MXPA98009291A - Proof of thermal change in microplates and apparatus for developing ligands and chemical optimization multivariable protei - Google Patents

Abstract

The present invention relates to a method for ordering the affinity of each of a multiplicity of different molecules of a target molecule that is capable of being denatured due to a thermal change. The method comprises contacting the target molecule with a molecule of the multiplicity of different molecules in each of a multiplicity of vessels, simultaneously heating the multiplicity of vessels, measuring in each of the vessels a physical change associated with the thermal denaturation of the white molecule that results from the heating in each of the containers, generating a curve of thermal denaturation for the target molecule as a function of the temperature for each of the containers and determining a midpoint of temperature (Tm) of the same, comparing the Tm of each of the thermal denaturation curves with the Tm of a denaturation curve obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules, and ordering the affinities of the multiplicity of molecules different from according to the change in Tm of each of the curves of thermal sniffing The present invention also provides a test apparatus that includes a means for adjusting the temperature to simultaneously heat a plurality of samples, and a medium that receives the emission of the spectrum from the samples while the samples are heating. In additional aspects of the invention, the receiving means may be configured to receive fluorescent emission, ultraviolet light, and visible light. The receiving means may be configured to receive the emission of the spectrum in a variety of ways, e.g. eg, one sample at a time, simultaneously of more than one sample, or simultaneously of all the samples. The means for adjusting the temperature can be configured with a temperature controller to change the temperature according to a predetermined profile

Description

Thermal change test in microplates and apparatus for ligand development and multivariable chemical optimization of proteins.

This application claims priority the provisional application U.S. do not. 60 / 017,860 published in May 9, 1996, which is incorporated completely by reference Declarations of Rights to Inventions Made Under Federally Sponsored Research and Development Imen e Part of the work done during the development of this invention used Government funds from the U.S. The Government of the U.S. You have certain rights to this invention.

Background of the Invention Field of the Invention The present invention relates generally to the screening of compounds and combinatorial libraries. More particularly, the present invention relates to a method and apparatus for performing tests, particularly REF tests: 28672 for thermal exchange. ürte related In recent years, pharmaceutical researchers have switched to combinatorial libraries as sources of new beneficial compounds for drug discovery. A combinatorial library is a collection of chemical compounds which have been generated, by chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" as reactants. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in each possible pathway for a given compound length (e.g., the number of amino acids in the polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixtures of chemical building blocks. In fact, one researcher observed that the systematic combinatorial mix of 100 interchangeable chemical building blocks resulted in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gordon, EM et al., J. Med. Chem. 37 : 1233-1251 (1994)).

The synthesis speed of the combinatorial library is accelerated by synthesis and evaluation of compounds automatically. For example, DirectedDiversityR is an iterative process based on the computer to generate chemical entities with defined physical, chemical and bioactive properties. The DirectedDiversity system is presented in U.S. Pat. 5,463,564, which is incorporated herein by reference.

Once the library has been constructed, it must be screened to identify compounds which possess some type of biological or pharmacological activity. To screen a library of compounds, each compound in the library is balanced with a target molecule of interest, such as an enzyme. A variety of proposals have been used to screen combinatorial libraries for targeted compounds. For example, in a coded library, each compound in a chemical combinatorial library can be made so that an oligonucleotide "tag" is bound to it. A careful record of the nucleic acid tag sequence is maintained for each compound. A compound which exerts an effect on the target enzyme is selected by amplifying branded nucleic acid using the polymerase chain reaction (PCR) From the tag sequence, the compound can be identified (Brenner, S. et al., Proc.Nat.Acid.Sci.USA 89: 5381-5383 (1992)). This proposition, however, consumes a lot of time because it requires multiple rounds of oligonucleotide amplification and subsequent electrophoresis of the amplification products.

A filamentous phage shows a library of peptides that can be screened by binding to a biotinylated antibody, receptor or other binding protein. The bound phage is used to infect bacterial cells and the determinant shown (eg, the peptide ligand) is then identified (Scott, J.K. et al., Science 249: 386-390 (1990)). This proposition suffers from different drawbacks. It takes time. Peptides that are toxic to the phage or to the bacteria can not be studied. In addition, the researcher is limited to researching peptide compounds.

In the International Patent Application WO 94/05394 (1994), Hudson, D. et al., Discloses a method and apparatus for synthesizing and screening a combinatorial library of biopolymers on a solid phase plate, in an array of 4 X 4 to 400 X 400. The library can be screened using a fluorescently labeled target or target molecule, arcade radio, or attached to an enzyme. The drawback to this proposition is that the target molecule must be labeled before it can be used to screen the library.

One challenge presented by the commonly available combinatorial library screening technologies is that they do not provide information about the relative binding affinities of different ligands for a receptor protein. This is true if the process for generating a combinatorial library involves the phage library that presents peptides (Scott, J.K. et al., Science 249: 386-390. (1990)), arrays of random synthetic peptides (Lam, K.S. et al. , Na t? Re 354: 82-84 (1991)), chemically encoded libraries (Brenner, S. et al., Proc. Na ti.

Acad. Sci. USA 89: 5381-5383 (1992)), the Hudson method (Int. Int. WO 94/05394), or more recently, combinatorial organic synthesis (Gordon, E. et al., J. Med. Chem. 37: 1385-1399 (1994)).

To acquire high-throughput quantitative binding data from screening of ligand affinities for a white enzyme, the researchers relied on enzyme activity tests. The enzymes themselves give for high screening performance because ligand binding effect can be monitored using kinetic tests. The experimental endpoint is usually a spectrophotometric change. Using a kinetic test, most researchers use a two-step approach to discover compounds. First, a large library of compounds is screened against the white enzyme to determine if any of the compounds in the library are active. These tests are usually performed at a single concentration (between 10"4-10-6 M) with one to three replicates, second, the promising compounds obtained from the first screening (eg, compounds which showed greater activity than a value). default) are usually re-tested to determine 50% of the inhibitory concentration (IC50), an inhibitory association constant (Ki), or a dissociation constant (Kd) .This two-step approach, however, is a intense, time-consuming and error-prone, each re-tested sample must be retrieved from the original or weighed test plate again, a concentration curve must be created for each sample and a separate set of test plates must be created for each sample. each test.

There are other problems associated with the high-throughput biochemical approach of combinatorial library screening.

Typically, a given test is not applicable to more than one receiver. That is, when a new receiver becomes available for testing, a new test must be developed. For many receivers, reliable tests are simply not available. Even when a test exists, it may not serve itself for automation. Additionally, if a K_ is the end point to be measured in a kinetic test, the inhibitor concentration to be used must first be assumed, the test performed, and then additional tests performed using at least six different concentrations of inhibitor. If one supposes too low, an inhibitor will not exert its inhibitory effect at the sub-optimal concentration tested.

In addition to the drawbacks for the kinetic screening proposal described above, it is difficult to use the kinetic approach to identify and order ligands that link outside the active site of the enzyme. Since the ligands that bind outside the active site do not prevent the binding of spectrophotometric substrates, there is no spectrophotometric change to be monitored. A more serious drawback to the proposed kinetic screening is that non-enzymatic receptors can not be fully tested.

Tests of thermal protein splitting, or thermal "change" have been used to determine if a given ligand binds to a target receptor protein. In a physical heat exchange test, a change in a biophysical parameter of a protein is monitored as a function of temperature increase. For example, in calorimetric studies, the measured physical parameter is the change in heat capacity with which a protein undergoes temperature-induced splitting transitions. Differential scanning calorimetry has been used to measure the affinity of a panel of azobenzene ligands of streptavidin (Weber, P. et al., J. Am. Chem. Soc. 16: 2717-2724 (1994)). Titration calorimetry is used to determine the binding constant of a ligand for a target protein (Brandts, J. et al., American Laboratory 22: 30-41 (1990)). The calorimetric approach requires, however, that the researcher have access to a calorimetric device. In addition, calorimetric technologies do not by themselves lead to high yields of combinatorial libraries, ** three searches per day are routine.

Similar to calorimetric technologies, spectral technologies have been used to monitor temperature-induced protein cleavage (Bouvier, M et al., Science 265: 398-402 (1994); Chavan, AJ et al., Biochemistry 33 : 7193-7202 (1994); Morton, A. et al., Biochemistry 1995: 8564-8575 (1995)). The spectral and calorimetric heat exchange studies described above have a common limitation. In each study, only one bonding reaction was heated and tested at the same time. The heating of a single sample and day test configuration, as is conventionally done, has impeded the application of heat exchange technologies for high-throughput screening of combinatorial libraries. Thus, a heat exchange technology is needed which can be used to screen combinatorial libraries, can be used to identify and order lead compounds, and is applicable to all receptor proteins.

Heat exchange tests have been used to determine when a ligand binds to DNA. Calorimetric, absorbance, circular dichroism and fluorescence technologies have been used (Pilch, DS et al., Proc. Nati Acad. Sci. USA 91: 9332-9336 (1994); Lee, M. et al., J. Med. Chem. 36: 863-870 (1993), Butour, J.-L et al., Eur. J. Biochem., 202: 975-980 (1991), Barcelo, F. et al., Chem Biol. Interactions 74: 315-324 (1990)). As conventionally used, however, these technologies have impeded the high throughput of screening nucleic acid receptors to give compounds which bind with high affinity. Thus, a thermal exchange technology is needed which can be used to identify and order the affinities of the leader compounds which bind to DNA sequences of interest.

When bacterial cells are used to overexpress exogenous proteins, the recombinant protein is frequently sequestered in bacterial cell inclusion bodies. For the recombinant protein to be used, it must be purified from the inclusion bodies. During the purification processes, the recombinant protein is denatured and must be renatured. It is impossible to predict the renaturation conditions that will facilitate and optimize appropriately the redoubling of a given recombinant protein. Usually, a number of renaturing conditions must be tested before a satisfactory set of conditions is discovered. In a study by Tachibana et al., Each of four disulfide linkages were simply removed, by site-directed mutagenesis of lysozyme in (Tachiba et al., Biochemistry 33: 15008-15016 (1994)). The mutant genes were expressed in bacterial cells and the recombinant proteins were isolated from inclusion bodies. Each of the isolated proteins was renatured under different temperatures and glycerol concentrations. The efficiency of protein recombination was estimated in a bacteriolytic test in which the bacteriolytic activity was measured as a renaturation temperature function. The thermal stability of each protein was studied using a physical heat exchange test. In this studio, however, only one reaction sample was heated and tested at the same time. The heating of a single sample and the configuration of the test prevents the application of heat exchange technologies to high screening performance of a multiplicity of conditions of protein redoubling, thus, a thermal exchange technology is needed which can be used to order efficiencies of different conditions of redoubling.

During the past four decades, X-ray crystallography and the resulting atomic models of proteins and nucleic acids have greatly contributed to an understanding of structural, molecular and chemical aspects of biological phenomena. However, crystallography analysis remains difficult because there are no reliable methodologies for obtaining quality protein crystals for X-rays. Conventional methods can not be used quickly to identify crystallization conditions that have the highest probability of promoting crystallization (Garavito , RM et al., J. Bioenergeti cs and Biomembranes 28: 13-27 (1996)). As well as the use of factorial design experiments and successive automated grid searches (Cox, MJ :, &Weber, PC:., J Appl. Cryst., 20: 366-373 (1987); Cox, MJ, & Weber, PC, J. Crystal Growth 90: 318-324 (1988)) does not facilitate rapid high-throughput screening of biochemical conditions that promote crystalization of X-ray quality protein crystals. In addition, different proteins are expected to require different conditions for its crystallization, as was the experience for its folding (mcPherson, A., In Preparation and Analysis of Protein Crystals, Wiley Interscience, New York, (1982)): conventional methods of determination of crystallization conditions are heavy , slow, and industrious. Thus, a fast high-performance technology is needed, which can be used to position the efficiencies of crystallization conditions.

High-throughput screening of combinatorial molecules or biochemical conditions that stabilize target proteins in heat-exchange tests would be facilitated by the simultaneous heating of many samples. To date, however, heat exchange tests have not used this route. Instead, the conventional approach to performing a heat exchange test has been to heat and test only one sample at the same time. That is, the researchers conventionally 1) heat a sample to a desired temperature in a heating apparatus; 2) assay a physical change, such as light absorption or change in secondary, tertiary or quaternary structure of the protein, 3) heat the samples to the next highest desired temperature; 4) Rehearse for a physical change; and 5) continue this process repeatedly until the sample has been tested at the highest desired temperature.

This conventional approach is disadvantageous for at least two reasons. First, this approach is labor intensive. Second, this approach limits the speed with which thermal change screening tests can be performed and thus precludes high-throughput rapid screening of combinatorial molecules that bind to a target receptor and biochemical conditions that stabilize target proteins. Thus, an apparatus capable of rapid high-performance heat exchange tests that will be available to all receivers, including reversibly folded proteins, is needed.

Brief Description of the Invention The present invention provides a multi-variable method for ordering the effectiveness of one or more of a multiplicity of different molecules or different biochemical conditions to stabilize a molecule which is capable of being denatured due to thermal change. The method comprises contacting the target molecule with one or more of a multiplicity of different molecules or different biochemical conditions in each of a multiplicity of containers, simultaneously heating the multiplicity of containers, measuring in each container a physical change associated with thermal denaturation of the white molecule resulting from the heating, generating a curve of thermal denaturation of the molecule resulting from the heating, generating a thermal denaturation curve for the target molecule as a function of the temperature for each of the containers, comparing each of the curves of denaturation a (i) each of the other denaturation curves and (ii) the thermal denaturation curve obtained for the target molecule under a reference group of biochemical conditions, and ordering the efficiencies of multiplicity of different molecules or the different bi-conditions ochemicals according to the change in each of the thermal denaturation curves.

The present invention provides a multivariable method to optimize the useful life of a molecule which is capable of being denatured due to thermal change. The method comprises contacting the target molecule with one or more of a multiplicity of different molecules or different biochemical conditions each in a multiplicity of containers, the simultaneous heating of the multiplicity of containers, the measurement in each of the containers of a physical change associated with the thermal denaturation of the white molecule resulting from heating, the generation of a denaturation curve of the target molecule as a function of the temperature of each of the containers, the comparison of the denaturation curves with (i) each of the other denaturation curves and (ii) with the thermal denaturation curve of the target molecule obtained as a reference for a group of biochemical conditions, and the ordering of the efficiency of a multiplicity of different molecules or of the different biochemical conditions according to the change in each of the curves d e denaturalization.

The present invention also provides a multivariable method for sorting the affinity of a combination of two or more of a multiplicity of different molecules with a target molecule which is capable of being denatured due to heat exchange. The method comprises the contact of the target molecule with a combination of two or more molecules of the multiplicity of different molecules in each of a multiplicity of containers, the measurement in each of such containers of a physical change associated with the thermal denaturation of the white molecule resulting from the heating, the generation of a curve of thermal denaturation of each of the containers, the comparison of each of the thermal denaturation curves obtained from the target molecule with (i) each of the denaturation curves obtained from the white molecule and (ii) with the curve for the target molecule in the absence of two or more different molecules, and the ordering of the affinities of the combination of two or more multiplicity of different molecules according to the change in each of the thermal denaturation curves.

The present invention also provides a multivariable method for ordering the efficiencies of one or more of a multiplicity of different biochemical conditions to facilitate the redoubling of a sample of a denatured protein. The method comprises placing one of the samples of redoubled proteins in each of a multiplicity of containers, in which each of the previously redoubled proteins has been redoubled according to a multiplicity of conditions, simultaneous heating of the multiplicity of containers, measurement in each of the containers a physical change associated with the thermal denaturation of the protein resulting from heating, the generation of a thermal denaturation curve for the protein as a function of the temperature for each of the containers, the comparison of each one of the thermal denaturation curves with (i) each of the thermal denaturation curves and (ii) with the thermal denaturation curve of the native protein under a group of reference biochemical conditions, and ordering the efficiencies of the multiplicity of different conditions of redoubling according to the change in the magnitude of the physical change of each of the thermal denaturation curves.

The present invention also provides an additional method for ordering the efficiencies of one or more of a multiplicity of different biochemical conditions to facilitate the redoubling of a sample of denatured protein, which comprises the determination of one or more combinations of a multiplicity of different conditions which promote the stability of the protein, the folding of the denatured protein under one or more combinations of said biochemical conditions that were identified as promoters of the stabilization of the protein, yield of the folded protein, the ordering of the efficacy of the multiplicity of reprogramming conditions mentioned according to bent protein yield, and the repetition of these steps until the identification of a combination of biochemical conditions that promote an optimal redoubling of the protein.

Using the thermal microplate test, one or more of the biochemical conditions that have an additive effect on the stability of the protein can be determined. Once a group of biochemical conditions is identified that facilitate an increase in the stability of the protein using the heat exchange test, the same group of conditions can be used in a recombination experiment with a recombinant protein. If the conditions that promote the stability of the protein in the heat exchange test are correlated with the conditions that promote the redoubling of the recombinant protein, these conditions can be further optimized by performing additional heat exchange tests until a combination of conditions is identified. that result in an additional increase in the stability of the protein. The recombinant protein is then redoubled under these conditions. This process is repeated until the optimum conditions of redoubling are identified.

The present invention also provides a method for ordering the efficacy of one or more of a multiplicity of different biochemical conditions that facilitate the crystallization of a protein which is capable of being denatured due to a thermal change. The method comprises contacting the protein with one or more of a multiplicity of biochemical conditions each in a multiplicity of containers, the simultaneous heating of the multiplicity of containers, the measurement of a physical change associated with the thermal denaturation of the resulting protein of the heating, the generation of a curve of thermal denaturation of the protein as a function of the temperature of each of the containers, the comparison of each of the denaturing curves with (i) each of the other thermal denaturation curves and (ii) with the thermal denaturation curve obtained using a group of reference biochemical conditions, and the ordering of the efficiencies of the multiplicity of different biochemical conditions according to the change in each of the denaturation curves.

The present invention also provides a method for ordering the affinity of each of a multiplicity of different molecules for a target molecule which is capable of being denatured due to a thermal change. The method comprises the contact of the target molecule with a molecule of a multiplicity of different molecules each in a multiplicity of containers, the simultaneous heating of the containers, the measurement in each of the containers of a physical change associated with the denaturing of the containers. the white molecule resulting from the heating, the generation of a thermal denaturation curve of the target molecule as a function of the temperature in each of the containers, the comparison of each of the thermal denaturation curves with the obtained thermal denaturation curve of the white molecule in the absence of the molecules of the multiplicity of different molecules, and the ordering of the affinities according to the change in each of the thermal denaturation curves.

The present invention also provides a method for testing a group or collection of a multiplicity of different molecules for a molecule which binds to a target molecule which is capable of being denatured due to a thermal change. The method comprises the contact of the target molecule with a collection of at least two molecules of a multiplicity of different molecules each in a multiplicity of containers, the simultaneous heating of the multiplicity of containers, the measurement in each of the containers of a physical change associated with thermal denaturation of the white molecule resulting from heating, generating a set of thermal denaturation curves of the target molecule as a function of temperature for each of the containers, the comparison of each of the thermal denaturation curves with the thermal denaturation curve obtained from the target molecule in the absence of any of the molecules of a multiplicity of molecules according to the change in each of the thermal denaturation curves, the selection of the collection of different molecules which contains a molecule with affinity for the target molecule, the division of the collection into small collections of molecules each into a multiplicity of containers, and the repetition of the steps until a single molecule is identified responsible for the thermal change in the multiplicity of molecules.

This invention also provides an improved method for generating exemplary compounds which comprises synthesis of a multiplicity of compounds and testing the ability of each compound to bind to a receptor molecule. The improvement comprises contact of the receptor molecule with a compound of a multiplicity of different compounds in each of one of a multiplicity of containers in a microplate, simultaneously heating the containers, measuring in each of the containers a physical change, result of the heating, which is associated with the thermal denaturation of a receptor molecule, the generation of a thermal denaturation curve for the receptor molecule as a function of the temperature in each of the containers, the comparison of each of the denaturation curves thermal with the curve of thermal denaturation obtained for the receptor molecule in the absence of any of the compounds in the multiplicity of compounds and ordering the affinities of each compound according to the change in each of the thermal denaturation curves.

The present invention also provides a manufacturing product which comprises a transporter having a multiplicity of containers within, each of the containers containing a target molecule which is capable of being denatured due to heating and at least one molecule selected from a multiplicity of different molecules present in a combinatorial library, where each of the different molecules is present in a container different from a multiplicity in the transporter.

The optimization of protein stability, binding to the ligand, folding of the protein, and protein crystallization are multivariable events. The optimization of multivariate problems requires a large number of parallel experiments to collect as much data as possible in order to determine which variables influence a favorable response. For example, multivariable optimization problems require large numbers of parallel experiments to collect as much data as possible in order to determine which variables influence the stability of the protein. In this regard, protein crystallization and quantitative analyzes of the structure-activity relationship have greatly benefited from mass screening procedures that employ array arrays of incremental changes in biochemical or chemical composition. Thus, this is largely similar to the quantitative relationships between structure and activity that are constructed to relate variations of functional chemical groups on ligands to their effect on binding affinity to give a therapeutic receptor, methods and apparatuses of the present invention facilitate the construction of a quantitative model that relates different biochemical conditions to experimental measurements of protein stability, ligand specificity, yield of folded protein and yield of crystallized protein.

The present invention offers a number of advantages over previous technologies that have been employed to optimize multivariable events such as stabilization, ligand binding, protein doubling, and protein crystallization. The main one of these advantages is that the present invention facilitates high screening performance.

Additionally, the present invention offers a number of advantages over previous technologies that have been used for screening in combinatorial libraries. The main one of these advantages is that the present invention facilitates high screening performance of combinatorial libraries for leading compounds. Many common library screening technologies simply indicate when a ligand binds or does not bind to a receptor. In that case, quantitative information is not provided. No information is provided about the relative binding affinity of a series of ligands. by contrast, the present invention facilitates the ordering of a series of compounds according to their relative affinity for a white receptor. With this information at hand, a structure-activity relationship can be developed for a group of compounds. The ease, reproducibility and rate of use of ligand-dependent changes at the midpoint of the splitting temperature (Tm) to order relative binding affinities make the present invention a powerful tool in drug discovery processes.

Typically, conventional kinetic screening approaches require at least six additional container tests at six different concentrations of inhibitor to determine a Ki. Using the present invention, the yield is increased approximately 6-fold over enzyme-based tests because a binding experiment can be performed in each container of a multi-container microplate. The kinetic screening approach is further limited by the usual compromise between dilution and signal detection, which usually occurs at a protein concentration of approximately 1 nM. In this aspect, the calorimetric approaches, both the calorimetric by differential search and the calorimetric by isothermal titration are at a worse disadvantage since they are limited to solitary bonding experiments, usually 1 per hour. In contrast, the present invention provides broad dynamic lag of measurable binding affinities, from ~ 10 ~ 4 to 10-15 M, in a single container.

The present invention does not require radiolabelled compounds. It does not require that the receptors be labeled with a fluorescent or chromophoric marker.

A very important advantage of the present invention is that it can be universally applied to any receptor that is a white drug. Thus, it is not necessary to invent a new test every time a new receiver is available to be tested. When the receptor under study is an enzyme, researchers can determine the order of affinity of a series of compounds more quickly and more easily than when using conventional kinetic methods. In addition, researchers can detect ligand binding to an enzyme, regardless of whether the binding occurs to the active site, to an allosteric cofactor binding site, or to a receptor subunit interface. The present invention is equally applicable to non-enzymatic receptors, such as proteins and nucleic acids.

In a further aspect of the present invention, a test apparatus is provided which includes a heating means for simultaneously heating a plurality of samples, and a receiving means for receiving spectral emissions of the samples while the samples are heating. In yet a further aspect of the present invention, a test apparatus is provided that includes a temperature adjusting means for simultaneously adjusting a temperature of a plurality of samples according to a predetermined temperature profile, and a receiving means for receiving spectral emissions of the samples while the temperature of the samples is adjusted according to the temperature profile.

In yet a further aspect, the present invention also provides a test apparatus that includes a movable platform on which a plurality of heat conducting blocks are disposed. The temperatures of the heat conducting blocks, and their samples, are adjusted by means of a temperature setting. Each of the plurality of heat conducting blocks is adapted to receive a plurality of samples. A light source is provided to emit an exciting wavelength of light for the samples. While the temperature of the samples begins to adjust, a sensor detects the spectral emission of the samples in response to the exciting wavelength of light. The movable platform moves between the heat conducting blocks to sequentially detect spectral emissions of the samples in each of the plurality of heat conducting blocks.

The test apparatus of the present invention provides the artisan with the opportunity to rapidly screen molecules and biochemical conditions that affect protein stability. The samples are heated simultaneously over a range of temperatures. During heating, spectral emissions are received. The test apparatus of the present invention also provides the craftsman with an opportunity to carry out conveniently and efficiently the methods of the present invention. The test apparatus of the present invention is particularly adapted to carry out heat exchange tests of molecules and biochemical conditions that stabilize target proteins.

Because the apparatus of the present invention comprises a heating means and a means of receiving spectral emissions, the apparatus of the present invention obviates the need to heat samples in one apparatus and then transfer them to another apparatus before taking the readings of the apparatus. spectral emission. As a result, the apparatus of the present invention facilitates temperature changes according to a predetermined temperature profile, rather than an increase in incremental temperature and intermediate cooling steps. Thus, more data points can be collected for a given sample and more accurate information can be obtained.

In addition, because the test apparatus of the present invention comprises a heating means and a means of receiving spectral emissions, the spectral measurements can be taken from the samples while they are heating. Thus using the test apparatus of the present invention, the artisan can study irreversible splitting proteins and reversible folding proteins.

