehydrogenase ?? Alpha Human Liver Dehydrogenase Alcohol, 20 ß-Hydroxysteroid Dehydrogenase L-lactate dehydrogenase NADH peroxidase . Agent of Comparison of the spectrum of activity ~ " As used here, the "activity spectrum comparison agent" is a means of computation or graph by which one can compare the comparison spectrum, derived from the observations made of the functional sounding library in the target protein, with the list of the functional reference spectrum. For example, the activity spectrum comparison agent may be a computer program with a worksheet that is readily available to people without experience in the art. For example, they can use Microsoft Excel (Microsoft Inc. Redmond, WA).

J. Functional Classification In the methods of the present invention, s indicates the function of the protein by means of the pattern of the ligands that bind to the protein. By using the activity spectrum comparison agent to compare the spectrum of observed white activity with the list of the functional reference spectrum, the protein can be classified functionally according to the ratio data obtained from known proteins. For example, the protein can be classified according to the group of ligands that stabilize the protein against thermal cleavage.

Thus, by comparing a graph of the degree to which each multiplicity of molecules or compound modifies the thermal stability of a protein (and through this is linked to the protein) a graph of the degree to which the same molecule modifies the thermal stability of a known protein (and by means of this binding to the protein), the classes of proteins to which the proteins belong are deduced.

Alternatively, the protein can be classified by comparing the activity spectrum of the target protein with the activity spectrum of the known, classified proteins. For example, one can consult the databases such as online PDR, Medline, SciFinder, STNExpress, in-house database, NAPRALERT Online, the In cycl opedi a of Common Na ture l Ingredi on ts Used in Foods, Drugs and Cosme ti cs, 2nd Edition, Leung and Foster, Eds. Wiley Interscience (1996), and the Ha ndbook of Enzyme Inhibitors, Part A and B, 2nd Edition, Ellner, Ed, ECH (1990).

Apparatus and Testing of Microplate Thermal Change In principle, any means to measure the effect of incubating a protein in the presence of a panel of probe ligands to determine which probe ligands can affect the stability of the target protein will be sufficient as a means to functionally classify proteins. Preferably, the microplate heat exchange test is used to determine the effect of one or more molecules or ligands on the thermal stability of a target protein. The microplate heat exchange test is a direct and quantitative technology to test the effect of one or more molecules on the thermal stability of a target protein.

The heat exchange test is based on the change that depends on the ligand in the thermal splitting curve of a receptor, such as a protein or a nucleic acid. When heated over a range of temperatures, a receiver will unfold. By making a graph of the degree of unfolding as a function of temperature, one obtains a curve of thermal splitting of the receiver. A useful reference point in the thermal splitting curve is the mid-point temperature (Tm), the temperature at half of the receptor molecules are unfolded.

The thermal exchange test is based on the ligand-dependent change at the midpoint to thermally induce the splitting curves, ΔTm, for the ligand receptor complex (relative to the non-complex receptor) as an experimentally observed variable that directly relates the binding affinity of the ligand, K1, due to the coupling of the ligand bond and the cleavage-free energy functions of the receptor (Sh_cellman, JA, Bi opol ymers 15: 999-1000 (1976); Brandts, JF Bi och emi s try 29: 6927-6940 (1990)). This strategy of thermal physical screening uses thermal stability in ligand-receptor mixtures as an indicator of binding affinity for ligand-receptor interactions. These tests have traditionally been conducted in a time in differential scanning calorimeters (DSC) that record the change in heat capacity as in proteins that experience the temperature induced by splitting transitions (Brandts et al., Bi ochemi s try 29: 6927-6940 (1990); and Weber, P. et al., J. Am. Ch em. Soc. 116: 2717-2724 (1994)). Alternatively, heat exchange tests can be developed one at a time, by using the temperature controlled optical instruments that record the absorbance (Chavan, A.J. et al., Bi ochemi s try 33: 7193-7202 (1994)); fluorescence (Chevan, A.J. et al., Bi or ch emi s try 33: 7193-7202 (1994)); or circular dichroism (Bouvier, M. et al., Sci in ce 265: 398-402 (1994)); Morton, A. et al., Bi ochemi s try 34: 8564-8575 (1995)) the changes that occur due to the transition of thermally induced cleavage of proteins.

There are several advantages to using the heat exchange assay as it does not require radiolabelled compounds, fluorescent or other chromophobic labels to help register the link. The assays take advantage of the thermal splitting of the molecules, a generally physicochemical process intrinsic to several, if not all, target biomolecules of drugs. General applicability is an important aspect in this trial as it is obvious the need to invent a new assay whenever a new therapeutic receptor protein is available. The assay is particularly well suited for measuring the binding of ligands to non-enzymatic targets, for example the growth factor / receptor interactions, where it is not usy possible to use the spectrophotometric assay. However, the simple configuration of the thermal exchange methods test, as it is conventionally developed, has limited the utility of this technique, especially for the high processing screening of the libraries of the compound.

We have been able to greatly accelerate the protein / ligand screening process by developing a ligand / receptor screening strategy generally applicable in the processing process in a 96 well (or high density) plate format that will identify and give a range of the main compounds based on the thermodynamic stabilization of the ligand-receptor complexes.

Ligand binding stabilizes the receptor (Schellman, J. Bi opolymers 14: 999-1018 (1975)). The degree of binding and the free energy of the interaction follow a parallel course as a function of the concentration of the ligand (Schellman, J. Bi ophysi ca l Chemi s try 45: 273-279 (1993)); Barcelo, F. et al., Ch em. Bi ol. In tera c ti ons 74: 315-324 (1990)). As a result of the stabilization of the ligand, more energy (heat) is required to unfold the receptor. Thus, the link of the ligand changes the curve of the thermal split. That is, the binding of the ligand increases the thermal stability of the protein. This property can be exploited to determine if a ligand bond binds with a receptor: a conversion, or "change", in the thermal splitting curve, and thus in the Tm, suggests that the ligand binds to the receptor.

The thermodynamic bases of the heat exchange assay have been described by Shellman, J.A. (Bi opolymers 15: 999-1000 (1976)), and also by Brands et al. (Bi och emi s try 29: 6927-6940 (1990)). Differential scanning calorimetry studies by Brands et al. (Bi och emi s try 29: 6927-6940 (1990)) have shown that for narrow link systems of 1: 1 stoichiometry, where there is a splitting transition, one can estimate the binding affinity to Tm with the following expression : Equation 1 Where KTm, = the association constant of the. ligand to Tm; Tm = the midpoint of the cleavage transition of the protein in the presence of the ligand; T0 = the midpoint of the splitting transition in the absence of the ligand; ? H1 the enthalpy of unfolding of the protein in the absence of the ligand to TQ; "CP" = the change in the capacity of the unfolding of the protein in the absence of the ligand; [Ltm_ = the concentration of the free ligand at Tm; and R = the gas constant.

This expression was found to be useful for the structure based on the design of azobenzene ligands for streptavidin where DSC scans of several ligand mixtures / is reptavidin facilitates the measurement of binding affinity to Tm (Weber, P. et al. , J. Am. Ch em. Soc. 116: 2717-2724 (1994)). These measurements were verified by the development of the mixture or isothermal titration calorimetric experiments that produces binding affinities consistent with those determined by DSC. The development and reproduction of the use of thermal splitting of the protein to stimulate the binding affinity of the ligand impressed us the potential of the prolongation of the method to become a more general drug discovery tool.

The parameters? HU and? Cpu are usually observed from the experiments and are specific for each protein. The calorimetric measurements of? HU and? Cpu are the most accurate estimates of these parameters due to the splitting data typically collected in calorimeters every 0.1 ° C. However, the parameters? HU and? Cp can also be estimated in the microplate heat exchange test, where the case of? HU no. is a "calorimetric enthalpy but a case of comparable van't Hoff enthalpy based on the split data collected at each 2.0 ° C using the current protocol, however, even in the absence of optimal data for? HU and? Cpu these parameters are specific constants for the protein involved in the screening of the compound and by means of this it is not changeable from well to well, causing no influence in the calculations of the relative values of the binding affinities, for example, KL to Tm.

However the parameters? HU and? Cpu it is also necessary to obtain estimates of Tm and T0 to solve KTm, in equation 1. This is achieved through the use of nonlinear least squares computation input of the split data for each individual well using the following equation: Equation 2 Equation 2 employs five input patterns? HU 'and? Cpu, Tm and f and yu, where yf and yu are the pre-transitional and post-transitional fluorescence levels, respectively. The inputs of the computer are determined by floating these parameters above the minimum of the sum of the squares of the residuals when using the Levenberg-Marquardt algorithm. The T0 values are obtained by means of wells that do not contain added ligand and are a reference group. The commercially available curve entry is easily available to a person with experience in the art. For example, Kaleidograph 3.0 (Synergi, Reading, PA) can be used.

It is also possible to calculate the equilibrium constant in association with the ligand at any temperature, KL to T, we know the equilibrium constant in association with the ligand at Tm, using equation 3, if we mix the calorimetric data for the enthalpy of link to T,? HL, and the change in heat capacity at the ligand bond,? CpL, (Brandts &Lin, 1990).

