MXPA98004456A - Method of analysis based on fluorescence, to identify ligan - Google Patents

Abstract

A novel method for analyzing chemical compounds to determine potential pharmaceutical effectiveness is described. The method described identifies possible therapeutic test ligands by placing them in the presence of target proteins and determining the ability of the test ligands to avoid partial denaturation of the target protein by measuring the fluorescence emission of a conformation-sensitive probe. This differs significantly from the known methods of testing novel pharmaceutical substances in that it is not necessary to know the biochemical function of the target protein and the existence of any known ligand of the target protein is necessary.

Description

METHOD OF ANALYSIS BASED E? FLUORESCENCE, TO IDENTIFY LIGANDS FIELD OF THE INVENTION This invention pertains to novel methods based on high performance fluorescence for pharmaceutical compounds, in particular those that bind to proteins involved in the pathogenesis of diseases or in the regulation of a physiological function.

BACKGROUND OF THE INVENTION Pharmaceutical substances can be developed from starting compounds that are identified by a randomly targeted process of analysis, such as a receptor. Large-scale analysis approaches can be complicated by many factors. First, many trials are laborious or costly to perform. The analysis may involve experimental animals, cell lines or tissue cultures that are difficult or expensive to acquire or maintain. They may require the use of radioactive materials and therefore have safety and waste disposal problems. These considerations often establish REF: 27570 practical limitations on many compounds that can be reasonably analyzed. Therefore, those who use random methods of analysis are often forced to limit their search to those compounds for which prior knowledge suggests that the compounds are likely to be effective. This strategy limits the scope of the tested compounds, and many useful medications are overlooked. In addition, the specificity of many biochemical assays can exclude a wide variety of useful chemical compounds, because the interactions between the ligand and the receptor protein are outside the scope of the assay. For example, many proteins have multiple functions, while most of the assays are able to observe only one such activity. With such a specific assay, many potential pharmaceutical substances may not be detected. Finally, in most of the existing biochemical analysis tests for drug discovery, the activity of the target protein must be exhibited. This requires that the system in question be well characterized before the analysis begins. Even when the sequence of a protein is known as in, for example, a newly cloned gel, the specific functions of the protein can not be revealed simply by analysis of its sequence, consequently, a biochemical analysis for therapeutic drugs directed against many proteins The objective must await a detailed biochemical characterization, a process that generally requires expensive research. Therefore, there is a need in the art for a high-throughput, cost-effective and rapid assay that allows the analysis of large amounts of compounds to determine their ability to bind to therapeutically or physiologically important proteins. In addition, there is a need in the art for methods of analysis that are independent of the biological activity of the target proteins and that detect compounds that bind to regions of the target proteins other than the biologically active domains.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a fluorescence-based method for identifying a ligand that binds to a target protein. According to the method, compounds that are not known to bind to the target protein are selected as test ligands. The target protein is incubated with each of the test ligands individually to produce a test combination, and in the absence of a test ligand, to produce a control combination. The test and control combinations are contacted with a conformation-sensitive fluorescence probe, ie, a probe that preferentially binds to a folded, denatured or globular molten state of a protein whose fluorescence properties are in no way altered by the folding state of the target protein. The combinations are treated to cause a detectable fraction of the target protein to exist in a partially or completely denatured state. After, the degree to which the target protein is presented in a folded state, a denatured state or a molten globular state, or combinations thereof, in the test and control combinations, is measured by observing the fluorescence emission intensity of the probe. When a difference in fluorescence intensity or other fluorescence property is present between the test and control combinations, this indicates that the target protein is present in the doubled state to a greater or lesser degree in the test combination than in the combination of control, and the ligand tested is a ligand that binds to the target protein. In a preferred embodiment, the steps of the method are repeated in a high throughput mode using a plurality of test compounds until a ligand is identified.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a gel profile of SDS-polyacrylamide of carbonic anhydrase after proteolysis in the absence and presence of increasing concentrations of acetazolamide. Figure 2 shows a gel profile of SDS-polyacrylamide of carbonic anhydrase after proteolysis in the absence and presence of 1.0 mM acetazolamide, in the absence or presence of a fungal extract. Figure 3 shows a graph showing a titration of the binding of radiolabeled human neutrophil elastase to nitrocellulose filters after proteolysis in the absence and presence of increasing concentrations of elastatinal. Figure 4 shows a graph depicting an ELISA detection titre of human neutrophil elastase after proteolysis in the presence of increasing concentrations of IC 200,355. Figure 5 shows a graph depicting the distribution of test ligands identified as elastase ligands of human neutrophils.

Figure 6 shows a graph representing the titration of a ligand for elastase of human neutrophils. Figure 7 shows a graph depicting the titration of five ligands to determine their ability to inhibit the enzymatic activity of human neutrophil elastase. Figure 8 shows a graph depicting a binding titre of human hemoglobin to nitrocellulose filters after proteolysis in the absence or presence of increasing concentrations of 2,3-diphosphoglycerate. Figure 9 shows a graph depicting an ELISA detection titre of human hemoglobin after proteolysis in the presence of increasing concentrations of 2,3-diphosphoglycerate. Figure 10 shows a graph representing the distribution of the test ligands identified as ligands of human hemoglobin S. Figure 11 shows a graph representing the titration of a ligand for human hemoglobin. Figure 12 shows a graph illustrating the effects of concentrations on, increase of guanidinium hydrochloride (GC1) in the fluorescence emission of bis-l-anilino-8-naphne sulfonate (bis-ANS) measured in the absence and presence of carbonic anhydrase I. Figure 13 shows a graph illustrating the time-dependent change in fluorescence emission intensity of bis-l-anilino-8-naphthalene sulfonate (bis -ANS) measured in the presence of carbonic anhydrase I and the indicated concentrations of hydrochloride of guanidinium (GC1). Figure 14 shows a graph illustrating the effect of increasing concentrations of a carbonic anhydrase ligand, acetazolamide, on the increase in fluorescence emission intensity of bis-1-anilino-8-naphthalene sulfonate measured in the presence of anhydrase carbonate I and guanidinium hydrochloride 2M.

DETAILED DESCRIPTION OF THE INVENTION All applications for patents, patents and literature references mentioned in this specification are incorporated in the presence as a reference in their entirety. In case of conflict, the present description, including the definitions, will prevail.

Definitions As used herein, the term "ligand" refers to an agent that binds to a target protein. The agent can bind to the target protein when the target protein is in its native conformation, or when it is partially or completely denatured. According to the present invention, a ligand is not limited to an agent that binds to a recognized functional region of the target protein, for example, the active site of an enzyme, the site of an antigen combination of an antibody, the site hormone binding of a receptor, a cofactor binding site, and the like. In the practice of the present invention, a ligand can also be an agent that binds to any surface or internal sequences or conformational domains of the target protein. Therefore, the ligands of the present invention encompass agents that in themselves and in addition to themselves may have no apparent biological function, beyond its ability to bind to the target protein in the manner described above. As used herein, the term "test ligand" refers to an agent, which comprises a compound, molecule or complex, which is tested for its ability to bind to a target protein. The test ligands can be virtually any agent, including, without limitation, metals, peptides, proteins, lipids, polysaccharides, nucleic acids, small organic molecules, and combinations thereof. Complex mixtures of substances such as natural product extracts can also be tested, which can include more than one test ligand, and the component that binds to the target protein can be purified from the mixture at a subsequent stage. Suitable compounds as test ligands can be found, for example, in libraries of natural products, fragmentation libraries (encompassing plants and microorganisms), combinational libraries, compound files and libraries of synthetic compounds. For example, libraries of synthetic compounds are commercially avble from Maybridge Chemical Co.(Trevillet, Corn all, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack NH), and Microsource (Nes) Milford, CT). A library of rare chemicals is available from Aldrich Chemical Company, Inc.

(Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of extracts of bacteria, fungi, plants and animals are available, for example, from Pan Laboratories (Bothell, WA) or from MycoSearch (NC), or can be produced easily. Additionally, natural and synthetically produced libraries and compounds are easily modified by conventional chemical, physical, and biochemical means (Blondelle et al., TibTech 14:60, 1996), preferably using automated equipment, to allow simultaneous analysis of a multiplicity of test compounds. As used herein, the term "target protein" refers to a peptide, protein or protein complex for which identification of a ligand or binding partner is desired. Target proteins include, without limitation, peptides or proteins that are known or considered to be involved in the etiology of a given disease, condition or physiological state, or in the regulation of a physiological function. Target proteins can be derived from any living organism, such as a vertebrate, particularly a mammal and even more particularly a human. For use in the present invention, it is not necessary to specifically identify the biochemical function of the protein. Target proteins include, without limitation, receptors, enzymes, oncogenic products, tumor suppressor gene products, viral proteins, and transcription factors, either in purified form or as part of a complex mixture of proteins and other compounds. In addition, the target proteins may be constituted of wild type proteins, or alternatively, mutant proteins or variants including those with stability, activity or other altered variant properties, or hybrid proteins to which extraneous amino acid sequences have been added, for example , sequences that facilitate its purification. As used herein, "test combination" refers to the combination of a test ligand and a target protein. "Control combination" refers to a target protein in the absence of a test ligand. As used herein, the "folded state" of a protein refers to the native or non-denatured form of the protein as it is present in its natural environment, or after isolation or purification, i.e., prior to exposure to conditions denaturing. Similarly, the "denatured state" refers to a situation in which the polypeptide has lost elements of its secondary and / or tertiary structure that are present in its "folded state". It will be recognized by those familiar with the art that it is difficult to experimentally determine at what point the polypeptide has been completely denatured, ie, it has lost all its elements of secondary and tertiary structure. Therefore, the term "denatured state", as used herein, encompasses partial or total denaturation. As used herein, "detectable moiety" refers to an amount that is determined empirically that varies based on the method used to differentiate a folded protein from a denatured protein. For example, when fluorescent probes are used to monitor the degree of folding of the target protein, conditions are chosen so that the changes in fluorescence are of an easily detectable magnitude. When protease sensitivity is used to monitor folding, conditions are chosen (for example, by adjusting the temperature or adding denaturants) so that approximately 80% of the target protein is digested within a convenient incubation period. When antibodies specific for the folded or denatured state of a target protein are used as the detection method, the conditions are chosen so that a sufficient amount of antibody is adhered to provide a detectable signal. The present invention encompasses high throughput analysis methods to identify a ligand that binds to a target protein. If the target protein to which the test ligand binds is associated with, or is causative of, a disease or condition, the ligand may be useful for diagnosis, prevention or treatment of the disease or condition. A ligand identified by the present method can also be one which is used in a purification or separation method, such that a method results in the purification or separation of the target protein from a mixture. The present invention also relates to ligands identified by the present method and their therapeutic uses (for diagnostic, prevention or treatment purposes) and uses in purification and separation methods. In accordance with the present invention, a ligand for a target protein is identified by its ability to influence the degree of folding or the folding or denaturation rate of the target protein. Experimental conditions are chosen so that the target protein is subjected to denaturation, either reversible or irreversible. If the test ligand binds to the target protein under these conditions, the relative amount of folded target protein: denatured or the folding or denaturing rate of the target protein in the presence of the test ligand will be different, ie the amount will be greater or less than that observed in the absence of the test ligand. Therefore, the present method encompasses the incubation of the target protein in the presence or absence of a test ligand, under conditions in which (in the absence of a ligand) the target protein would be partially or totally denatured. This is followed by analysis of the absolute or relative amounts of folded target protein relative to the denatured or fold or denaturation rate of the target protein. An important feature of the present invention is that it will detect any compound that binds to any sequence or domain of the target protein, not only to sequences or domains that are intimately related to biological activity or function. The sequence, region or binding domain may be present on the surfaces of the target protein when it is in its folded state, or it may be buried inside the protein. Some binding sites only become accessible to the ligand binding when the protein is partially or completely denatured. In the practice of the present invention, the test ligand is combined with a target protein, and the mixture is maintained under appropriate conditions for a sufficient time to allow binding of the test ligand to the target protein. The experimental conditions for each target protein are determined empirically. When multiple test ligands are tested, the incubation conditions are chosen so that most of the ligand: target protein interactions are expected to proceed to completion. In general, the test ligand is present in a molar excess relative to the target protein. The target protein may be in insoluble form or, alternatively, may be bound to a solid phase matrix. The matrix may comprise, without limitation, spheres, membrane filters, plastic surfaces or other suitable solid supports. For each target protein, appropriate experimental conditions are chosen, for example, temperature, time, pH, salt concentration and additional components, so that the detectable fraction of the protein is present in denatured form in the absence of the test ligand. For a target protein that is irreversibly denatured, the conditions of the preferred experimental conditions allow a detectable amount of the protein to be denatured during a convenient incubation period in the absence of the test ligand. To adjust or optimize the ratio of folded protein: denatured or the folding or denaturing rate, denaturing conditions may be required which include the use of elevated temperatures, the addition of chaotropes or denaturing agents such as urea or guanidinium or guanidinium salts such as guaninium thiocyanate, detergents or combinations thereof. In addition, mutant proteins containing stabilizing or destabilizing amino acid substitutions can be used in relation to the wild-type version of the protein to manipulate the folded: denatured protein ratio. The time necessary for the binding of the target protein to the ligand will vary based on the test ligand, test protein or other conditions used. In some cases, the binding will occur instantaneously (for example, essentially simultaneous with a combination of the test ligand and the target protein), while in other cases, the test-target protein ligand combination is maintained for a long time, by example of up to 12-16 hours, before the union is detected. When many test ligands are used, an incubation time is chosen so that it is sufficient for most protein: ligand interactions. In the case of target proteins that are irreversibly denatured, the rate of denaturation must also be taken into account to determine the appropriate time for binding to the test ligand. The binding of a test ligand to the target protein is determined by comparing the absolute amount of folded or denatured target protein in the absence and presence of the test ligand, or, alternatively, by determining the proportion of folded target protein: denatured or the speed of folded or denatured target protein in the absence and presence of the test ligand. If a test ligand binds to the target protein (ie, if the test ligand is a ligand for the target protein), the target protein may be significantly more folded, and less denatured, (and therefore, a higher proportion). elevated of folded target protein with respect to denaturation) than that which is present in the absence of a test ligand. Alternatively, the binding of the test ligand can result in a target protein significantly less folded, and more denatured than that which is present in the absence of a test ligand. Similarly, binding of the test ligand can cause the rate of folded or denatured target protein to change significantly. In any case, the determination of the absolute amounts of folded and denatured target protein, the proportion of folded protein: denatured or the folding or denaturing rates can be carried out using one of the known methods as described below. These methods include, without limitation, measuring the fluorescence emission of specific probes for conformation, proteolysis of the target protein, binding of the target protein to appropriate surfaces, binding of specific antibodies to the target protein, binding of the target protein to molecular chaperons. , binding of the target protein to immobilized ligands, and aggregation measurement of the target protein. Other physico-chemical techniques can also be used, either alone or together with the previous methods; These include, without limitation, measurements of circular dichroism, intrinsic ultraviolet spectroscopy and fluorescence, and calorimetry. A preferred embodiment involves comparing the fluorescence emission of a specific probe for conformation in the presence of a target protein after incubation in the absence and presence of a test ligand. Typically, this involves contacting the test and control combinations with the fluorescence probe before treatment, (such as, for example, elevated temperature) which is used to manipulate the degree of folding. However, it will be recognized by those familiar with the art that each target protein may have unique properties that revert to a particular mass detection method suitable for the purposes of the present invention. For the purposes of a high throughput analysis, the experimental conditions described above are adjusted to obtain a threshold ratio of test ligands identified as "positive" compounds or ligands from the total compounds analyzed. This threshold is established according to two criteria. First, the number of positive compounds must be manageable in practical terms. Secondly, the number of positive compounds should reflect ligands with an appreciable affinity towards the target protein. A preferred threshold is obtained when it is shown that 0.1% to 1% of the total test ligands are ligands of a given target protein. The binding to a given protein is a prerequisite for pharmaceutical substances designed to directly modify the action of that protein. Therefore, if it is demonstrated, by use of the present method, that a test ligand binds to a protein that reflects or alters the etiology of a condition, it may indicate the potential ability of the test ligand to alter the function of the protein and to be an effective pharmaceutical substance or an initial compound for the development of such a pharmaceutical substance. Alternatively, the ligand can serve as the basis for the construction of hybrid compounds that contain an additional component that has the potential to alter the function of the protein. In this case, the binding of the ligand to the target protein serves to fix or orient the additional component so that its pharmaceutical effects are carried out. For example, a known compound that inhibits the activity of a family of related enzymes can be made specific for a family member by conjugation of the known compound to a ligand, identified by the methods of the present invention, which binds specifically to that member in a different site than recognized by the known compound. The fact that the present method is based on physical-chemical properties common to most proteins allows its wide application. The present invention can be applied in large-scale systematic high-throughput procedures that allow cost-effective analysis of many thousands of test ligands. Once a ligand has been identified by the methods of the present invention, it can be further analyzed in greater detail using known methods specific to the particular target protein used. For example, the ligand can be tested for binding to the target protein directly, for example, by incubating radiolabeled ligand with unlabeled target protein, and subsequently separating the ligand bound to unbound ligand protein. In addition, the ligand can be tested to determine its ability to alter, positively or negatively, a known biological activity of the target protein. In a preferred embodiment of the present invention, binding of the test ligand to a target protein is detected by the use of molecular probes whose fluorescence emission characteristics are sensitive to the conformation of the target protein. Certain fluorescent compounds show only a weak fluorescence emission when they are free in an aqueous solution (Semisotnov, et al, Biopolymers 31: 119, 1991), but fluoresce much more strongly when bound to organized hydrophobic surfaces. The binding of these compounds to fully folded globular proteins is typically weak, since the hydrophobic residues are buried predominantly in the interior of the protein. In addition, the binding of these compounds to random helical conformations (such as those found in completely denatured polypeptides) is also disadvantaged, because, in these conformations, the hydrophobic residues, although exposed, are not sufficiently well organized to support the binding of high affinity of the probes. However, the probes typically bind with superior affinity and stoichiometry to compact conformations of unfolded protein, often referred to as "fused pellets," which are characterized by their compact condition relative to the unfolded, random helical states, the presence of a substantial secondary structure and the lack of a single total conformation. The initially melted globular states were identified as compact denatured states that are stably presented for various proteins under specialized conditions such as low pH, moderate concentrations of denaturant and heat (Ptitsyn et al., Mol. Biol. 17: 569, 1983). . The molten globule has been identified as a common intermediary state in the protein folding process (Ptitsyn et al., FEBS Letts, 262: 20, 1990). Without wishing to be bound by any theory, it is considered that the molten globule states show hydrophobic surfaces organized sufficiently to support the attachment of fluorescent probes useful as affinities in the micromolar range. Fluorescent molecules useful in the practice of the present invention (hereinafter referred to as "probes") include, without limitation, 1-anilino-8-naphthalene sulfonate (ANS), bis-1-anilino-8-naphthalene sulfonate ( bis-A? S) and 6-propionyl -2- (?,? -dimethyl) -aminonaphthalene (Prodan) (Molecular Probes, Eugene, OR).