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

Brief Description of the Figures The present invention is described with reference to the accompanying drawings. In the drawings, similar reference numbers indicate identical or functionally similar elements. Additionally, the digit (s) at the left end of a reference number identify the figures in which the reference number appears first.

FIGURE 1 shows the results of a microplate heat exchange test for ligands which bind to the active site of human -bombombina (with turbidity as an experimental signal).

FIGURE 2 shows the results of a microplate heat exchange test for ligands which bind to the fibroblast growth factor (aFGF) (with turbidity as the experimental signal).

FIGURE 3 shows the results of a microplate heat exchange test for ligands which bind to the active site of α-thrombin (with fluorescence emission as an experimental signal). The lines drawn between the points represent a non-linear fit of the least squares curve using the equation shown below in the figure. There are five adjustment parameters for this equation of y (T) vs. T: (l) and f, the pretranscriptional fluorescence of the native protein; (2) yu, the posttransitional fluorescence of the native protein without redoubling; (3) Tm, the temperature at the midpoint of the non-doubling transition; (4)? HU, the van't Hoff enthalpy change of non-doubling; and (2)? Cpu, the change in the heat capacity of the protein without redoubling. The nonlinear least-squares fit of the curve was performed using the KaleidaGraph ™ 3.0 software (Synergy Software, Reading PA), which allows the five adjustment parameters to float while Marquart methods are used for the minimization of the sum of squares. residuals FIGURE 4 shows the results of a microplate thermal change test of ligands which bind to the D (II) domain of FGF receptor 1 (D (II) FGFR1) (with fluorescence emission as an experimental signal). The lines drawn between the points represent the nonlinear fit of the curve by the least squares method using the equation of the bottom of the figure, as described in Figure 3.

FIGURE 5 shows the results of a miniaturized microplate heat exchange test for Factor D in the absence of any ligand.

FIGURE 6 shows the results of the microplate heat exchange test for factor Xa in the absence of any ligand.

FIGURE 7 shows the results of the microplate heat exchange test for the ligand that binds to the catalytic site of human a-thrombin.

FIGURE 8 shows the results of a miniaturized miniaturized microplate heat exchange test that binds to the D (II) domain of FGF receptor 1.

FIGURE 9 shows the results of a miniaturized microplate heat exchange test for urokinase in the presence of glu-gly-arg chloromethyl ketone.

FIGURE 10 shows the results of a heat exchange test in a miniaturized microplate of human -trombin in which the test volume is 2 μL. The thermal denaturation curves of three experiments are shown.

FIGURE 11 shows the results of a miniaturized microplate heat exchange test of human α-thrombin in which the test volume is 5 μL. The thermal denaturation curves of five experiments are shown.

FIGURE 12 shows the results of a single-temperature microplate heat exchange test in the presence of four different compounds in four separate experiments.

FIGURE 13 shows the results of a microplate heat exchange test of the intrinsic tryptophan fluorescence of human α-thrombin. In this test, the fluorescence of the blank was not subtracted from the fluorescent sample.

FIGURE 14 shows the results of a microplate heat exchange test of the intrinsic tryptophan fluorescence of human α-thrombin. In this test, the fluorescence of the blank was' subtracted from the fluorescent sample.

FIGURE 15 shows the results of a microplate heat exchange test of a single binding ligand that interacts with three different classes of binding sites of human α-thrombin.

FIGURE 16 shows the results of a heat exchange test in multiligand microplate binding with interaction with human a-thrombin.

FIGURES 17A-D show the results of a microplate thermal change test of the effect of pH and various concentrations of sodium chloride on the stability of α-thrombin. In Figure 17A, the fluorophore is 1.8-ANS. In Figure 17B, the fluorophore is 2,6-ANS. In Figure 17C, the fluorophore is 2,6-TNS. In Figure 17C the fluorophore is bis-ANS.

FIGURE 18 shows the results of the microplate heat exchange test of the effect of calcium chloride, ethylenediaminetetraacetic acid, dithiothreitol, and glycerol on the stability of human α-thrombin.

FIGURE 19 shows the test results of the microplate thermal change of the effect of pH and concentration of sodium chloride on the stability of receptor 1 of human D (II) FGF.

FIGURE 20 shows the results of a microplate thermal change test of the effect of various biochemical conditions on the stability of receptor 1 of human D (II) FGF.

FIGURE 21 shows the results of a microplate thermal change test of the effect of various biochemical conditions on the stability of receptor 1 of human D (II) FGF.

FIGURE 22 shows the results of a microplate thermal change test of the effect of various biochemical conditions on the stability of receptor 1 of human D (II) FGF.

FIGURE 23 shows the results of a microplate thermal change test of the effect of various biochemical conditions on the stability of human D (II) FGF receptor 1.

FIGURE 24 shows the results of a microplate heat exchange test of the effect of various biochemical conditions on the stability of human D (II) FGF receptor 1.

FIGURE 25 shows the results of a microplate thermal change test of the effect of various biochemical conditions on the stability of human urokinase.

FIGURE 26 is a schematic diagram of a thermodynamic model for the binding of free energies of protein recombination and ligand binding.

FIGURE 27 is a schematic diagram of a method for screening conditions that optimize the redoubling of proteins.

FIGURE 28 shows the results of microplate thermal change tests of a-thrombin stability using various fluorophores.

FIGURE 29 shows a schematic diagram of one embodiment of a test apparatus of the present invention.

FIGURE 30 shows a schematic diagram of an alternative embodiment of a test apparatus of the present invention.

FIGURE 31 shows a schematic diagram of a test apparatus according to another embodiment of the present invention.

FIGURES 32A-E illustrate one embodiment of a thermal electrical state for the test apparatus of the present invention. Figure 32 A shows a side view of the thermal electrical state. Figure 32B shows a view superior of the thermal electric state. Figures 32 C-E show three configurations of inserts that can be anchored to the thermal electric state. In one embodiment, the inserts are accommodated in a microtiter plate. In such an embodiment, the test samples are contained within the containers of the microtitre plate.

FIGURE 33 is a schematic diagram illustrating a top view of another embodiment of the test apparatus of the present invention.

FIGURE 34 is a schematic diagram of a top view of another embodiment of the test apparatus of the present invention shown in Figure 33 with a camera installed.

FIGURE 35 is a schematic diagram of a side view of the embodiment of the test apparatus shown in Figures 33 and 34.

FIGURES 36A and 36B illustrate a temperature profile e as the temperature profile is implemented using the automated test apparatus of the present invention.

FIGURE 37 shows an exemplary computer system suitable for use with the present invention.

FIGURE 38 shows a flow diagram illustrating one embodiment for the implementation of the present invention.

FIGURE 39 shows a flow chart illustrating an alternative embodiment for the implementation of the present invention.

FIGURE 40 shows a comparison of the results of a microplate heat exchange test of the denaturation of human α-thrombin performed using a fluorescence scanner (scanner) and a CCD camera.

FIGURES 41A and 41B show photographs of a thermal change test of the denaturing of human α-thrombin performed using a CDD camera; Figure 41A: bottom V of the microplate. Figure 41B: dimple of the microplate.

FIGURE 42 shows a comparison of the results of microplate heat exchange tests of the α-thrombin denaturation performed using a fluorescence scanner (scanner) and a CCD camera.

Detailed Description of the Preferred Modalities In the following description, reference will be made to various terms and methodologies known to those skilled in the biochemical and pharmacological arts. Publications and other materials appearing below such as known terms and methodologies are hereby incorporated by reference in their entirety as if they were made known in their entirety.

Panorama of the Methods of the Present Invention The present invention provides a method for ordering a multiplicity of different molecules depending on their ability to bind to a target molecule which is capable of cleavage due to a thermal change. In one embodiment of this method, the target molecule will be contacted with a molecule of a multiplicity of different molecules in each of a multiplicity of containers. The containers are then heated simultaneously, at intervals, over a range of temperatures. After each heating interval, a physical change associated with the thermal denaturation of the target molecule is measured. In an alternate mode of this method, the containers are heated continuously. A curve of thermal denaturation is plotted as a function of the temperature for the target molecule in each of the containers. Preferably, the mean temperature point, Tm of each thermal denaturation curve is then identified and compared to the Tm of the thermal denaturation curve obtained for the target molecule in the absence of any of the molecules in the containers. Alternatively, a complete thermal denaturation curve can be compared to another complete thermal denaturation curve using computer analytical tools.

The term "combinatorial library" refers to a plurality of molecules or compounds which are formed by combination, in each of the possible pathways to give a compound length, a group of chemical or biochemical building blocks which may or may not be related in structure. Alternatively, the term may refer to a plurality of chemical or biochemical compounds which are formed by selective combination of a group of chemical building blocks. Combinatorial libraries can be constructed according to methods familiar to those skilled in the art. For example, see Rapoport et al., Immunology Today 16: 43-49 (1995); Sepetov, N.F. et al., Proc. Nati Acad. Sci. U.S.A. 92: 5426-5430 (1995); Gallop, M.A. et al., J. Med. Chem. 9: 1233-1251 (1994); Gordon, E.M. et al., J. Med. Chem. 37: 1385-1401 (1994); Stankova, M. et al., Peptide Res. 7: 292-298 (1994); Erb, E. et al., Proc. Nati Acad. Sci. U.S.A. 91: 11422-11426 (1994); DeWitt, S.H. et al., Proc. Nati Acad. Sci. U.S.A. 90: 6909-6913 (1993); Barbas, C.F. et al., Proc.

Nati Acad. Sci. U.S.A. 89: 4457-4461 (1992); Brenner, S. et al. Proc. Nati Acad. Sci. U.S.A. 89: 5381-5383 (1992); Lam, K.S. et al., Nature 354: 82-84 (1991); Devlin, J.j. et al., Science 245: 404-406 (1990); Cwirla, S.E. et al., Proc. Nati Acad. Sci. U.S.A. 87: 6378-6382 (1990); Scott, J.K. et al., Science 249: 386-390 (1990). Preferably the term, "combinatorial library" refers to a Directed Diversity library, as a group shown in U.S. Pat. 5,463,564. Regardless of the way in which a combinatorial library is constructed, each molecule or compound in the library is cataloged for future reference.

The term "compound library" refers to a plurality of molecules or compounds which were not formed using the combinatorial proposal of chemistry or building block biochemistry. Instead, a library of compounds is a plurality of molecules or compounds which are accumulated and stored for use in future ligand-receptor binding assays. Each molecule or compound in the compound library is cataloged for future reference.

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

The term "multivariable" refers to more than one experimental variable.

The term "screening" refers to the testing of a multiplicity of molecules or compounds by their ability to bind to a target molecule which is capable of being denatured.

The term "ordering" refers to the ordering of the affinities of a multiplicity of molecules or compounds by a white molecule, according to the ability of the molecule or compound to change the thermal denaturation curve of the target molecule in the absence of any molecule or compound.

The term "ordering" also refers to the ordering of the efficiencies of a multiplicity of biochemical conditions in the optimization of protein stabilization, protein folding, protein crystallization, or shelf life of a protein, in the context of optimization of the stabilization of the protein, optimization of the folding of the protein, optimization of the crystallization of the protein, and optimization of the useful life of the protein, the term "ordering" refers to the ordering of the efficacies of one or more combinations of biochemical conditions to change the thermal denaturation curve of the target molecule under a reference group of conditions.

The term "condition reference group" refers to a group of biochemical conditions under which a thermal denaturation curve for a target molecule is obtained. The thermal denaturing curves obtained under conditions other than the reference conditions are compared to each other and to the thermal denaturation curve obtained for the target molecule under reference conditions.

As discussed above, ordered molecules, compounds, or biochemical conditions can be ordered by their ability to stabilize a target molecule according to the change in the complete thermal denaturation curve.

The term "leader molecule" refers to a molecule or compound, of a combinatorial library, which show relatively high affinities for a target molecule. The terms "lead compound" and "leader molecule" are synonymous. The term "relatively high affinity" refers to affinities in the Kd in the range of 10"4 to 10" 15 M.

The term "white molecule" encompasses peptides, proteins, nucleic acids, and other receptors. The term encompasses enzymes and proteins which are not enzymes. The term encompasses monomeric and multimeric proteins. Multimeric proteins can be homomeric or heteromeric. The term encompasses nucleic acids comprising at least two nucleotides, such as oligonucleotides. The nucleic acids can be single chain, double chain or triple chain. The term encompasses a nucleic acid which is a synthetic oligonucleotide, a portion of a recombinant DNA molecule, or a portion of chromosomal DNA. The term white molecule also encompasses portions of peptides, proteins and other receptors which are capable of acquiring secondary, tertiary or quaternary structure through bending, coiling or twisting. The white molecule can be substituted with substituents including but limited to cofactors, coenzymes, prosthetic groups, lipids, oligosaccharides, or phosphate groups. The term "capable of denaturing" refers to the loss of the secondary, tertiary or quaternary structure by unfolding unwinding or unraveling. The terms "white molecule" and "receptor" are synonymous.

Examples of target molecules are included, but are not limited to those discussed in Faisst, S. et al., Nucleic Acids Research 20: 3-26 (1992); Pi entel, E., Handbook of Growth Factors. Volumes I-III CRC Press, (1994); Gilman, A.G. et al., The Pharmacological Basis of Therapeutics, Pergamon Press (1990); Lewin, B., Genes V, Oxford University Press (1994); Roitt, I., Essential Immunology, Blackell Scientific Publ. (1994); Shimizu, Y., Lymphocyte Adhesion Molecules, RG Landes (1993); Hyams, J.S. et al., Microtubules. Wiley-Liss (1995); Montreuil, j. et al., Glycoproteins, Elsevier (1995); Woolley, P., Lipases: Their Structure Biochemistry and Applications, Cambridge University Press (1994); Kurjan, J., Signal Transduction: Prokariotic and Simple Eukariotic Systems, Academic Press (1993); Kreis, T-, et al., Guide Book to the Extra Cellular Matrix and Adhesion Proteins, Oxford University Press (1993); Schlesinger, M.J., Lipid Modifications of Proteins, CRC Press (1992); Conn, P.M., Receptors: Model Systems and Specific Receptors, Oxford University Press (1993); Lauffenberger, d.A. et al., Receptors: Models for Binding Trafficking and Signaling, Oxford University Press (1993); Webb, E. C, Enzyme nomenclature, Academic Press (1992); Parker, M.G., Nuclear Hormone Receptors; Molecular Mechanisms, Cellular Functions Clinical Abnormalities Academic Press Ltd (1991); Woodgett, J.R., Protein Kinases. Oxford University Press (1995); Balch, W.E. et al., Methods in Enzymology, 257, Pt. C. Small GTPases and Their Regulators: Proteins Involved in Transport, Academic Press (1995); The Chaperonins, Academic Press (1996); Peleen, L., Protein Kinase Circuitry in Cell Cycle Control, RG Landes (1996); Atkinson, Regulatory Proteins of the Complement System, Franklin Press (1992); Cooke, d.T. et al., Transport and Receptor Proteins of Plant membranes: Molecular Structure and Function, plenum Press (1992); Schumaker, V.N., Advances in Protein Chemistry: Lipoproteins, Apolipoproteins, and Lipases, Academic Press (1994); Brann, m. , Molecular Biology of G-Protein-Coupled Receptors: Applications of Molecular Genetics to Pharmacology, Birkhauser (1992)trum.

; Konig, W., Peptide and Protein Hormones: Structure, Regulations, Activity-A Reference Manual, VCH Publ. (1992); Tuboi, S. et al., Post-Translational Modification of Proteins, CRC Press (1992); Heilmeyer, L.M., Cellular Regulation by Protein Phosphorilation Springer-Verlag (1991); Takada, Y., Integrin: The Biological Problem, CRC Press (1994); Ludlow, J.W., Tumor Suppressors: Involvement in Human Disease, Viral Protein Interactions, and Growth Regulation, RG Landes (1994); Schlesinger, M.J., Lipid Modification of proteins, CRC Press (1992); Nitsch, R.M., Alzheimer's Disease: Amyloid Proteins Precursor, signal Transduction, and Neuronal Transplantation, New York Academy of Sciences (1993); Cochrane, C.G. et al., Cellular and Molecular Mechanisms of Inflammation, Vol. 3: Signal Transduction in Inflamatory Cells, part A, Academic Press (1992); Gupta, S. et al., Mechanism of Lymphocyte Activation and Immune Regulation IV: Cellular Communications, Plenum Press (1992); Authi K.S. et al., Mechanism of Platelet Activation and Control, Plenum Press (1994); Grunicke, H., Signal Transduction Mechanism in Cancer, R.G. Landes (1995); Latchman, D.S., Eukariotic Transcription Factors, Academic Press (1995).

The term "white molecule" refers more specifically to proteins involved in the blood coagulation cascade, fibroblast growth factors, and fibroblast growth factor, urokinase, and factor D receptors.

The term "molecule" refers to compounds which are tested for binding affinity for the target molecule. This term involves compounds of any structure, including, but not limited to, nucleic acids and peptides. More specifically the term molecule encompasses compounds in a combinatorial or compound library. The term "molecule" and "ligand" are synonymous.

The term "heat change" and "physical change" involve 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 the polar properties of light.

Specifically, the term refers to fluorescent emission, fluorescent energy transfer, ultraviolet or visible light absorption, changes in light polarization properties, changes in fluorescence emission polarization properties, changes in turbidity, and changes in enzymatic activity . The fluorescent emission may be intrinsic to the protein or may be due to a fluorescent reporter molecule (below). For a nucleic acid, the fluorescence may be due to ethidium bromide, which is an intercalating agent. Alternatively, the nucleic acids can be labeled with a fluorophore (below).

The term "contacting a white molecule" refers generally to depositing the target molecule in solution with the molecule to be screened by linkage. In a less general way contacting refers to the turns, rotational movements, agitation or vibration of a solution of the target molecule and the molecule to be screened for binding. More specifically, contacting refers to the mixing of the target molecule with the molecule to be tested by binding. Mixing is accompanied, for example, by repeated taking and discharging through a pipette end. Preferably contacting refers to the binding equilibrium between the target molecule and the molecule to be tested by linkage. Contact can occur in the container (infra) or before the target molecule and the molecule to be screened are placed inside the container.

The target molecule can be contacted with a nucleic acid before initiating contact with the molecule to be screened by binding, the target molecule can be complexed with a peptide before starting to contact the molecule to be screened by linkage. The target molecule can be phosphorylated or dephosphorylated before starting contact with the molecule to be screened by linkage.

A carbohydrate moiety can be added to the target molecule before the molecule contacts the molecule to be screened by linkage. Alternatively, a carbohydrate moiety can be removed from the target molecule before the molecule is screened by linkage.

The term "container" refers to some vessel or chamber in which the receptor and the molecule to be tested by link will be deposited. The term "container" encompasses reaction tubes (e.g., test tubes, vials, etc.). Preferably, the term "container" refers to a well in a multi-container microplate or microtitre plate. The term sample refers to the content of a container.

A "thermal denaturation curve" is a graph of the physical change associated with denaturation of a protein or nucleic acid as a function of temperature. See, for example, Davidson et al. , Na ture Structure Biology 2: 859 (1995); Clegg, R.M. et al. , Proc. Na ti. Acad. Sci. U. S. A. 90: 2994-2998 (1993).

The "midpoint of temperature, Tm" is the midpoint of temperature of a denaturation curve. The Tm can actually be determined using methods well known to those skilled in the art. See, for example, Weber, P.C. et al., J. Am. Chem. Soc. 116: 2717-2724 (1994); Clegg, r.M. et al., Proc. Nati Acad. Sci. U.S.A. 90: 2994-2998 (1993).

The term "fluorescence probe molecule" refers to a fluorophore, which is a fluorescent molecule or a compound which is capable of binding to a unfolded or denatured receptor and, after being excited by light at a defined wavelength, emits energy fluorescent. The term fluorescent probe molecule encompasses all fluorophores. More specifically, for proteins, the term encompasses fluorophores such as thioinosine, and N-ethenoadenosine, formicin, dansyl derivatives, fluorescein derivatives, 6-propionyl-2- (dimethylamino) -naphthalene (PRODAN), 2-anilinonaphthalene, and N-derivatives. arylamino-naphthalene sulfonate such as l-anilinonaphthalene-8-sulfonate (1,8-ANS), 2-anilinonaphthalene-6-sulfonate (2,6_ANS), 2-aminonaphthalene-6-sulfonate, N, -dimethyl-2-aminonaphthalene-6 -sulfonate, N-phenyl-2-aminonaphthalene, N-cyclohexyl-2-aminonaphthalene-6-sulfonate, N-phenyl-2-aminonaphthalene-6-sulfonate, N-phenyl-N-methyl-2-aminonaf-taleno-6 -sulfonate, N- (o-toluyl) -2-aminonaphthalene-6-sulfonate, N- (m-toluyl) -2-aminonaphthalene-6-sulfonate, N- (p-toluyl) -2-aminonaphthalene-6-sulfonate , 2- (p-toluidinyl) -naphthalene-6-sulfonic acid (2,6-TNS), 4- (dicyanovinyl) julolidino (DCVJ), 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN), 6-hexadecanoyl-2- (((2- (trimethylammonium) ethyl) methyl) amino) naphthalene chloride (PATMAN), nyl red, Nf enyl-1-naphthylamine, 1,1-dicyano-2- [6- (dimethylamino) naphthalene-2-yl] propene (DDNP), 4,4'-dianilino-1, l-binaphthyl-5, 5-disulfonic acid (bis-ANS), and Dapoxil ™ derivatives (Molecular Probes, Eugene, OR). Preferably for proteins the term refers to 1,8-ANS or 2,6-TNS.

A double-stranded oligonucleotide can be used in resonance energy transfer fluorescence tests. A strand of the oligonucleotide will contain the donor fluorophore. The other strand will contain the acceptor fluorophore. For a nucleic acid to "contain" an acceptor or donor fluorophore, the fluorophore can be incorporated directly into the oligonucleotide sequence. Alternatively, the fluorophore can be anchored either at the 5 'or 3' end of the oligonucleotide.

A donor fluorophore is one that, when excited by light, will emit fluorescent energy. The energy emitted by the donor fluorophore is absorbed by the acceptor fluorophore.

The term "donor fluorophore" encompasses all fluorophores including, but not limited to, carboxyfluoroscein, iodoacetimidofluorocescein, and fluoroscein isothiocyanate. The term "acceptor fluorophore" encompasses all fluorophores including, but not limited to, iodoacetimidoenosine and tetramethylthrhodamine.

The term "transporter" encompasses a platform or other object, in any form, which by itself is capable of supporting at least two containers. The conveyor can be made of any material, including, but not limited to glass, plastic or metal. Preferably the conveyor is a multi-well microplate. The term microplate and 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 has a plurality of containers.

The term "biochemical conditions" encompasses any component of a physical, chemical or biochemical reaction. Specifically, the term refers to the conditions of temperature, pressure, protein concentration, pH, ionic strength, salt concentration, time, electric current, potential difference, cofactor concentration, coenzyme, oxidizing agents, reducing agents, detergents, metal ions, ligands or glycerol.

The term "denatured protein" refers to the protein which has been treated to remove the secondary, tertiary or quaternary structure. The term "native protein" refers to the protein that has the degree of secondary, tertiary or quaternary structure that the protein has complete chemical and biological functions. A native protein is one that has not been heated and has not been treated with denaturing or chemical agents such as urea.

The term "denatured nucleic acid" refers to the nucleic acid which has been treated to remove folded, chipped or curled structures. The denaturation of a complex nucleic acid of three strands is completed when the third strand is removed from the two complementary strands. The denaturation of a double-stranded DNA is completed when the base pairing between the two complementary strands has been interrupted and has resulted in DNA molecules that assume a random form. The denaturation of single-stranded RNA is completed when the intramolecular hydrogen bonds have been interrupted and the RNA assumes a random form, not bound by hydrogen.

The terms "doubling", "redoubling" and "renaturation" refers to the acquisition of the correct secondary, tertiary or quaternary structures of a protein or a nucleic acid, which provides the complete chemical or biological function of the biomolecule.

The term "efficacy" refers to the effectiveness of a particular set of biochemical conditions in facilitating the redoubling or renaturation of an unfolded protein or a denatured protein.

The terms "spectral measurement" and "spectrophotometric measurement" refer to the measurement of changes in light absorption. Turbidity measurements, visible light absorption measurements, and ultraviolet light absorption measurements are examples of spectral measurements.

The term "polarimetric measurement" refers to the measurement of changes in the properties of polarization of light and fluorescence emission. Circular dichroism and optical rotation are examples of polarization properties of light which can be measured polarimetrically. The measurements of circular dichroism and optical rotation are made using a spectropolarimeter. "Non-polarimetric" measurements are those that are not obtained using a spectropolarimeter.

The term "collection" refers to a group or group of at least one molecule to be tested for binding to a target molecule or receptor.

A host is a bacterial cell that has been transformed with recombinant DNA for purposes of expression of a protein which is heterologous to the host bacterial cell.

The heat exchange test is based on the ligand-dependent change in thermal denaturation curve of a receptor such as a protein or a nucleic acid. When heated over a range of temperatures, a receiver will unfold. By plotting the degree of denaturation as a function of temperature, one obtains a curve of thermal denaturation for the receiver. A point that is used as reference in the curve of thermal denaturation the midpoint of temperature (Tm), the temperature at which the receiver is half of its denaturation.

The ligand binding stabilizes the receptor (Schellman, J., Biopolymers 14: 999-1018 (1975)). The degree of binding and interaction-free energy follows a parallel course as a function of the concentration of the ligand (Schellman, J., Biophysical Chemistry 45: 273-279 (1993); Barcelo, F. et al., Chem. Biol. Interactions 74: 315-324 (1990)). As a result of stabilization by ligand plus energy (heat) is required to unfold the receiver. Thus, the link to the ligand changes the thermal denaturation curve. This property can be exploited to determine when a ligand binds to a receptor: a change, in the thermal denaturation curve, and thus in the Tm suggests that a ligand bound to the receptor.