Equation 3 where K = the association constant of the ligand at any temperature, T. KTm ,. = constant of association of the ligand to Tm. Tm = the midpoint of the splitting transition of the protein in the presence of 1 igand. ? H1 the enthalpy of the bond of the ligand © temperature, T? CpL = the change in the binding capacity of the ligand. R = the gas constant The second exponential term in equation 3 is usually so small to be ignored so that the approximate values of KL to T can be obtained by using only the first exponential term, and equation 3 is reduced to equation 4: Equation 4 The parameter "HT" can be measured using isothermal titration calorimetry, using a calorimetric apparatus such as Omega (MicroCal, Northampton, MA). When calorimetric data are not available,? HTmL can be estimated to be approximately "" -10.0 kcal / mol, which is an average bond enthalpy (Wiseman et al., Ana I. Bi och., 179: 131-137 (1989)). ).

Preferably, fluorescence spectrometry is used to record the thermal split. The fluorescence methodology is more sensitive than the absorption methodology. The use of intrinsic protein fluorescence and fluorescence test molecules in fluorescence spectroscopy experiments is well known to those skilled in the art. See, for example, Bashford, C.L. et al., Spectrophotometry and Spectrofl uorometry: A Pra cti cal Approa ch, IRL Press Ltd., pub., p. 91-114 (1987); Bell, J.E., Spectros copy i n Bi och emi s try, Vol. 1, CRC Press, pub., Pp. 155-194 (1981); Brandts, L et al., Ann Rev. Bi och em. 41: 843 (1972).The microplate heat exchange test is further described in US Patent Application No. 08 / 853,464, filed on May 9, 1997 and international patent application No. PCT / US 97/08154 (published on November 13, 1997 as WO 97/42500), which is incorporated herein by reference in its entirety.

The spectral readings, preferably the fluorescence readings, can be taken from all the samples in a simultaneous transporter. Alternatively, the readings can be taken in the samples in groups at least two at the same time.

A fluorescence imaging system, for example, a fluorescence emission imaging system, can be used to record the thermal cleavage of a target molecule or receptor. Fluorescence imaging systems are well known to those skilled in the art. For example, the ALPHAIMAGER ™ Gel Documentation and Analysis system (Alpha Innotech, San Leandro, CA) employs a high-performance coupled charge (CCD) camera with 768 x 494 pixels of resolution. The camera with attached charge is interconnected with a computer and the images analyzed are the Image analysis software ™. The CHEMIIMAGER ™ (Alpha Innotech) is a cooled coupled charge device that performs all ALPHAIMAGER ™ functions and also captures images of chemiluminescence samples and other low intensity samples. The CHEMIIMAGER ™ coupled charging device includes a Pentium processor (1.2 Gb hard disk, 16 MB RAM), AlphaEase ™ analysis program, deep-light cabinet, and a trans-illuminator of UV light and white light. For example, the MRC-1024 UV / Visible Laser Confocal Image System (Biorad, Richmond, CA) facilitates the simultaneous imaging of more than one fluorophore over a wide range of illumination wavelengths (350 to 700 nm) The Doc 1000 Fluorescent Gel Documentation System (BioRad, Richmond, CA) can clearly display sample areas as large as 20 x 20 cm, or as small as 5 x 4 cm. At least two 96 well microplates can be fixed in an area of 20 x 20 cm. The Gel Doc 1000 system facilitates the development of_the experiments based on time.

The fluorescence imaging system, for example, a fluorescence emission imaging system, can be used to record the splitting of the receptor in a microplate thermal change assay. In this embodiment, a plurality of samples is heated simultaneously between 25 to 110 ° C. The reading of the fluorescence emission is taken for each of the pluralities of simultaneous samples. For example, fluorescence in each well of a 96-well or 384-well microplate can be recorded simultaneously. Alternatively, the fluorescence readings can be taken continuously and simultaneously for each sample. At lower temperatures, all samples register a low level of fluorescence. While the temperature is increased, the fluorescence in each sample increases. The wells containing ligands bind to the target molecule with a high affinity change the thermal split curve at higher temperatures. As a result, wells containing ligands that bind to the target molecule with less high affinity fluorescence, at a given temperature above the Tm of the target molecule in the absence of any ligand, than wells that do not have high affinity ligands . If the samples are heated in later stages, the fluorescence of all the plurality of samples the image is obtained simultaneously in each heating stage.

If the samples are heated continuously, the fluorescence emission of all the plurality of samples is taken simultaneously during the heating.

A heat exchange test can be developed in a volume of 100 μL volumes. For the following reasons, however, it is preferable to develop a heat exchange test of 1-10 μL. First, a protein with less than about 10- to 100-fold is required for the miniaturized assay. Thus, only -4 to 40 pmoles of protein (0.1 μg for a 25 μDa protein) is required for the assay (for example, 4 μL working volume with a concentration of the target molecule from about 1 to about 4 μM). Thus, 1 mg of protein can be used to make 1,000 to 10,000 in the miniaturized format. This is particularly advantageous when the white molecule is available in negligible amounts.

Second, approximately ligands with less than 10- to 100-fold are required for the miniaturized assay. This advantage is very important for researchers when screening valuable combinatorial libraries where the libraries of the compounds are synthesized in insignificant quantities. In the case of human a-thrombin, the ideal concentration of the ligand is approximately 50 μM, which translates into 25-250 pmoles of ligand, or 10-100 ng (assuming a MW of 500 Da) of ligand per assay in the miniaturized format.

Third, the small volume of work permits the potential to use large testing arrangements because the miniaturized test can be set in a much smaller area. For example, plates with 384 wells (16 x 24 array) or 864 wells (24 x 36 array) have the same dimensions as 96 well plates (8.5 x 12.5 cm). The plate with 384 wells and the plate with 864 wells allow the user to develop 4 and 9 times several tests, respectively, can be developed using a plate with 96 wells. Alternatively, plates with more wells may be used, such as 1536 wells (32 x 48 array, Matrix Technologies Corp.). A plate with 1536 wells will provide sixteen times the processing provided by a plate with 96 wells.

Thus, using the plate configuration with 1536 wells, the assay speed can be increased by about 16 times, relative to the speed in any assay can be developed using the 96-well format.

The 8 x 12 trial setup (in a 96 well plate) facilitates the development of 96 assays / r, to approximately 2300 assays / 24 hours. The 32 x 48 assay setup facilitates the development of approximately 1536 HR assays, or approximately 37,000 assays / 24 hours can be developed using a 32 x 48 assay setup.

The assay volume can be 1-100 μL. Preferably, the assay volume is 1-50 μL. More preferably, the assay volume is 1-25 μL. Even more preferably, the assay volume is 1-10 μL. Even more preferably, the assay volume is 1-5μL. Even more preferably, the assay volume is 5 μL. More preferably, the assay volume is 1 μL or 2 μL.

Alternatively, the test developed in polycarbonate plates with V-bottom, polystyrene, or polypropylene or dimpled plates. The dimpled plate is a plate containing a plurality of wells with a round bottom that is maintained at a total volume of 15μL.

The microplate heat exchange test is developed (a) to contact a protein with one or more of a multiplicity of different molecules in each multiplicity of the containers; (b) heating the multiplicity of containers of step (a), preferably simultaneously; (c) measuring in each of the containers a physical change associated with the thermal splitting of the white molecule caused by the heating; (d) generating a thermal splitting curve of the target molecule as a function of the temperature for each of the containers; and (c) comparing each of the splitting curves in step (d) to (1) each of the thermal splitting curve and (2) the thermal splitting curve obtained for the protein in the absence of any multiplicity of different molecules; and (f) determining whether any multiplicity of the different molecules modifies the thermal stability of the protein, if a change in stability is indicated by a change in the thermal splitting curve.

Step (d) further comprises determining a midpoint temperature (Tm) of the thermal split curve. Step (e) may further comprise comparing the Tm for each of the curves in step (d) to (1) the Tm of each of the thermal splitting curve and for (2) the Tm of the splitting curve obtained thermal for the target proteins in the absence of any of the different molecules.

To practice the methods of the present invention using spectroscopy or luorescence imaging, step (a) comprises contacting the target protein with a fluorescence probe molecule present in each multiplicity of vessels and step (c) comprising (cl) : excite the fluorescence probe molecule, in each multiplicity of vessels, with light; and (c2) measuring the fluorescence of each multiplicity of vessels. Fluorescence, for example, fluorescent emission, can be measured from each multiplicity of containers one container at a time, from one subgroup of the multiplicity of containers simultaneously, or from each multiplicity of containers simultaneously.

To generate a spectrum of activity, the molecules are arranged according to the degree to which the target protein stabilizes against thermal splitting. After the molecules are accommodated, the activity spectrum of the target protein of the molecules in the functional test library is compared to one or more lists of functional reference spectra.