It will be understood that any fluorescent compound can be used to the extent that fluorescent properties are substantially altered upon binding to a protein and that it preferentially binds to the molten globule or other denatured forms of the particular target protein. The only limitations are that the relative binding affinities and binding stoichiometry of the probe should be of a magnitude that ensures that a change in fluorescence observed by conformational change between the native and molten globular states can be readily detected. Preferably, the concentration of the probe used in the practice of the invention is low enough to avoid substantial destabilization of the folded form. For example, the native DnaK protein binds bis-ANS with a stoichiometry of 1: 1 and a dissociation constant of 7.0 μM; whereas DnaK in the molten globule state binds bis-ANS with a stoichiometry of 3: 1 and dissociation constants of 2.0, 5.1, and 39 μM (Shi et al., Biochemistry 33: 7536, 1994). Exposure of protein DnaK μM bis -ANS 60 μM results in conformational changes in DnaK consistent with the conversion from a native conformation to a molten globular. However, lower concentrations of bis -ANS can be used as an indicator of conformational change, according to the present invention. For example, the addition of 1 μM DnaK to 1 μM bis -ANS improves the fluorescence of bis -ANS by almost 30-fold as a result of the binding of the molten globular state of DnaK. The addition of a ligand, ATP, to the mixture reduces the fluorescence enhancement by about five times. In this embodiment, a target protein is contacted with a probe in the presence or absence of a test compound (i.e., combinations of both test and control), under conditions in which at least a portion of the target protein is partially denatured. After an appropriate incubation period, usually one to ten minutes, the test and control combinations are irradiated, with light of an appropriate wavelength to excite the probe, and the emission of fluorescence is measured at an appropriate wavelength. for the particular probe. If the test compound is a ligand of the target protein, the amount of protein in the molten globule state (for intermediate folding) must be reduced by the presence of the test compound, which will be reflected in a decrease in the binding of the probe and a corresponding reduction in the intensity of the fluorescence emission.

In another embodiment, the binding of the test ligand to the target protein is detected by the use of proteolysis. This assay is based on the increased susceptibility of the denatured polypeptides to protease digestion in relation to the folded proteins. In this case, the test ligand-target protein combination, and a control combination lacking a test ligand, are treated with one or more proteases that act preferentially on the denatured target protein. After an appropriate incubation period, the intact target protein level is determined, ie, without proteolysis, using one of the methods described below, eg, gel electrophoresis and / or immunoassay. There are two possible results that indicate that the test ligand has been bound to the target protein. An absolute amount can be observed whether significantly greater or significantly lower intact or degraded protein in the presence of ligand, compared to its absence. Proteases useful in the practice of the present invention include, without limitation, trypsin, chymotrypsin, V8 protease, elastase, carboxypeptidase, proteinase K, thermolysin, and subtilisin (all of which can be obtained from Sigma Chemical Co., St.