The thermodynamic bases for the heat exchange test were described by Schellman, J.A. . { Biopolymers 15: 999-1000 (1976)) and also by Brandts et al. (Biochemistry 29: 6927-6940 (1990)). Differential scanning calorimetry studies by Brandts et al., (Biochemistry 29: 6929-6940 (1990)) showed that for very narrow 1: 1 stoichiometry systems, in which there is a splitting transition, the binding affinity to Tm of the following expression: (equation i; Where KL = the association constant to the ligand at Tm; Tm = the midpoint for the cleavage transition of the protein in the presence of the ligand; To = The midpoint for the splitting transition in the absence of the ligand: ? HUTO = the enthalpy of unfolding of the protein in the absence of the ligand to To; ? CPU = the change in heat capacity. Over the unfolding of the protein in the absence of the ligand; [T-] = The concentration of free ligand at Tm; and R = the gas constant.

The parameters? HU and? CPU are usually observed from differential scanning calorimetry experiments and are specific for each receiver. To calculate the link constant of equation 1, one should have access to a differential scanning calorimetry instrument to measure? HU and? CPU for the receiver of interest.

These parameters can also be located for the receptor of interest, or a receiver closely related to it, in the literature. In these situations, equation (1) will allow the exact measurement of KL and Tm.

It is also possible to calculate the association constant at any temperature, KL to T, using equation 2. to use equation 2, the calorimetry data for the enthalpy of T bond,? HL, and the change in heat capacity after the link to the ligand,? CPL must be known. (Brandts et al., Biochemistry 29: 6927-6940 (1990)). [equation 2) where KLT = the association constant to the ligand at some temperature T; KL1 ™ = the association constant to the ligand at Tm; Tm = the midpoint for the cleavage transition of the protein in the presence of the ligand; ? HLT = the enthalpy of binding to the ligand in the absence of the ligand at T; -CpL = the change in the heat capacity on the link to the ligand; Y R = the gas constant.

The second exponential term in equation 2 is usually small enough to be ignored so that approximate values from KL to T can be obtained by using only the first exponential term: T J (equation 3) One does not need, however, to calculate the link constants according to equations 1-3 to order the affinities of a multiplicity of different ligands for a receiver. On the contrary, the present invention provides a method for ordering ligand affinities according to the degree to which the thermal denaturation curve is changed by the effect of the ligand. Thus, it is possible to obtain estimates from KL to TM in the absence of exact values of? HU,? CPU, and? HL.

The present invention is particularly useful for • Screen a combinatorial or compound library. To be successful in high screening performance, samples on a multi-container hauler or platform are better. A multi-container hauler facilitates the heating of a plurality of samples simultaneously. In one embodiment, a multi-container microplate, for example a microplate with 96 containers or with 384 containers, which can accommodate 96 or 384 different samples, is used as a carrier.

In one embodiment, a sample is contained in each container of a multi-container microplate. The control container contains the receptor, but it does not have the molecule to be tested by binding. Each of the other samples contains at least one molecule to be tested per link. The curve of thermal denaturation for the receiver in the control container is the curve against which the curves of all the other experiments are compared.

The speed of screening is accelerated when the sample contains more than one molecule to be tested per link. For example, the screening rate is increased 20 times when the sample contains a set of 20 molecules. Samples containing a binding molecule must then be divided into samples that contain a small collection of molecules to be tested per link. These divided collections should then be assayed by binding to the target molecule. These steps must be repeated until a single molecule responsible for the original heat exchange is obtained.

The denaturation of the receptor can be measured by light spectrophotometry. When a protein in solution is denatured in response to heating, the receptor molecule is added and the solution becomes turbid. The thermally induced aggregation after denaturation is the rule rather than the exception. In general, aggregation complicates calorimetric experiments. Aggregation, however, is an advantage when using a spectrophotometric technology, because changes in turbidity can be measured by monitoring the change in absorbance of visible light or ultraviolet light of a defined wavelength.

The denaturation of a nucleic acid can be monitored using light spectrophotometry. The change in hyperchromicity, which is the increase in light absorption by polynucleotide solutions due to the loss of ordered structure, is monitored as a function of the increase in temperature. Changes in hyperchromicity are typically tested using light spectrophotometry tests.

In another embodiment, however, fluorescence spectrophotometry is used to monitor thermal denaturation. The fluorescence methodology is more sensitive than the absorption methodology.

The use of fluorescence-intrinsic proteins and fluorescence in probe molecules in spectroscopy experiments is well known to those skilled in the art. See, for example, Bashford, C.L. et al., Spectrophotometry and Spectrofluorometry: a Practical Approach, pp. 91-114, IRL Press Ltd. (1987); Bell, J.E., Spectroscopy in Biochemistry, Vol. I, pp. 155-194, CRC Press (1981); Brand, L. et al., Ann. Rev. Biochem. 41: 843 (1972).

If the target molecule or receptor to be studied is a nucleic acid, fluorescence spectrophotometry can be performed using a displacement test of ethidium bromide (see, M. et al., J. Med. Chem. 36: 863-870 (1993)). ). In this regard, the linkage to the ligand displaces the ethidium bromide and results in a decrease in the fluorescence emission of ethidium bromide. An alternative approach is to use fluorescent resonance emission transfer. In this latter approach, the transfer of fluorescent energy, from a donor fluorophore of one strand of an oligonucleotide to an acceptor fluorophore of the other strand, is monitored by measurement of the fluorescent energy of the acceptor fluorophore. The denaturation prevents the transfer of fluorescent energy.

The fluorescence resonance emission transfer methodology is well known to those skilled in the art. For example, see Ozaki, H et al., Nucleic Acids Res. 20: 5205-5241 (1992); Clegg, R.G., Proc. Nati Acad. Sci. U.S.A.90: 2994-2998 (1993); Clegg. R. G. et al., Biochemistry 31: 4846-4856 (1993).

The element on which the sample holder is heated may be any element capable of heating samples rapidly and in a reproducible manner. In the present invention, a plurality of samples are heated simultaneously. The plurality of samples can be heated in a single heating element. Alternatively, the plurality of samples may be heated to a temperature in a heating element, and then moved to another heating element for another temperature. Warming can be achieved at regular or irregular intervals. To generate a smooth denaturation curve, the samples should be heated uniformly, in intervals of 1 to 2 ° C. The temperature range at which samples can be heated is from 25 to 110 ° C. The spectral readings are taken in each heating step. The samples can be heated and read by the equipment continuously. Alternatively, after each heating step, the samples could be cooled to a lower temperature than the spectral readings were taken. Preferably, the samples are heated continuously and the spectral readings are taken while the samples are being heated.

The spectral readings can be taken from all the samples on the conveyor simultaneously. Alternatively, the readings can be taken in samples of groups of at least two at a time. Finally the readings can be taken in a sample each time.

In one embodiment, the thermal denaturation is monitored by fluorescence spectrometry using a test apparatus such as that shown in Figure 29. The instrument consists of a scanner. and a control software system. The system is able to quantify the emission of soluble fluorescence and that associated to the cell. The fluorescence emission is detected in a photomultiplier tube in a light-proof detection chamber. The software runs on a personal computer and the scanner's action is controlled through the software. A precision X-Y mechanism scans the microplate with a sensitive fiber optic probe to quantify the fluorescence in each well. The microplate and samples can remain stationary during scanning of each sample line, and then the fiber optic probe moves to the next line. Alternatively, the microplate and the samples can move their position to a new sample line under the fiber optic probe. The scanning system is capable of scanning 96 samples in less than one minute. The scanner is capable of having a plurality of excitation filters and emission filters to measure the most common fluorophores. Thus, the fluorescence emission readings can be taken in a sample each time, or in a subset of samples simultaneously. An alternative embodiment of this test apparatus is shown in Figure 33. The test apparatus of the present invention will be described in more detail later.

The present invention is also directed to a manufacturing product which comprises a conveyor having a multiplicity of containers in it. The manufacturing product can be used to screen a combinatorial library of guide compounds which bind to the receptor of interest. The combinatorial library can be screened using the method according to the present invention.

In the manufacturing product, each of the containers contains a uniform amount of receptor of interest. In addition, each of these containers contains a compound different from a combinatorial library at a concentration which is at least two times higher than the concentration of the receptor. Preferably, the manufacturing product is a multi-well microplate or a multiplicity of multi-well microplates. If the receptor is a protein, each container may contain a fluorescent probe molecule. If the receptor is a nucleic acid, each container may additionally contain ethidium bromide. Alternatively, the nucleic acid can be labeled with a fluorophore.

Prior to its use, the manufacturing product may be stored in any form necessary to maintain the integrity of the recipient of interest. For example, the manufacturing product can be stored at a temperature between -90 ° C and room temperature. The receptor and compound can be stored in lyophilized form, in liquid form, in powder form, or stored in glycerol. The manufacturing product can be stored either in the light or in the dark.

The heat conducting element or block on which the sample conveyor can be any element capable of heating samples quickly and reproducibly. The plurality of samples can be heated in a single heating element. Alternatively, the plurality of samples may be heated to a given temperature on a heating element, and then moved to heating to another heating element at another temperature. Warming can be achieved at regular or irregular intervals. To generate a smooth denaturation curve, the samples should be heated uniformly, in intervals of 1 to 2 ° C. The temperature range at which samples can be heated is from 25 to 110 ° C.

In the present invention, a plurality of samples are heated simultaneously. If the samples are heated at discrete temperature intervals, stepwise, the spectral readings are taken after each heating step. Alternatively, after each heating step, the samples may be cooled to a temperature prior to taking the spectral readings. Alternatively, the samples can be heated continuously and the spectral readings are taken during heating.

The spectral readings can be taken from all the samples of a conveyor simultaneously. Alternatively, the readings can be taken in samples of groups of at least two at a time.

The present invention also provides a method for generating guide compounds. After a compound or library of compounds has been screened using the heat exchange test, the compounds which bind to the target receptor are chemically modified to generate a second library of compounds. This second library is screened using the heat exchange test. This process of screening and generating a new library continues until compounds are obtained that bind to the target receptor with affinities in the Kd range from 10"4 to 10 ~ 15 M.

A fluorescence emission image analyzer system can be used to monitor the thermal denaturation of a target molecule or receptor. The image analyzer 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-resolution charge camera coupled with a resolution of 768 x 494 pixels. The attached load chamber uses a computer as an interface and the images are analyzed with the Image Analysis software ™. The Chemilmager ™ (Alpha Innotech) is a cooled charge-coupled device that performs all functions of the Alphalmager ™ and also captures images of chemiluminescent samples and other low-intensity samples. The Chemilmager ™ charge-coupled includes a Pentium processor (1.2 Gb hard disk, 16 Mb) Alpha analysis software Ease ™, compact light cabin, and transilluminator of white light and UV light. For example, the MRC-1024 UV / Visible Confocal Laser image analyzer system (BioRad, Richmond, CA) facilitates the simultaneous analysis of more than one fluorophore over a wide range of wavelengths (350 a 700). The Gel Doc 1000 Fluorescent Gel Documentation System (BioRad, Richmond, CA) can clearly show samples of areas as large as 20 x 20 cm, or as small as 5 x 4 cm. At least two microplates of 96 containers can be placed in an area of 20 x 20 cm. The Gel Doc 1000 system also facilitates the realization of experiments based on time.

A fluorescent thermal image analysis system can be used to monitor the unfolded receiver in a microplate thermal change test. In this embodiment, a plurality of samples are heated simultaneously between 25 and 110 ° C. A fluorescence emission reading is taken simultaneously for each of the plurality of samples. For example, fluorescence emission from each 96-well or 384-well microplate can be monitored simultaneously. Alternatively, the fluorescent emission readings can be taken continuously and simultaneously for each sample. At low temperatures, all samples have a low level of fluorescence emission. When the temperature increases, the fluorescence in each sample also increases. Wells which contain ligands that bind to the target molecule with high affinity change the curve of thermal denaturation at high temperatures. As a result, wells containing ligands which bind to the target molecule with high affinity fluoresce less, at a temperature up to Tm of the target molecule in the absence of any ligand, than wells that do not contain high affinity ligands. . If the wells are heated in incremental steps, the fluorescence images of all the plurality of samples in each heating step are analyzed. If the samples are heated continuously the fluorescence emission of all the plurality of samples, the image analysis is done simultaneously during the heating.

In a heat exchange test, it can be done in 100 μL volumes. For the following reasons, however, it is preferable to perform a heat exchange test in a volume of 10 μL. First, about 10 times less protein is required for the miniaturized test. Thus, only 5 to 40 moles of protein (0.1 μg to 1.0 μg for a 25 kDa protein) are required for the test (eg 5 to 10 μL of working volume with a concentration of the target molecule of 1 to about 4 μM). Thus, 1 mg of protein can be used to perform 1000 to 10000 tests in the miniaturized format. This is particularly advantageous when the target molecule is available in small amounts.

Second, approximately 10 times less ligand is required for the miniaturized test. This advantage is very important for researchers who are screening combinatorial libraries for which libraries of compounds are synthesized in small quantities. In the case of human a-thrombin, the ideal ligand concentration is around 50 μM, which translates into 250 pmoles of ligand or 100 ng (assuming a molecular weight of 500 Da) of ligand per test in the miniaturized format .

Third, smaller work volumes allow for the potential to use larger experiment arrays because the miniaturized test can be placed in a much smaller area. For example plates with 384 wells (16 x 24 array) or 864 wells (24 x 36 array) have approximately the same dimensions as the 96-well plates (about 8.5 x 12.5 cm). The 384 well plate and the 864 well plate allow the user to perform 4 and 9 times more experiments, respectively, than those that can be done using a 96 well plate.

Alternatively, 1536-well plates (38x46 arrays, Matrix Technologies Corp.) can be used. A plate of 1536 wells will provide 16 times the effort made with a 96-well plate.

Thus, using the 1536-well plate configuration, the test is made 16 times faster, in relation to the speed at which the test can be done using a 96-well format. The 8 x 12 arrangement (in a 96-well plate) performs 96 tests / hr, or about 2300 tests in 24 hours. The 32 x 48 arrangement facilitates the completion of about 1536 tests / hr or about 37,000 tests / 24 hours can be performed using a 32 x 48 configuration.

The test volume can be 1-100 μL.

Preferably, the test volume is 1-50 μL. More preferably, the test volume is 1-25 μL. More preferably still the test volume is 1-10 μL. More preferably still, the test volume is 5 μL. More preferably, the test volume is 1 or 2 μL.

Preferably, the test is performed on polycarbonate plates with bottom V or perforated polycarbonate plates. A perforated plate is a plate containing a plurality of wells with a rounded bottom with a total volume of 15 μL.

An alternative to take the spectral readings in a range of temperatures around Tm of the therapeutic target to obtain a complete curve of non-thermal splitting for the ligand / target complex, to identify changes in Tm is to perform the test using only temperature close to the Tm of the white molecule. In this embodiment, the samples that emit less fluorescence, relative to the control sample (containing a target molecule but not the candidate ligand), indicate that the candidate ligand binds to the target molecule.

In this embodiment, the magnitude of a physical change associated with the thermal denaturation of the target molecule resulting from heating is determined by the generation of a thermal denaturation curve of the target molecule as a function of temperature in a range of one or more discrete or fixed temperature. The physical change associated with thermal denaturation, eg, fluorescence emission, it is measured. The magnitude of the physical change in the fixed or discrete temperature for the target molecule in the absence of any ligand is noted. The magnitude of the physical change in the presence of each of a multiplicity of different molecules, for example, combinatorial compounds, is measured. The magnitude of the physical change associated with the thermal denaturation of the target molecule in the presence of each of a multiplicity of molecules is compared to the magnitude of the physical change obtained from the target molecule at the discrete or fixed temperature in the absence of any of a multiplicity of different molecules. The affinities of a multiplicity of different molecules are arranged according to the change in the magnitude of the physical change.

The fixed or discrete temperature at which the physical change is measured can be any temperature that is useful for discriminating change in thermal stability. Preferably, the fixed or discrete temperature is the midpoint Tm of the thermal denaturation curve for the target molecule in the absence of any of the multiplicity of different molecules tested to bind to the target molecule.

The configuration of a single temperature is particularly advantageous if one is interested in testing a relatively high affinity ligand series, which are the preferred compounds as candidates in clinical trials. In cases where it is preferred that the requirements be less strong for binding affinity, it is however possible to increase the concentration of ligand to 500 μM to identify ligands with Kd's of 2.5 μM or higher affinity.

The one-temperature mode offers numerous advantages. First, the speed of the test is increased by a factor of 10. Thus, as the 96-well test plate (8 x 12 array) provides about 96 tests per hour, the variation of a single temperature will facilitate about 1000 tests per hour. Using a 1536 well plate (32 x 48 array), so large that the aliquots of the sample can be handled at the same speed for the 32 x 48 array system as the 8 x 12 system, about 15,000 tests can be performed per hour.

Another alternative method for detecting thermal split transitions for the microplate heat exchange test is through the intrinsic fluorescent tryptophan (Trp) of the target protein. Many fluorescence emission readers, such as CytoFluorlI, use tungsten lamps as their light source. These lamps do not give enough light near 280 nm to allow excitation of the intrinsic Trp residues of the proteins which have absorption maxima near 280 nm. However, the Biolumin 960 (Molecular Dinamics) uses a Xenon-Are lamp. The Xenon-Are lamp provides excitation at 280 nm and emission measurement at 350 nm. The methods and test apparatus of the present invention are not limited to the tests of ligand protein interactions. Test methods and apparatus can be used to rapidly test any multivariate system related to protein stabilization. For example, the methods and test apparatus of the present invention can be used to simultaneously test the binding of more than one compound or ligand to a target molecule. Using this approach, the addictive effect of the binding of multiple ligands can be appreciated. Positive and negative cooperativity can be determined. To achieve this method, the heat exchange test is performed for a target molecule, such as a protein, in the absence of any ligand, in the presence of a single ligand, and in the presence of two or more ligands. A thermal denaturation curve is generated for the protein alone and for each of the combinations of protein and ligand (s). The temperature of the midpoint Tm is then determined for each curve. Each Tm is then compared with each of the other Tm's of the other curves. Alternatively, each complete thermal denaturation curve is compared with each of the other thermal denaturation curves. In these two forms, the additive contribution of more than one ligand to interact in the binding or in the stability of the protein can be determined.

In a similar manner, the additive contributions of one or more biochemical conditions for the stability of the protein can be determined. Thus, the present invention can be used to quickly identify conditions that optimize the stability of the protein, and also the shelf life of a protein.

In addition, the methods and test apparatus of the present invention can be used to order the efficacy of various biochemical conditions for the redoubling or denaturing of a split or denatured protein. This embodiment points out the need in the art for a reliable method for screening for effective re-blending or renaturation conditions.

For example, expression of recombinant DNA in a bacterial cell usually results in the sequestration of a recombinant protein in bacterial inclusion bodies (Marston, F.A.O., Biochem. J. 240: 1-12 (1986)). Although other expression systems can be used instead of bacterial expression systems, expression in bacterial cells remains the method of choice for the production of high levels of recombinant proteins (Rudoplh, R., Protein Engineering: Priciples and Practices, pp 283-298 John Wiley and Sons (1995)). In many cases, the recovery of the recombinant protein requires that the protein be isolated from the inclusion bodies. The purification of the protein from the inclusion bodies is a process that requires the denaturation of the recombinant protein. As a result, the recombinant protein must be renatured or redoubled under appropriate conditions to generate the protein in its native, fully functional form.

In each of these cases, the denatured protein must be renatured or refolded to be useful for later use or study. Unfortunately, one can not easily predict the exact conditions under which a given protein or protein fragment should be renatured. Each protein is different. One must always resort to testing a number of different combinations of renaturation conditions before one can know which group of conditions is optimal. Thus, it is desirable to have a reliable and rapid method for ordering the efficiencies of various renaturation conditions.

Recombinant DNA technology has allowed the biosynthesis of a wide variety of heterologous polypeptides of interest in relatively large amounts through the recruitment of the bacterial protein expression apparatus. However, the promise of an abundant and inexpensive supply of rare bent sister proteins of high therapeutic value expressed in E. coli has failed due to the overwhelming -predominance of unfolded or partially unfolded white proteins in the inclusion body of insoluble protein. For recent reviews see Rudolph, R., & Lilie, H PHASE BJ. 10: 49-56-995); Sadana, A., Biotechnology &Bioengineerieng 48: 481-489 (1995); Jaenicke, R., Phli. Trans Royal Soc. London Ser. B-Biol. Sci. 34897-105 (1995)) the reasons for the autoaggregation reaction to prevail in E. coli. they have focused on the relatively high concentration of the heterologous protein (as high as 30% of the cell's weight) found in varying degrees in partially split states. Thus, at the elevated protein concentrations of an overproducing strain of E. coli, the exposed hydrophobic residues of the split protein are more likely to find other molecules with similarly exposed groups (inter-molecular reaction) than the sample collapses in the polypeptide conformation where the hydrophobic residues are packed in an appropriate orientation (intramolecular transition state) to proceed to the native state completely bent (see Figure 26). From this perspective, inclusion bodies of insoluble protein appear to be kinetically trapped in the products of side reactions that impede the preferred folding process of the protein.

Techniques for the isolation of inclusion bodies, the purification of recombinant protein from the inclusion bodies, and the techniques for redoubling or renaturation are well known to those skilled in the art. For example, see Sambrook, J. et al., Molecular Cloning: a Laboratory Manual, pp 17.37-17.41., Cold Spring Harbor Laboratory Press (1989); Rudolph, R et al., FASEB J. 10: 49-56 (1995).

Another impediment to producing large amounts of correctly doubled proteins in E. coli is that the reduced redox potential of the E. coli cytosol prevents the formation of disulfide bonds in vivo. The formation of disulfide bonds is an important co- and post-translational event in the biosynthesis of many cellular proteins that is frequently coupled in the doubling of the protein. In addition, it has been shown that cis trans proline isomerization reaction is the speed limiting step for the correct folding of certain proteins (Lin, L.-N., &; Branddts, J.F., Biochemistry 22: 564-573 (1983)). As a result, partially doubled intermediates accumulate in sufficient quantity in vivo that they aggregate and precipitate into protein masses.

The cells employ a class of host proteins called molecular chaperons that assist in doubling the protein in vivo apparently to prevent many of the unproductive side reactions discussed above with respect to inclusion body formation, e.g. ex. the aggregation and the formation of inappropriate disulfide bonds. However, the chaperone machinery of E. coli. which is understood in part by proteins, GroEL and GroES, presumably becoming overwhelmed by the massive over-expression. Despite many attempts to correct this deficiency of the chaperones by co-expression of chaperones with the protein of interest (Rudolph, R., &Lilie H., The FASEB J. 10: 49-56 (1995)) have been reported positive results in only one case (Goloubinoff, P ,, et al., Nature 342: 884-889 (1989)).

Two hypotheses have been proposed to explain how GroEl and GroES assist in the folding of proteins in vivo. In the first hypothesis, the Anfinsen box hypothesis, the function of a molecular chaperon is to provide a protected environment where the folding of the protein to its native state can proceed without interference by cell aggregation conditions (Martin, et al. , Na ture 352: 36-42 (1991), Ellis, RJ, Currente Bi olgy 4: 633-635 (1994)). In the second hypothesis, the "iterative reinforcement" hypothesis, the function of the chaperon is to partially unfold the misfolded proteins (ie, kinetically trapped intermediates) with some of the energy of ATP hydrolysis being channeled into the conformational energy of the substrate polypeptide , forcing the polypeptide to a higher energy state which could again try to redouble correctly after being released into the solution. (Todd, MJ et al., Science 265: 659-666 (1994); Jackson, et al., Biochemistry 32: 2554-2563 (1993); Weissman, JS, et al., Cell 78: 693-702 (1994 ); Weissman, JS &Kim, PS, Science 253: 1386-1393 (1991)).

The in vivo results discussed above in many forms are in many ways consistent with recent experiences with the redoubling of heterologous recombinant proteins expressed in E. coli. That is, while the primary amino acid sequence of a protein may contain sufficient information to determine its native bent conformation (Anfinsen, CB Science 181: 223-230 (1973)), the biochemical conditions in which the folding reactions take place may influence strongly the partition between unfolded, aggregated and correctly folded forms.

For example, the pH can influence the bending reaction of its effect on electrostatic interactions to strong ones added in the fourth term of equation (4).

? Gdobl =? Gconf + S? Gi / int + S? Gi, s +? Wel + (? Gunion) Equation (4) Where? Gconf = Free conformational energy (thermal order / disorder); ? gi / int = weak interactions (hydrogen bonds, van der Walls interactions, salt bridge, link cofactors, etc.); ? gi / S = weak interactions with the solvent (hydrophobic effect, ion hydration, etc.); ? Wel = strong electrostatic interactions. AG binding- free energy of ligand binding.

When the pH of the protein solution decreases below the pl of the protein, the functional groups of the polypeptide become increasingly protonated, to the point where the electrostatic repulsion forces eventually remove the other terms in the free energy equation ( equation (4)), and the protein is not sufficiently capable of adopting the native conformation. Another important biochemical parameter for the folding of the protein is the solvent, water, which repels the aromatic and aliphatic side chains (and possibly the main chain to some extent). to minimize its exposed surface area. The influence of the solvent on the bending reaction is added in the third term of the free energy equation (equation (4)). It is known that certain salts increase the hydrophobic interactions in the side chains of the protein in aqueous solutions. The effect depends on the nature of the ions following the Hofmeister series: Cations: Mg2 + > Li + > Na + > K + > NH4 +. Anions; S042_ > HP042_ > acetate > citrate > tartrate > C1"> N03" > C103"> I" > C104 ~ > SCN. "Hofmeister stabilizing anions such as S042 ~ and HP042 ~ at 0.4 M have been found to increase the yield of correctly folded proteins (Creigton, TE, In: Proteins: Structures and Molecular Properties, Freeman, New York, 1984). )). This favorable result for the native conformation of the proteins has been attributed to the "salting" effect of the cations and anions which leads to the preferential hydration of the protein (Creighton, TE, In: Proteins: Structures and Molecular Properties , Freeman, New York, (1984).