Heating apparatuses suitable for practicing the methods of the present invention are well known to a person without experience in the art. For example, the ROBOCYCLER® Gradient Temperature Cycler (Stratagéne, La Jolla CA) can be used (see US Patent No. 5,525,300). Alternatively, a heating block with a temperature gradient may be used (see U.S. Patent No. 5,255,976). The fluorescence can be read using a fluorescence spectroscopy apparatus. For example, the CytoFluor II apparatus (PerSeptive Biosystems, Framingham, MA) can be used.

The element on which the sample conveyor is heated can be any element capable of heating the samples rapidly and in a reproducible manner. In the present invention, a plurality of samples are heated simultaneously. The plurality of samples is heated in a simple heating element. Alternatively, the plurality of samples may be heated to a given temperature in the heating element, and then moved to another heating element to heat to another temperature. The heating can be done at regular or irregular intervals. To generate a slight bend curve, the samples should also be heated, at intervals of 1 to 2 ° C. The temperature range through the samples can be heated from 4 to 110 ° C. The spectral readings, and the fluorescence readings in particular, are taken after each heating step. The samples can be heated and read by the spectrum apparatus, for example, a fluorescence imaging camera, continuously. Alternatively, after each heating step, the sample can be cooled to a lower temperature to take the spectral readings. Preferably, the samples are heated continuously and the spectral readings are taken while the samples are heated.

The readings of the spectra, for example, the fluorescence can be taken in all the samples simultaneously. Alternatively, the readings can be taken in the group samples at least two at a time. Finally, the readings can be taken from one sample at a time.

Preferably, the instrument used for the development of the microplate heat exchange test consists of an explorer and a programming system for the control. Fluorescence, e.g., fluorescence emission, can be detected by a photomultiplier tube within a light-proof detection chamber. The programming system runs on a personal computer and the action of the browser is controlled through the programming system.

Exemplary is an apparatus 200 in Figure 2. A precision X-Y mechanism scans the microplate with a sensitive fiber optic probe to quantify the fluorescence in each well. The microplate and the samples can remain stationary during the scan of each row of samples, and the fiber optic probe then moves to the next row. Alternatively, the microplate and the samples can be moved to the position of a new row of samples under the fiber optic probe. The scanning system is capable of scanning 96 samples in one minute. The scanner is capable of maintaining a plurality of excitation filters and a plurality of emission filters for measuring the most common fluorophores. Thus, the fluorescence emission readings can take one sample at a time, - or a group of subgroups of samples simultaneously.

The element or block of heat conductor in which the heated sample is transported can be any element capable of heating the samples rapidly and reproducing them. The plurality of samples can be encouraged in a simple heating element. Alternatively, the plurality of samples may be heated to a given temperature in a heating element, and then moved to another heating element to heat to another temperature. Warming can be achieved at regular or irregular intervals. To generate a slight bend curve, the samples should be heated regularly, in intervals of 1 to 2 ° C. The temperature ranges through the samples can be heated from 4 to 110 ° C.

Preferably, a plurality of samples is heated simultaneously. If the samples are heated at discrete intervals, in a stepwise manner, the spectral readings are taken after each heating step. Alternatively, after each heating step, the samples can be cooled to a lower temperature before taking the spectral readings. Alternatively, the samples can be heated continuously and the spectral readings are taken during heating. (The test apparatus may be configured to contain a simple heating conductor block.Alternatively, the test apparatus may be configured to contain a plurality of conductive blocks on a mobile platform.The platform shall be a convertible platform that is can be converted, for example, by a linear sliding device with servomotor.A linear sliding device is the model SA A5M400 (IAI America, Torrance, CA). In this mode, the sensor receives the spectral emissions of each sample in a heat conducting block. The platform is then moved from place to another heat conducting block and its accompanying sensors under the sensor in order to receive spectral emissions of each sample in the heating block. The platform is moved until the spectral emissions of the samples are received in all the heating conductor blocks.

Alternatively, the platform may by means of a rotating platform, as shown in Figure 2, be rotatable, for example, with a servomotor shaft. In a later embodiment, the sensor receives the spectral emissions of each sample in a heat conducting block. The platform is then rotated to another heat conducting block and its accompanying samples under the sensor in order to receive the spectral emissions of each sample in the heating block. The platform is rotated until the spectral emissions of the samples are received in all the heat conducting blocks.

In the apparatus 200, a plurality of heat conducting blocks 204, each including a plurality of wells for a plurality of samples 210, is mounted on a rotatable platform or carousel 206. The platform or carousel 206 may be composed of a heat conductive material, such as the material which is composed of heat conducting blocks 204. The shaft 208 is rotatably connected to the base 202. The rotating platform 206 is mounted axially about the axis 208. The rotation of the shaft 208 it is controlled by means of a servo motor 210. The servomotor is controlled with a control computer 250 in a manner well known to a person skilled in the relevant art. The control computer 250 causes the servo motor 210 to rotate the shaft 208 by means of rotating the rotary platform 206. In this manner, the heat conducting blocks 204 are sequentially placed under the fiber optic probe 212.

Each plurality of the heat conducting blocks 204 can be controlled independently with the temperature controller 214. Thus, the temperature of a first heat conducting block 204 can be higher or lower than the temperature of a second heat conducting block 204. Similarly , the temperature of a third heat-conducting block 204 may be greater or less than the temperature of the first or second heat-conducting block 204.

The temperature controller 214 is connected to the heat conducting block 204 by a thermoelectric connection 230. Under the action of the temperature controller 214, the temperature of the heat conducting block 204 can be increased, decreased, or kept constant. The temperature controller 214 can be configured to adjust the temperature of the rotating platform 206. In this configuration, when the rotating platform 206 is heated, the heat conducting blocks 204 are also heated. Alternatively, the temperature of each heat conducting block 204 can be controlled by a circulating water system as noted above. Particularly, the temperature of the heat conductor block 2 CM can be changed by the temperature controller 214 according to the predetermined temperature profile. Preferably, the temperature computing controller 214 is implemented using a computer system.

As used herein, the term "temperature profile" refers to a change in temperature over time. The term "temperature profile" encompasses the continuous changes made up and down in temperature, both linear and non-linear changes. The term also encompasses any protocol of temperature change, including protocols characterized by incremental increases or decreases in temperature during which the increase or decrease in temperature is interrupted for periods during which the temperature is held constant. In the apparatus shown in Figure 2, the temperature profile can be pre-determined by the programmable computer controller of the temperature 214. For example, the temperature profiles can be stored in memory in the temperature control apparatus. _214, or input to the temperature controller 214 by an operator.

The assay apparatus 200 also includes a light source 218 when emitting an exciting wavelength of light. The exciting light from the light source 218 excites the samples 216 with the exciting light. Any suitable light source can be used. The excitation light causes a spectral emission of the samples 216. The spectral emission can be an electromagnetic radiation or any wavelength in - the electromagnetic spectrum. Preferably the spectral emission is fluorescent, ultraviolet, or visible light. More preferably, the spectral emission is the fluorescent emission.

A sensor is adhered to a sensor armature 226. An experimental sensor is a fiber optic probe 212. Fiber optic probe 212 includes a fiber optic cable to transmit exciting light to samples 216, and a capable fiber optic cable of receiving a spectral emission of the samples 216. The electromagnetic radiation is transmitted from the exciting light source 218 to the optical fiber probe 212 by means of the exciting light input fiber optic cable 228.

A servo controller of the exciting light filter 258 controls the opening of the exciting light filter 256. The exciting light source 218 and the exciting light filter servo controller 258 are communicated and operatively connected to the exciting light control computer 254 The control computer 254 controls the wavelength of the exciting light transmitted to the samples 216 by controlling the servo driver of the exciting light filter 258. The exciting light is transmitted through the optical fiber input wire of the exciting light. 228 to the optical test probe 212 for the transmission of the samples 216.

The spectral emission of the samples 216 is received by the optical fiber probe 212 and transmitted to a spectral emission filter 238 by means of an optical fiber output cable 250. A servo controller of the spectral emission 240 controls the aperture of the spectral emission 240. Spectral emission filter 238, by this means controlling the wavelength of the emission of the spectrum is transmitted to the photomultiplier tube 220. The servo controller of the spectral emission 240 is controlled by a control computer 242.

The spectral emission of the samples 216 is transmitted from the photomotive tube 220. The electrical output 244 connects the photomultiplier tube 220 with the electrical connection 224. The electrical connection 224 connects the electrical output 244 to the computer 222. Operated by the appropriate programming system, the computer 222 processes the signal of the spectral emission of the samples 216. Exemplifying the programming system is a graphical interface that automatically analyzes the fluorescence data obtained from samples 216. This programming system is well known to people sine experience in art. For example, the multi-well fluorescence plate reader CytoFluor ™ II (PerSeptive Biosystems Framingham, MA) uses the Cytocalc ™ Data Analysis System (PerSeptive Biosystems Framingham, MA). Another suitable programming system includes, Microsoft Excel or any compatible programming system.

A sensor armature relates to the movement means 260 moves the armature of the sensor 226 in the direction 234 and 236. A second means of relative movement 232 moves the armature of the sensor 226 in the direction 246 and 248 so that the probe fiber optic 212 can be moved to detect the spectral emissions of the samples 216.