Louis, MO). The most important criterion for selecting a protease or proteases for use in the practice of the present invention is that the protease or proteases must be able to digest the particular target protein under the chosen incubation conditions and that this activity is preferentially directed towards the denatured protein To avoid "false positive" results caused by test ligands that directly inhibit the protease, more than one protease can be used simultaneously, particularly proteases with different enzymatic action mechanisms, or in parallel assays. In addition, cofactors that are necessary for the protease or protease activity are provided in excess to avoid false positive results because the test ligands can sequester these factors. Typically, a purified target protein is first taken to a final concentration of 2-100 μg / ml in a buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM calcium acetate and 0.034 mg / ml bovine serum albumin. Subsequently proteinase K and thermolysin are added, proteases with different mechanisms of action, up to a final concentration of 2-10 μg / ml. Subsequently, parallel incubations are carried out for different periods of time ranging from 5 minutes to 1 hour, at temperatures ranging from 20 ° C to 65 ° C. The reactions are suspended by the addition of phenylmethylsulfonyl chloride (PMSF) to a final concentration of 1 mM and ethylenediaminetetraacetic acid (EDTA) to a final concentration of 20 mM. Subsequently, the amount of intact protein remaining in the reaction mixture at the end of the incubation period is determined by any of the following methods: polyacrylamide gel electrophoresis, ELISA or binding to nitrocellulose filters. The above protocol allows the selection of appropriate conditions that result in digestion of approximately 80% of the target protein, indicating that a significant degree of denaturation has occurred. If a known ligand for the target protein is available, the ligand is included in the reaction mixture at a concentration of 20-200 μM, and the experiment is repeated. Typically, at least a two-fold increase or decrease in the level of intact target protein is observed, indicating that the binding of a known ligand changes the ratio of folded target protein: denatured and / or the folding or denaturing rate . Once the conditions for a high throughput test as described above are set, the protocol is repeated simultaneously with a large number of test ligands at concentrations ranging from 20 to 200 μM. The observation of an increase or decrease of at least two times in the level of intact protein means a "functional" (successful) compound, that is, a ligand that binds to the target protein. Preferred conditions are those in which between 0.1 and 1% of the test ligands are identified as "functional" compounds using this procedure. In another embodiment, the relative amount of denatured and folded target protein in the presence and absence of the test ligand is determined by measuring the relative amount of target protein that binds to an appropriate surface. This method takes advantage of the increased propensity of the denatured proteins to adhere to surfaces, which is due to an increased surface area and decreased masking of the hydrophobic residues resulting from denaturation. If a test ligand binds to a target protein (i.e., it is a ligand for the target protein), it can stabilize the folded form of the target protein and decrease its binding to a solid surface. Alternatively, a ligand can stabilize a denatured form of the protein and increase its binding to a solid surface. In this embodiment, the target protein, the test ligand and a surface that preferentially binds denatured protein are combined and maintained under conditions appropriate for binding of the target protein to a ligand and binding of the denatured target protein to the surface. Alternatively, the target protein and the test ligand can be pre-incubated in the absence of the surface to allow binding. The surfaces suitable for this purpose include, without limitation, microtiter plates constructed from a variety of treated or untreated plastics, treated plates for tissue culture or for high protein binding, nitrocellulose filters and PVDF filters. The determination of the amount of surface bound target protein or of the amount of target protein remaining in the solution can be carried out using standard methods known in the art, for example, determination of radioactivity or immunoassay. If a significantly greater or lesser amount of target protein is bound to the surface in the presence of a test ligand, compared to the absence of the test ligand, the test ligand is a ligand for the target protein. Similarly, the proportion of target protein bound to surface: soluble will be significantly greater or less in the presence of a test ligand than in its absence, if a test ligand is a ligand for the target protein. In another embodiment, the degree to which the folded and denatured target protein is present in the test combination is determined by the use of antibodies specific for either the denatured state or the folded state of the protein, i.e. denaturing ("DS") or specific for naturalization ("NS") respectively. (Bryer, 1989, J. Biol. Chem., 264 151: 13348-13354). Specific DS and NS polyclonal and monoclonal antibodies can be prepared for particular target proteins by methods that are well known in the art (E. Harlow &; D. Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988; Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1987). For DS antibodies, animals can be immunized with a peptide from a region of the protein that is buried in the region of the protein when it is in its native state. If the three-dimensional structure of the protein is unknown, antibodies are prepared against several peptides and then analyzed for denatured state preferential binding. For NS antibodies, the intact non-denatured protein is used as an immunogen, and the resulting antibodies are analyzed for preferential binding to the native protein and purified for use in the present invention. DS or NS antibodies can be used to detect ligand-induced change at the level of the folded target protein, denatured target protein, the folded: denatured ratio or the folding or denaturation rate. In one approach, a test combination containing the DS antibody, the target protein and the test ligand are exposed to a solid support, for example a microtiter plate coated with the denatured target protein or a peptide fragment thereof, under appropriate conditions to bind the target protein with its ligand and the binding of the DS antibody to denature the target protein. A control combination is processed in the same way as the test solution, which is equal to the test combination except that it does not contain the test ligand. By comparing the amount of antibody bound to the plate or the amount remaining in solution in the test and control combinations, the difference in the folding of the target protein is detected. The amount of antibody bound to the plate or remaining in solution can be measured as described in the following. In a second approach, a test combination containing the DS antibody, the test ligand and the target protein is exposed to a solid support coated with a second antibody termed a solid-phase antibody, which can not bind to the protein target simultaneously with the DS antibody, and is specific for the target protein, but is specific for the folded state, (NS antibody), or is unable to differentiate between the native and denatured states ("non-differentiating" or "ND" antibody). The resulting test combination or solution is maintained under conditions appropriate for binding of the target protein with a ligand of the target protein and for binding of the antibodies to the proteins it recognizes. A control combination which is the same as the test solution except that it does not contain a test ligand is processed in the same way as the test solution. In both combinations, the denatured (unfolded) target protein binds to the DS antibody and inhibits binding to the antibody in the solid phase. The ability of the test ligand to bind the target protein can be gauged by determining the amount of target protein that binds the solid phase antibody in the test solution and comparing it to the amount to which the target protein binds with the antibody in question. solid phase, in the absence of test ligand, which in turn reflects the amount of target protein in the folded state. The amount of target protein can be detected to the plate via the second antibody or remnant in solution by the methods described below. This approach can be used in a manner comparable to the NC antibody as the soluble antibody, and the DS or ND antibody in the solid phase. In a third approach, the test solution containing the target protein and the test ligand is exposed to a solid support, for example, a microtiter plate that has been coated with DS or NS antibody and kept under appropriate conditions for binding of the target protein to its ligand and for the binding of the antibody to the target protein. Alternatively, the antibody may be present on the surfaces of spheres. The ability of the test ligand to bind the target protein is gauged by determining the degree to which the target protein remains in solution (not bound to the antibody) or on the solid surface (bound to the antibody), or the ratio of the two, in the presence and absence of the test ligand. Alternatively, the antibody can be present in solution and the target protein can be attached to a solid phase, such as a plate surface or sphere surface. In another embodiment, molecular chaperons are used to determine the relative levels of folded and denatured protein in a test combination. Chaperones encompass known proteins that bind denatured proteins as part of their normal physiological function. They are generally involved in the assembly of oligomeric proteins, in ensuring that certain proteins fold correctly, in facilitating localization of proteins and in preventing the formation of proteinaceous aggregates during physiological stress (Hardy, 1991, Science, 251: 439-443 ). These proteins have the ability to interact with many denatured or partially denatured proteins without specific recognition of defined sequence motifs. A molecular chaperone, found in E. coli, is a protein known as SecB. It has been shown that SecB is involved in exporting a subset of proteins that are otherwise unrelated. Competing experiments have shown that SecB binds tightly to all of the denatured proteins tested, which include outer proteins from its particular export subgroup, but does not appear to interact with the folded protein. Other chaperones suitable for use in the present invention include, without limitation, heat shock proteins 70s, heat shock protein 90s, GroEI and GroES (Gehting et al., Nature 355: 33, 1992). In this embodiment, a test combination containing the test ligand and the target is exposed to a solid support, for example to a myrotein plate or other suitable surface coated with a molecular chaperone, under conditions appropriate for protein binding target with its ligand and molecular chaperone binding to the denatured target protein. The denatured target protein in the solution will have a greater tendency to bind to the surface coated with molecular chaperone relative to the ligand-stabilized target protein. Thus, the ability of the test ligand to bind target protein can be determined by determining the amount of target protein that remains unbound, or the amount bound to the surface coated with chaperone. Alternatively, a competition assay for molecular chaperone binding can be used. A test combination containing purified target protein, the test ligand and a molecular chaperone can be exposed to a solid support, eg, a microtiter well coated with denatured target protein, under conditions appropriate for binding of the target protein with its ligand and molecular chaperone binding to the denatured target protein. A control combination is processed in the same way, which is the same as the test combination except that it does not contain the test ligand. The denatured target protein in its section will bind to the chaperone and in this way its binding to the denatured target protein bound to the support is inhibited. The binding of a test ligand to the target protein will result in a difference in the amount of denatured target protein and, therefore, more or less chaperone will be available to bind to the solid phase denatured target protein as compared to the case in that there is an absence of binding of the test ligand. Therefore, the binding of the test ligand can be determined by determining the chaperone bound to the surface or in solution in the test combination and in the control combination, and by comparing the results. In this trial, chaperones are generally not provided in excess, so that competition for their union can be measured. Alternatively, a test combination containing the target protein, the test ligand and a molecular chaperone can be exposed to a solid support, for example a microliter well that has been coated with antiserum or a monoclonal antibody specific for the folded target protein. (NS antibody) and unable to bind the target protein that is attached to the chaperone. The denatured target protein will bind to the chaperone in solution and will therefore be inhibited to bind to the antibody in the solid phase. By detecting the target protein in the solution or attached to the well walls and comparing the degree of either or both of them in an appropriate control (the same combination without the test ligand), the capacity of the ligand of test to bind target protein. If the test ligand is a ligand for the target protein, more target protein will bind to the antisera or monoclonal antibodies bound to the surface of the 'container in the test combination, compared to the control combination, and correspondingly more will be present. or less unbound target protein (in solution) in the test combination, compared to the control combination. In another embodiment, a known ligand, cofactor, substrate or analogue thereof of the target protein is used to determine the presence of the folded target protein. The greater the fraction of the protein in folded form, the greater the amount of protein that is available to bind to a ligand that binds exclusively to the folded state. Consequently, if a protein has a known ligand, it is possible to increase or decrease the binding of the protein to the known ligand by adding a test ligand that binds to another site of the protein. For example, the binding of dihydrofolate reductase to methotrexate, a folic acid analog, can be used to determine the level of folding of this enzyme. In this approach, the ligand, cofactor, substrate or analog thereof which is known to bind to the target protein is immobilized on a solid substrate. Then a solution containing the target protein and the test ligand is added. An increase or decrease in the amount of target protein that binds to the immobilized compound relative to an identical assay in the absence of the test ligand indicates that the test ligand binds to the target protein. The amount of target protein bound to the solid substrate can be determined by sampling the solid substrate or by sampling the solution. In another modality, the amount of denatured target protein in a test combination is determined by measuring protein aggregation. For proteins that are irreversibly denatured, the denatured protein often forms insoluble aggregates.