Glycerol alters the solvation properties of water in favor of the native conformation of proteins. The mechanism by which it occurs is the exclusion of co-solvent and the preferential hydration of the protein, not dissimilar to the effect of salt or salts of the Hofmeister series (Timasheff &Arakawa, In: Protein Structure, A Practical Approach, TE Creigton, ed., IRL Press, Oxford, UK (1989), pp. 331-354).

Another example of how the environment influences the doubling of prptein is the effect that ligands and known factors have on the yield of folded protein. The binding ligands have the effect of changing the equilibrium from an unfolded state to a native-complex protein complex through the coupling of the binding-free energy to the bending reaction. The role of metal ions in the redoubling of bovine carbonic anhydrase II has been described (Bergenhem &Carlsson, Biochem Biophys, Acta 998: 277-285 (1989).) Other biochemical parameters that have been shown to affect bending Proteins are: protein concentration, temperature, redox regulators of glutathione (GSH, GSSG), the presence of detergents, and the presence of other additives, such as glycerol, arginine-HCl, polyethylene glycol (PEG), and organic solvents.

During incubation under redoubled conditions, the recombinant proteins can be immobilized on a solid phase support. This configuration resembles the "Anfinsen box" hypothesis for the GroEl and GroES function where a split protein is temporarily immobilized in a protected environment where doubling to the native state can proceed without any interference from competing aggregation reactions. The conformation of the protein folded into solid supports has two new reports in the literature. A polyhistidine protein TIMP-2 having tag sequences could be redoubled by dialysis while still bound to a chelated metal column (Black, A. et al., FEBS Lett 360: 52-56 (1995)). A polyionic fusion peptide anchored to the carboxy or amino terminus of α-glucosidase allowed bending while bound to a heparin resin Sepharose at about 5 mg / mL (Rudolph &Lilie, FASEB J. 10: 49-56 (1995 )). The methodology for immobilizing and redoubling an α-glucosidase with polionic arginine tag sequences is described in Stempfer, G. et al. , Nature Biotechnology 14: 329-334 (1996).

In the present invention, the heat exchange test is used to order the efficiency of various re-blending or renaturation conditions. Each of a multiplicity of aliquots of a protein of interest, which is incubated under a variety of different bending conditions, is placed in a container in a multi-container conveyor. An aliquot of the native, fully functional protein of known concentration is placed in the control container. Samples can be placed on any multi-container conveyor. Preferably, each sample can be placed in a well of a multi-well microplate.

By considering the many biochemical variables that can influence the results of the protein folding reaction, the optimization of protein folding is a multivariable optimization problem, not unlike protein crystallization and quantitative activity and structure relationships (QSAR) in drug discovery. Multivariable optimization problems require a large number of parallel experiments to collect as much data as possible that influence a favorable response. In this case, protein and QSAR crystallization analyzes have greatly benefited from mass screening protocols that employ matrix arrays of incremental changes in chemical or biochemical composition.

The present invention can be used to order the efficiencies of the redoubling or renaturation conditions. Such conditions include, but are not limited to, the concentration of glycerol, the concentration of protein, the use of agents which catalyze the formation of disulfide bridges, temperature, pH, ionic strength, type of solvent, use of thiols such as glutathione. reduced (GSH) and oxidized glutathione (GSSG), chaotropes such as urea, guanidinium chlorides, alkyl urea, organic solvents such as carbonic acid amides, L-arginine HCl, Tris regulator, polyethylene glycol, non-ionic detergents, zwitterionic detergents, micelles mixed and detergent in combination with cyclodextrin. The present invention can be used interchangeably if a denaturing agent is removed from the protein using dialysis, column chromatography techniques, or suction filtration.

Using a heat exchange test spectrum, the conditions which facilitate the optimal redoubling can be determined quickly. In this modality, the samples of renatured protein and the sample of. Control protein (eg, a sample of native protein in its fully functional form) is heated in a temperature range. In discrete temperature intervals, the spectral readings are taken. Alternatively, the spectral readings can be taken during a predetermined continuous temperature period. A curve of thermal denaturation is constructed for each sample. The Tro for the thermal denaturation curve of the fully functional native protein is determined. The relative efficiencies of the doubling conditions are arranged according to the magnitude of the physical change associated with the splitting at the Tm of the fully functional native protein, in relation to the magnitude of the physical change of a known quantity of functional protein to that Tro The magnitude of the physical change used to measure the extent of unfolding ( (reflected in the ordinate, or y-axis, of a thermal denaturation curve) corresponds to the amount of protein correctly folded.

The present invention provides a method for the screening of biochemical conditions that facilitate and optimize protein folding. To screen the conditions for a given protein, it is first necessary to determine the thermal profile of the split protein for a protein of interest. This is achieved by the generation of a denaturation curve using the microplate heat exchange test. Several conditions can be optimized, including optimization of pH, dependence on ionic strength, concentration of salts of the Hofmeister series, glycerol concentration, sucrose concentration, arginine concentration, dithiothreitol concentration, concentration of metal ions, etc.

Using the microplate heat exchange test, one or more biochemical conditions can be determined that have an additive effect on the stability of the protein. Once a group of biochemical conditions facilitating an increase in protein stability have been identified using the heat exchange test, the same group of conditions can be used in the recombinant protein folding experiments. See Figure 27. If the conditions that promote the stability of the protein in the heat exchange test are correlated with the conditions that promote the folding of the recombinant protein, the conditions can be further optimized by performing additional heat exchange tests until a combination is identified. of stabilization conditions that result in additional stability of the protein. The recombinant protein is then doubled under these conditions. This process is repeated until the optimum bending conditions are identified. It is expected that the stability of the protein correlates with the yield of the folded protein. The performance of the correctly folded protein can be determined using any suitable technique. For example, the yield of the correctly folded protein can be calculated by passing the redoubled protein through an affinity column, for example a column in which a protein ligand is anchored, and quantifying the amount of protein that is present in the sample. In this way, the additive contributions of the conditions of doubling to the correct doubling can be estimated. The transition state of the redoubling protein resembles more the native form of the protein than the denatured form. This has been demonstrated for many proteins (Fersht, A.R., Curr., Op. Struct. Biol. 7: 3-9 (1997)).

The methods and apparatus of the present invention provide a rapid yield approach for screening combinations of biochemical conditions that favor protein folding. The method does not require cumbersome and time consuming steps that require conventional protein folding approaches. For example, using the method of the present invention, it is not necessary to dilute the protein in large volumes and low concentrations (~ 10 μg / mL) to avoid the problems of aggregation associated with conventional methods of recombinant protein redoubling. The suppression of protein aggregation will allow the screening of biochemical parameters that change the equilibrium of protein folding (between doubled and unfolded forms of proteins) to the correct native conformation.

As well as protein stabilization, protein folding, ligand selection, and drug design, the selection of conditions that promote crystallization that promote crystallization is another multivariable optimization problem that is solved using the methods and apparatus of the present invention.

The test methods and apparatus of the present invention are also useful for determining the conditions that facilitate the crystallization of proteins. The crystallization of molecules of a solution is a process of reversible equilibrium, and the kinetic and thermodynamic parameters are a function of the chemical and physical properties of the solvent system and the solute of interest (McPerson, A., In: Preparation and Analysis of Protein Crystals, Wiley Interscience (1982), Weber, PC, Adv. Protein Chem 41: 1-36 (1991)). Under supersaturation conditions the system is driven towards equilibrium where the solute is partitioned between the soluble and solid phase instead of between the unfolded and native states. The molecules of the crystalline phase are packaged in ordered, periodic three-dimensional arrays that are energetically dominated by the same types of cohesive forces that are important for protein folding, e.g. ex. van der Waals interactions, electrostatic interactions, hydrogen bonds, and covalent bonds (Moore, W.J., Physical Chemistry, 4th Ed., Prentice Hall, (1972), pp. 865-898).

Thus, in many ways crystallization can be seen as a high-level variation of protein folding where whole molecules are packaged to maximize cohesive energies instead of single amino acid residues. In addition, for protein crystallization and protein folding, the composition of the solvent can make important contributions to the extent of the partition between soluble (split) and crystalline (native) forms. The cohesive interactions present in protein macromolecules and the role played by the solvent in the modulation of these interactions in the folding and crystallization of proteins are complex and have not been understood so far. In this aspect, the biochemical conditions that promote the stability and folding of proteins also promote crystallization.

For example, biochemical conditions that were found to increase the stability of the 1 D (II) FGF receptor (Figures 19-24) correlate with the conditions that facilitate crystal crystallization of X-ray diffraction quality protein. The conditions that were employed for obtaining protein crystals D (II) FGFR1 (Lewankowski, Myslik, Bone, Springer R., BA and Pantoliano, MW, unpublished results (1997)) are shown in Table 1. The protein crystals were obtained in the range of 7.4 to 9.2 in the presence of Li2SO4 salts of Hofmeister (65 to 72%). those crystallization conditions correlated with the optimum pH of about 8.0 in Figure 23. Other salts of the Hofmeister series such as Na2SO4, (NH4) S04 and Mg2SO4 were also found to be useful in decreasing the amount Li2SO4 required as a precipitant. Clearly, those conditions for successive FGFRl crystallizations correlate closely with the stabilization conditions they identified using the microplate heat exchange test.

Conditions that were identified as facilitators of the stabilization of human a-thrombin also facilitated the crystallization of the human a-thrombin protein. Figures 17A-D and 18 show the results of heat exchange testing conditions that facilitate the stability of human α-thrombin. Table 2 contains a summary of the conditions identified by three different investigators that facilitate the crystallization of X-ray diffraction-quality human a-thrombin crystals (Bode, W., et al., Protein Sci. 1: 426-471 (1992 ); Vijayalakshmi, J. et al., Protein Sci. 3: 2254-22271 (1994); and Zdanov, A. et al., Proteins: Struct. Funct. Genet., 17: 252-265 (1993)).

The conditions summarized in Table 2 correlate closely with the conditions identified in the microplate heat exchange test thus facilitating the stability of human a-thrombin. Crystals formed close to the optimum pH of about 7.0. Additionally, there is a clear preference for the presence of 0.1 to 0.5 m NaCl (50% of conditions) or 0.1 to 0.2 m NaHP04. This is consistent with the recent discovery of the Na + binding site (Dang et al., Nature Biotechnology 15: 146-149 (1997)) and the results of the microplate heat exchange test in Figures 17A-D and 18. All the samples of human a-thrombin described in Table 2 that yielded good crystals are complexed with a ligand, therefore additional stabilization of the native structure of this protein in addition to the acquired one of the biochemical conditions.

Table 1. Crystallization conditions of D (II) FGFR1 High performance elucidation of conditions that promote the stability of a given protein and thus the formation of X-ray quality protein crystals.

When a protein is more stable, it has less thermodynamic mobility that inhibits its packing within the lattice of the crystal. With low mobilities, the protein fits better within the glass lattice. Using conventional crystallization methods, the experiments are set at room temperature for weeks at a time. All the time, protein unfolding occurs. Using the method of the present invention, conditions that stabilize a protein are examined over a range of temperatures. Conditions that change the curve of thermal splitting at high temperatures will greatly decrease the splitting that occurs while the crystallization process occurs. cp Complete View of the Test Apparatus The test apparatus of the present invention is directed to an automated temperature adjustment and spectrum emission receptor that simultaneously adjusts the temperature of a multiplicity of samples over a defined temperature range and receives the emission of the spectrum from the samples . The test apparatus of the present invention is particularly useful for carrying out the microplate thermal inversion tests for the stabilization of the protein. The apparatus of the present invention can be used to practice all methods of the present invention.

The test apparatus of the present invention restores the separate heating devices and the receiving devices of the emission of the spectrum. In contrast to other devices, the test apparatus of the present invention can be configured to simultaneously set the temperature of a multiplicity of samples and receive spectrum emissions of the samples during temperature adjustment according to a predetermined temperature profile.

After denaturing by venting, the reversibly wound proteins are partially or completely re-rolled after denaturation by heating. Rewinding mainly excludes measurements in a thermal displacement test. Using the test apparatus of the present invention, however, reversibly wound proteins can be tested in a thermal shift test. That is, because in the test apparatus of the present invention, the spectrum measurements are taken while the protein is being heated. And the re-winding of the protein does not occur.

In such a configuration, the test apparatus of the present invention includes a detector that is positioned on a mobile heat conducting block in which a sample array is placed. A relative movement means, such as a servo-driven armature, is used to move the detector such that the detector is sequentially positioned on each sample in the sample array. The detector receives the spectral emissions of the samples.

The test apparatus of the present invention can be configured to contain a plurality of heat conducting blocks in a mobile platform. The platform could be a portable platform that can be moved, for example by a servo-driven linear slide device. An example of the linear slip device is the SA A5M400 model (IAI America, Torrance, CA). In this mode the detector receives the spectrum emissions of each of the samples in a given block that conducts the heat. The platform is then moved to place another heat conductor block and its accompanying samples below the detector so that it receives spectrum emissions from each of the samples in the heating block. The platform is moved until the spectral emissions of the samples are received in all heat conducting blocks.

Alternatively, the platform could be rotated by means of a rotating platform that could be, for example, by a servo-driven shaft. In the last mode, the detector receives the spectrum emissions of each of the samples in a given heat conductor block. The platform is then rotated to place another heat conductor block and its accompanying samples under the detector so that it receives spectrum emissions from each of the samples in the heating block. The platform is rotated until the spectral emissions of the samples are received in all heat conducting blocks.

System Description Figure 29 shows a schematic diagram of one embodiment of a test apparatus 2900 of the present invention. The test apparatus 2900 includes a heat conducting block 2912 that includes a plurality of wells 2920 for a plurality of samples 2910. The heat conducting block 2912 is composed of a material having a relatively high thermal conductivity coefficient, such as aluminum , stainless steel, bronze, Teflon, and ceramics.

Accordingly, the heat conducting block 2912 can be heated and cooled to a uniform temperature but will not be sufficiently thermal conductive to require excess heating or cooling to maintain a temperature.

The test apparatus 2900 also includes a light source 2906 for emitting a wavelength -exciting 2916, shown generally at 2916, for the samples. The light source 2906 excites the samples 2910 with the exciting light 2916. Any suitable light source can be used. For example, a halogen-tungsten lamp can be used. Alternatively, a Xenon arc lamp, such as Biolumin 960 (Molecular Dynamics) can be used.

Alternatively, a mercury lamp can be used (Hg) high pressure. High-pressure mercury lamps emit light of higher intensity than lamps of Xenon (Xe). The light intensity of a high pressure mercury lamp is concentrated in specific lines, and they are only useful if the mercury lines are of wavelengths suitable for the excitation of particular fluorophores.

Some fluorescent plate readers employ lasers for excitation in the visible region of the electromagnetic spectrum. For example, the Fluorlmager ™ (Molecular Dynamics, Palo Alto, CA) is such a device. This technology is useful when using fluorescent dyes that absorb energy at approximately 480 nm and emit energy at approximately 590 nm. Such a dye could then be excited with the 488 nm illumination of standard argon, argon / krypton lasers. For example, 1, l-dicyano-2- [6- (dimethylamino) naphthalen-2-yl] propene (DDNP) is such a dye. The advantage of using a laser is that a laser is characterized by very high light intensity, which results in an improved signal for the noise ratio.

The exciting light 2916 causes an emission of the spectrum 2918 of the samples 2910. The emission of the spectrum 2918 can be electromagnetic radiation of any wavelength in the electromagnetic spectrum. Preferably, the emission of the spectrum 2918 is fluorescent, ultraviolet or visible light. More preferably, the emission of the spectrum 2918 is fluorescent emission. The emission of the spectrum 2918 is received by means of a photomultiplier tube 2904. The photomultiplier tube 2904 is communicatively and operatively coupled to a computer 2914 by means of an electrical connection 2902. The computer 2914 functions as a means of analyzing data to analyze the emission of the spectrum as a function of temperature.

As discussed above, the medium receiving the spectrum or detector of the test apparatus of the present invention may comprise a photomultiplier tube. Alternatively, the medium that receives the spectrum or detector may include a coupled charge device or a charge device camera coupled. In yet another alternative, the medium that receives the spectrum or detector may include a diode array.

An alternate embodiment of the test apparatus of the present invention is shown in Figure 30. In the embodiment shown in Figure 30, a coupled device (CCD) camera 3000 is used to detect the emission of spectrum 2918 from samples 2910. The CCD 3000 camera can be any CCD camera suitable for fluorescent image emissions. For example, suitable CCD cameras are available at Alpha-Innotech (San Leandro, CA), Stratagene (La Jolla, CA), and BioRad (Richmond, CA). To measure the fluorescent emission in the microplate thermal slip test, an alternative for a fluorescent plate reader is a charge coupled device (CCD). For example, high-resolution CCD cameras can detect very small amounts of electromagnetic energy, whether originating from distant stars, diffracted by crystals, or emitted by fluorophores. A CCD is made of semi-conductor silicon. When photons of light fall on it, free electrons are released. As an electronic imaging device, a CCD camera is particularly suitable for the image that emits fluorescence because it can detect very weak objects, produces sensitive detection over a wide range of the spectrum, produces low levels of electromagnetic noise, and detects signals over a wide dynamic range - that is, a charge coupled device can simultaneously detect bright objects and weak objects. In addition, the output is linear in such a way that the amount of electrons collected is directly proportional to the number of photons received. This means that the brightness of the image is a measure of the actual brightness of the object, a property not produced by, for example, photographic emulsions.

When a fluorescent imaging camera or a CCD camera is used, the exciting light 2916 may be a suitable lamp that is positioned over the plurality of samples 2910. Alternatively, the exciting light 2916 may be a suitable lamp that is positioned below the plurality of samples 2910. In another alternative embodiment, the exciting light 2916 can be released to each sample 2910 by means of a plurality of fiber optic cables. Each fiber optic cable is disposed through a plurality of tunnels in the conductor block 2912. Thus, each of the samples 2910 receives exciting light 2916 through a fiber optic cable.

As shown in Figure 30, the source 2906 excites the samples 2910 with exciting light 2916. The exciting light 2916 causes the emission of the spectrum 2918 of the samples 2910. The emission of the spectrum 2918 is filtered by an emission filter 3002. The emission filter 3002 filters the wavelengths of the 2918 spectrum emission that are not to be monitored or received by the CCD 3000 camera. The CCD 3000 camera simultaneously receives the emission of the filtered 2918 spectrum from all 2910 samples. simplicity and ease of understanding, only spectral emissions from a row of samples 2910 are shown in Figure 30. CCD camera 3000 is communicatively and operatively coupled to computer 2914 via electrical connection 2902.

Now with reference to Figure 31, one embodiment of the test apparatus 2900 is shown in more detail. As shown in Figure 31, many components of the apparatus are attached to a base 3100. A relative moving means of the heat conducting block 3128 is used to move the heat conducting block 2912 in the 3150 and 3152 directions. of relative movement of the heat conducting block 3128 communicatively and operatively connects to a servo controller 3144. Activation of the relative movement means of the heat conducting block 3128 by a servo controller 3144 moves the heat conducting block 2912 in the directions 3150 and 3152. Servo controller 3144 is controlled by means of a control computer 3142. Alternatively, computer 2914 could be used to control servo controller 3144.

A detector is removably attached to a detector armature 3120. An example of a detector is a fiber optic probe 3122. The fiber optic probe 3122 includes a fiber optic cable capable of transmitting received excitation light 2916 to samples 2910, and a fiber optic cable capable of receiving the emission of the spectrum 2918 of the samples 2910. The electromagnetic radiation is transmitted from the exciting light source 2906 to the optical fiber probe 3122 by the entrance of the exciting light to the fiber cable Optical 3108. In one embodiment of the present invention, a spectrum receiving means comprising the photomultiplier tube 2904 is used to detect the emission of the spectrum of the samples 2910. In this embodiment, the electromagnetic radiation is transmitted from the optical fiber probe 3122 to the tube-photomultiplier 2904 by means of a fiber optic cable 3110. In an alternative embodiment of the present invention, the CCD 3002 camera is used a to detect the emission of the spectrum of the samples 2910. In this modality, the fiber optic cable 3110 is not required.

A temperature sensor 3124 is removably attached to the armature of the detector 3120. The temperature detector 3124 communicatively and operably links to a temperature controller 3162. The temperature detector 3124 monitors the temperature of the heat conductor block 2912 and feeds the temperature backup information to the temperature controller 3162. The temperature controller 3162 is connected to heat the conductor block 2912 by means of a thermoelectric connection 3164. Under the action of the controller of temperature 3162, the temperature of the heat conducting block 2912 may be increased, decreased, or kept constant. Particularly, the temperature of the heat conducting block 2912 can be changed by the temperature controller 3162 according to a predetermined temperature profile. Preferably, the temperature controller computer 3162 is implemented using a computer system such as that described below with respect to Figure 37.

As used herein, the term "temperature profile" refers to a change in temperature over time. The term "temperature profile" encompasses continuous upward or downward changes in temperature, linear or non-linear changes. The term also encompasses any protocol for progressive temperature change, the protocols characterizing the differential increase or decrease in temperature during which temperature increases or decreases are interrupted for periods during which the temperature remains constant. In the apparatus of the present invention, the temperature profile can be pre-determined by programming the computer of the temperature controller 3162. For example, the temperature profiles can be stored in a memory device of the temperature controller 3162, or input to the controller of temperature. 3162 temperature by an operator.

A means of relative movement of the armor of the detector 3130 is used to move the armature of the detector 3120 in the directions 3154 and 3156. A servo controller of the armature of the detector 3118 is fixedly connected to the housing of the exciting light filter 3160. The activation of the servo controller of the armature of the detector 3118 moves the probe of 3122 optical fiber at addresses 3154 and 3156. It would be apparent to one of skill in the relevant art how to configure the servo controllers to move the heat conducting block 2912 and the armature of the detector 3120. It should be understood that the present invention is not limited to the use of servo controllers for the movement of the heat conductor block 2912 and the armature of the temperature detector 3120, and other suitable means known to one skilled in the art, such as a motor, can also be used.

Servo controllers 3118 and 3144 connect communicatively and operatively to the controller's computer 3142. The controller's computer 3142 controls the movement of the detector's 3120 armature in addresses 3154 and 3156. In addition, the 3142 controller's computer controls the movement of the relative movement means of the heat conducting block 3128 in the directions 3150 and 3152.

In the test apparatus of the present invention, the exciting light source 2906 is used to excite the samples 2910. The exciting light source 2906 communicatively and operably connects to the exciting light filter 3104, which is contained within the housing of the exciting light filter 3160. The exciting light filter 3104 filters all the wavelengths of the light from the exciting light source 2906 except for the wavelengths of the light that it is desired that is released by the fiber optic probe 3122 to the samples 2910. A servo controller of the exciting light filter 3106 controls the opening of the exciting light filter 3104. The exciting light source 2906 and the servo controller of the exciting light filter 3106 communicate communicatively and operatively to the computer of the exciting light filter 3106. exciting light controller 3102. The controller computer 3102 controls the wavelength of the exciting light transmitted to the samples 2910 by controlling the servo controller of the exciting light filter 3106. The exciting light 2916 is transmitted through the fiber optic cable of the exciting light input 3108 to fiber optic probe 3122 for transmission to samples 2912.

The emission of spectrum 2918 from samples 2910 is received by fiber optic probe 3122 and transmitted to a spectrum emission filter 3144 by the output of fiber optic cable 3110. The spectrum emission filter 3114 is contained within a housing of the emission filter of the spectrum 3166. The housing of the emission filter of the spectrum 3166 is arranged in the housing of the photomultiplier tube 3168. The housing of the photomultiplier tube 3168 contains the photomultiplier tube 2904. A servo emission controller of the spectrum 3112 controls opening the emission filter of the spectrum 3114, thereby controlling the wavelength of the emission of the spectrum 2918 that is transmitted to the photomultiplier tube 2904. The servo emission controller of the spectrum 3112 is controlled by a computer of the controller 3170.

The emission of the spectrum 2918 of the samples 2910 is transmitted from the photomultiplier tube 2904. The electrical output 3140 connects the photomultiplier tube 2904 to the electrical connection 2902. The electrical connection 2902 connects the electrical output 3140 to the computer 2914. Directed by suitable programs , computer 2914 processes the signal of the spectrum emission of samples 2910. Program example is a graphical interface that automatically analyzes the fluorescence results obtained from samples 2910. Such a program is well known to those skilled in the art. For example, the CytoFluor ™ II Multipoint Plate Fluorescence Reader (PerSeptive Biosystems, Framingham, MA) uses the Cytocalc ™ Data Analysis System (PerSeptive Biosystems, Framingham, MA). Another suitable program includes, MicroSoft Excel or any comparable program.