As discussed above, the spectral receiving means or sensor of the test apparatus of the present invention may comprise a photomultiplier tube. Alternatively, the spectral reception means or sensor may include a charge coupled apparatus (CCD). In yet another alternative, the spectral receiving means or sensor may include a diode array. A CCD is made of semiconductor silica. When photons of light fall on it, free electrons are released.

In addition, a CCD camera can be used for image fluorescence, such as fluorescence emission. High-resolution CCD cameras can detect very small amounts of electromagnetic energy, whether it originates from distant stars, is diffracted by crystals, or is emitted by fluorophores. As an electronic imaging apparatus, a CCD camera is particularly suitable for the fluorescence emission image because it can detect very blurry objects, providing sensitive detection over a wide spectral range, providing low levels of electromagnetic noise, and detecting signals over a wide dynamic range - that is, a charge coupled device can simultaneously detect bright objects and fuzzy objects. In addition, the output is linear so that the amount of electrons collected is directly proportional to the number of photons received. This measures that the brightness of the image is a measure of the brightness - the real object, a property not provided, for example, the photographic emulsions. Suitable CCD cameras are available by Alpha-Innotech (San Leandro, CA), Stratagene (La Jolla, CA), and BioRad (Richmond, CA).

Apparatus useful for practicing the microplate heat exchange assay are also described in US Patent Application No. 08 / 853,459, filed May 9, 1997, and in US Patent Application No. PCT / US97 / 08154 (published on November 13, 1997 as publication No. WO 97/42500), which are hereby incorporated by reference in their entirety.

Having now generally described the invention, it will be easier to understand with reference to the following specific examples which are included herein for purposes of illustration only and is not intended to be limited unless otherwise specified.

Example 1 Wide Utility of the White Cross of the Microplate Thermal Change Test A number of different therapeutic protein targets have been tested in the microplate heat exchange assay, for recording, and are listed in Table 2. They include a variety of different proteins, with a wide variety of in vi vo function. Several serine proteases, a DNA binding protein (lac repressor), two growth factors (basic fibroblast growth factor (bFGF) and acid fibroblast growth factor (aFGF)), and a growth factor receptor have been included. (domain II of the fibroblast growth factor receptor 1 (D (II) FGFR1)).

Table 2 Therapeutic Targets Analyzed by the Microplate Thermal Change Test White PM Assay / mg (in 10 uL format) a-Thrombin 37.0 kDa 1430 0.7ug / assay (20 pmol) D Factor 25.0 kDa 1000 l.Oug / assay (40 pmol) Factor Xa 4b.0 JDa 1667 0.6ug / assay (7 pmol) DFGF 17.5 kDa 2000. 0.5ug / assay (29 pmol) D (II) FGFR1 13.5 kDa 588 1.7ug / assay (126 pmol) lac Repressor 77.0 kDa 1200 0.8ug / assay (10 pmol) Urokinase 28.0 kDa 714 1.4ug / assay (50 pmol) Protein NFkB 65.0 kDa 3030 0.33ug / assay (5 pmol) Receiver GLP1 26.0 kDa MHCII. 45.0 kDa Factor 500 kDa 400 2.5ug / assay (20 pmol) and yielding aFGF 18.0 kDa The molecular weights of the target protein range is from 13.5 kDa to approximately 500 kDa. On average, it is possible to conduct 1322 assays per 1.0 mg of protein using 10 μL of assay volume. The number of tests that can be performed can be doubled if the 5 μL test format is used.

All microplate heat exchange assays are developed in 96-well V-bottom polycarbonate plates using 200 μM of 1,8-ANS as a fluorescent probe to monitor thermal cleavage transitions for protein / ligand mixtures. Switches in fluorescence emission at 460 nm were recorded with a CytoFluor II fluorescence plate reader (perseptive Biosystems) (excitation at 360 n), and the temperature was increased by 2 ° C with the Gradient Temperature Cycler (Stratagene, La Jolla, CA).

Other different proteins have been tested using the microplate heat exchange assay, including the following proteins of the following classes: serine proteases (thrombin, Factor Xa, Factor D, urokinase, trypsin, chymotrypsin, subtilisin); cell surface receptors (receptor 1 FGF, MHC Class II, GLP1 receptor, β-2 adrenargic receptor, fibronectin receptor (libllla)); growth factors (aFGF, bFGF); proteins that bind DNA (repressors l to c, NF-K-B, helicase); motor proteins (myosin, helicase); oxido-reductases (horse radish peroxidase, cytochrome c, lactate dehydrogenase, lactoperoxidase, malate dehydrogenase, cholesterol oxidase, glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate carboxylase, dihydrofolate reductase); modifications of carbohydrate (cellulase, α-amylase, hyalurinidase, β-glucoside, invertase); immunoglobulins (IgG Fab, IqG Fe); DNases (DNase I, DNase II) RNase (RNase A); intracellular calcium receptors (calmodulin, S100 protein); hydrolase neurotransmitters (acetyleolyesterase); radial free rapero (superoxide dismutase); protein that binds biotin. { streptavidin); proteins that bind oxygen (myoglobin); and protease inhibitors (trypsin inhibitor).

Example 2 MuI i-Ligand Linking Interactions with a Simple White Protein The recent universal utility of the microplate heat exchange assay technology is also illustrated for the multi-ligand binding interactions that occur several times within a single protein molecule. The ability to evaluate the binding of different types of ligands with a simple protein without the re-use of the assay is a great advantage of this technology and easily leaves to itself the task of assigning the function "to a protein that is not known in Place of the main sequence Knowledge of the binding of the different ligands will help in the evaluation of the function of a sample protein driven by genomic information.

As previously demonstrated, the microplate heat exchange assay can be used to screen the ligands for binding at the single sites on the target proteins. However, based on the next addition of ligand binding free energy and protein cleavage, it is possible to employ the microplate heat exchange assay to analyze the binding interactions of mu ti t-ligands in the target protein. In principle, if the binding free energy of different ligands in the same protein are closely additive then one can analyze the multi-ligand binding systems non-cooperatively or cooperatively [positively or negatively].

With respect to this, human thrombin is an ideal system to test the usefulness of the assay for the analysis of muI t-ligand binding interactions because it has at least four different binding sites: (1) the binding site catalytic; (2) the fibrin binding site (exosite I); (3) the heparin binding site (exositium II); (4) the Na + binding site, located around 15A of the catalytic site.

First, the binding of the individual ligands was determined with 3DP-4660, Hirugen (hirudin 53-64) (Sigma), heparin 5000 (CalBiochem), linked to the catalytic site, the fibrin binding site and the binding site of heparin, respectively of thrombin.

A solution of cattle thrombin was diluted to 1 μM in 50 M Hepes, pH 7.5, 0.1 M NaCl, 1 mM CaCl2 and 100 μM 1.8-ANS. Each thrombin ligand included single in several combinations of the thrombin solutions of μM a final concentration of 50 μM each, except for heparin 5000, which was 200 μM. One lOOμL of thrombin or thrombin / ligand (s) was placed in the wells of a 96-well V-bottom polycarbonate microtiter plate. The contents were mixed by repeating the capture and discharge at the tip of a 100 μL pipette. Finally, a drop of mineral oil (Sigma, St. Louis, MO) was added at the top of each reaction well to reduce the evaporation of the samples at elevated temperatures. The plate was subjected to 3 minutes of heating in a thermal block RoboCycler® Gradient temperature Cycler (Stratagene, La Jolla CA), with which a temperature gradient is created through the microplate, followed by 30 seconds of cooling to 25 minutes. ° C, and the subsequent reading in the fluorescence plate reader. The data was analyzed by non-linear least squares input.

The results of these individual binding reactions are shown in Figure 3. JS1 order of binding affinity placement was from 3DP-4660 > hirugen > heprin 500, which correspond to Kd of 15 nM, 185 nM and 3434 nM, respectively, for the ligand bonds in each Tm (see Equation (1)).

Next, the binding of the combinations of two ligands was studied. The data is shown in Figure 4. The results in Figure 4 revealed thermal splitting changes that were slightly smaller than expected for the entire addition. For example, Hirugen only gave a? Tm of 508 ° C, and 3DP-4660 only gave a? Tm of 7.7 ° C, but together gave a? Tm of 12.2 ° C, and not the 13.5 change that should be expected if the Link energies were totally additive. This result would mean that the binding affinity of one or both ligands decreases when both ligands bind to thrombin, it would be an example of negative cooperativism between the fibrin binding site and the catalytic binding site. This result is consistent with the thrombin literature, where the kinetics of the hydrolysis of several chromogenic substrates has been found to depend on the ligands that bind to exosite I. Indeed, an increase of 60% was observed in the Km of the hydrolysis of D-phenylalanylpipecolyl arginyl-p-nitroaniline when Hirugen was present (Dennis et al., Eur. J. Bi or chem 188: 61-66 (1990)). However, there is also structural evidence of cooperation between the catalytic site and the exosite I. A comparison of the isomorphic structure of PPACK-binding thrombin (PPACK is an inhibitor of the catalytic site of thrombin) and the thrombin that binds the hirugen revealed conformational changes that occur in the active site as a result of the hirugen binding in exosite I (Vi j ayalakshmi et al., Pro t ein Sci in ce 3: 2254-2271 (1994)). Thus, the apparent cooperation observed between the catalytic center and the exosite I are consistent with the functional and structural data in the literature.