The degree of protein aggregation can be measured by techniques known in the art, including, without limitation, light scattering, centrifugation and filtration. In this approach, the target protein and the test ligand are incubated, and the amount of aggregation protein is measured with respect to time or after a fixed incubation time. The extent of protein aggregation in the test mixture is compared to the same measurement for a control assay in the absence of the test ligand. If the test ligand binds to the target protein, the rate of denaturation of the target protein will be lower or higher than in the absence of the test ligand. For measurements with respect to time, the rate of appearance of the folded protein will be lower or higher if the test ligand is a ligand for the target protein compared to the case where it is not. For fixed time measurements, there will be more or less denatured protein and corresponding less or more aggregated protein if the test ligand is a ligand for the target protein, as compared to the case where it is not. Therefore, the ability of a test ligand to bind target protein can be determined by determining the degree of protein aggregation in the presence and absence of the test ligand.

The modalities described in the above are summarized in the following table.

TABLE DETERMINATION OF TARGETED AND UNNATURALIZED OBJECTIVE PROTEIN Protein detection methods The embodiments described in the foregoing require a final step to detect and / or quantify the level of target protein or digestive products thereof, or antibodies, in order to quantify the relative amounts of folded or denatured target protein after exposure to the test ligands. In the practice of the present invention, methods known in the art are used to detect the presence or absence of protein, small peptides or free amino acids. The method used will be determined by the product to be detected (proteins, peptides, free amino acids). For example, techniques for detecting protein size can be used in order to determine the degree of proteolytic degradation of the target protein, for example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, high performance liquid chromatography and Similar. Measurements of radioactivity, fluorescence or enzymatic activity can detect the presence or absence of products, either in solution or on a solid support. Immunological methods including, for example, ELISA and radioimmunoassay can detect the presence or absence of a known target protein in solution or on a substrate. The above methods are described in, for example, Harlow, E. and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, 1988; S.F.Y. Li, Capillary Electrophoresis, Elsevier Press, 1993; Bidlingmeyer, Practical HPLC Methodology and Applications, John Wiley and Sons, Inc., 1992; and Cantor, C.R. and P.R. Schimmel, Biophysical Chemistry, WH Freeman and Co., 1980. In a preferred embodiment, gel electrophoresis is used to detect the presence or absence of protein, and can also be used to detect the size of the protein. This latter method is especially useful in conjunction with proteolysis, since the presence of a greater or lesser amount of undigested target protein in the test combination compared to the control combination indicates that the test ligand has been bound to the target protein. The following examples are designed to illustrate the invention, without limitation thereto.

Example 1: Methotrexate binding protects dihydrofolate reductase (DHFR) from proteolytic digestion by proteinase K The following is combined and incubated at 54 ° C for minutes: DHFR (100 μg / ml), Proteinase K, (80 μg / ml), Tris-HCl 0.1 M, pH 7.5, and methotrexate at 10"10 to 10" 4 M. Samples are extracted and DHFR quantified undigested by means of ELISA as follows: (a) protease incubations are diluted 50-fold with buffered saline with Tris (TBS); (b) 50 μl of diluted samples are transferred to wells of an ELISA plate and incubated at 60 minutes at room temperature; (c) wells from the plate are carefully washed with TBS plus 0.1% Tween-20 (TBST); (d) 50 μl of rabbit serum is added to each well diluted 250 times in TBST plus 5% dry milk without fat, and incubated for 30 minutes at room temperature, (e) the wells of the plate are washed as in subsection (c) above; (f) 50 μl of goat alkaline phosphatase conjugate and antibodies against rabbit IgG are added, diluted 500 times in TBS plus 5% milk, and incubated for 30 minutes at room temperature; (g) the wells of the plate are washed as in part (c); and (h) 0.1 ml of 1.0 mg / ml p-nitrophenyl phosphate in 0.1% diethanolamine is added. The color development is proportional to the bound conjugate of antibody and alkaline phosphatase. ELISA analysis shows that methotrexate protects DHFR from digestion at concentrations of 10"8M and above.For the same methods, it is shown that nicotinamide dinucleotide phosphate (NADPH) and dihydrofolate, at concentrations of 10" 5M and above, inhibit the proteolysis of DHFR, in separate experiments.

Example 2: The binding of methotrexate, NADPH and dihydrofolate protect dihydrofolate reductase (DHFR) from proteolytic digestion by proteinase K in the presence of a mixture of amino acids The following are combined and incubated at 54 ° C for 5 minutes: DHFR (2.1 μg / ml), proteinase K (80 μg / ml), TriS-HCl 0.1M (pH 7.5), 10 SM of all the 20 common amino acids and either zero or 10"SM of ligand.The ligands used were the methotrexate inhibitor and the substrate dihydrofolate and NADPH.The samples are extracted and undigested DHFR is quantified by ELISA as follows: (a) incubations are diluted protease 50-fold with saline buffered with Tris (TBS), (b) transfer 50 μl of diluted samples to the wells of an ELISA plate and incubate for 60 minutes at room temperature, (c) carefully wash the wells of the plate with TBS plus 0.1% Tween-20 (TBST); (d) 50 μl of serum is added to each well against rabbit DHFR diluted 250 times in TBST plus 5% dry milk without fat, and incubated for 30 minutes at room temperature, (e) the wells of the plate are washed as in part (c) above, (f) they are added to each well 50 pl of alkaline phosphatase conjugate of goat and antibodies against rabbit IgG, diluted 500 times in TBST plus 5% milk, and incubated 30 minutes at room temperature; (g) the wells of the plate are washed as in part (c); and (h) 0.1 ml of 1.0 mg / ml p-nitrophenyl phosphate in 0.1% diethanolamine is added. The color development is proportional to the conjugate of antibody and bound alkaline phosphatase. The ELISA analysis shows that methotrexate and substrates protect DHFR from digestion in relation to the absence of ligands that bind to DHFR. Therefore, specific binding can be detected in the presence of a complex mixture of compounds that do not bind to the target protein.

Example 3: Methotrexate binding inhibits the binding of DHFR to microliter plates The following was combined in a volume of 60 μl and incubated in a microliter plate Falcon 3072"treated for tissue culture" at 20 or 47 ° C: 100 mg DHFR, 50 mM Tris-Cl (pH 7.5), and methotrexate, 10"10 to 10" 4 M. Subsequently, 50 μl of each sample is transferred to wells of an ELISA plate, and the DHFR that remains in solution is quantified by ELISA, as follows: (a) 50-well samples are incubated. μl for 60 minutes at room temperature; (b) the wells of the plate are carefully washed with TBS plus 0.1% Tween-20 (TBST); (c) 50 μl of serum against rabbit DHFR diluted 250 times in TBST plus 5% non-fat dry milk is added to each well and incubated for 30 minutes at room temperature; (d) the pozos of the plate are washed as in subsection (b) above; (e) 50 μl of goat alkaline phosphatase conjugate and antibodies against rabbit IgG are added to each well, diluted 500 times in TBST plus 5% milk, and incubated 30 minutes at room temperature, - (f) washed the wells of the plate as in part (b); and (g) 0.1 ml of 1.0 mg / ml of D-nitrophenyl phosphate in 0.1% diethanolamine is added. The color development is proportional to the conjugate of bound alkaline phosphatase antibody. Analysis by ELISA reveals that methotrexate inhibits the binding of DHFR to Falcon 3072 plates at concentrations of 10"7M and above.