Figures 32A-C illustrate one embodiment of a thermal electric stage or heat conducting block for the test apparatus of the present invention. Figure 32A shows a side view of the heat conducting block 2912 and a heat conducting cable 3206. Figure 32B shows a top view of the heat conducting block 2912 and the heat conducting cable 3206. The heat conducting cable 3206 is a adjusting element that adjusts the temperature of the heat conducting block 2912. By means already known to a person skilled in the art, the temperature controller 3162 causes the heat conducting cable 3206 to increase or decrease in temperature, thereby changing the temperature of the heat conducting block 2912. For example, an example of a temperature controller is a resistance device that converts electrical energy into heat energy. Alternatively, the heating element may be a water circulation system, such as that disclosed in U.S. Pat. do not. 5,255,976, the content of which is incorporated herein by reference. In another alternative, the temperature adjusting element can be a heat conductive surface on which the heat conducting block 2912 is exposed. Particularly, the temperature of the heat conducting cable 3206 can be changed by the temperature controller 3162 in accordance with a predetermined temperature profile. Temperature controller 3162 is preferably implemented using a computer system as described later with respect to Figure 37. Alternatively, computer 2914 could be used to implement temperature controller 3162. An example set of specifications for the temperature controller 3162 and the heat conducting block 2912 is as follows: resolution 0., 1 ° C accuracy + 0. .5 ° C stability 0., 1 ° C repetitivity 0., 1 ° C.

The temperature controller 3162 changes the temperature according to a temperature profile as discussed below with respect to Figures 36A and 36B.

The temperature of the heat conducting block 2912 can be controlled so that a uniform temperature is maintained across the temperature conducting block. Alternatively, the temperature of the heat conducting block 2921 can be controlled so that a temperature gradient is established from one end of the heat conducting block to the other. Such a technique is disclosed in U.S. Patents. us. 5,255,976 and 5,525,300, the integrity of both is incorporated herein by reference.

The heat conducting block 2912 is preferably configured with plurality of wells 2920 for the samples 2910 to be tested. In one embodiment each of the wells 2920 is configured to receive a container containing a plurality of samples 2910. Alternatively, the heat conducting block 2912 is configured to receive a container containing a plurality of samples 2910. An example container for contain the plurality of samples 2910 is a microtiter plate.

In yet another alternate embodiment, the heat conducting block 2912 is configured to receive a heat conducting adapter that is configured to receive a container containing one or more samples 2910. The heat conducting adapter is disposed in the heating conductor block 2912 , and the sample container 2910 fits into the heat conducting adapter. Figures 32C-E show three examples of configurations of a heat conducting adapter. A 3200 adapter is configured with wells with a rounded bottom. An adapter 3202 is configured with square bottom wells. An adapter 3204 is configured with V-shaped wells. For example, the adapter 3200 can receive a plurality of round bottom containers, each containing a sample. Similarly, adapter 3202 may receive a plurality of square bottom containers, and adapter 3204 may contain a plurality of V-shaped bottom containers. Adapters 3200, 3202, and 3204 may also receive a transport of a multiplicity of containers round background. An example of transport is a microtitre plate having a plurality of wells, each well containing a sample. When the heat conducting block 2912 is heated, the heat conducting adapters 3200, 3202, or 3204 are also heated. In this way, the samples contained in the containers that are fitted with the 3200, 3202, or 3204 adapters are also heated. The 3200, 3202 and 3204 adapters can accept standard microplate geometries.

Another embodiment of the test apparatus of the present invention is shown in Figure 33. In this embodiment, a plurality of heat conducting blocks 2912 is mounted on a turntable or carousel 3306. Alternatively, the platform may be a movable platform. The platform or carousel 3306 can be composed of a heat conducting material, such as the material of which the heat conducting block 2912 is composed. Although six heat conducting blocks are shown in Figure 33, this number is an example and will be understood as that any number of heat conducting blocks can be used. As shown in Figure 33, a shaft 3308 is rotatably connected to the base 3100. The rotating platform 3306 is axially mounted to rotate about the shaft 3308. The rotation of the shaft 3308 is controlled by means of a servo controller 3312. The servo controller 3312 is controlled by means of a computer of the controller 3314 in a manner well known to a person skilled in the relevant art. The controller computer 3314 causes the servo controller 3312 to rotate the axis 3308 thereby rotating the turntable 3306. In this way, the heat conducting blocks 2912 are sequentially placed under the fiber optic probe 3122.

Each of the plurality of heat conducting blocks 2912 can be independently controlled by the temperature controller 3162. Thus, the temperature of a first heat conducting block 2912 may be higher or lower than the temperature of a second heat conducting block. 2912. Similarly, the temperature of a third heat conductor block 2912 may be higher or lower than the temperature of the first or second heat conductor block 2912.

In a manner similar to that described above for Figure 31, the relative movement means 3130 is also used to move the armature of the detector 3120 in the directions 3150 and 3152 so that the optical fiber probe 3122 can be moved to detect the emission of the spectrum of the samples 2910. A second means of relative movement of the armature of the detector 3316 is used to move the armature of the detector 3120 in the directions 3154 and 3156.

The temperature of the heating blocks 2912 is controlled by the temperature controller 3162. The temperature controller 3162 is connected to the turntable 3306 by means of the connection 3164 to the heat conducting blocks 2912. Under the action of the temperature controller 3162, the temperature of heat conducting blocks 2912 may be increased or decreased. Alternatively, the temperature controller 3162 can be configured to adjust the temperature of the turntable 3306. In such a configuration, when the turntable 3306 is heated, the heat conducting blocks 2912 are also heated. Alternatively, the temperature of each of the heat conducting blocks 2912 can be controlled by means of a water circulation system such as that indicated below.

In a manner similar to that illustrated in Figure 31, the exciting light source 2906 is used to excite the samples 2910. The exciting light source 2906 communicatively and operably connects to the exciting light filter. 3104, which is contained within the housing of the exciting light filter 3160. The exciting light filter 3104 filters all the wavelengths of light from the exciting light source 2906 except for the wavelengths of the light that is desired to be will be released by the fiber optic probe 3122 to the samples 2910. A servo controller of the exciting light filter 3106 controls the opening of the exciting light filter 3104. The exciting light source 2906 and the exciting light filter servo controller 3106 they communicate communicatively and operatively to the computer of the exciting light controller 3102. The computer of the controller 3102 controls the wavelength of the exciting light transmitted to the samples 2910 by controlling the servo controller of the exciting light filter 3106. The exciting light 2916 is transmits through the exciting light input of the fiber optic cable 3108 to the fiber optic probe 3122 for transmission to the samples as 2912.

The emission of the spectrum 2918 of the samples 2910 is received by means of the optical fiber probe 3122 and transmitted to the emission filter of the spectrum 3114 by the optical fiber cable 3110. The servo controller of. The emission of the spectrum 3112 controls the opening of the emission filter of the spectrum 3114 and thus controls the wavelength of the emission of the spectrum that is transmitted to the photomultiplier tube 2904. In a manner similar to that explained for Figure 31, the servo controller of the 3112 spectrum emission is controlled by the 3170 controller computer.

The test apparatus of the present invention can detect the emission of the spectrum of the samples 2910 one sample at a time or simultaneously from a subset of samples 2910. As used herein, the term "sample subset" refers to the minus two of the samples 2910. It should be used to detect the emission of the spectrum simultaneously from the subset of samples in one embodiment of the test apparatus of the present invention comprising the photomultiplier tube 2904, a plurality of exciting light filters 3104, input of exciting light of fiber optic cables 3108, exciting light output of fiber optic cables 3110, and light emitting filters 3114.

The spectrum emission signal is transmitted from the photomultiplier tube 2904 to the computer 2914. The photomultiplier tube 2904 communicatively and operably couples to the computer 2914 via the electrical connection 2902. The connection 2902 is connected to the photomultiplier tube 2904 through the electrical output 3140. Computer 2914 functions as a means of analyzing data to analyze the emission of the spectrum as a function of temperature.

Figure 34 illustrates a top view of the test apparatus shown in Figure 33 with a housing 3400 covering the apparatus. A door 3402 opens to reveal the samples 2910. The door 3402 can be a hinged door that opens by rotating. Alternatively, the door 3402 may be a sliding door that opens sliding. A side vita of the test apparatus shown in Figures 33 and 34 is illustrated in Figure 35. Cover 3400 is disposed at the top of base 3100. Cover 3400 can be made of any suitable material. For example, cover 3400 can be made of plaxiglas, fiberglass, or metal.

Figures 36A and 36B illustrate a temperature profile and how the temperature profile is implemented using the test apparatus of the present invention. Figure 36A illustrates a temperature profile 3600 showing the temperature of heat conducting blocks 2912 as a function of time. The heat conducting blocks 2912 and the samples 2910 are heated in a continuous manner according to the temperature profile 3600. Alternatively, the turntable 3306 may be heated together with the light conducting blocks 2912. Preferably, the temperature profile 3600 is linear, with temperatures in the range of about 25 ° C to about 110 ° C.

Alternatively, the temperature profile 3600 can be characterized by the stepped differential increase in temperature, at which the heat conducting blocks 2912 and samples 2910 are heated to a predetermined temperature, maintained at that temperature for a predetermined period of time, and heated to a higher pre-determined temperature. For example, the temperature can be increased from 0.5 ° C to 20 ° C per minute. Although the temperature range of about 25 ° C to about 110 ° C is exposed, it will be understood that the range of temperature at which a given target molecule, eg, a protein, will be heated to generate a thermal denaturation curve can be easily determined by one skilled in the art, the duration in which the 3600 temperature profile is performed will vary, depending on how many samples are to be tested and how quickly the detector receiving the 2918 spectrum emission can detect the emission of 2918 spectrum of samples 2910. For example, an experiment in which each of six heat conducting blocks 2912 contains a total of 96 samples 2910 (for a total of 576 samples), and in which samples are searched using A fluorescent reading device that has a simple fiber optic probe, and in which the temperature profile is 38 ° C and 76 ° C, would take approximately 38 minutes to develop using the apparatus shown in Figure 33.

While heating according to the temperature profile 3600, the emission of the spectrum 2918 from each sample 2910 in a first heat conducting block 2912 is received through the fiber optic probe 3122. As illustrated in Figure 36B, after that the emissions of all the samples 2910 have been received in the first heat conducting block 2912, the platform 3306 is rotated to move the next heat conducting block 2912 under the fiber optic probe 3122 and the emission of the 2918 spectrum Samples 2910 is received by fiber optic probe 3122. This process is continued until the reception of the spectrum emissions of all samples is complete in all heat conducting blocks 2912. The emission of the spectrum from samples 2910 in each heat conducting block 2912 can be received one at a time, simultaneously from a subgroup of samples, simultaneously from one row of samples at a time, or all the samples at the same time.

Izpplementación of the Program of Computation of the Preferred Modalities The present invention could be implemented using computer elements, programming, or a combination thereof, and could be implemented in a computer system or other processing system. A flow chart 3800 for the implementation of one embodiment of the present invention is shown in "Figure 38. The flow diagram 3800 begins with a start step 3802. In a step 3804, the temperature profile 3600 is started. example, the temperature controller 3162 causes the temperature of the heat conducting block 2912 to increase. In a step 3806, a detector such as the fiber optic probe 3122 or the CCD camera 3000 moves over a sample 2910, sample row 2910, or all of the samples 2910. In a step 3808, the exciting light is transmitted to the samples 2910 using the exciting light 2906. In a step 3810, the emission of the spectrum is received by the detector of the sample 2910. In one step of decision 3812, it is determined if the 2918 spectrum emission of all the samples, rows of samples, has been received in a heat conducting block 2912. If the 2918 spectrum emission has not been received in all samples or rows of samples , the dete ctor is moved over the next sample or row of samples in step 3814. The processing then continues in step 3808 to transmit the exciting light 2916. The processing then continues in step 3810 to receive the 2918 spectrum emission of the 2910 samples .

If the emission of the spectrum 2918 has been received in all samples or rows of samples, processing continues to a decision step 3816. In decision step 3816, it is determined whether the 2918 spectrum emission has been received from the samples in all heat conducting blocks. If not, the turntable 3306 is rotated in a step 3818 to place the next heat conductor block 2912 and samples 2910 contained therein below the detector. Steps 3806 to 3818 are followed until the emission of spectrum 2918 has been received from all samples in all heat conducting blocks 2912. Processing then continues to step 3820, wherein temperature profile 3600 is completed and the processing ends in step 3822.

A flow chart 3900 for the implementation of an alternate embodiment of the present invention is shown in Figure 39. In this embodiment, a detector for simultaneously receiving the emission of spectrum 2918 from all samples 2910 in heat conducting block 2912, such as a CCD camera 3000, it is positioned on the heat conductor block 2912. The flow diagram 3900 starts with the initial step 3902. In a step 3904, the temperature profile 3600 is started. For example, the temperature controller 3162 causes the temperature of the heat conducting block 2912 to increase. In a step 3906, the exciting light is transmitted to the samples 2910 using the exciting light source 2906. In a step 3908, the emission of the spectrum is received by a camera of CCD 3000 from samples 2910. In a decision step 3910, it is determined if the emission of spectrum 2918 has been received from all heat conducting blocks 2912. If not, the platform-rotator 3306 is rotated in a step 3912 to place the next heat conductor block 2912 and the samples 2910 contained therein under the CCD camera 3000. Steps 3906 to 3912 are followed until the emission of the 2918 spectrum has been received from Samples 2910 in all heat conducting blocks 2912. Processing then continues to step 3914. In step 3914, temperature profile 3600 is completed and processing is completed in step 3916.

As stated above, the present invention could be implemented using computer elements, programming, or a combination thereof, and could be implemented in a computer system or other processing system. An example of computer system 3702 is shown in Figure 37. The computer of controllers 3102, 3142, 3162, 3170, or 3314, can be implemented using one or more computer systems such as computer system 3702.

After reading this description, it will be apparent to a person skilled in the relevant art how to implement the invention using other computer systems and / or computing architectures. The computer system 3702 includes one or more processors, such as the processor 3704. The processor 3704 is connected to a collective communication line 3706.

The computer system 3702 also includes a main memory 3708, preferably random access memory (RAM), and may also include a secondary memory 3710.

The secondary memory 3710 may include, for example, a hard disk drive 3712 and / or a removable storage unit 3714, which represents a floppy disk drive, a magnetic tape drive, an optical disk drive, etc. The removable storage unit 3714 reads from and / or writes to a removable storage unit 3716 in a well-known manner. The removable storage unit 3716 represents a flexible disk, magnetic tape, optical disk, etc. which is read by and written by the removable storage unit 3714. As will be appreciated, the removable storage unit 3716 includes a usable storage medium of computer which has computer programs and / or results stored therein.

In alternative modalities, the secondary memory 3710 could include other similar means to allow the computer programs or other instructions to be loaded into the computer system 3702. Such means may include, for example, a removable storage unit 3718 and an interface 3720. Examples such may include a program cartridge and cartridge interface (such as that found in video devices), a removable memory circuit (such as EPROM, or PROM) and associated plug, and other removable storage units 3718 and 3720 interfaces which allow the computer program and results to be transferred from the removable storage unit 3718 to the computer system 3702.

The computer system 3702 can also include a communication interface 3722. The communication interface 3722 allows the program and results to be transferred between the computer system 3702 and the external devices. Examples of the 3722 communication interface may include a modem, a network interface (such as an Ethernet card), a communications port, a slot and PCMCIA card, etc. The program and results transferred via communication interface 3722 are in the form of signals 3724 that may be electronic, electromagnetic, optical or other signals capable of being received via a communications interface 3722. These signals 3724 are provided to the communication interface via a channel 3726. This channel 3726 carries the signals 3724 and can be implemented using cable, optical fibers, a telephone line, a cellular telephone link, an RF link and other communication channels. In the test apparatus of the present invention, an example of channel 3726 is the electrical connection 2902 which carries signal 3724 or emission of spectrum 2918 to computer 2914.

In this document, the terms "computer program medium" or "usable computer medium" are used to refer generally to the medium such as the removable storage device, 3716 and 3718, a hard disk installed in the hard disk device 3712, and signals 3724. These computer program products are means for providing programs to computer systems 3702.

The computation programs (also called logical computing control) are stored in the main memory 3708 and / or secondary memory 3710. The computation programs can also be received via the communication interface 3722. Such computer programs, when executed, they allow computer system 3702 to develop the features of the present invention as discussed herein. In particular, the computer programs, when executed, allow the processor 3704 to develop the features of the present invention. Therefore, such computer programs represent controllers of the computer system 3702.

In a mode where the invention is implemented using programming, the programming could be stored in a computer program product and loaded into the computer system 3702 using removable storage device 3714, hard device 3712 or communication interface 3722. Logical control (programming), when executed by processor 3704, causes processor 3704 to perform the functions of the invention as described herein.

In another embodiment, the invention is implemented primarily in a computer element using, for example, computer components such as application-specific integrated circuits (ASICs). The implementation of the elements of the computer that the machine expresses to perform the functions described here will be evident to experts in the art.

In yet another embodiment, the invention is implemented using a combination of computer elements and programming.

The test apparatus of the present invention is particularly suitable for carrying out the methods of the present invention. To conduct a microplate thermal slide test using the method and apparatus of the present invention, the samples are placed in a heat conducting block, heated according to a predetermined temperature profile, stimulated with an exciting wavelength of light , and the emission of the spectrum of the samples is detected while the samples are heated according to the pre-determined temperature profile.

It will be understood that the test apparatus of the present invention is not limited to use with the methods of the present invention or is limited to conducting tests on biological polymers, proteins or nucleic acids. For example, the test apparatus of the present invention can be used to incubate samples at a predetermined temperature.

Alternatively, the test apparatus of the present invention can be used to develop the polymerase chain reaction, the thermal cycle steps, the thermal stability test of a compound, such as a drug, to determine the conditions that stabilize a compound , or to determine the conditions that facilitate the crystallization of a compound.

Having generally described the invention, it will be more readily understood by reference to the following specific examples which are included herein for purposes of illustration only and are not intended to be limited unless otherwise specified.

Example 1 Sorting of Ligands That Link the Active Site of Human a-thrombin Using the DirectedDiversity® computer-controlled process (see U.S. Patent 5,463,564), scientists at 3-Dimensional Pharmaceuticals, Inc. have generated a combinatorial library of compounds directed to the human a-thrombin active site. Approximately 400 compounds were synthesized and tested by a conventional spectrophotometric kinetic test in which succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (Bachem, King of Prussia, PA) served as the substrate. Five of these compounds, which are characterized by the K s that extend almost four orders of magnitude in the binding affinity, were used to test the range and detection limits of the heat exchange test. These five compounds are properly listed in Table 3, together with the Ki of each respective compound, as measured by the kinetic test (last column). The Ks for these compounds are in the range of 7.7 nM for 3dp-4026 to 20.0 μM for 3dp-3811.

A stock solution of human a-thrombin (1.56 mg / mL) from Enzyme Research Labs was first diluted to 0.5 mg / mL (11 μM) with 50 mM Hepes, pH 7.5, 0.1 M NaCl (test buffer, unless mentioned otherwise), and stored on ice. The five ligands (recrystallized solids characterized by mass spectrometry and RNM) were exactly weighed to be 1.5 to 2.0 mg and dissolved in 1.0 mL of 100% DMSO so that the concentration was between 1.8 and 3.8 mM. A 96-well Costar microplate in V-bottom was then placed in such a way that 100 μL of 11 μM human a-thrombin solution was pipetted into wells Al to A6. This was followed by the addition of 2 μL of 3dp-3811 in well A2, 2 μL of 3dp-3959 in well A3, 2 μL of 3dp4077 in well A4, 2 μL of 3dp-4076 in well A5, 2 μL of 3dp4026 in well A6, and 2 μL of 100% DMSO in control well Al. The content was mixed by repeated intakes and discharges using a 100 μL pipette tip. Finally, a drop of mineral oil (Sigma, St. Louis, MO) was added at the top of the wells to reduce the evaporation of the samples at elevated temperatures. The microplate was then placed in the heating block 4 of a RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, CA), set at 25 ° C, for 1 minute. The plate was then placed in a spectrophotometer SPECTRAmax ™ 250 (set at 30 ° C) and absorbance at 350 nm was measured for each sample. This reading served as a target or reference that the other readings were compared to higher temperatures. The test was started by establishing the heating block 1 at 38 ° C, programming the temperature cycle to move the microplate to the heating block 1, and keeping the microplate there for 3 minutes. After equilibration at 38 ° C, the plate was moved to the 25 ° C block (Block 4) for 30 seconds, inserted into the spectrophotometer, and the absorbance was read at 350 nm. The microplate was then placed in the temperature cycle and moved to heating block 2, which had been pre-equilibrated at 40 ° C. After 3 minutes at 40 ° C, the plate was returned to 25 ° C (in block 4) for 30 seconds, and returned to the spectrophotometer for an absorbance measurement at 350 nm. This process was repeated 18 more times until the temperature had increased to 76 ° C in 2 ° C increments. After subtracting the absorbance of the target (A350 at 25 ° C), turbidity, reflected in the absorbance value, was plotted as a function of temperature. The thermal denaturation curves for this experiment are shown in Figure 1.

The control (in the Al well), which contained only 11 μM of a-thrombin in 2% DMSO, was found to undergo a thermal denaturation transition starting at ~50 ° C, as reflected in the large increase in A350 . The midpoint in this transition was observed to be at ~ 55 ° C. This result was consistent with calorimetric differential search measurements for bovine prothrombin 1, which revealed a denaturation transition at Tm = 58 ° C (Lentz, B.R. et al., Biochemistry 33: 5460-5468 (1994)). The thermal denaturation curves for all tested inhibitor compounds exhibit a change in the transition to higher temperatures. 3d? - 4026 showed the highest change in Tm: ~ 9 ° C. This result is consistent with the fact that, among the compounds tested, 3dp4026 exhibited the greatest binding affinity, as judged by the kinetic measurements with succinyl-Ala-Ala-Pro-Arg-p-nitroanilide as a substrate. In truth, the order of arrangement of the changes in Tm, showed in Figure 1, parallel to the order of binding affinity as measured by conventional enzymology. These results indicate that by simply observing the change in Tm for a series of compounds relative to the control, a series of compounds can be easily and correctly ordered in order to increase the binding affinity for the protein of interest.

It was possible to take another step of the microplate thermal change test and estimate the binding affinity of each ligand to Tm. This was done by substituting T0, Tm,? HU and Cpu in equation (1). If? HU and? Cpu can not be measured because a calorimetric device is not available, assumptions can be made to? HU and? Cpu for the therapeutic destination. In the case of human a-thrombin, it was possible to use? HU = 200.0 kcal / mol, a measured value for the closely related protein of bovine prothrombin-1 (Lentz, BR et al., Biochemistry 33: 5460-5468 (1994 )). A value of? Cpu = 2.0 kcal / mol- ° K was used to calculate KL to Tm since similar proteins of this size have been shown to produce similar values. The binding affinities to Tm of the five test ligands parallel to the Ki 's measured with a spectrophotometric substrate (Table 3).

Table 3. Change Test > Thermal in Microplate for Ligand Link for the Active Site of the Human Thrombin. Tur i dez as an Experimental Sign 1 • Protein [Ligand tm? T? D to tma Kd to Ki / ligand] (° K) (° K) (nM) 310 ° K (310 ° K) C (μM) (nM) (nM) Thrombin swims 327.15 0.0 (TH) TH / 3dp- 37 328.15 1.0 14400 5880 2000 3811 TH / 3dp- 76 332.15 5.0 660 224 250 3959 TH / 3dp- 48 333.15 6.0 160 51.7 46 4077 TH / 3dp- 60 334.15 7.0 76.3 23.6 26 4076 TH / 3dp- 67 336.15 9.0 12.3 3.5 7.7 4026 a Calculations for Kd to Tm were made using equation (1) with? HT0U = 200.0 kcal / mol, as observed for prothrombin 1 by Lenz, B.R. et al., Biochemistry 33: 5460-5468 (1994), and an estimate of? Cpu - 2.0 kcal / mol- ° K; and Ka = 1 / Ka. b The estimates for Kd at T = 310 ° K were made using equation (3), where? HTL was estimated to be - 10.0 'kcal / mol. c K was measured by classical enzymatic methods that are seen in the dependence of [inhibitor] enzymatic hydrolysis of the spectrophotometric substrate succinyl-Ala-Ala-Pro-Arg-p-nitroanilide at 310 ° K (Hepes 50 nM, pH 7.5, 0.2 M NaCl, 0.05% ß-octylglucoside).

Example 2 Sorting of Ligands Linking to the Heparin Linkage Site of Human a-thrombin Tests for ligands that bind to the binding site of human a-thrombin heparin are more difficult to perform than tests for ligands that link the active site of human a-thrombin. At the heparin binding site, substrate is not hydrolysed, so the spectrophotometric signal can not be extended for instrumental detection. The activity of heparin is usually estimated in biological coagulation time tests. Alternatively, the binding affinity of heparin for human a-thrombin can be painstakingly determined by conducting tests of 15 to 20 simple points, in which the concentration of low MW heparin varies in two sections, and monitoring the shutdown of the fluorescent probe, p-aminobenzamidine, linked to the active site of human a-thrombin (Olson, ST et al., J. Biol. Chem. 266: 6342-6352 (1991)). Thus, the binding of heparin to human a-thrombin represents the type of test found with the vast majority of non-enzymatic receptor / ligand binding events, which are commonly observed for hormone / receptor interactions, DNA / DNA interactions. , neurotransmitter / receptor interactions, etc. Several heparin-like compounds sulphated oligosaccharides and naphthalene sulfates were tested by means of the microplate heat exchange test. Using the microplate heat exchange test, it was possible to use a single compound per well to rapidly order the compounds in order of increasing binding affinity, with K s that are in the range of three orders of magnitude (see Table 4). Like the experiment of Example 1, the heat exchange test results in accordance with the results obtained by an alternative method, which requires a series of laborious fluorescence quench tests (15 to 20 single determinations) over a wide range of heparin concentrations of low MW (Olson, ST et al., J. Biol. Chem. 266: 6342-6352 (1991)). These results confirm that by simply observing the change in Tm for a series of compounds, relative to the control, a series of compounds can be easily and correctly ordered in ascending order of binding affinity for the protein of interest.