One would expect that if the binding energies of all three ligands were totally additive, a? Tm of 17.7 ° C could be seen. However, when the three ligands were present together, the ΔTm was 12.9 ° C. This result implies another negative cooperation involving the binding of the ligand in the three binding sites of the protein. There is evidence in the literature that is consistent with this assumption. For example, thrombin, in a ternary complex with heparin and fibrin monomer, has decreased activity towards chromogenic substrates tri-peptides and pro-thrombin (Hogg &; Jackson, J. Bi ol. Chem. 265: 248-255 (1990)), the reactivity markedly reduced with the anti-thrombin (Hogg &Jackson, Proc. Na ti.Ac d.Sci.U.S.A.86: 3619-3623 (1989)) . Also, recent observations by Hotchkiss et al., (Blood 84: 498-503 (1994)) indicates that the ternary complex is also formed in plasma and markedly comprises the anticoagulant activity of heparin.

A summary of the results of the binding of mult i-ligands to thrombin is shown in Table 3. From the results in Figures 3 and 4 • and in Table 3, the following conclusions are made. First, in the presence of heparin 5000, huridin 53-65 bind thrombin approximately 21 times less closely than in the absence of heparin; and in the presence of heparin 5000, 3DP-4660 binds to thrombin approximately 10 times less than in the absence of heparin.

Second, in the presence of huridin 53-65, thrombin binds heparin approximately 18 times less than in the absence of huridin 53-65; and in the presence of huridin 53-65, thrombin binds 3DP-4660 approximately 3 times less than in the absence of huridin 563-65.

Third, in the presence of 3DP-4660, thrombin binds heparin approximately 25% more closely than in the absence of 3DP-4660; and in the presence of 3DP-4660, thrombin binds hirudin approximately 2.3 times more closely than in the absence of 3DP-4660.

Table 3 Assay for Linkage of Ligands in an Active Site, Exositium, and Heparin Linkage Site of Thrombin Protein / Ligand [Ligand] TB? TB K at Ta * Kd a (μM) (° K) (° K) (nM) 298 ° Kto (nM) Thrombin (TH) None 323.75 0.0 TH / Heparin 5000 200 327.95 4.2 3434 470 TH / Huridin 53-65 50 329.52 5.8 185 23 TH / 3dp-4660 50 331.40 7.7 29 3 TH / Heparin 5000 200 327.95 TH / Hep. / Hir. 50 330.57 2.6 4254 478 TH / Heparin 5000 200 327.95 TH / Hep. / 3dp 4660 50 333.20 5.3 350 32 TH / Huridin 53-65 50 329.52 -TH / Hir. / Hep. 200 330.57 1.1 75422 846"TH / Huridin 53-65 50 329.52 TH / Hir ./3dp-4660 50 335.97 6.5 117 9 TH / 3dp-4660 50 331.40 TH / 3dp-4660 / Hep. 20Ü 333.20 1.8 38205 351 TH / dp -4660 50 331.40 TH / 3dp-4660 50 335.97 4.6 731 54 Calculations for Kd to Tm were made using equation (1) with? HTmu = 200.0 kcal / mol, as observed for pre-thrombin 1 by Leintz et al., Biochemistry 33: 54-60-5468 (1994), and an estimate? Cpu = 2.0 kcal / mol - ° K; and Kd = 1 / Ka. Estimates for Kd at T = 298 ° K were made using equation (3) with? HTm ?, it was estimated to be -10.0 kcal / mol.

Thus, the microplate heat exchange assay offers several advantages for analyzing the interactions of multi-ligand binding in functional genomic classification studies. For example, the same assay can simultaneously detect the binding of different types of ligands. which bind in a multiple binding site on a white protein. Each binding interaction of the identified ligand helps the user assign a function of a protein. When the functions are added together, one obtains the response curve that is characteristic of a particular class of proteins.

For example, if one considers that the information obtained here for thrombin, and for the time being forgets that this protein is known, the binding data of heparin may suggest an extracellular role for this protein since heparin and other sulfated oligosaccharides are compounds of the cellular matrix of the tissues of the major organisms. The ligand with catalytic binding site, 3DP-4660, is a non-peptide mimic of a peptide having an arginyl side chain in the Pl position, substrate characteristics and serine protease inhibitors similar to trypsin. Similarly, analogs of the transition state of boroarginine, which have an arginine group in the Pl position for this synthetic mimic peptide, were found to be specific inhibitors for serine proteases, thrombin, trypsin and plasmin (Tapparelli et al., J. Bi ol. Chem. 268: 4734-4741 (1993)) with the observed specificity: Kd -10 nM (thrombin), Kd -1,000 nM (trypsin), Kd -10,000 nM (plasmin). Thus, the combined knowledge of the heparin bond with the observed link to the boroarginin transition state analogue would rapidly focus on assigning this protein to an extracellular proteolytic function in the absence of any other information.

In addition, the microplate heat exchange assay, in a high processing manner, can be used to detect cooperation at the ligand link. Information about the cooperation of the ligand link can be collected and analyzed very quickly, in a few hours, instead of several months, as was required when conventional methods were used to classify the function of the protein.

Example 3 Screening of the Functional Sounding Library against Human Xa Factor Xa A functional sound library is shown in Figure 5. A 96-well plate (Plate 1) contained 94 compounds (and two control wells) and incubated several compounds that are considered useful in providing information about ligand binding preferences. , and therefore the probable function of proteins. For example, cofactors such as NAD and ATP were found in wells A4 and A5, respectively. This particular plate contained conditions for a large metal ion bond to help probe a target protein with metal ion cofactors.

In order to validate functional sounding screening, two known proteins were incubated with the compounds of Plate 1 and then tested using the microplate heat exchange assay. For example, the spectrum of activity obtained for Factor Xa (Enzyme Research Labs) is shown in Figure 6.

Factor Xa is sold by Enzyme research Labs (South Bend, IN). The reactions were prepared in a polycarbonate microtiter plate of 96 wells with V bottom. The final concentration of Factor Xa was μM (55ng / mL) in 200 mM Tris-HCl, pH 8. The final concentration of 1, 8-ANS was 100 μ.

The final concentration of each of the molecules tested for binding is shown in Figure 6. The content was mixed by taking and repeatedly discharging at the tip of a 100 μL pipette. Finally, a drop of mineral oil (Sigma, St Louis MO) was added at the top of each reaction well to reduce the evaporation of the samples at elevated temperatures.

The microplate reactions were heated simultaneously, in two degrees of increase, from 40 to 70 ° C, using a RoboCycler® Gradient Temperature Cycler. (Stratagene, La Jolla, CA). After each heating step, before the fluorescence scan, the sample was cooled to 25 ° C. Fluorescence was measured using a CytoFluor II fluorescence microplate reader (PerSerptive Biosystems, Framingham, MA). The 1,8-ANS was excited with light at a wavelength of 360 nm. The fluorescence emission was measured at 460 nm.

Here we found six conditions that stabilized this enzyme with a? Tm greater than 1.0 ° C: (1) 0.5 M (NH4) S04, (2) 0.5 MgSO4, (3) 0.5 M Li2S04, (4) 0.5 M KCl (5) ) 0.1 M tripolyphosphate, since tri-polyphosphate is a polyelectrolyte that mimics heparin and other sulphated oligosaccharides, and its binding to proteins suggests the presence of a site that binds heparin, something that is well known is Factor Xa. Similarly, Ca2 + is known to bind to the Gla domain of Factor Xa, which is consistent with the stabilization effect seen for 0.1 M CaCl2. -_ Some of the metal ions found have a strong destabilizing effect on Factor Xa. For example, [Co (NH3) 6] Cl3, BaCl2, CdCl2, YC12 and NiS04 were observed to destabilize Factor Xa from 6 to 17 ° C. The reason for this destabilization is unknown. It is possible that these metal ions preferentially bound to the split form of Factor Xa. Some interference with the fluorescence probe is also possible.

Example 4 Library of the Functional Sounding Screening against the Human D (I) FGFRl The compounds in Plate 1 of the functional probe library were also used to generate an activity spectrum for D (II) FGFR1. The D (II) FGFR1 was cloned and expressed in Recombinant D (II) FGFR1 of E. col i was renatured from the inclusion of bodies essentially as described (Wetmore, DR et al., Proc. Soc. Mtg., San Diego, CA .._ (1994)), except that a hexa-histidine tag was included. in the N-terminus to facilitate recovery by chromatographic affinity on a Ni2 + chelate column (Janknecht, R. et al., Na ti.Ac d.Sci. USA 88: 8972-8976 (1991)). In addition, D (II) FGFR1 was purified in a column with heparin-sepharose (Kan, M. et al., Sci in ce 259: 1918-1921 (1993), Pantoleano, MW et al., Bi ochemi s try 33: 10229-10248 (1994)). The purity was > 95%, as judged by SDS-PAGE. The protein D (II) FGFR1 was concentrated up to 12 mg / mL (-1 mM) and stored at 4 ° C.