Example 4: Inhibition of the binding increase of denatured specific antibody (1) ELISA plates are coated by incubation for 60 minutes with the following mixture: 4 μg / ml irreversibly denatured target protein or peptide fragments thereof, in Tris buffered saline (Tris-Cl, 10 mM, pH 7.5, NaCl 0.2M; TBS). (2) Plates are washed 3 times with TBS plus 0.1% Tween-20 (TBST). (3) The following mixture is incubated (total volume 50 μl) in the wells of the microliter plate for 60 minutes: (a) Antibody specific for the denatured state of the target protein at a sufficient concentration to provide 50% of the maximum binding (in the absence of the competition target protein) ). (b) Target protein at a sufficient concentration to obtain 90% inhibition of antibody binding to the plate. The concentration of appropriate target protein differs for each target protein. The concentration depends, in part of the stability of the folded form of the target protein. In some cases it may be desirable to reduce the stability of the target protein by high temperature, inclusion of chemical denaturing agents of the protein or introduction of destabilizing amino acid substitutions in the target protein. (c) Test ligands 10"9 to 10" s M. (d) Dry milk without 5% fat, in TBST. (4) The plates are washed 3 times with TBST. (5) Add 50 μl of goat alkaline phosphatase conjugate against IgG, conjugate in an appropriate dilution in TBST plus 5% non-fat dry milk and incubate for 30 minutes at room temperature. (6) The plates are washed three times with TBST. (7) 0.1 ml of 1.0% / D-nitrophenyl phosphate in 0.1% diethanolamine is added and the amount of color development is recorded by means of a plate reader in ELISA. The ELISA analysis will reveal that the plaque has bound more or less antibody to the plaque when a successful binding has been presented between the test-target protein ligand compared to the absence of such binding.

Example 5: Inhibition or improvement of chaperone binding (1) ELISA plates are coated by incubation for several hours with 4 μg / ml of chaperone in TBS. (2) The plates are washed three times with TBST. (3) Subsequently, the following mixture (total volume, 50 μl) is incubated in the coated wells of 10 microtiter plates for 60 minutes: (a) target protein at a concentration sufficient to saturate approximately 50% of the available binding sites present in the chaperone proteins. Denaturing conditions can be used in cases where the folded form of the target protein is otherwise too stable to allow appreciable binding to chaperones. (b) Test ligands 10"9 to 10" SM, in TBST. (4) Aliquots are transferred in well solutions to wells of a new ELISA plate and incubated for 60 minutes at room temperature. (5) The wells of the plate are washed 3 times with TBST. (6) Add 50 μl of antibody specific for the target protein to the appropriate dilution in TBST, plus 5% dry milk without fat, and incubate for 30 minutes at room temperature. (7) The wells of the plate are washed 3 times with TBST. (8) 50 μl of goat alkaline phosphatase conjugate against rabbit IgG is added to each well at an appropriate dilution in TBST plus 5% non-fat dry milk, and incubated for 30 minutes at room temperature. (9) The plate wells are washed three times with TBST. (10) 0.1 ml of 1.0 mg / ml p-nitrophenyl phosphate in 0.1% diethanolamine is added. The color development (proportional to the bound alkaline phosphatase antibody conjugate) is monitored with an ELISA plate reader. The ELISA analysis will reveal that the target protein in the solution at higher or lower concentration when the ligand binding test-target protein has occurred compared to when it has not occurred.

Example 6: Improvement or inhibition of binding to a known ligand (1) The following mixture (total volume, 50 μl) is incubated in wells coated with a microliter plate for 60 minutes: (a) Ligand known to bind to the target protein, covalently bound to solid spheres such as Sephadex. This ligand may be a small molecule or a macromolecule. (b) Target protein at a concentration well below the saturation of the ligand so that only 10% of the protein binds to the ligand sites. The solution conditions are such that most of the target protein is present in its denatured state. (c) Test ligands 10"9 to 10" 5 M (d) in TBST plus the necessary denaturant such as urea. (2) Aliquots of the supernatant from the wells (free of spheres) are transferred to wells of a new ELISA plate and incubated for 60 minutes at room temperature. (3) The wells of the plate are washed 3 times with TBST. (4) Add 50 μl of antibody specific for the target protein to the appropriate dilution in TBST, plus 5% dry milk without fat, and incubate for 30 minutes at room temperature. (5) Be well washed plate 3 times with TBST. (6) 50 μl of goat alkaline phosphatase conjugate is added to each well against rabbit IgG at an appropriate dilution in TBST plus 5% milk, and incubated for 30 minutes at room temperature. (7) The wells of the plate are washed three times with TBST. (8) 0.1 ml of l.O mg / ml of p-nitrophenyl phosphate in 0.1% diethanolamine is added. The color development (proportional to the bound alkaline phosphatase antibody conjugate) is monitored with an ELISA plate reader. The ELISA analysis will reveal a higher or lower concentration of the target protein in the solution when a successful binding has occurred between the test ligand and the target protein.

Example 7: Low throughput assay for carbonic anhydrase ligands The ligand binding test to carbonic anhydrase I (Sigma) is performed using proteolysis as a probe of the folding of the target protein, and denaturing gel electrophoresis is used as a method for detection of intact protein remaining after digestion with proteases. To validate the assay, acetazolamide, a known carbonic anhydrase ligand, is tested. Although acetazolamide is a known inhibitor of carbonic anhydrase activity, these experiments make use of this property and the enzymatic activity of the protein is not measured. In addition, the sensitivity of the method to interfere with an extract of natural product is examined.

The reaction mixtures contain 13.3 μg / ml of carbonic anhydrase, 0.05 M Tris-HCl, pH 7.5, 0.01 M calcium acetate, 2.5 μg / ml proteinase K, 10% DMSO and acetazolamide (Sigma) in concentrations ranging from 0.0 to 1.0 mM. The reactions are incubated at 54 ° C for 15 minutes, and then cooled in ice. Phenylmethylsulfonyl fluoride (PMSF) is then added from a 20 mM concentrated solution in ethanol, to a final concentration of 1 mM, and EDTA is added from a 0.5 M concentrated solution to a final concentration of 20 mM. 0.01 ml of SDS charge buffer (10% sodium dodecyl sulfate (SDS), 0.5 M dithiothreitol, 0.4 M Tris-HCl buffer, pH 6.8, 50% glycerol) are added and the samples are heated at 95 ° C for 3 hours. minutes The samples are analyzed by SDS-polyacrylamide gel electrophoresis using a gradient gel of 4-15% polyacrylamide (BioRad), which is then stained with Coomassie blue dye. As shown in Figure 1, the binding of the known carbonic anhydrase acetazolamide ligand ligand results in stabilization of carbonic anhydrase against proteinase K proteolysis at a concentration of acetazolamide of 1 x 10"5 M. It has been reported that the dissociation constant for this interaction is 2.6 x 10 'M (Matsumoto, K. et al. (1989), Chem. Pharm. Bull, 37: 1913-1915).

A mycotic methanolic extract is included in reactions that were otherwise identical to those described above, so that the final concentration of a small aggregated molecule will be equal to its concentration in the source culture. The presence of the extract does not induce a false signal and decreases the response to acetazolamide 1.0 mM (Figure 2).

Example 8: High performance assay for HIV Rev protein Reaction mixtures (0.03 ml of total volume) contain 30 μg / ml of HIV Rev proteins that have been produced in E. coli, 0.05 M Tris-HCl, pH 7.5, 0.01 M calcium acetate, 2.5 μg / ml proteinase K, 10% DMSO and variable amounts of tRNA as a known ligand. The reactions are incubated on ice for 15 minutes. After the addition of PMSF and EDTA as described in Example 7 above, the samples are prepared for gel electrophoresis and analyzed as described in Example 7. The results show that, in the absence of AR? T, the protein Rev is almost completely degraded by proteinase K under these conditions. However, in the presence of AR? T, a protein fragment of lower molecular weight is stabilized against proteolysis. Therefore, the binding of a known ligand to the HIV Rev protein is detectable using the methods of the present invention.

Example 9: High performance analysis of ligands for human neutrophil elastase.