A literature search did not alternatively locate the measured link that results from the other ligands, which could certify the difficulty of these experiments. However, the literature revealed that pentosan polysulfate (PS04) Sigma, St. Louis, MO), dextran S04 (Sigma, St. Louis MO), and suramin (CalBiochem, LaJolla, CA) have been found to have anticoagulant properties . Indeed, pentosan polysulfate and suramin were previously tested in clinical trials for anti-angiogenic activity, but were ruled out due to toxic effects, many of which are described as coagulation abnormalities (Pluda, JM et al., J Nati, Cancer Inst. 85: 1585-1592 (1993), Stein, CA., Cancer Res. 53: 2239-2249 (1993)). The affinities of PS04 pentosan and suramin to Tm, as measured by the heat exchange test, were found to be 7 times and 5700 times higher, respectively, than the affinity of heparin 5000 (Table 4). These results suggest that these ligands could alter coagulation rates by interfering with the heparin-mediated binding of human a-thrombin to anti-thrombin III (AT III), a protein cofactor for human a-thrombin activity.

The results in Table 4 revealed another advantage of the microplate heat exchange test for libraries of screened compounds: the process is blind and unbiased in the sense that the ligand binding is detected regardless of whether it is on the site active, a binding site of the allosteric cofactor, or in an interfacial protein subunit. The ability to detect ligands that bind with high affinity to external sites an active site of the enzyme will greatly facilitate the discovery of guide molecules.

Table 4. Microplate Thermal Change Test for Linkage of Ligands to the Heparin Linkage Site of Human a-thrombin. Turbidity as an Experimental Sign.

Protein [Ligand tn? Tm Kd to Tma Kd to Ki / ligand] (°?) - (° K) (nM) 298 ° Kb (298 ° K) (μM) (nM) (nM) See Literatu do ra Thrombin swims 329.15 0.0 (TH) TH / Hepar 61 329.65 0.5 38.300 7,570 an S04 TH / Hepar 50 330.15 1.0 19,700 3,810 in 3000 TH / Hepar 44 330.15 1.0 17,200 3,490 5,400c in 5000 TH / Pents 40 332.15 3.0 2,425 427 san PS04 TH / Dextr 48 336.15 7.0 68.8 10.1 an S04 TH / South 102 340.15 11.0 3.02 0.37 amine a Calculations for K a to T m were made using equation (1) with? HT0U = 200.0 kcal / mol, as observed for pre-thrombin 1 by Lenz, BR et al., Biochemistry 33: 5460-5468 (1994), and an estimate of? Cpu = 2.0 kcal / mol- ° K; and Kd = 1 / Ka. Thrombin, human a-thrombin (Factor lia), from Enzyme Research Labs (South Bend, IN) was diluted to 0.5 mg / mL (11 μM) using 50 mM Hepes, pH 7.5, 0.1 M NaCl (3 times dilution) ). All the ligands were dissolved in the same buffer. b The estimates for Kd at T = 298 ° K were made using equation (3), where? HTL was estimated to be - 10.0 kcal / mol. c Olson, S.T. et al., J. Biol. Chem. 266: 6342-6352 (1991).

Example 3 Ordering of Ligands aFGF The second therapeutic receptor tested in the microplate heat exchange test was the acid fibroblast growth factor (aFGF), a growth factor that plays a key role in angiogenesis (Folkman, J. et al., J. Biol. Chem. 267: 10931-10934 (1992)). A synthetic gene for this protein was purchased from R &D Systems (Minneapolis, MN), and cloned and expressed in E. coli using methods similar to those described for basic fibroblast growth factor. (bFGF) (Thompson, L.D. et al., Biochemistry 33: 3831-3840 (1994); Pantoliano, M.W. et al., Biochemistry 33: 10229-10248 (1994); Springer, B.A. et al., J. Biol. Chem. 269: 26879-26884 (1994)). The recombinant aFGF was then purified by heparin-sepharose affinity chromatography as described (Thompson, L.D. et al., Biochemistry 33: 3831-3840 (1994)). AFGF is also known to bind heparin / heparan, which is a cofactor for mitogenic activity. Heparin-like molecules, such as PS04 pentosan and suramin, inhibit the biological activity of the growth factor. A microplate thermal test of these compounds was established in a manner similar to that described above for human a-thrombin. The change in turbidity, as a function of temperature, for each of the ligands suramin, heparin 5000, and PS04 pentosan, are shown in Figure 2. The results are summarized in Table 5. The affinity constants cover a large clearly link affinity range, with the PS04 pentosan showing the highest affinity. The binding affinity order of the ligand of PS04 pentosan, heparin 5000 and suramin was parallel to that found for bFGF, as measured using isothermal titration calorimetry (Pantoliano, M.W. et al., Biochemistry 33: 10229-10248 (1994)). The lack of link affinities measured alternatively for these compounds probably certifies the difficulty of making these measurements using tests that do not monitor physical changes that depend on temperature.

The results in Table 5 are consistent with the results in Tables 3 and 4. By simply observing the change in Tm for a series of compounds relative to the control, a series of compounds can be easily and correctly ordered in ascending order of binding affinity. for the protein of interest.

Table 5. Microplate Thermal Change Test for Link of Ligands for aFGF. Turbidity as a Signal Experimental.

Protein [Ligand Tm? Tm¡ ^ a Trea Kd a Ki / ligand] (° K) (° K) (nM) 298 ° Kb (298 ° K) C (μM) (nM) (nM) See Literatu do ra aFGF nothing 317.15 0.0 aFGF / EEE 50 317.15 0.0 > 50,000 EE aFGF / Der 50 318.15 1.0 37,000 12,7000 kill S04 aFGF / EEE 50 322.15 5.0 10,076 3,040 EEEEE aFGF / ß- 47 329.15 12.0 1055 213 1500 CD 14 S04 aFGF / South 200 330.15 13.0 3220 622 amine aFGF / Hep 50 331.15 14.0 576 106 470 Arina 5000 aFGF / Hep 61 333.15 16.0 357 60 aran S04 aFGF / Pen 100 336. 15 19. 0 208 31 tosan PS04 a Calculations for Ka to Tm were made using equation (1) with? HT0U = 60.0 kcal / mol estimated, and an estimate of? Cp = 0.95 kcal / mol-CK; and Kd = 1 / Ka. All ligands, except β-CD 14 S04, were purchased from Sigma and used without further purification. ß-CD 14 S04 was purchased from American Maize Products Co. (Hammond, IN). The aFGF was diluted to 0.25 mg / mL in 50 mM Hepes, pH 7.5, 0.1 M NaCl. All ligands were dissolved in the same buffer. b The estimates for Kd at T = 298 ° K were made using equation (3), where? HTL was estimated to be - 10.0 kcal / mol. c No binding affinity data were found for these ligands in the literature, but the affinities for these ligand bonds to bFGF are shown, as measured by isothermal titration calorimetry ((Thompson, LD et al., Biochemistry 33: 3831-3840 (1994); Pantoliano, MW et al., Biochemistry 33: 10229-10248 (1994)).

Example 4 Sorting of Ligands bFGF The microplate heat exchange test was used to evaluate the ligands for binding to the heparin binding site of the basic fibroblast growth factor (bFGF). The gene for bFGF was purchased from R &D Systems and cloned and expressed in E. coli as described above (Thompson, LD et al., Biochemistry 33: 3831-3840 (1994); Pantoliano, MW et al., Biochemistry 33: 10229-10248 (1994); Springer, BA et al., J. Biol. Chem. 269: 26879-26884 (1994)). It was found that PS04 pentosan and suramin bind to bFGF with binding affinities of 55 nM and 3.5 μM, respectively. This result for PS04 compared very well with the 88 nM affinity observed for the PS04 to bFGF binding, as determined by isothermal titration calorimetry.

Example 5 Sorting of Human a-thrombin ligands using Fluorescence Emission Because the fluorescence measurements are more sensitive than the absorbance measurements, a fluorescence heat exchange test was used to evaluate the binding of ligand to human a-thrombin. The fluorescence emission spectrum of many fluorophores is sensitive to the polarity of their surrounding environment and therefore are effective tests of phase transitions for proteins (eg, from the native to the split phase). The most studied example of these medium-dependent fluorophores is 8-anilinonaphthalene-1-sulfonate (1,8-ANS), for which it has been observed that changes in the emission spectrum for shorter wavelengths (blue changes) decrease as the polarity of the solvent. These blue changes are usually accompanied by an increase in fluorescence quantum production of the fluorophore. In the case of ANS, quantum production is 0.002 in water and decreases to 0.4 when ANS binds to serum albumin.

ANS was used as the fluorescence test molecule to monitor the denaturation of the protein. In the fluorescence test, the final concentration of human a-thrombin was 0.5 μM, which is 20 times more diluted than the concentrations used in the turbidity tests. This concentration of human a-thrombin is in the range used for the kinetic screening tests.

ANS was excited with light at a wavelength of 360 nm. The fluorescence emission was measured at 460 nm using a CytoFluor II fluorescence microplate reader (PerSeptive Biosystems, Framingham, MA). The temperature was increased as described above for the turbidity tests (see Example 1). The fluorescence plot as a function of temperature is shown in Figure 3 for human a-thrombin alone, and for the human 3dp-4026 / a-thrombin complex. The denaturing transition for human a-thrombin was clearly observed at 57 ° C, a temperature that is only slightly higher than that observed in the turbidity experiment. The result of the fluorescence test is not less, according to the Tm of 58 ° C observed for prothrombin 1 of the differential search calorimetry experiments. Importantly, it was found that 3dp-4026 (at 67 μM) changes the denaturation transition to ~66 ° C to give a change in Tm of 9 ° C which is identical to that found using turbidity as the detection signal (Table 3) .

The results in Figure 3 and Table 4 illustrate several important points. First, at least a 20-fold increase in sensitivity can be gained by changing an absorbance system to a fluorescence emission detection system. This can be critical for receptor proteins for which supplies are limited.

Second, in the fluorescence tests, the denaturation transition signal is much cleaner than the turbidity test signal. In turbidity tests, higher protein concentrations lead to precipitation of denatured protein. The precipitated protein contributes to the noise signal.

Third, the changes in the Tm measurements of the microplate heat exchange tests are reproducible from one detection system to another.

Example 6 Ordering of the Ligands for Domain D (II) of FGFRl The microplate heat exchange test was used to test the binding of heparin 5000 and PS04 pentosan to the known heparin binding site in the D (II) domain of fibroblast growth factor receptor 1 (FGFR1). D (II) FGFR1 is a 124 residue domain that is responsible for most of the binding free energy for bFGF: D (II) FGFR1 was cloned and expressed in E. coli. The recombinant D (II) FGFR1 was renatured from the inclusion bodies essentially as described (Wetmore, DR et al., Proc. Soc. Mtg., San Diego, CA (1994)), except that a hexa- histidine was included in the N-terminus to facilitate recovery by affinity chromatography on a Ni2 + chelate column (Janknecht, R. et al., Nati. Acad. Sci. USA 88: 8972-8976 (1991)). D (II) FGFR1 was further purified on a heparin-sepharose column (Kan, M. et al., Science 259: 1918-1921 (1993); Pantoliano, MW et al., Biochemistry 33: 10229-10248 (1994)). ). The purity was > 95%, as judged by SDS-PAGE. Protein D (II) FGFR1 was concentrated to 12 mg / mL (μL mM) and stored at 4 ° C.

The protein D (II) FGFR1 was dissolved in a solution of ANS up to a concentration of 1.0 mg / mL (70 μM). The quantum production for the ANS binding to the denatured form of D (II) FGFR1 was less than the quantum production for the ANS binding to human a-thrombin. Because the fluorescence of ANS is very dependent on the environment (see Lako icz, IR, Principies of Fluorescence Spectroscopy, Plenum Press, ew York (1983)), the quantum production observed for the denaturation of different proteins will vary. (II) FGFRl, the signal for the turbidity version of the test, however, was almost detectable. Despite the decreased sensitivity for D (II) FGFR1, ANS rescued this system for the microplate test. A similar result was obtained for Factor Xa, except that the fluorescence quantum production for the ANS binding to denatured Factor Xa was almost as good as it was for human a-thrombin. It was found that the fluorescence quantum production for the ANS binding to the denatured bFGF was as high as the quantum production for the binding of ANS to human a-thrombin.

The binding results of D (II) FGFR1, as determined by the microplate heat exchange test, are shown in Figure 4 and Table 6. As previously demonstrated for the other receptor proteins described above, the heat exchange test in microplate facilitated the ordering of ligand binding affinities for D (II) FGFR1.

Table 6. Microplate Thermal Change Test for Link of Ligands for D (II) FGFRl. Fluorescence Emission as an Experimental Signal.

Protein [Ligand Tm? Tm K_ to Tma Kd to Ki / ligand] (° K) (° K) (nM) 298 ° Kb (298 ° K) C (μM) (nM) (nM) Observe - Literatu do ra D (II) None 312.8 0.0 FGFRl D (II) 150 317.9 5.1 30.0 13.6 85.3 FGFRl / He parina 5000 D (II) 156 319.4 6.6 19.1 4.9 10.9 FGFRl / PS 04 Pentosan a Calculations for Kd to Tm were made using equation (1) with an estimate of? HT0U = 60.0 kcal / mol, and an estimate of? Cpu = 0.95 kcal / mol- ° K; and Kd = 1 / Ka. The D (II) FGFR1 was diluted to 1.0 mg / mL (70 μM) in 50 mM Hepes, pH 7.5, 0.1 M NaCl with 136 μM of ANS present. All ligands were dissolved in the same buffer and diluted 50 times in the protein solution. b Estimates for Kd at T = 298 ° K were made using equation (3), where? HTL = -12.1, and -7.48 kcal / mol for PS04 pentosan and heparin 5000, respectively, as determined by titration calorimetry isothermal (Pantoliano, MW et al., Biochemistry 33: 10229-10248 (1994)). c The published data of binding affinity for these ligands for D (II) -D (III) FGFR1 as determined by titration calorimetry (Pantoliano, M.W. et al., Biochemistry 33: 10229-10248 (1994)).

Example 7 Thermal Change Test in Factor D Microplate To further demonstrate the utility of the cross-target of the microplate heat exchange test, another enzyme, Factor D, was tested for its ability to withstand transitions without thermal coiling. Factor D is an essential serine protease involved in activating the alternative pathway of the complement system, the main effector system of host defense against invading pathogens. Factor D was purified from the urine of a patient with Fanconi syndrome (Narayana et al., J. Mol. Biol. 235: 695-708 (1994)) and diluted to 4 μM in test buffer (50 mM Hepes , pH 7.5, 0.1 M NaCl). The test volume was 10 μL and the concentration of 1.8-ANS was 100 μM. The experiment was carried out using perforated round bottom plates (an array of 8 x 12 wells). The protein was heated in two degree increments between 42 ° C to 62 ° C, using a Robocycler ™ temperature cycle. After each heating step, and before the fluorescence search using the reading of the fluorescence plate of CytoFluor II ™ the sample was cooled to 25 ° C (see Example 1). The nonlinear fit of the least squares curve and other data analyzes were performed as described for Figure 3. The results of the Factor D microplate heat exchange test are shown in Figure 5 and reveal a transition without winding thermal that occurs about 324 K (51 ° C) for the unbound form of the protein. The non-reversible ligands of significant affinity are shown for Factor D. The results of Figure 5 show that the microplate heat exchange test can be used to screen a library of compounds for Factor D ligands. The results in Figure 5 also show that the microplate heat exchange test is generally applied to any white molecule.

Example 8 Thermal Change Test in Factor Xa Microplate Human Xa Factor, a key enzyme in the blood coagulation pathway, was chosen, another test of the cross-target utility of the microplate heat exchange test. Factor Xa was purchased in Enzyme research Labs (South Bend, IN) and diluted to 1.4 μM in test buffer (50 mM Hepes, pH 7.5, 0.1 M NaCl). The test volume was 100 μL and the concentration of 1, 8-ANS was 100 μM. The protein was heated in two degree increments between 50 ° C to 80 ° C using a Robocycler ™ temperature cycle. After each heating step, before searching for fluorescence using the reading of the CytoFluor II ™ fluorescence plate, the sample was cooled to 25 ° C (see Example 1). The results of a heat exchange test of Factor Xa are shown in Figure 6. A transition without thermal winding was observed at 338K (65 ° C). The result analysis was described as described for Figure 3. The results in Figure 6 show that the microplate heat exchange test for protein stability is generally applied to any target molecule.

Example 9 Miniaturization of the Thermal Change Test in Linking Microplate of Ligands for Human a-Trsmbina A miniaturized form of the microplate heat exchange test was developed to minimize the amount of valuable therapeutic protein and ligands required for the test. In the first attempt to decrease the test volume, the test volume decreased from 100 μL to 50 μL without adversely affecting the fluorescence signal. When the test volume was further reduced by a factor of ten, to 5 μL, favorable results were obtained for human a-thrombin. As shown in Figure 7, the non-coiling transition of human a-thrombin could be easily observed at its usual Tm. More importantly, an active inhibitor site was observed to change the Tm of the transition without winding by 8.3 ° K to produce an estimate of the E ^ of 15 nM and the Tm. Ka to Tm was calculated using the relation: -H: To 1.? for Tm. To? Tm = .LTm \ where KLTm = Ka to Tm (associated constant of ligand to Tm) Tm = 332.2 ° K (midpoint of the transition without coiling in the absence of a ligand) To = 323.9 ° K ? HuTo = 200.0 kcal / mol, (enthalpy of unwinding for pre thrombin observed by Lentz et al., 1994) ? Cpu = 2.0 kcal / mol (estimated change in the heat capacity of human a-thrombin without coiling) L ^ = 50.0 μM The Kd at temperatures close to 25 or 37 ° C will be of higher affinity if the link enthalpy, Hb, is negative for this ligand. Using a spectrophotometric test, one Kj. apparent approximately 8 nM was observed at 37 ° C (310 ° K).

The measurements shown in Figure 7 were obtained using the CytoFluor II fluorescence plate reader (perSeptive Biosystems, Framingham, MA). In the experiment, the light excitation wavelength was 360 nm and the emission was measured at 460 nm. The microplates used for this miniaturized test were the 96 well V bottom plate of conventional polycarbonate (Stratagene, or Costar) or polycarbonate plates containing 15 μL holes in an 8 x 12 array (Costar píate lids). In the reaction, the human a-thrombin concentration was μM in test buffer (50 mM Hepes, pH 7.5, 0.1 M NaCl). The test volume was 5 μL and the concentration of 1,8-ANS was 100 μM. The protein was heated in two degree increments between 44 ° C to 64 ° C using a Robocycler ™ temperature cycle. After each heating step, and before the fluorescence search using the CytoFluor II ™ fluorescence plate reader, the sample was cooled to 25 ° C for 30 seconds (see Example 1). The non-linear least squares fit and other data analyzes were developed as described for Figure 3.

Example 10 Miniaturization of the Thermal Change Test in Linking Microplate of Ligands for D (II) FGFRl The recombinant D (II) FGFR1 was purified with inclusion bodies and purified by affinity chromatography on heparin sepharose. A stock solution of D (II) FGFR1 (15 mg / mL, 1.1 mM) was diluted to 50 μM in test buffer (50 mM Hepes, pH 7.5, 0.1 M NaCl). The test volume was 10 μL and the concentration of 1.8-ANS was 250 μM. The transition without coiling in the absence of ligands was found to be approximately 312 K (39 ° C) as shown in Figure 8. In the presence of heparin that looks aposulate (300 uM), the transition without coiling was observed to increase by approximately 8 K to approximately 320 K. Using this midpoint of temperature Tm, it is possible to estimate the binding affinity of aposulate for D (II) FGFRl which is approximately 18 μM at Tm (Table 6). These results demonstrate the ability of the microplate heat exchange test to estimate ligand binding affinity for a non-enzyme target molecule.

Example 11 Miniaturization of the Thermal Change Test in Urokinase Microplate Another white molecule analyzed was the human urokinase plasminogen activator (u-Pa). U-PA enzymatically converts plasminogen into active plasmin protease. U-PA is involved in tissue remodeling, cell migration and metastasis. The gene for u-PA was obtained from ATCC (Rockville, MD) and modified to appropriately express the active enzyme in E. coli. u-PA was cloned, overexpressed in E. coli, and purified using procedures similar to those described by Winkler et al. (Biochemistry 25: 4041-4045 (1986)). The last step of the purification of u-PA was developed in the presence of the active site inhibitor glu-gly-arg-chloromethyl ketone (CMK) and therefore the u-PA used for this experiment was the CMK-u-PA complex. The experiment was developed in the miniaturized format in 5 μL of well volume. One μL of concentrated CMK-u-PA (13 g / L, 371.4 μM) was added to 4 μL of 62.5 mM MOPS, pH 7, 125 mM NaCl, and 250 μM of 1.8-ANS, in multiple wells of a plate titration of 96-wells of polycarbonate V bottom. A thermal denaturation curve was generated as previously described for thrombin, aFGF, D (II) FGFR1, Factor D, and Factor Xa, by differential heating of the microplate followed by a fluorescence reading after each temperature increase. The analysis and non-linear least squares fit of the results of this experiment show that the Tro for CMK-u-PA under these conditions is 81 ° C, which is considerably higher than the view for thrombin, aFGF, D ( II) FGFR1, Factor D, and Factor Xa (55.44, 40, 51, 55, and 65 ° C, respectively). This experiment demonstrates the utility of the present invention in the determination of the Tm for relatively thermostable proteins or proteins stabilized by the high affinity binding of ligands and further demonstrates the ability to develop such an experiment in a miniaturized format.

Example 12 Additional Miniaturization of the Thermal Change Test in a-Thrombin Human Microplate A stock of thrombin was diluted to 1 μM in 50 mM Hepes, pH 7.5, 0.1 M NaCl and 100 μM 1.8-ANS. An electronic multi-channel pipette was used to deliver 2 μL or 5 μL of diluted thrombin solution into the wells of a 96-well polycarbonate titration plate. The plate was subjected to 3 minutes of heating thermal block capable of establishing a temperature gradient across the microplate, followed by 30 seconds of cooling at 25 ° C, and subsequent reading on the CytoFluor II fluorescence plate reader. The results were analyzed by non-linear least-squares adjustment and plotted as shown in Figures 10 and 11. Each curve represents a replicated experiment. Standard deviations for Tm determinations are very good for experiments using volumes of 5 μL or 2 μL (+/- 1.73 and +/- 0.90 K, respectively), demonstrating the ability of the present invention to operate at very low volumes. In fact, the volume that could be used in the present invention seems to be limited only by the available technology to deliver exactly small volumes.

The test volume was reduced to 2 μL, as shown for human a-thrombin (1.0 μM) in Figure 11. The reproducible pipetting of 2 μL in a 96 well array requires the use of pipetting tools such as the multichannel pipette available from Matrix Technologies Corp. (Lo ell, MA) that has accuracy of ± 2.0% or 0.15 μL and ± 2.5% or 0.15 μL accuracy for volumes of 0.5 to 12.5 μL.

Example 13 Simple Temperature Mode of the Microplate Thermal Change Test The results of a simple temperature test are shown in Figure 12. Compounds 3DP-3811, 3DP-3959, 3DP-4076, and 3DP-4660 link the active site of human a-thrombin. The Ks (enzymatically determined) of these four human a-thrombin compounds are 20,000 nM, 250 nM, 25 nM, and 8 nM, respectively, each of these four compounds are equilibrated with human a-thrombin in volumes Separate test of 5 μL in a 96-well plate. The final concentration of the ligand was 50 μM.

For ligands that bind to human a-thrombin with higher affinity, low levels of fluorescence emission were observed, relative to the control reaction (human a-thrombin alone) at 55 ° C. The result of the sample containing the ligand weakly linked 3DP-3811 was little different from the result obtained for the control sample. The decrease in fluorescence emission for 3DP-4076 was not as great as expected, given its high affinity (K ± of 25 nM) for human a-thrombin. This result could be due in part to the lower solubility of sodium chloride salts of this compound.

The results of Figure 12 clearly demonstrate the utility of the simple temperature mode of the microplate heat exchange test to rapidly identify ligands with binding affinities (Kd's) of 250 nM or better when the concentration of the ligand is 50 μM.

Example 14 Thermal Change Test in Fluorescence Microplate of Tryptophan Fluorescence of Intrinsic Protein Human a-thrombin Trp fluorescence was tested in a microplate heat exchange test. 100 μL samples contained 2 μM of human a-thrombin. The samples were exposed to light from a Xenon Arc lamp at 280 nm. The emission was detected at 350 nm using the BioLumin (Molecular Dynamics). The temperature cycle, between 44 ° C and 66 ° C, was developed as described in previous examples. The results of the test are shown in Figures 13 and 14. A small increase in fluorescence emission was observed at 350 nm with temperature increase. However, this increase in fluorescence emission was detected barely above the fluorescence level in the blank wells that did not contain protein (Figure 13). Subtracting an average from the target improved the signal for the noise ratio (Figure 14), but the transition without observed coiling was different from that typically observed in tests employing 1, 8-ANS. In contrast to the transition observed using 1.8-A? S, the transition in Figure 14 seems wider and has a mean temperature point Tm at 334.4 ± 5.1 ° K, about five degrees higher than the Tm observed for a -Rombine of human in tests developed with 1.8-A? S.

Example 15 Test of Link Interactions Multi-Ligando As previously demonstrated, the heat exchange test can be used for the screening of ligands to bind to simple sites on the target proteins. In view of the highlighted physical principles on which the microplate thermal change test is based, the next additivity of the free energy of ligand bond and protein without winding, it is possible to use the microplate thermal change test to analyze the interactions of multi-ligand binding with a white protein. If the free energy of the different ligand binding link the same protein is almost additive, then the multi-ligand binding systems can be analyzed, if the ligands bind in a cooperative (positive) or a non-cooperative (negative) manner.