The reactions were prepared in a polycarbonate microtiter plate with 96 wells with a V background. The final concentration of D (II) FGFR1 was 50 μM in 200 mM tris. HCl, pH 8 in each well of a polycarbonate microtiter plate with 96%. The final concentration of 1,8-ANS was 100 μM. The final concentration of each of the molecules tested for binding is shown in Figure 7. The contents were mixed by repeated taking and discharging at the tip of a 100 μL pipette. Finally, a drop of mineral oil was added (Sigma, St. Louis MO. ) at the top of each reaction well to reduce the evaporation of samples at elevated temperatures.

The reactions of the microplate were heated simultaneously, in two degrees of increase, from 25 to 60 ° C, using a RoboCycler® Gradient Temperature Cycler (Stratagene, La Jolla, CA). After each heating step, before the fluorescence scan, the sample was cooled to 25 ° C. Fluorescence was measured using a CytoFluor II fluorescence microplate reader (PerSeptive Biosystems, Framingham, MA). The 1,8-ANS was excited with light with a wavelength of 360 nm. The fluorescence emission was measured at 460 nm.

The resulting activity spectrum was shown in Figure 7. A large number of compounds were found to stabilize D (II) FGFR1. For example, all sugars, D (+) - glucose, D (+) - sucrose, xylitol and sorbitol were found to all stabilize (and presumably bind) to D (II) FGFR1. This result may be consistent with the known heparin binding properties of this protein. The tri-polyphosphate, a mimic of heparin polyelectrolyte, produces a large change: approximately 11 ° C. This result is consistent with the heparin binding properties of this protein (Pantoleano, M. W. et al., Bi och emi s try 33: 10229-10248 (1994)).

Thus, in a situation where a user does not know anything about this protein (as is typically the case when a new gene is cloned the function of the encoded protein is unknown), the information obtained is screened only the compounds on Plate 1 could provide a user with some evidence that D (II) FGFR1 could be classified as a protein that binds heparin.

Example 5 Identification of White Proteins Containing DNA Linkages The repressor l a c is usually the tetrameric protein, a dimer of dimers. However, this protein has been shown to bind DNA in its di eric state. Lewis et al. resolved the crystal structure of Lac repressor by binding to its cognate DNA ligand (Lewis et al., 1996, Sci in ce 271: 1247-1254). A genetically altered dimer, one that is unable to form a tetramer, and a 21-meroligonucleot gone synthetic, the palindromic sequence of the native l a c operator, were obtained by Dr.

Mitch Lewis at the University of Pennsylvania.

The linkage of the synthetic operator l a c with the repressor l a c mutant was tested using the microplate heat exchange test.

The final concentration of repressor l a c was 60 μM in 200 mM tris. HCl, pH 8. Reactions were prepared in a polycarbonate microtiter plate with 96 wells with V-bottom. The final concentration of 1,8-ANS was 100 μM. The final concentration of each of the molecules tested for binding is shown in Figure 7. The contents were mixed by repeated taking and discharging at the tip of a 100 μL pipette. Finally, a drop of mineral oil (Sigma, St Louis MO) was added at the top of each reaction well to reduce the evaporation of the samples at elevated temperatures.

The microplate reactions were heated simultaneously, in two degrees of increase, from 25 to 75 ° C, using a ROBOCYCLER® Gradient Temperature Cycler (Stratagene, La Jolla, CA). After each heating step, before the fluorescence scan, the sample was cooled to 25 ° C. Fluorescence was measured using a CytoFluor II fluorescence microplate reader (PerSerptive Biosystems, Framingham, MA). The ANS was excited with light at a wavelength of 360 nm. The fluorescence emission was measured at 460 nm.

In the presence of 80 μM of the synthetic operator DNA, the Tm for the transition from the repressor cleavage l to c was changed to 5.6 ° C (Figure 8). The Kd calculated at a Tm is 6 μM. Using approximations for "HL" ~ (-10.0 Kcal / mol), the Kd calculated at 25 ° C is 1.2 μM and the Kd calculated at a physiological temperature (37 ° C) is 3.4 μM. the fluorescence probe, 1,8-ANS, does not bind to DNA alone (for example, there is no fluorescence signal for the control reaction where no repression was incubated).

These results show that the microplate heat exchange assay can be used as an assay for DNA / protein interactions.

Example 6 ATP Link Assays Adenosine triphosphate (ATP) and ATP analogue linkage can be assayed using the microplate heat exchange assay. Bovine muscle myosin (Sigma), the 3 '-5' cAMP-dependent protein of the bovine heart (Sigma), and chicken muscle pyruvate kinase (Sigma) each of these were dissolved in the Shock absorber A to generate the solutions with a final concentration of 2 mg / mL. Magnesium chloride (MgCl2), adenosine triphosphate, adenosine triphosphate -? - S (ATP -? - S), aluminum trifluoride (A1F3), and sodium fluoride (NaF) were dissolved in the Shock absorber A (50 mM HEPES, pH 7.5, 100 M NaCl) at the concentrations used in each experiment. Dapoxil ™ 12800 solution was prepared by diluting a 20 mM storage of Dapoxil 12800 ™ (sodium salt of 5- (4"-dimethylaminophenyl) -2- (4'-phenyl) oxazole sulfonic acid, Molecular Probes, Inc. ) in dimethyl sulfoxide to the appropriate concentration in Shock Absorber A.

In the ATP and the ATP -? - S reactions, each sample contained 12 μL of the protein storage solution (2 mg / mL), 9.6 μL of each ATP or ATP -? - S (50 mM), 4.8 μL of MgCl2 (100 mM) and 21.6 μL of a 222 uM solution of Dapoxil 12800 in Buffer A. In the reactions of ATP, aluminum trifluoride, and sodium fluoride, each sample contained 12 μL of the storage solution of the protein (2 mg / mL), 9.6 uL of ATP (50 mM), 9.6 mL of aluminum trifluoride (50 mM) + sodium fluoride (50 mM), 4.8 μL of 100 mM MgCl2, and 12 μL of a solution of 400 uM of Dapoxil 12800 in Shock Absorber A.

For the thermal shift test, four 10 μL aliquots of each test mixture were supplied in four wells located in different quadrants of a 384 well MJ Research thermocycle plate. Then 10 μL of mineral oil was added to each of the wells to prevent evaporation. Each data at the point shown was collected by heating the plate at the temperature shown for three minutes, for example, the plate was heated to a given temperature, and then allowed to cool to 25 ° C for one minute, followed by illumination with V and the collection of the data. Then the plate was heated to the next higher temperature, and something similar. The illumination with V was developed using an illumination with long wavelength at 200-420 nm, having a peak at 365 nm. The fluorescence image was projected using a CCD camera having a band pass filter centered at 550 nm.

Figure 9 shows the results of an ATP microplate heat exchange assay with bovine muscle myosin data were plotted as the fluorescence intensity as a function of temperature. The Tm of the thermal splitting curve (without ATP) was 49. 3 ° C (well microplate K2). The Tm of the thermal splitting curve for bovine muscle myosin bound to ATP ((+) ATP) was 51.4 ° C (well microplate K16). Thus the? Tm for the ATP binding was 2.1 ° C. The Kd was 440 μM.

Figure 10 shows the result of a microplate heat exchange assay of ATP -? - S with the kinase protein dependent on 3 A 5'-cAMP. The data was plotted as the fluorescence intensity as a function of temperature. The Tm of the control thermal splitting curve (without ATP -? - S) was 46.2 degrees centigrade- (microplate well E14). The Tm of the thermal splitting curve for the 3 A 5 '-cAMP-dependent kinase protein binding ATP -? - S ((+) ATP -? - S) was 51.8 ° C (well microplate M15). So? Tm for the ATP -? - S link was 5.6 ° C. The Kd was 200 μM. The results, including the results of pyruvate kinase, are summarized in Table 4.

Table 4. Summarizes the results of enzymes that bind ATP. The value in the parentheses is the standard deviation.

ATP -? - S ATP ATP + AIF3 (10 mM) (10 mM) (10 mM) Protein Reference? Tm? Tm? Tm Myosin 49.4 0.0 (± 0.2) 2.2 (± 0.4) 2.8 (± 0.4) Protelna 44.7 5.6 (± 1.7) 7.5 (± 0.7) 8.2 (± 1.4) kinase 3 '-5' cAMP Piruvate 54.5 0.8 (± 0.11) -0.44 (± 0.1) -0.27 (± 0.2) kinase Example 7 Folic Acid Link Assay The folic acid linkage can be assayed using the microplate thermal exchange assay. Hydrofolate reductase from bovine liver (DHFR, Sigma), chicken liver hydrofolate reductase (DHFR, Sigma), pigeon liver arylamine acetyltransferase (ArAcT. Sigma), pig liver formimino glutamic transferase (FGT, Sigma) ) each was dissolved in Shock Absorber A (50 mM HEPES, pH 7.5, 100 mM NaCl) to generate storage solutions up to the final concentration of 2 mg / mL. Solutions of dihydrofolic acid (FAH2), methotrexate, nicotinamide adenine dinucleotide phosphate (NADP), were prepared by dissolving solid material in Shock Absorber A immediately before use. The Daproxil ™ 12800 solution was prepared by diluting a 20 mM stock of Dapoxil ™ 12800 in dimethyl sulfoxide to an appropriate concentration in Shock Absorber A.