In carrying out the present invention, the ability to perform the binding assay on large amounts of compounds is critical in determining its utility in the discovery of compounds with potential pharmaceutical utility. Two different approaches have been successfully implemented in a high throughput mode of analysis, and each of these has been applied to two target proteins: human neutrophil elastase (HNE) and human hemoglobin, both hemoglobin A (HbA) and hemoglobin (HbS) (described in Example 10 below). Notably, these target proteins differ from each other in numerous important aspects: HbS is an intracellular tetrameric protein that contains a prostatic group critical for its function. It is known to exist in two conformations with different structural and functional properties. In contrast, HNE is monomeric, lacks a prostate group and is secreted. HNE has enzymatic activity (proteolysis) and does not seem to undergo any conformational change. For a high throughput analysis with both target proteins, proteolysis is used as the probe for the folding of the target protein. The two high-throughput modes differ in the methods used to detect the residual target protein after proteolysis. The two methods of detection are: 1) capture of radiolabelled protein in nitrocellulose filters followed by quantification of bound radioactivity, and 2) measurement of the protein by enzyme-linked immunosorbent assay (ELISA). Each of these methods is used successfully with both hemoglobin and HNE.

A) Union to nl trocel slab of radiolabeled HNE: 0.1 mg of HNE (Elastin Products) are labeled by reaction with 12 sodium iodide (Amersham) in the presence of Iodogen (Pierce), according to the manufacturer's protocols (Pierce). Reaction mixtures are prepared in a final volume of 0.05 ml containing radiolabelled HNE (20,000 cpm, corresponding to approximately 10 μg), 0.025 mg / ml bovine serum albumin, 50 mM Tris-HCl, pH 7.5, calcium acetate 10 M, 2.5 μg / ml thermolysin (Boeringer Mannheim), 2.5 μg / ml proteinase K (Merck), 10% DMSO and the test compound at a concentration of 200 μM. The control mixtures were identical, except that the test compound is omitted. The mixtures are incubated at 20 ° C for 15 minutes, then at 65 ° C for 30 minutes, after which they are placed on ice. Subsequently 0.12 ml of 50 mM sodium acetate buffer, pH 4.5, are added to each mixture. After an additional 15 minutes of incubation on ice, the samples are filtered through nitrccellulose membrane sheets using Schleicher and Schuell Minifold. Each well of the apparatus is then washed once with 0.2 ml of 50 mM sodium acetate buffer, pH 4.5, and twice with 0.5 ml of 50 mM sodium phosphate, pH 5.5, containing 2.0% SDS and Triton X- 100 to 1.0%. After the filter is dried, the bound radioactivity is determined by scintillation counting using a Wallac MicroBeta apparatus. To validate the assay, a known ligand for HNE, elastatinal, at concentrations ranging from 1 to 5 mM is included in the assay. As shown in Figure 3, the inclusion of elastatinal increases the retention of marked HNE in the nitrocellulose filters, indicating that HNE is protected from proteolysis.

B) Quantification by ELISA of HNE: The reaction mixtures in a final volume of 0.05 ml contain 2 μg / ml of HNE, 0.020 mg / ml of bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 10 mM calcium acetate, 7.5 μg / ml of terinolysin ( Boeringer Mannheim), 7.5 μg / ml proteinase K (Merck), 10% DMSO and the test compound at a concentration of 20 or 200 μM. The control mixtures were identical except that the test compound is omitted. The mixtures are incubated at 20 ° C for 15 minutes, then at 63 ° C, 30 minutes and then placed on ice. Subsequently, 0.1 ml of rabbit antibody against HNE (Calbiochem) is added to each reaction at a dilution of 1: 10,000 in TBST (10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween-20) containing 5 ml. % of dry milk without fat (Carnation). After 10 minutes of incubation at room temperature, the mixtures are transferred to 96-well Immulon-4 plates (Dynatech) which has been coated with HNE by overnight incubation with 0.1 ml per well of 0.2 μg / ml of HNE in buffer of 50 mM sodium borate, pH 8.5, and 3 mM sodium azide, and then washed thoroughly with TBST. The. plates are subsequently incubated at room temperature for one hour, after which they are carefully washed with TBST. 0.1 ml of goat antibody is added to each well against rabbit IgG conjugated with alkaline phosphatase (Calbiochem) diluted 1: 1000 in TBST containing 5% dry milk without fat, and the plates are incubated at room temperature for l-2 hours. Subsequently the plates are washed carefully with TBST and finally with TBST lacking Tween. 0.1 ml per ml of p-nitrophenyl phosphate is added to each well (0.5 mg / ml) in diethanolamine IX substrate buffer (Pierce) The plates are incubated at room temperature until color develops, after which the absorbance of each well is measured at 405 nm using a BioRad 3550-UV microplate reader. To validate the assay, a known ligand for HNE, ICI 200,355, is included in the assay at concentrations ranging from 0.01 to 10 μM. As shown in Figure 4, the inclusion of the ligand causes an inhibition of antibody binding to the plate, indicating an increased level of immunoreactive HNE in the reaction mixtures.

C. Results of the performance analysis: 3,600 compounds were examined for interaction with HNE using proteolysis and detection by ELISA as above (figure 5). Of these, 24 inhibited the proteolysis of HNE by proteinase K to a degree of 50% or greater when tested at a concentration of 20 μM (positive functional compounds). Six additional compounds were found that increase the extent of proteolysis at least twice when tested at 20 μM (compounds with negative functionality). The dependence of the concentration of the effects of the functional compounds was measured. The functional compounds show maximum mean effects at concentrations as low as 8 μM; Figure 6 shows an example. The maximum inhibition usually, but not always, is close to 100%. The functional compounds are tested for their ability to inhibit the enzymatic activity of HNE. Since the compounds identified in the binding assay can be bound anywhere on the surface of the protein, it is expected that only a small fraction will inhibit the enzymatic activity of HNE. The compounds, inhibitors of the proteolysis of Suc- (Ala) 3-pNA (Elastin Products), a chromogenic synthetic substrate, are tested according to the method of Bieth, J. Spiess, B. and Wermuth, CG (1974, Biochemical Medicine, 11: 350-357). Two positive functional compounds and one negative functional compound inhibit the proteolytic activity of HNE significantly in these assays (Figure 7).

Example 10: High performance analysis of ligands for human hemoglobin A) Neocellulose binding of radiolabelled hemoglobin 0.2 mg of HbS or HbA (Sigma) are radiolabelled by reaction with 1 mCi of 12SI-Bolton-Hunter reagent (Amersham) in 100 mM sodium borate buffer, pH 8.5, on ice, for one hour. The labeling is stopped by addition of borate buffer containing 200 mM glycine. The mixture is then fractionated by size on a column of execelulose GF-5 (Pierce) in 50 mM sodium phosphate buffer, pH 7.5, containing 0.25% gelatin. For the binding assay, the reaction mixtures in a final volume of 0.05 ml contain radiolabeled hemoglobin (20,000 CPM), 0.063 mg / ml unlabeled hemoglobin, 0.034 mg / ml bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 10 mM calcium acetate, 2.5 μg / ml thermolysin (Boeringer Mannheim), 2.5 μg / ml proteinase K (Merck), 10% DMSO and test compound. The control mixtures were identical, except that the test compound is omitted. The mixtures are incubated at 20 ° C for 15 minutes, then at 40 ° C for 30 minutes and placed on ice. Subsequently 0.12 ml of 50 mM sodium acetate buffer, pH 4.5, is added to each mixture. After an additional 15 minutes of incubation on ice, the samples are filtered through nitrocellulose membrane sheets using the Schleicher and Schuell Minifold. Each well of the apparatus is subsequently washed once with 0.2 ml of 50 mM sodium acetate buffer, pH 4.5, twice with 0.5 ml of 50 mM sodium phosphate buffer, pH 5.5, containing 2.0% SDS and Triton XlOO at 1.0%. After the filter is dried, the bound radioactivity is determined by scintillation counting using the Wallac MicroBeta apparatus. To validate the assay, a known ligand for hemoglobin, 2,3-diphosphoglycerate, at concentrations ranging from 10"S to 10" 1 M is included in the reaction mixture. As shown in Figure 8, 2.3 - Diphosphoglycerate significantly increases hemoglobin filter retention.