The binding ligand for human a-thrombin was tested in a microplate heat exchange test. Human a-thrombin has at least four different ligand binding sites: (1) the catalytic binding site; (2) the fibrin binding site (exosite I); (3) the heparin binding site (exositium II); and (4) the Na + binding site, located ~ 15A of the catalytic site. First, the independent binding of three individual ligands was tested: 3DP-4660, Hirugen (hirudin 53-64) (Bachem), and heparin 5000 (CalBiochem). These ligands are linked to the catalytic site, the fibrin binding site and the heparin binding site, respectively.

A thrombin stock solution was diluted to 1 μM in 50 mM Hepes, pH 7.5, 0.1 M NaCl, 1 mM CaCl 2, and 100 μM 1,8-ANS. Each thrombin ligand was included alone and in various combinations for thrombin solutions of 1 μM final concentrations of 50 μM each, except for heparin 5000, which was 200 μM. 100 μL of thrombin solution or thrombin / ligands was distributed in the wells of a 96 well V bottom polycarbonate microtiter plate. The plate was subjected to 3 minutes of heating in a thermal block capable of establishing a temperature gradient across the microplate, followed by 30 minutes of cooling at 25 ° C, and subsequently reading in a fluorescence plate reader. The results were analyzed by adjustment of least squares.

The results of these individual binding reactions are shown in Figures 15 and 16. The ordering of the binding affinity order was 3DP-4660 > Hirugen > heparin 5000, which corresponds to the Kd values of 15 nM, 185 nM and 3434 nM, respectively, for the binding of ligands to each Tm (see equation (4)).

The results reveal changes without thermal coiling that are slightly smaller than would be expected if the bond free energies were completely additive. For example, Hirugen only presents a? Tm of 5.8 ° C, and 3DP-4660 only presents a? Tm of 7.7 ° C. In combination, however, Hirugen and 3DP-4660 have a? Tm of 12.2 ° C. This result means that the binding affinity of one or both ligands decreases when both ligands are linked, and is an example of negative cooperativity in the bond between fibrin and catalytic binding sites. Such a negative cooperative effect is consistent with that of the literature of human a-thrombin, in which the kinetics of hydrolysis of several chromogenic substrates was found to be dependent on the ligands that bind exosite I. Indeed, a decrease of 60% in K_ it was observed for the hydrolysis of D-phenylalanylpipecolyl arginyl-p-nitroanilide when Hirugen was present (Dennis et al., Eur. J. Biochem 188: 61-66 (1990)). In addition, there is also structural evidence of cooperativity between the catalytic site and the exosite I. A comparison of the isomorphic structures of human a-thrombin bound to PPACK (an inhibitor of the catalytic site of human a-thrombin) and Hirugen revealed conformational changes that occur in the active site as a result of the Hirugen binding to exosite I (Vijayalkshmi et al., Protein Science 3: 2254-2271 (1994)). Thus, in the microplate heat exchange test, the apparent cooperativity observed between the catalytic center and the exosite I is consistent with the functional and structural results of the literature.

Similarly, when the binding of the three ligands was tested, a Tra of 12.9 ° C was observed (Figure 16). If the free bond energies were completely additive, one might expect to observe a? Tm of 17.7 ° C. The observed result indicates that negative cooperativity is also presented by ligand binding at the three protein binding sites. This result is consistent with the literature. In a ternary complex with heparin and fibrin monomer, human a-thrombin has decreased its activity towards chromogenic substrates tri-peptides and pro-thrombin (Hogg &Jackson, J. Biol. Chem. 265: 248-255 (1990) ), and markedly reduce the reactivity with anti-thrombin (Hogg &Jackson, Proc.Na.I.Acid.Sci.USA 86: 3619-3623 (1989)). Also, recent observations indicate that ternary complexes are also formed in plasma and markedly compromise the anticoagulant activity of heparin (Hotchkiss et al., Blodd 84: 498-503 (1994)). A summary of these multi-ligand binding results are shown in Table 7.

The results of Figure 15, Figure 16, Table 7 illustrate the following advantages of using the microplate heat exchange test to develop multivariable analyzes. First, the same microplate heat exchange test can be used to simultaneously detect the binding of multiple ligands at multiple binding sites on a target protein. Second, the microplate heat exchange test can be used to detect the same ligand bonds for two or more sites in a therapeutic target. Third, the microplate heat exchange test provides the detection of cooperativity in ligand binding. The information on the cooperativity in ligand link can be collected and analyzed very quickly. Thus, multi-ligand binding experiments that would take months to perform using alternative technologies take only hours to develop them using the microplate heat exchange test.

Table 7. Microplate Thermal Change Test for Link of Ligands for the Active Site, Exositio, and Heparin Link Site of Human a-thrombin Protein / [Ligand] Tm? Tm Kd to T. »P ^ to 298 ° b ligand (μM) (° K) (° K) (nM) (nM) Thrombin swims 323.75 0.0 (TH) TH / Hepari 200 327.95 4.2 3434 470 na 5000 TH / Hirudi 50 329.52 5.8 185 23 na 53-65 TH / 3dp- 4660 50 331.40 7.7 29 3 TH / Hepari 200 327.95 na 5000 TH / Hep. / H 50 330.57 26 4254 478 go. TH / Hepari 200 327.95 na 5000 TH / Hep. 50 333.20 5.3 350 32 3dp-4660 TH / Hirudi 50 329.52 na 53-65 TH / He. / H 200 330.57 11 75422 8467 go. TH / Hirudi 50 329.52 na 53-65 TH / Hir. 50 335.97 6.5 117 9 3dp-4660 TH / 3dp- 50 331.40 4660 TH / 3dp- 200 333.20 1.8 38205 351 4660 / Hep TH / 3dp- 50 331.40 4660 TH / 3dp- 50 335.97 4.6 731 54 4660 / Hir a Calculations for Kd to Tm were made using equation (1) with? HT0U = 200. 0 kcal / mol, as observed for pre-thrombin 1 by Lentz et al., (1994), and an estimate of? Cpu = 2.0 kcal / mol- ° K; and Kd = 1 / Ka. b The estimates for Kd at T = 298 ° K were made using equation (3), where? HTL is estimated to be -12.1 kcal / mol.

Example 16 Critical Biochemical Conditions that Increase the Stability of Human a-Thrombin The microplate heat exchange test, with four different fluorophores, was used to simultaneously screen for the effects of multiple pH values, sodium chloride concentrations, and oxide-reduction compounds on human a-thrombin stability. The thrombin solution was diluted to 1 μM in 50 mM Hepes, pH 7.5, 0.1 M NaCl or 0.5 M, 10 mM EDTA, 10 mM CaCl2, 10 mM dithiothreitol, 10 mM 10 CaCl, and 100 μM 1.8-ANS, 10% of glycerol (v / v), or 0.1% polyethylene glycol (w / v) (PEG) 6000. The reaction volume was 100 μL.

The results of these multivariable experiments are shown in Figures 17A-D and Figure 18. Figures 17-D summarize the stability results collected in a single 96-well plate for human a-thrombin. In Figure 17A, the fluorophore is 1.8-ANS. In Figure 17B, the fluorophore is 2,6-ANS. In Figure 17C, the fluorophore is 2,6-TNS. In Figure 17D, the fluorophore is bis-ANS. The results in Figures 17A-D show an optimum pH of about 7.0 and an increase in stability with increasing NaCl concentration. A ΔTm of about 12 ° C was observed when the NaCl concentration was increased from 0 to 0.5 M. Figure 18 shows a stabilizing effect of 10% glycerol and a destabilizing effect of dithiothreitol. From Figures 17A-D and 18 it is evident that the 1,8-ANS and 2,6-ANS fluorophores are more effective in the heat exchange test. in microplate.

The stabilizing effect of NaCl is particularly interesting as there are recent reports in the literature of a weak Na + binding site (Kd of 30 ± 3 mM in 5 mM Tris buffer pH 8.0, 0.1% PEG, 25 ° C) approximately 15 Á of the catalytic center of thrombin (Dang et al., Nature Biotechnology 25: 146-149 (1997)). Using equation (1), it is possible to estimate the NaCl bond that is ~ 6 mM close to Tm (53 ° C) in Hepes 50 mM buffer pH 8.0 (zero NaCl and 0.10 M).

Additional stabilization that occurs at a NaCl concentration greater than 0.10 M could come from additional binding events of Na + and / or Cl ", added to the complete structure of human a-thrombin, alternatively, the source of this additional stabilization. could come from a less specific salting effect that is usually observed in NaCl of 0.5 to 2 M and is due to the preferential hydration of proteins induced by salts (Timasheff &Arakawa, En: Protein Structure, A Practi cal Approach, TE Creighton , ed., IRL Press, Oxford, UK (1989), pp 331-354)).

The stabilizing effect of glycerol in proteins has been attributed to the balance between the preferential exclusion of glycerol (eg preferential hydration of proteins) and the specific binding to polar regions on the surface of proteins (Timasheff &Arakawa, En: Protein). Structure, A Practical Approach, TE Creighton, ed., IRL Press, Oxford, UK (1989), pp 331-354)).

Ejenplo 27 Biochemical Screening Conditions that Increase the Stability of Receptor 1 of D (II) FGF The microplate heat exchange test was used to simultaneously screen for the effects of multiple biochemical conditions on the stability of receptor 1 of D (II) FGF. The tests were performed by mixing 1 μL of D (II) FGFR1 (from a stock solution concentrated in 50 mM Hepes pH 7.5) with 4 μL of each biochemical condition in the wells in a 96-well polycarbonate microtiter plate. The final concentration of the protein after mixing was 100 μM and the final concentration of 1,8-ANS was 200 μM. The biochemical conditions were tested as follows: The tested pH was 5 (Na acetate), 6 (MES), 7 (MOPS), 8 (HEPES), and 9 (CHES), with the final concentrations of 50 M buffer.

The salt concentrations tested were 0.1 or 0.5 M NaCl. The additives were tested in 50 mM MOPS, pH 7.0, 0.1 M NaCl, at final concentrations of 1 mM (EDTA, dithiothreitol), 10 mM (CaCl2, MgCl2, MgSO4, NiS04 ), 50 mM (arginine), 100 mM ((NH4) S04, LiSO4, Na2SO4, ZnSO, 5% w / v (polyethylene glycol 6000), and 10% v / v glycerol.

The thermal denaturing profiles were generated as previously described for thrombin, aFGF, Factor D, and Factor Xa, by differential heating of the microplate followed by a fluorescence reading after each temperature increase. The results were analyzed by nonlinear least-squares adjustment as previously described.

The results of these multi-variable experiments are shown in Figures 19-24. As the picture shows 19, stability increased with increasing concentration of NaCl A? Tm of about 5 ° C was observed as the NaCl concentration increased from 0.1 to 0.5 M. As shown in Figure 20, MgSO4 and arginine stabilized the protein. As shown in Figure 21, 10% glycerol stabilized the protein. In addition, the salts of the Hofmeister series such as Li2S04, Na2S04, (NH4) S04 and Mg2S04 all had stabilizing effects (Figure 21). As shown in Figure 22, dithiothreitol destabilizes the protein. These results are not very different from those of human a-thrombin. As shown in Figure 23, an optimum pH of about 8.0 was observed. The stabilizing effects relative to EDTA, CaCl2, MgCl2, MgSO4, arginine, (NH4) S04, LiSO4, Na2SO4, ZnSO4), glycerol, polyethylene glycol 6000, and dithiothreitol are shown in Figure 24.

Example 18 Biochemical Screening Conditions that Increase the Stability of Urokinase The microplate heat exchange test was used to simultaneously screen the effects of multiple biochemical conditions on the stability of human urokinase. This experiment was carried out by mixing 1 μL of urokinase (from a concentrated stock solution 371 5 M in 20 mM Tris pH 8) with 4 μL of each biochemical condition in the wells in a 96-well polycarbonate microtiter plate. The final concentration of the protein after mixing was 74 μM and the final concentration of 1.8 -ANS was 200 μM. The biochemical conditions were tested as follows: The tested pH was 5 (acetate), 6 (MES), 7 (MOPS), 8 (HEPES), and 9 (CHES) with the final concentrations of 50 mM buffer. The concentrations tested were? ACl 0.1 or 0.5 M. The glycerol was tested at 10% v / v in 50 mM MOPS, pH 7,? ACl 0.1.

The thermal denaturation profiles were generated as previously described for thrombin, aFGF, Factor D, D (II) FGFR1, and Factor Xa, by differential heating of the microplate followed by a fluorescence reading after each temperature increase. The results were analyzed by nonlinear least-squares adjustment as previously described.

The results of these multi-variable experiments are shown in Figure 25. An optimum pH of 7.0 was observed. Increasing the concentrations of sodium chloride stabilized the protein. 10% glycerol also stabilized the protein. These results are consistent with the results reported in the literature (Timasheff &Arakawa, In: Protein Structure, A Practical Approach, T.E. Creighton, ed., IRL Press, Oxford, UK (1989), pp 331-354)).

Figures 17-25 illustrate the advantage of using the microplate heat exchange test to simultaneously screen multi-variable biochemical conditions that optimize the stability of the protein. Using the methods and apparatus of the present invention, large arrays of biochemical conditions can be rapidly screened for conditions that influence the stability of the proteins. Thus, the present invention can be used to quickly identify the biochemical conditions that optimize the shelf life of the protein.

Example 19 Biochemical Conditions of Crzibado that Facilitate the Curl of the Protein Factorial experiments were developed to identify the biochemical conditions that increase the performance of correctly rolled His6-D (II) -FGFR1. His6-D (II) -FGFR1 is the protein of receptor 1 of D (II) FGF, to which a polyhistidine binds to the N-terminus. The results are summarized in Table 8. When the final concentration of guanidinium hydrochloride was 0.38 M, a re-coiled protein that produced 13.5 ± 0.2% was obtained at pH 8.0 and 0.5 M NaCl. This yield could be increased to 15.5 ± 0.3% if glycerol was present at 7% (v / v). A further increase in re-coiled His6-D (II) -FGFR1 that produced approximately 18% was observed when the pH was increased to 8.9. In fact, increasing the pH from 8.0 to 8.9 improved the yields in all the experiments. These results show that a pH between 8 and 9, and 7% glycerol, are two important conditions that facilitate the winding of D (II) -FGFR1. Each of these conditions increased the yield of the rolled protein from approximately 15 to 20% over the initial conditions at pH 8.0 and 0.5 M NaCl.

Importantly, the effects of pH and glycerol appear to be almost additive. The increased yield of the re-coiled protein at pH 8.9 and 7% glycerol was found to be 17.8%, 32% higher than the yield obtained at a pH of 8.0 and 0.5 M NaCl (13.5 ± 0.2% yield). The close addition of the re-wound determinants has important consequences since it suggests that the individual free energy components comprising the total free wind energy can be combined increasing to optimize the production of rolled up protein.

Table 8. Factorial Experiment to Optimize the Production of Rolled Protein to Immobilize His6-D (II) -FGFR1 to a Final Concentration of Gdn-HCl of 0.3 * Ma a The rewinding was started by diluting a suspension of 3.2 mL of Ni2 + NTA / 6M Gdn-HCl up to 50 mL in the respective winding dampeners (dilution 1: 15.6) in such a way that the final concentration of Gdn-HCl was 0.38 M . b Performance is based on the measured values of A280 for fractions eluted on a Heparin-Sepharose column. The concentration of immobilized protein was 1.2 mg / mL, as measured by a Bio-Rad protein test. Since the column size was 21 mL, 25.2 mg of D (II) FGFRl was bound to the resin.

The results of a second round of rewinding experiments at a final concentration of Gdn-HCl of 0.09 M revealed that Gdn-HCl is an even more important factor affecting the curl of His6-D (II) -FGFRl (Table 9) . A pH of 8.0 and 0.5 M NaCl, decreasing the concentration of Gdn-HCl to 0.09 M, doubled the yield of the rolled protein, relative to the yield obtained at pH 8.0, 0.5 M NaCl, and 0.38 M Gdn-HCl (Table 9). According to the results obtained at a concentration of 0.38 M, the yield of His6-D (II) -FGFR1 re-wound in 0.09 M Gdn-HCl also increased in the presence of glycerol. This suggests that the improved yield of His6-D (II) -FGFR1 in glycerol (5 to 10%) and lower concentrations of Gdn-HCl are additive. In addition, the results of Table 9 reveal that the Hofmeister salt Na2S04 increases the yield of the re-coiled protein almost as much as 5 to 10% glycerol.

Table 9. Factorial Experiment to Optimize the Production of Rolled Protein to Immobilize His6-D (II) -FGFR1. Gdn-HCl Final 0.09 Ma a The rewinding was started by diluting a suspension of 7.5 mL of Ni2 + NTA / 6M Gdn-HCl up to 50 mL in the respective winding buffers (dilution 1: 6.7) in such a way that the final concentration of Gdn-HCl was 0.09 M . b The yield is based on the measured values of A280 'for fractions eluted in a Heparin-Sepharose column. The concentration of immobilized protein was 1.6 mg / mL, as measured by a Protein Bio-Rad test. Since the size of the column was 20 mL, 32 mg of D (II) FGFRl was bound to the resin.

In the comparison of the biochemical conditions that increase the performance of the His6-D (II) -FGFR1 rolled bound to Ni2 + NTA (Tables 8 and 9) and the conditions that increase the total stability of His6-D (II) -FGFRl (Figures 19-24), it is clear that there is a 'strong correlation between protein curl results and protein stability results. Glycerol, the salts of the Hofmeister series, and pH 8.5 to 8.9 improve the performance of the rolled protein and the total stability of the His6-D (II) -FGFR1 protein.

These results are consistent with the model of the wound protein in Figure 26. If aggregation of uncoiled His6-D (II) -FGFR1 is suppressed when immobilized with Ni2 + NTA, and there are two simple equilibrium states between U and N , then the factors that influence the relative position of the balance between U and N should be the same if one starts from U (in the rewinding experiment) or starts from N (in the protein stability screen of the heat exchange test in microplate). Since thermodynamics is independent of the path, only the initial and final states of this reaction should be important. Since similar biochemical conditions facilitate the stability and production of the coiled protein, the simple model for the coiled protein exposed in Figure 26 is accurate for this protein. Thus, the microplate heat exchange test can serve as a rapid and general method for biochemical screening conditions that optimize the curl of the protein.

Example 20 Figure 28 shows the results of the microplate heat exchange tests using each of four fluorescence test molecules: bis-ANS, 2,6-TNS, 1,8-TNS, and 2,6-ANS. The thrombin solution was diluted to 1 μM in 50 mM Hepes, pH 7.5, and 0.1 M NaCl.

Example 21 Comparison of the Results of the Arrangement for a Search of Fluorescence and a Charging Device Coupled to the Camera A documentation of Gel and Analysis System ((Alpha Innotech Corp., San Leandro, CA) was used to develop a microplate heat exchange test. This system uses a CCD camera to detect the fluorescent emission of stained gels, spot spot tests, and 96-well plates. The exciting light source was a UV wavelength trans light box located directly below the CCD camera. The 96 well plate to be tested was placed in the trans light box in the focal view area of the CCD camera (21 x 26 cm).

A solution of 2 μM human a-thrombin was prepared in 50 mM Hepes, pH 7.5, 0.1 M NaCl by diluting a stock solution of '34 μM (1:17) of purified human a-thrombin (Enzyme Research Labs, Madison, Wl). The human a-thrombin solution also contained 100 μM 1,8-ANS. 100 μL of the human-1, 8-ANS α-thrombin solution was aliquoted into each of the twelve single-row wells (row A) of a V-bottom polycarbonate microplate (Costar). A gradient block (RoboCycler ™, Stratagene) was used to heat the twelve samples, from 44 to 66 ° C, through the rows of the microplate. p. e. a temperature gradient of 2 ° C per well was established. Thus, the Al well was at 66 ° C and the Al2 well was at 44 ° C. The control solution containing 1,8-ANS 100 μM in the same buffer (without protein) was placed in each of wells Bl to B12. After adding a drop of mineral oil to each well to avoid evaporation, the plate was heated in the gradient block for 3 min. The content of each well was then allowed to reach room temperature and transferred to a flat bottom microplate. In this experiment, filters were not used for the narrowest exciting wavelength -360 nm and the emission wavelength ~ 460 nm, which are the optimal wavelengths for the 1.8 ANS fluorophore. The flat bottom plate was then placed near the UV transillumination box and the CCD camera was used to measure the amount of light emitted. The plate was also read using a conventional fluorescence plate reader (CytoFluor II), to compare the results obtained by the two different detection methods. The results of the two detection methods are plotted in Figure 40. The results in Figure 40 show that the camera is useful as a fluorescence emission detector to monitor the unwinding of a protein in the microplate heat exchange test.

Example 22 Upgrade of Microplate Thermal Change Using a Charge Device Attached to the Camera An emission filter was used to block all the deviated light output of the emission region for 1,8-ANS (~ 460 nm). In addition, the miniaturized 5 μL form of the microplate thermal change test was used to test the CCD camera detection method in this configuration. V-bottom plates and polycarbonate holes were tested. The experiment was essentially the same as described in Example 21, except that the volume of the test was 5 μL in the 96-hole or V-bottom well plates. The temperature range was 44 to 66 ° C (right to left) for the bottom plate in V, and 46 to 70 ° C (right to left) for the hole plate. Photographs of the CCD images are shown in Figure 41. The image of the V-bottom well microplate is shown in Figure 41A. The image of the hole plate is shown in Figure 41B. The results obtained from the plate in Figure 41A are shown in Figure 42. The results in Figure 42 show that the results obtained using a CCD camera compare very well with the results obtained using a fluorescence plate reader that employs a photo-multiplier tube (PMT) for fluorescence detection.

All publications and patents mentioned above are incorporated herein by reference in their entirety.

While the above invention has been described in some detail for purposes of clarity and understanding, it will be evident to one skilled in the art to read this disclosure that various changes may be made in form and detail without departing from the true scope of the invention and claims. annexed.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it refers.

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

Claims (220)

1. A multivariable method for ordering the efficiency of one or more of a multiplicity of different molecules or different biochemical conditions to stabilize a white molecule that is capable of unwinding due to a thermal change, characterized in that it comprises (a) contacting the target molecule with one or more of the multiplicity of different molecules or one or more the multiplicity of different biochemical conditions in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the target molecule resulting from the heating; (d) generating a thermal unwinding curve for the target molecule as a function of the temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) for (i) each of the other thermal unwinding curves and (ii) the thermal unwinding curve obtained for the white molecule under a reference group of biochemical conditions; Y (f) order the efficiencies of the multiplicity of the different molecules or the multiplicity of the different biochemical conditions according to the change in each of the thermal unrolling curves.

2. The method of claim 1, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a curve of thermal denaturation;

3. The method of claim 1, characterized in that step (d) further comprises determining a mean temperature point (Tm) of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) to (i) the Tm of each of the other thermal unwinding curves and (ii) the Tm of the unwinding curve obtained for the molecule white in the absence of any of the different molecules or obtained under a reference group of biochemical conditions; Y wherein step (f) comprises (fl) order the efficiencies of the multiplicity of the different molecules or the multiplicity of the different biochemical conditions according to the change in the Tm of each of the thermal unwinding curves.

4. The method of claim 1, characterized in that the target molecule is a protein.

5. The method of claim 1, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the wells.

6. The method of claim 4, characterized in that step (a) comprises contacting the protein with one or more different molecules or different biochemical conditions, in the presence of a fluorescence test molecule present in each of the multiplicity of wells and wherein step (c) comprises (cl) exciting the fluorescence test molecule in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

7. The method of claim 6, characterized in that the fluorescence is fluorescence emission.

8. The method of claim 1, characterized in that the target molecule is a nucleic acid.

9. The method of claim 8, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

10. The method of claim 8, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

11. The method of claim 10, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

12. The method of claim 10, characterized in that step (a) comprises contacting the oligonucleotide with the multiplicity of different molecules, or with the multiplicity of different biochemical conditions, in each of the multiplicity of wells, and wherein the step (c) comprises (cl) excite the donor fluorophore, in each of the multiplicity of wells, with light; Y (c2) measuring the fluorescence of the acceptor fluorophore in each of the multiplicity of wells.

13. The method of claim 12, characterized in that the fluorescence is fluorescence emission.

14. The method of claim 6, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells one well at a time.

15. The method of claim 6, characterized in that step (c2) further comprises measuring the fluorescence of a subset of the multiplicity of wells simultaneously.

16. The method of claim 6, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

17. The method of claim 4, characterized in that step (c) comprises (c) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

18. The method of claim 17, characterized in that the fluorescence is fluorescence emission.

19. A multivariable method for ordering the affinity of one or more combinations of a multiplicity of different molecules for a molecule or different molecules for a target molecule that is capable of unwinding due to a thermal change, characterized in that it comprises (a) contacting the target molecule with a combination of two or more different molecules of the multiplicity of different biochemical conditions in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the target molecule resulting from the heating; (d) generating a thermal unwinding curve for the target molecule as a function of the temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) for (i) each of the other thermal unwinding curves obtained for the target molecule and (ii) the thermal unwinding curve for the target molecule in the absence of any of the molecules in the multiplicity of different molecules; Y (f) order the affinities of the combinations of the different molecules for the target molecule according to the change in each of the thermal unwinding curves.

20. The method of claim 19, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a curve of thermal denaturation;

21. The method of claim 19, characterized in that step (d) further comprises determining a mean temperature point (Tm) of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the unwinding curve obtained for the white molecule in the absence of any of the different molecules in the multiplicity of different molecules; Y wherein step (f) comprises (fl) order the efficiencies of the combinations of different molecules according to the change in the Tm of each of the thermal unwinding curves.