Each test sample contained 12 ~~ μL of the protein storage solution (2mg / mL), 4.8uL of dihydrofolic acid (FAH2) or methotrexate storage solution (lmM), and 31.2 μL of a solution of 154 μM of Dapoxil ™ 12800 in Shock Absorber A. Each sample contained 12 μL of the protein storage solution (2 mgs / mL), 4.8 μL of NADP storage solution (50 mM), and 31.2 μL of a solution of 154 μM Dapoxil ™ 12800 in Shock absorber A.

For the thermal change test, four aliquots of 10 μL of each test sample were placed in four wells located in different quadrants of a thermocyclic plate with 384 MJ Research wells. Then it was added μL of mineral oil to each of the four wells to prevent evaporation. Each data point shown was collected by heating the plate to the temperature shown for three minutes, followed by incubation at 25 ° C for one minute, followed by UV light illumination and data collection. The results are shown in Table 5.

Table 5. Results of the proteins that bind methotrexate, FAH2 and NADP. The value in the parentheses is the standard deviation.

Methotrexate FAH2 NADP (100 μM) (100 μM) (5 mM) Protein Reference? Tm? Tm? Tm tffi DHFR 52.47 7.0 (± 0.1) -0.64 (± 0.2) 3.2 (± 0.13) DHFR 56.6 8.6 (± 0.2) 2.5 (± 0.2) 3.8 (± 0.4) Arylamine 49.8 1.0 (± 0.4) -1.8 (± 0.5) 2.8 (± 0.4) Acetyltransferase Acid 47.2 0.9 (± 0.5) 3.26 (± 0.4) 0.0 (± 0.2) Formimino L-glutamic trans ferasa ~ Example 8 Methotrexate / NADP Linkage Assay (H) The ability to measure the temperature change of the methotrexate and NADPH bond, separately and simultaneously, is an orthodox example of the utility of the present invention in measuring multi- ligand binding interactions. In this case, the binding sites of two ligands are close, and this cooperates positively in the binding of two ligands, as shown by the fact that the heat exchange of both ligands are simultaneously linked are 2-4 degrees greater than the total of the changes of each link of the ligands separately (Table 6).

The binding of methotrexate (MTX) and NADPH can be assayed using the microplate heat exchange assay. The bovine liver hydrofolate reductase (DHFR, Sigma), and chicken liver hydrofolate reductase (DHFR, Sigma), each were dissolved in Buffer A_ (50 mM HEPES, pH 7.5, 100 mM NaCl) to generate solutions of storage until the final concentration of 2 mg / mL. The storage solutions of the ligands were prepared by dissolving solid material in Shock Absorber A immediately before use. The nicotinamide adenine dinucleotidephosphate reduced (NADPH) solutions, 100 mM), NADP (100 mM), and methotrexate, (1 mM) were further diluted in Shock Absorber A to twice the final assay concentration (2x storages): methotrexate (200 μM), NADP (20mM), NADPH (20 mM), methotrexate + NADP (200 μM + 20 mM), methotrexate + NADPH (200 μM + 20 M). The Dapoxil ™ 12800 solutions were prepared by diluting a 20 mM stock of Dapoxil ™ 12800 in dimethyl sulfoxide to an appropriate concentration in Shock Absorber A. 5 μL of each protein storage solution was added to 25 μL of 2 x buffer storage solution. Ligand was mixed with 20 μL of a 250 μM Dapoxil ™ 12800 solution in Shock Absorber A.The final concentration of the ligand was 10 mM NADP; 10 mM NADPH; and 100 μM MTX.

For the thermal change test, four aliquots of 10 μL of each test sample were placed in four wells located in different quadrants of a thermocyclic plate with 384 MJ Research wells. Then 10 μL of mineral oil was added to each of the four wells to prevent evaporation. Each data point shown was collected by heating the plate to the temperature shown for three minutes, followed by incubation at 25 ° C for one minute, followed by UV light illumination and data collection.

Figure 11 shows the result of a microplate heat exchange microplate test of methotrexate with dihydrofolate reductase. The data is plotted as the fluorescence intensity as a function of temperature. The Tm of the control thermal splitting curve (without MTX) was 47.2 ° C (microplate well Ml). The Tm of .J.a thermal splitting curve for DHFR that binds methotrexate ((+) MTX) was 56.4 ° C (well microplate G6). Thus, the? Tm for the methotrexate that is bound was 9.2 ° C. The Kd was 24 nM.

Figure 12 shows the result of a NADPH microplate heat exchange test with dihydrofolate reductase. The data is plotted as the intensity of fluorescence as a function of temperature. The Tm of the control thermal splitting curve (without NADPH) was 50.8 ° C (well microplate G8). The Tm of the thermal splitting curve for DHFR to bind the NADPH ((+) NADPH) was 53.8 ° C (well microplate B20). Thus the? Tm for the NADPH binding was 3 ° C. The Kd was 0.7 μM.

Table 6. Complex ligands with DHFR of? Tm. The values in parentheses is the standard deviation a The value shown in the sum of the individual? T of the protein incubated separately with each ligand. b The value shown in the sum of the? T observed when the protein was incubated simultaneously with both ligands.

Example 9 Hydrofolic acid is a substrate of dihydrofolate reductase (DHFR). Methotrexate is a folic acid analogue that binds to DHFR. As evidence that the method of the present invention is reliable, it has been shown that the method can be used to detect the binding of dihydrofolic acid to DHFR. DHFR from bovine liver was combined with 80 compounds for screening for protein function, and binding with methotrexate, but several compounds were not detected.

Each well of the plate of the compounds of the microfuente # 198104 contained one of the 80 different compounds with a concentration of 10 mM in dimethyl sulfoxide. Each solution of the compound was diluted in Buffer A (50mM HEPES, pH 7.5, 100mM NaCl) to a final concentration of 200 μM in separate wells in a polystyrene plate with 384 wells. 5 μL of the solution contained in each well was transferred to an MJ Research polypropylene plate containing 5 μL of DHFR from bovine liver (at a concentration of 0.5 mg / mL and Dapoxil ™ 12800 dye at a concentration of 200 μM, producing a final concentration of 100 μM of ligand, 0.25 mg / mL of DHFR, and 100 μM of Dapoxil in the volume of 10 L of each well. μL of mineral oil was added to each well to prevent evaporation. The thermal splitting profiles were then measured in each of the wells from 25 to 70 ° C, when collecting the data in the points at each temperature, it was separated by an increment of one degree. Each data point was collected by heating the plate at the temperature shown for 3 minutes, followed by incubation at 25 ° C for one minute, followed by illumination with UV light with long wavelength and data collection using a CCD camera.

Data were collected with four replicates of 80 compounds in the quadrants of a plate with 384 wells. The four quadrants consist of: wells A2 to Hll (first quadrant), wells Al 4 to H23 (second quadrant), wells 12 to Pll (third quadrant), and wells 114 to 123 (fourth quadrant). Columns 1, 12, 13, and 24 consist of reference wells containing only DHFR and dimethyl sulfoxide.

Wells F2, F14, N2 and N14 contained methotrexate. The link was revealed when entering into the programming system as a red well. Methotrexate changed the Tm from 5.13 ± 0.19 (average of 4 quadrants), and the other compounds in the plate had little or no effect (shown as wells close to the target). These results indicate that DHFR binds methotrexate. - All publications and patents mentioned above are incorporated in their entirety by reference.

Since the above invention has been described in detail for the purpose of clarity and understanding, it will be appreciated by a person skilled in the art in reading this discussion that various changes in form and detail can be made without departing from the scope of the invention and the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (30)

Claims

1. A method for functionally classifying a protein, characterized in that the method comprises: (a) screening one or more functionalities of different molecules for its ability to modify the stability of the protein, wherein the modification of the stability of the protein indicates that the molecule it binds to the protein; (b) generate the activity spectrum of the protein of the screening step (a), where the spectrum of activity reflects a subset of molecules, of the multiplicity of different molecules, which modify the stability of the protein and therefore are ligands that bind to the protein; (c) comparing the spectrum of activity of the protein with one or more list of the functional reference spectrum; and (d) classifying the protein according to the group of molecules in the multiplicity of different molecules that modify the stability of the protein.

2. The method of claim 1, characterized in that the screening step (a) comprises: (a) contacting the protein with one or more of the multiplicity of different molecules in each multiplicity of the containers; (a2) treating the protein in each multiplicity of vessels to cause the protein to unfold; (a3) measuring in each of the containers a physical change associated with the unfolding of the target molecule; (a4) generating a cleavage curve of the target molecule for each vessel; _y (a5) comparing each of the splitting curves in step (d) with (1) each of the splitting curves and with (2) the splitting curve obtained from the protein in the absence of any multiplicity of different molecules; and (a6) determine if any of the multiplicity of different molecules modifies the stability of the protein, where a change in stability is indicated by the change in the splitting curve.