B) Quantification of hemoglobin by ELISA: The reaction mixtures in a final volume of 0. 05 ml contains 0.063 mg / ml hemoglobin, 0.034 mg / ml bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 10 mM calcium acetate, 7.5 μg / ml thermolysin (Boeringer Mannheim), 7.5 μg / ml Proteinase K (Merck), 10% DMSO and the test compound, at a concentration of 20 or 200 uM. The control reactions were identical, except that the test compound is omitted. The mixtures were incubated at 20 ° C for 15 minutes, then at 44 ° C for 30 minutes and then placed on ice. To each mixture is added 0.05 ml of a 0.1 M sodium borate buffer containing 20 mM EDTA and 1 mM PMSF. After 10 minutes of incubation on ice, the mixtures are transferred to 96-well uncoated plates of Immuulon-4 (Dynatech). Subsequently the plates are incubated at 4 ° C during the night to allow the binding of the protein to the plate. The plates are washed thoroughly with TBST, and 0.1 ml of rabbit antibody against human hemoglobin (Calbiochem) diluted 1: 500 is added to each well. The plates are incubated at room temperature for 1 hour, and then carefully washed with TBST. Subsequently, 0.1 ml of alkaline phosphatase conjugated with goat antibody against rabbit IgG (Calbiochem) diluted 1: 1000 in TBST plus 5% dry milk without fat is added to each well, and the plates are incubated at room temperature for 1-2 hours. Subsequently the plates are washed carefully with TBST finally with TBST lacking Tween. 0.1 ml per ml of p-nitrophenyl phosphate (0.5 mg / ml) in 1X diethanolamine substrate buffer (Pierce) is added to each well. The plates are incubated at room temperature until color develops and the absorbance of each well is measured at 405 nm, using a BioRad 3550 UV microplate reader. To validate the assay, a known ligand for hemoglobin, 2,3-diphosphoglycerate is included in the reaction. As shown in Figure 9, this compound increases the detection of immunoreactive hemoglobin.

C) Results of the performance analysis: 4,000 compounds were examined for interaction with HbS using proteolysis and detection by ELISA as in the previous (figure 10). Of these, it was found that in 23, they inhibit proteolysis to a degree of 20% or more when tested at a concentration of 20 μM (compounds with positive functionality.The concentration dependence of the effects of functional compounds was measured. The functional compounds showed maximum mean effects at concentrations ranging from 2.0 μM (For example, see Figure 11).

Example 11: Low yield fluorescence-based assay for carbonic anhydrase ligands The binding of ligand to carbonic anhydrase I was tested using specific fluorescent probes for conformation as indicators of folding of the target protein. Reaction mixtures in a final volume of 0.1 ml contained 2 μM human carbonic anhydrase (Sigma), 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2.0 μM bis-1-anilino-8-naphthalene sulfonate (bis- ANS) (Molecular Probes, Inc., Eugene, OR). The fluorescence emission of bis-ANS at 450 nm was measured after excitation at 365 nm. Measurements were made using a Dynatech fluorescence microplate reader. First, the effects of increasing concentrations of guanidinium hydrochloride were examined (GC1) on the fluorescence of bis-ANS. Figure 12 shows the fluorescence intensity of bis-ANS (in arbitrary units) measured 3 minutes after the addition of GC1. In mixtures lacking carbonic anhydrase, the fluorescent yields of bis -ANS were low and were affected by the presence of GC1. (Figure 12, black boxes.) In contrast, carbonic anhydrase increased the fluorescence emission of bis-AβS in a manner that is possible at the concentration of GCl.In the absence of GCl, the fluorescence is increased fivefold by carbonic anhydrase The addition of between 0.5 M and 2 M of GCl increases the fluorescence proportionally, up to a maximum increase of five times to 2 M GCl An extra addition of GCl causes the fluorescence to decline The figure shows the time dependence of the fluorescence increase induced by GCL in the mixtures described above These data indicate that the fluorescence emission intensity of bis-ANS is affected by the folding state of carbonic anhydrase To validate the use of this assay in the analysis of carbonic anhydrase ligands , acetazolamide, a known ligand of carbonic anhydrase, was tested for its ability to affect the fluorescence emission of bis-ANS in mixtures Figure 14 shows the emission of bis-ANS one minute after exposure of carbonic anhydrase to 2M GCl and increasing concentrations of acetazolamide. The fluorescence emission was reduced by acetazolamide in a concentration dependent manner. The data indicate that the binding of acetazolamide to carbonic anhydrase prevents GCl-induced conversion of the folded form of the protein to a molten globular folded intermediate and that this effect can be used as a measure of ligand binding.

Example 12: High performance assay, based on fluorescence, for ligands Fluorescence probes, selective for conformation, are used in a high throughput assay format for measuring ligand binding, according to the present invention. Mixtures of a fluorescent probe, a target protein and test compounds are provided (and parallel control wells lacking test compounds) in individual wells of 96-well microtiter plates. After an appropriate incubation period, fluorescence was determined in each well using a fluorescence plate reader (such as, for example, Dynatech, Chantilly, VA). An increase or decrease in the fluorescence intensity in a well relative to a control well lacking test compounds indicates that the binding of the test compound to the folded or denatured state has occurred., respectively, of the target protein. The conditions for each target protein are determined by systematically monitoring the change in the fluorescence of the probe as conditions vary from stabilizing to destabilizing. A substantial portion of the observed fluorescence intensity must be due to interaction of the probe with the molten globular state of the target protein in order that a measurable change in fluorescence occurs upon stabilization of the pleated state by a ligand. If necessary, denaturing conditions such as elevated temperature or addition of urea, guanidine or organic solvents are used to increase the fraction of target protein present in the molten globular state. If necessary, the presence of a molten globular state is verified by biophysical measurements that include NMR, viscometry, intrinsic fluorescence and size exclusion chromatography. Under appropriate conditions, increased fluorescence is observed as the target protein is converted from the native state to a molten globular state, while decreasing the fluorescence that is observed upon conversion from a molten globular state to a random coil. In the case of some target proteins, the molten globular state may predominate even under "stabilizing" conditions (ie, in the absence of denaturing conditions listed above). In such cases, a relatively high increase in the fluorescence of the probe is observed by the target protein even under stabilizing conditions, and decrease in fluorescence is observed as the conditions become more destabilizing. The presence of a molten globular state can be verified as described in the above. The reversibility of the conformational change from native to globular or molten was also characterized. If the transition is reversible, tests can be established under equilibrium conditions. In this case, an incubation time is chosen which is long enough to allow the binding of the probe to the molten globular state of the target protein (in the absence of the test compounds) to reach equilibrium. If the transition is irreversible, an incubation time is chosen so that a measurable change, although not complete, is present in the increase in fluorescence. Such conditions can also be used in the case of reversible conformational changes. 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 description of the invention. Hg described the invention as above, property is claimed as contained in the following:

Claims (8)

1. A method of fluorescence-based analysis for identifying a ligand that binds to a predetermined target protein, the method is characterized in that it comprises the steps of: (a) selecting as test ligands a plurality of compounds that are not known to bind to the target protein; (b) incubating the target protein with each of the test ligands to produce a test combination, and in the absence of a test ligand, to produce a control combination; (c) contacting the test and control combinations with a conformation-sensitive fluorescent probe; (d) treating the test and control combinations under conditions that cause the target protein to denature to an appropriate extent; (e) measuring the fluorescence emission of the conformation-sensitive fluorescence probe in the test and control combinations; and (f) comparing the measurements made in step (d) between the test and control combinations, in which, if the fluorescence emission intensity is greater or less in the test combination than in the control combination, the Ligand test is a ligand that binds to the target protein.

2. The method according to claim 1, characterized in that it further comprises repeating steps (b) - (f) with a plurality of test ligands until a ligand is identified that binds to the target protein.

3. The method according to claim 1, characterized in that the fluorescence probe binds preferentially to the folded, denatured or globular molten state of the protein.

4. The method according to claim 3, characterized in that the probe is selected from the group consisting of 1-anilino-8-naphthalene sulfonate (ANS), bis-l-anilino-8-naphthalene sulfonate (bis-ANS) and 6-propionyl-2- (N, N-dimethyl) -aminonaphthalene (Prodan).

5. The method according to claim 4, characterized in that the fluorescence probe is bis-ANS.

6. The method according to claim 1, characterized in that the treatment comprises increasing the temperature at which the test and control combinations are exposed, contacting the test and control combinations with a protein denaturant, or combinations thereof.

7. The method according to claim 1, characterized in that the target protein contains stabilizing or destabilizing mutations in relation to the wild-type version of the protein.

8. The method according to claim 1, characterized in that the test ligand is selected from the group consisting of metals, peptides, proteins, lipids, polysaccharides, nucleic acids, small organic molecules, and combinations thereof.