3. A method for functionally classifying a protein, characterized in that the method comprises: (a) screening one or more functionalities of different molecules known to bind a particular class of proteins for their ability to modify the stability of the protein, wherein the modification of the stability of the protein indicates that the molecule binds to the protein; (b) generate a spectrum of activity of the protein of the screening step (a), where the activity spectrum reflects a subgroup of molecules, of the multiplicity of different molecules, which modify the stability of the protein and therefore they are ligands that bind to the protein; and (c) classifying the protein as a member of the protein class if one or more of the multiplicity of different molecules modify the stability of the protein.

4. The method of claim 3, characterized in that the screening step (a) comprises: (a) contacting the protein with one or more of the multiplicity of different molecules in each multiplicity of the containers; (a2) treating the protein in each multiplicity of vessels to cause the protein to unfold; (a3) measuring in each of the containers a physical change associated with the unfolding of the target molecule; (a4) generating a cleavage curve of the target molecule for each vessel; and (a5) comparing each of the splitting curves in step (d) with (1) each of the splitting curves and with (2) the splitting curve obtained from the protein in the absence of any multiplicity of different molecules; and (aß) determine if any of the multiplicity of different molecules modifies the stability of the protein, where a change in stability is indicated by the change in the splitting curve.

5. A method for functionally classifying a protein, characterized in that the method comprises: classifying the protein according to the group of molecules in a multiplicity of different molecules that modify the stability of the protein.

6. A method for functionally classifying a protein that is capable of unfolding due to thermal change, characterized in that the method comprises: (a) screening one or more of the multiplicity of different molecules for their ability to change the thermal cleavage curve of the protein, wherein the change in the thermal cleavage curve of the protein indicates that the molecule binds to the protein; (b) generate a spectrum of activity of the protein of the screening step (a), where the spectrum of activity reflects a subset of molecules, of the multiplicity of different molecules, which change the thermal cleavage curve of the protein and by they are therefore ligands that bind to the protein; (c) comparing the spectrum of activity of the protein with one or more of the lists of the functional reference spectrum; and (d) classifying the protein according to the group of molecules that change the thermal splitting curve of the protein.

7. The method of claim 6, characterized in that the screening step (a) comprises: (a) contacting the protein with one or more of the multiplicity of different molecules in each multiplicity of the containers ^ (a2) heating the multiplicity of the containers of step (al); (a3) measuring in each of the containers a physical change associated with the unfolding of the white molecule caused by the heating; (a4) generating a thermal splitting curve of the target molecule as a function of the temperature for each of the containers; and (a5) comparing each of the splitting curves in step (d) with (1) each of the thermal splitting curves and with (2) the splitting curve obtained from the protein in the absence of any multiplicity of different molecules; and (aß) determine if any of the multiplicity of different molecules changes the thermal splitting curve of the protein.

8. The method of claim 7, characterized in that the step of comparing (a5) comprises locating the molecules at the multiplicity of different molecules for the protein according to the capacity of each multiplicity of the different molecules to change the thermal splitting curve of the protein .

9. The method of claim 7, characterized in that the heating step (a2), the multiplicity of the containers are heated simultaneously.

10. The method of claim 7, characterized in that step (a4) further comprises determining a midpoint temperature (Tm) of the thermal split curve; and wherein step (a5) further comprises comparing the Tm of each of the splitting curves in step (a4) with (1) the Tm of each of the thermal splitting curves and with (2) the Tm of the thermal splitting curve obtained for the target protein in the absence of any other molecule.

11. The method of claim 7, characterized in that step (a3) comprises measuring the absorbance of light by the contents of each of the containers.

12. The method of claim 7, characterized in that step (al) comprises contacting the protein with a fluorescence probe molecule present in each multiplicity of vessels and wherein step (a3) comprises (i) exciting the molecule of fluorescence probing, with light, in each multiplicity of vessels; and (ii) measuring the fluorescence of each multiplicity of vessels.

13. The method of claim 12, characterized in that step (a3) (ii) further comprises measuring the fluorescence of each multiplicity of containers one container at a time.

14. The method of claim 12, characterized in that step (a3) (ii) further comprises measuring the fluorescence of a. sub-group of the multiplicity of recipients simultaneously.

15. The method of claim 12, characterized in that step (a3) (ii) further comprises measuring the fluorescence of each multiplicity of containers simultaneously.

16. The method of claim 7, characterized in that step (a3) comprises (i) exciting light with the tryptophan residues in the protein, in each multiplicity of vessels; and «(ii) measuring the fluorescence of each multiplicity of vessels.

17. The method of claim 7, characterized in that the multiplicity of containers in step (al) comprises a multiplicity of wells in a microplate.

18. A method for unionally classifying a protein that is capable of unfolding due to thermal change, characterized in that the method comprises: (a) screening one or more multiplicities of different molecules known to bind a particular kind of protein for its ability to change the curve of thermal splitting of the protein, where the thermal splitting curve of the protein indicates that the molecule binds to the protein; (b) generating the activity spectrum of the protein of the screening step (a), where the spectrum of activity reflects a subgroup of molecules, of the multiplicity of different molecules, which change the thermal cleavage curve of the protein and by they are therefore ligands that bind to the protein; (c) classify the protein as a member of the protein class if one or more of the multiplicities of the different molecules change the thermal splitting curve of the protein.

19. The method of claim 18, characterized in that the screening step (a) comprises: (a) contacting the protein with one or more of the multiplicity of different molecules in each multiplicity of the containers; (a2) heating the multiplicity of the containers of step (al); (a3) measuring in each of the containers a physical change associated with the thermal splitting of the white molecule caused by the heating; (a4) generating a thermal splitting curve of the target molecule for the target molecule as a function of the temperature for each vessel; and (a5) compare each of the splitting curves in step (a4) with (1) each of the thermal splitting curves and with (2) the thermal split curve obtained from the protein in the absence of any multiplicity of different molecules; and (aß) determine if any of the multiplicity of different molecules changes the thermal splitting curve of the protein.

20. The method of claim 19, characterized in that the step of comparing (a5) comprises locating the molecules at the multiplicity of different molecules for the protein according to the ability of each multiplicity of the different molecules to change the thermal splitting curve of the protein

21. The method of claim 19, characterized in that the heating step (a2), the multiplicity of the containers are heated simultaneously.

22. The method of claim 19, characterized in that step (a4) further comprises determining a midpoint temperature (Tm) of the thermal split curve; and wherein step (a5) further comprises comparing the Tm of each of the splitting curves in step (a4) with (1) the Tm of each of the thermal splitting curves and with (2) the Tm of the thermal splitting curve obtained for the target protein in the absence of any other molecule ^.

23. The method of claim 19, characterized in that step (a3) comprises measuring the absorbance of light by the contents of each of the containers.

24. The method of claim 19, characterized in that step (al) comprises contacting the protein with a fluorescence probe molecule present in each multiplicity of vessels and wherein step (a3) comprises (i) exciting the molecule of fluorescence probing, with light, in each multiplicity of vessels; and (ii) measuring the fluorescence of each multiplicity of vessels.

25. The method of claim 24, characterized in that step (a3) (ii) further comprises measuring the fluorescence of each multiplicity of containers one container at a time.

26. The method of claim 24, characterized in that step (a3) (ii) further comprises measuring the fluorescence of a subgroup of the multiplicity of containers simultaneously.

27. The method of claim 24, characterized in that step (a3) (ii) further comprises measuring the fluorescence of each multiplicity of containers simultaneously.

28. The method of claim 19, characterized in that step (a3) comprises (i) exciting light with the tryptophan residues in the protein, in each multiplicity of vessels; and (ii) measuring the fluorescence of each multiplicity of vessels.

29. The method of claim 18, characterized in that the multiplicity of containers in step (al) comprises a multiplicity of wells in a microplate.

30. A method for functionally classifying a protein capable of unfolding due to thermal change, characterized in that the method comprises: classifying the protein according to the group of molecules in a multiplicity of different molecules that change the thermal splitting curve of the protein. High Processing Method for as car Functionally Identified Proteins Using a Genomic Method Summary of the Invention The present invention provides a method for functionally classifying a protein that is capable of unfolding due to thermal change. The method comprises screening one or more functionalities of different molecules for its ability to change the thermal splitting curve of the protein, where the change of the thermal splitting curve indicates that the molecule binds to the protein or affects the stability in a protein. measurement means; generate the spectrum of activity of the protein where the activity spectrum reflects a group of molecules, the multiplicity of different molecules, which change the thermal splitting curve of the protein and therefore are ligands that bind to the protein, compare the spectrum of activity of the protein with one or more list of the functional reference spectrum; and classify the protein according to the group of molecules in the multiplicity of different molecules that change the thermal splitting curve of the protein.