22. The method of claim 19, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the wells.

23. The method of claim 19, characterized in that the target molecule is a protein.

24. The method of claim 19, characterized in that step (a) comprises contacting the target molecule with the combinations of the multiplicity of different molecules, in the presence of a fluorescence test molecule present in each of the multiplicity of wells, and wherein step (c) comprises (cl) excite the fluorescence test molecule in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

25. The method of claim 24, characterized in that the fluorescence is fluorescence emission.

26. The method of claim 24, characterized in that the target molecule is a nucleic acid.

27. The method of claim 26, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

28. The method of claim 19, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

29. The method of claim 19, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

30. The method of claim 29, characterized in that step (a) comprises contacting the oligonucleotide with the combinations of different molecules, in each of the multiplicity of wells, and wherein step (c) comprises (cl) excite the donor fluorophore, in each of the multiplicity of wells, with light; Y (c2) measuring the fluorescence of the acceptor fluorophore in each of the multiplicity of.

31. The method of claim 30, characterized in that the fluorescence is fluorescence emission.

32. The method of the claim. 24 or 30, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells one at a time.

33. The method of claim 24 or 30, characterized in that step (c2) further comprises measuring the fluorescence of a subset of the multiplicity of wells simultaneously.

34. The method of claim 24 or 30, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

35. The method of claim 19, characterized in that the multiplicity of different molecules comprises a combinatorial library.

36. The method of claim 35, characterized in that the combinatorial library is a DirectedDiversityR library.

37. A multi-variable optimization method for ordering the efficiencies of one or more combinations of a multiplicity of different biochemical conditions to facilitate the winding or rewinding of an unrolled protein, characterized in that it comprises (a) placing a sample of the coiled or rewound protein in each of the multiplicity of wells in a microplate, where the coiled or re-wound protein has been previously coiled or re-wound according to one or more combinations of the multiplicity of the different biochemical conditions. (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the protein resulting from the heating; (d) generating a thermal unwinding curve for the protein as a function of temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) for (i) each of the other thermal unwinding curves and for (ii) the thermal unwinding curve for the protein in its native form under a group of reference of biochemical conditions; Y (f) order the efficiencies of the combinations of the different winding or rewinding conditions according to the change in each of the thermal unwinding curves.

38. The method of claim 37, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

39. The method of claim 37, characterized in that it also comprises (g) generating combinations of biochemical conditions that increase the magnitude of the physical change, in relation to the magnitude of the physical change of each of the thermal unwinding curves in stage (f); Y (h) repeating steps (a) to (g) until, that a combination of biochemical conditions is determined to promote the maximum winding or re-winding of the protein.

40. The method of claim 37, characterized in that step (e) further comprises (el) evaluate the performance of the rolled up protein.

41. The method of claim 37, characterized in that step (d) further comprises determining a mean temperature point (Tm) of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the unwinding curve obtained for the protein and its native form under a reference group of biochemical conditions; Y wherein step (f) comprises (fl) order the efficiencies of the combinations of the different winding or rewinding conditions according to the change in the Tm of each of the thermal unwinding curves.

42. The method of claim 37, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the wells.

43. The method of claim 37, characterized in that step (a) comprises contacting the protein with the combination of different biochemical conditions in the presence of a fluorescence test molecule in each of the multiplicity of wells and wherein the step ( c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

44. The method of claim 43, characterized in that the fluorescence is fluorescence emission.

45. The method of claim 43, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells one at a time.

46. The method of claim 43, characterized in that step (c2) further comprises measuring the fluorescence of a subset of the multiplicity of wells simultaneously.

47. The method of claim 37, characterized in that step (c) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

48. The method of claim 47, characterized in that the fluorescence is fluorescence emission.

49. A multi-variable optimization method for ordering the efficiencies of one or more combinations of a multiplicity of different biochemical conditions to facilitate the winding or rewinding of an unrolled protein, characterized in that it comprises (a) determining one or more combinations of a multiplicity of different biochemical conditions that promotes the stability of the protein. (b) rolling the unrolled protein under one or more combinations of the biochemical conditions in step (a) which promotes the stability of the protein; (c) evaluate the performance of the rolled protein. (d) order the efficiencies of the combinations of the different winding or rewinding conditions according to the production of the rolled up protein; Y (e) repeating steps (a) - (d) until a combination of biochemical conditions is identified that promotes optimal winding or rewinding of the protein.

50. The method of claim 49, characterized in that it also comprises (f) repeating steps (a) to (e) until a combination of biochemical conditions is determined to promote the maximum winding or rewinding of the protein.

51. The method of claim 49, characterized in that step (a) further comprises the method of any of claims 41-47.

52. The method of claim 51, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

53. In a recombinant DNA method of producing a protein, which comprises expressing an expression of a heterologous protein to a host, obtaining the protein in an inclusion body in the host, and recovering the functional protein of the inclusion body by re-coiling or renaturing the protein under conditions of rewinding or renaturation, the improvement, characterized in that it comprises ordering the efficiencies of the multiplicity of different groups of rewinding or renaturation conditions by the method of claim 37 or 49.

54. A multi-variable optimization method for ordering the efficiencies of one or more combinations of a multiplicity of different biochemical conditions to facilitate the crystallization of a protein that is capable of unwinding due to a thermal change, characterized in that it comprises (a) contacting the protein with a combination of the multiplicity of different biochemical conditions in each of the multiplicity of wells in a microplate. (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the protein resulting from the heating; (d) generating a thermal unwinding curve for the protein as a function of temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) for (i) each of the other thermal unwinding curves and for (ii) the thermal unwinding curve for the protein under a reference group of conditions biochemicals; Y (f) order the efficiencies of the multiplicity of the different biochemical conditions according to the change in each of the thermal unwinding curves.

55. The method of claim 54, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

56. The method of claim 54, characterized in that it also comprises (g) generating combinations of biochemical conditions that increase the magnitude of the physical change, in relation to the magnitude of the physical change of each of the thermal unwinding curves in stage (f); Y (h) repeating steps (a) to (g) until a combination of biochemical conditions is determined to promote maximum crystallization of the protein.

57. The method of claim 54, characterized in that step (d) further comprises determining a temperature midpoint (TJ of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the thermal unwinding curve obtained for the protein and its form under a reference group of biochemical conditions; Y wherein step (f) comprises (fl) order the efficiencies of the combinations of the different biochemical conditions according to the change in the Tm of each of the thermal unrolling curves.

58. The method of claim 54, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the multiplicity of wells.

59. The method of claim 54, characterized in that step (a) comprises contacting the target molecule with the combination of different biochemical conditions, in the presence of a fluorescence test molecule in each of the multiplicity of wells and wherein the stage (c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; and (c2) measuring the fluorescence of each of the multiplicity of wells.

60. The method of claim 59, characterized in that the fluorescence is fluorescence emission.

61. The method of claim 59, characterized in that step (c2) further comprises measuring the fluorescence of each of the mutiplicity of wells one at a time.

62. The method of claim 59, characterized in that step (c2) further comprises measuring the fluorescence of a subgroup of the multiplicity of wells simultaneously.

63. The method of claim 59, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

64. The method of claim 54, characterized in that step (c) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

65. The method of claim 64, characterized in that the fluorescence is fluorescence emission.

66. A method for ordering the affinity of each of a multiplicity of different molecules for a white molecule that is capable of unwinding due to a thermal change, characterized in that it comprises (a) contacting the target molecule with a molecule of the multiplicity of different molecules in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the target molecule resulting from the heating; (d) generating a thermal unwinding curve for the target molecule as a function of the temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) with each thermal unwinding curve obtained for the target molecule in the absence of any of the molecules at the multiplicity of different molecules; Y (f) order the affinities of the combinations of the different molecules for the target molecule according to the change in each of the thermal unwinding curves.

67. The method of claim 66, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a curve of thermal denaturation.

68. The method of claim 66, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the multiplicity of wells.

69. The method of claim 66, characterized in that step (d) further comprises determining a temperature midpoint (TJ of the thermal unwinding curve; wherein step (e) comprises (the) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the thermal unwinding curve obtained for the target molecule in the absence of any of the molecules in the multiplicity of the different molecules; Y wherein the step '(f) comprises (fl) order the affinities of the multiplicity of the different molecules for the target molecule according to the change in the Tm of each of the thermal unwinding curves.

70. The method of claim 66, characterized in that the target molecule is a protein.

71. The method of claim 70, characterized in that step (a) comprises contacting the target molecule with the multiplicity of different molecules, in the presence of a fluorescence test molecule in each of the multiplicity of wells, and wherein the stage (c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

72. The method of claim 71, characterized in that the fluorescence is fluorescence emission.

73. The method of claim 70, characterized in that step (c) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

74. The method of claim 73, characterized in that the fluorescence is fluorescence emission.

75. The method of claim 66, characterized in that the target molecule is a nucleic acid.

76. The method of claim 75, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

77. The method of claim 75, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

78. The method of claim 77, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

79. The method of claim 78, characterized in that step (a) comprises contacting the oligonucleotide with the multiplicity of different molecules, in each of the multiplicity of wells, and wherein step (c) comprises (cl) excite the donor fluorophore, in each of the multiplicity of wells, with light; Y (c2) measuring the fluorescence of the acceptor fluorophore in each of the multiplicity of wells.

80. The method of claim 79, characterized in that the fluorescence is fluorescence emission.

81. The method of claim 70 or 79, characterized in that step (c2) further comprises measuring the fluorescence of each of the mutiplicity of wells one at a time.

82. The method of claim 70 or 79, characterized in that step (c2) further comprises measuring the fluorescence of a subgroup of the multiplicity of wells simultaneously.

83. The method of claim 70 or 79, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

84. The method of claim 66, characterized in that the multiplicity of different molecules comprises a combinatorial library.

85. The method of claim 84, characterized in that the combinatorial library is a DirectedDiversityR library.

86. A combinatorial method for the generation of guide compounds, comprising synthesizing a multiplicity of compounds for binding to a receptor molecule, characterized in that the enhancement comprises (a) contacting the receptor molecule with a compound of the multiplicity of compounds in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the receptor molecule resulting from the heating; (d) generating a thermal unwinding curve as a function of the temperature for each of the wells; (e) comparing each of the thermal unwinding curves of step (d) with each thermal unwinding curve obtained for the receptor molecule in the absence of any of the compounds at the multiplicity of different compounds; Y (f) order the affinities of the multiplicity of the different compounds for the receptor molecule according to the change in each of the thermal unwinding curves.

87. The method of claim 86, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a curve of thermal denaturation.

88. The method of claim 86, characterized in that step (d) further comprises determining a temperature midpoint (TJ of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the thermal unwinding curve obtained for the white molecule in the absence of any of the compounds in the multiplicity of the different compounds; Y wherein step (f) comprises (fl) order the efficiencies of the multiplicity of the different compounds for the receptor molecule according to the change in the Tm of each of the thermal unwinding curves.

89. The method of claim 86, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the multiplicity of wells.

90. The method of claim 86, characterized in that the target molecule is a protein.

91. The method of claim 90, characterized in that step (a) comprises contacting the target molecule with the multiplicity of different molecules, in the presence of a fluorescence test molecule present in each of the multiplicity of wells, and wherein stage (c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

92. The method of claim 91, characterized in that the fluorescence is fluorescence emission.

93. The method of claim 86, characterized in that step (c) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

94. The method of claim 93, characterized in that the fluorescence is fluorescence emission.

95. The method of claim 86, characterized in that the target molecule is a nucleic acid.

96. The method of claim 95, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

97. The method of claim 95, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

98. The method of claim 97, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

99. The method of claim 98, characterized in that step (a) comprises contacting the oligonucleotide with the multiplicity of different molecules, in each of the multiplicity of wells, and wherein step (c) comprises (cl) excite the donor fluorophore, in each of the multiplicity of wells, with light; Y (c2) measuring the fluorescence of the acceptor fluorophore in each of the multiplicity of wells.

100. The method of claim 99, characterized in that the fluorescence is fluorescence emission.

101. The method of claim 93 or 98, characterized in that step (c2) further comprises measuring the fluorescence of each of the mutiplicity of wells one at a time.

102. The method of claim 93 or 98, characterized in that step (c2) further comprises measuring the fluorescence of a subset of the multiplicity of wells simultaneously.

103. The method of claim 93 or 98, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

104. The method of claim 86, characterized in that it further comprises selecting one or more compounds from the multiplicity of wells by their maximum binding affinity for the receptor molecule and generating a second library of compounds having high affinity for such a receptor molecule.

105. The method of claim 86, characterized in that the multiplicity of compounds comprises a combinatorial library.

106. The method of claim 105, characterized in that the combinatorial library is a DirectedDiversityR library.

107. A processing product, characterized in that it comprises a vehicle having a multiplicity of containers in which, each of the containers contains (a) a white molecule that is capable of denaturing due to heating; Y (b) a molecule selected from a multiplicity of different molecules present in a combinatorial library, each of the different molecules present in a different multiplicity of vessels in the vehicle.

108. The processing product of the claim 107, characterized in that the target molecule is a protein.

109. The processing product of the claim 108, characterized in that it comprises a fluorescence test molecule in the multiplicity of vessels.

110. The processing product of the claim 107, characterized in that the target molecule is a nucleic acid.

111. The processing product 110, characterized in that the nucleic acid is a double-stranded oligonucleotide fluorescently labeled.

112. The processing product of claim 111, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

113. The processing product of claim 107, characterized in that the multiplicity of the containers comprises a multiplicity of wells in a microplate.

114. The processing product of claim 107, characterized in that the combinatorial library is a DirectedDiversityR library.

115. A method for ordering the affinity of each of at least two of a multiplicity of different molecules for a white molecule, which is capable of unwinding due to a term change, characterized in that it comprises (a) contacting the target molecule with a collection of at least two molecules of the multiplicity of different molecules in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the receptor molecule resulting from the heating; (d) generating a set of thermal unwinding curves for the target molecule as a function of the temperature for each of the wells; (e) comparing each of the thermal unwinding curves of step (d) with each obtained thermal desenililation curve for the target molecule in the absence of any of the molecules at the multiplicity of different molecules; Y (f) order the affinities of the multiplicity of the different molecules for the target molecule according to the change in each of the thermal unwinding curves. (g) selecting the collection of different molecules that contains a molecule with affinity for the target molecule; (h) dividing the collection into smaller collections of molecules in each of a multiplicity of wells; Y (i) repeating steps (b) to (h) until a single molecule of the multiplicity of different molecules is identified that causes a change in the thermal unwinding curve for the target molecule obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules.

116. The method of claim 115, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a curve of thermal denaturation.

117. The method of claim 114, characterized in that step (d) further comprises determining a temperature midpoint (TJ of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unwinding curves and for (ii) the Tm of the thermal unwinding curve obtained for the white molecule in the absence of any of the molecules in the multiplicity of the different molecules; Y wherein step (f) comprises (fl) order the affinities of the collections of different molecules according to the change in the Tm of each of the thermal unwinding curves.

118. The method of claim 115, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the multiplicity of wells.

119. The method of claim 115, characterized in that the target molecule is a protein.

120. The method of claim 115, characterized in that step (a) comprises contacting the target molecule with the multiplicity of different molecules, in the presence of a fluorescence test molecule present in each of the multiplicity of wells, and wherein stage (c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

121. The method of claim 120, characterized in that the fluorescence is fluorescence emission.

122. The method of claim 120, characterized in that step (c2) further comprises measuring the fluorescence of a subgroup of the multiplicity of wells simultaneously.

123. The method of claim 115, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

124. The method of claim 119, characterized in that step (c) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

125. The method of claim 93, characterized in that the fluorescence is fluorescence emission.

126. The method of claim 115, characterized in that the multiplicity of compounds comprises a combinatorial library.

127. The method of claim 126, characterized in that the combinatorial library is a DirectedDiversityR directed diversity chemical library.

128. The method of claim 115, characterized in that step (c) comprises simultaneously measuring the physical change in all the multiplicity of wells.

129. A method for ordering the affinity of each of a multiplicity of different molecules for a white molecule that is capable of unwinding due to a thermal change, characterized in that it comprises (a) determining the magnitude of a physical change associated with the thermal unwinding of the target molecule resulting from heating by generating a thermal unwinding curve for the target molecule as a function of temperature over a range of one or more discrete temperatures, measuring a physical change associated with the thermal unwinding of the target molecule resulting from the heating, observing the magnitude of the physical change in each of the discrete temperatures within the temperature range; (b) contacting the target molecule with a molecule of the multiplicity of different molecules in each of a multiplicity of wells in a microplate; (c) simultaneously heating the multiplicity of wells of step (b) to one or more discrete temperatures within the temperature range; (d) measuring in each of the wells a physical change associated with the thermal unwinding of the target molecule resulting from the heating and determining the magnitude of the physical change therein at one or more of the discrete temperatures; (e) comparing the magnitude of the physical changes in step (d) at one or more discrete temperatures with the magnitude of the physical change obtained for the target molecule at the same or more discrete temperatures in the absence of any of the molecules in the multiplicity of different molecules; and (f) order the affinities of the multiplicity of different molecules for the target molecule according to the change in the magnitude of the physical change of each of the wells.

130. The method of claim 129, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

131. The method of claim 129, characterized in that the discrete temperature is the midpoint of temperature (TJ for the thermal unwinding curve for the target molecule in the absence of any of the molecules at the multiplicity of different molecules.

132. The method of claim 129, characterized in that step (d) comprises measuring the absorbance of light by the contents of each of the multiplicity of wells.

133. The method of claim 129, characterized in that the target molecule is a protein.

134. The method of claim 129, characterized in that step (b) comprises contacting the target molecule with the multiplicity of different molecules, in the presence of a fluorescence test molecule present in each of the multiplicity of wells, and wherein stage (d) comprises (d) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (d2) measure the fluorescence of each of the multiplicity of wells.

135. The method of claim 132, characterized in that the fluorescence is fluorescence emission.

136. The method of claim 133, characterized in that step (d) comprises (cl) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

137. The method of claim 136, characterized in that the fluorescence is fluorescence emission.

138. The method of claim 129, characterized in that the target molecule is a nucleic acid.

139. The method of claim 129, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

140. The method of claim 129, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

141. The method of claim 140, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

142. The method of claim 134 or 136, characterized in that step (d2) further comprises measuring the fluorescence of each of the mutiplicity of wells one at a time.

143. The method of claim 134 or 136, characterized in that step (d2) further comprises measuring the fluorescence of a subgroup of the multiplicity of wells simultaneously.

144. The method of claim 134 or 136, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

145. The method of claim 129, characterized in that step (c) comprises (di) excite the tryptophan residues in the protein, in each of the multiplicity of wells, with light; Y (d2) measure the fluorescence of each of the multiplicity of wells.

146. The method of claim 145, characterized in that the fluorescence is fluorescence emission.

147. The method of claim 129, characterized in that the multiplicity of different molecules comprises a combinatorial library.

148. The method of claim 147, characterized in that the combinatorial library is a DirectedDiversityR directed diversity chemical library.

149. A multi-variable optimization method for ordering the efficiency of one or more different molecules or different biochemical conditions to optimize the shelf life of a protein that is capable of unwinding due to a thermal change, characterized in that it comprises (a) contacting the protein with one or more of the multiplicity of different molecules or different biochemical conditions in each of the multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the protein resulting from the heating; (d) generating a thermal unwinding curve for the protein as a function of temperature for each of the wells; (e) comparing each of the unwinding curves in step (d) for (i) each of the other thermal unwinding curves and for (ii) the thermal unwinding curve obtained for the protein in the absence of any of the different molecules or under a reference group of biochemical conditions; Y (f) order the efficiencies of the multiplicity of the different molecules or the biochemical conditions according to the change in each of the thermal unwinding curves.

150. The method of claim 149, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

151. The method of claim 149, characterized in that it also comprises (g) generating combinations of biochemical conditions that increase the magnitude of the physical change, in relation to the magnitude of the physical change of each of the thermal unwinding curves in stage (f); and (h) repeating steps (a) to (g) until a combination of biochemical conditions is determined to promote maximum shelf life.

152. The method of claim 149, characterized in that step (d) further comprises determining a temperature mid point (TJ of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unrolling curves and for (ii) the Tm of the obtained thermal unrolling curve for the white molecule in the absence of any of the molecules in the multiplicity of the different molecules; Y wherein step (f) comprises (fl) order the affinities of the collections of different molecules according to the change in the Tm of each of the thermal unwinding curves.

153. A multivariable method for ordering the efficiency of one or more combinations of two or more of a multiplicity of different biochemical conditions to stabilize a white molecule, which is capable of unwinding due to a term change, characterized in that it comprises (a) contacting the target molecule with a combination of two or more of the multiplicity of different biochemical conditions in each of a multiplicity of wells in a microplate; (b) simultaneously heating the multiplicity of wells of step (a); (c) measuring in each of the wells a physical change associated with the thermal unwinding of the target molecule resulting from the heating; (d) generating a thermal unwinding curve for the target molecule as a function of the temperature for each of the wells; (e) comparing each of the unrolling curves of step (d) for (i) each of the other unwinding curves and for (ii) the unwinding curve obtained for the white molecule under a reference group of conditions biochemicals; Y (f) order the efficiencies of the combinations of different biochemical conditions according to the change in each of the thermal unwinding curves.

154. The method of claim 153, characterized in that the unwinding is denaturing, and wherein the thermal unwinding curve is a thermal denaturation curve.

155. The method of claim 153, characterized in that step (d) further comprises determining a temperature midpoint (TJ of the thermal unwinding curve; wherein step (e) comprises (el) comparing the Tm of each of the unwinding curves in step (d) for (i) the Tm of each of the other thermal unrolling curves and for (ii) the Tm of the obtained thermal unrolling curve for the white molecule under a reference group of biochemical conditions; and wherein step (f) comprises (fl) order the efficiencies of the combinations of the different biochemical conditions according to the change in the Tm of each of the thermal unrolling curves.

156. The method of claim 153, characterized in that the target molecule is a protein.

157. The method of claim 153, characterized in that step (c) comprises measuring the absorbance of light by the contents of each of the wells.

158. The method of claim 156, characterized in that step (a) comprises contacting the protein with the combinations of the different biochemical conditions, in the presence of a fluorescence test molecule present in each of the multiplicity of wells, and in wherein step (c) comprises (cl) excite the fluorescence test molecule, in each of the multiplicity of wells, with light; Y (c2) measure the fluorescence of each of the multiplicity of wells.

159. The method of claim 158, characterized in that the fluorescence is fluorescence emission.

160. The method of claim 158, characterized in that the target molecule is a nucleic acid.

161. The method of claim 158, characterized in that step (c) comprises measuring the change in the hyperchromicity of the nucleic acid.

162. The method of claim 153, characterized in that the target molecule is a fluorescently labeled double-stranded oligonucleotide.

163. The method of claim 162, characterized in that one strand of the oligonucleotide contains a donor fluorophore and the other strand of the oligonucleotide contains an acceptor fluorophore.

164. The method of claim 153, characterized in that step (a) comprises contacting the oligonucleotide with the combination of different biochemical conditions, at each of the multiplicity of wells, and wherein step (c) comprises (cl) excite the donor fluorophore, in each of the multiplicity of wells, with light; Y (c2) measuring the fluorescence of the acceptor fluorophore in each of the multiplicity of wells.

165. The method of claim 164, characterized in that the fluorescence is fluorescence emission.

166. The method of claim 158, characterized in that step (c2) further comprises measuring the fluorescence of each of the mutiplicity of wells one at a time.

167. The method of claim 158, characterized in that step (c2) further comprises measuring the fluorescence of a subset of the multiplicity of wells simultaneously.

168. The method of claim 158, characterized in that step (c2) further comprises measuring the fluorescence of each of the multiplicity of wells simultaneously.

169. The method of claim 156, characterized in that step (c) comprises (cl) exciting the tryptophan residues in the protein, in each of the multiplicity of wells, with light; and (c2) measuring the fluorescence of each of the multiplicity of wells.

170. An apparatus for detecting fluorescence emissions of a plurality of heated samples, characterized in that it comprises a first heat conductor block configured to receive a first plurality of samples; a temperature controller coupled to the first heat conducting block; a light source arranged adjacent to the first heat conducting block; a fluorescence emission detector disposed adjacent to the first heat conducting block; Y a computer program product that contains a useful means of computation that has the modality of logical control in the middle, the logical control comprises recording means of thermal unrolling results to cause a computer system to record the thermal unrolling results received from the fluorescence emission detector, means of generating the thermal curve to cause the computation system to generate the thermal curves from the results of thermal unwinding, and means of comparison of the thermal curve to cause the computer system to compare the thermal curves.

171. The apparatus according to claim 170, characterized in that the temperature controller comprises: a temperature profile controller.

172. The apparatus according to claim 170, characterized in that the temperature controller comprises: a temperature gradient controller.

173. The apparatus according to claim 170, characterized in that the temperature controller comprises: a temperature profile controller; and a temperature gradient controller.

174. The apparatus according to claim 170, characterized in that it further comprises: a second heat conducting block configured to receive a second plurality of samples.

175. The apparatus according to claim 174, characterized in that the temperature controller comprises: means for independently controlling the respective temperatures of the first and second heat-conducting blocks.

176. The apparatus according to claim 170, characterized in that the fluorescence emission detector is configured to receive the fluorescence emissions of one sample at a time.

177. The apparatus according to claim 170, characterized in that the fluorescence emission detector is configured to receive the fluorescence emissions of two or more samples at a time.

178. The apparatus according to claim 170, characterized in that the fluorescence emission detector is configured to receive the fluorescence emissions of the entire first plurality of samples at the same time.

179. The apparatus according to claim 170, characterized in that it further comprises: A first heat conducting adapter disposed within the first heat conducting block, the first heat conducting adapter is configured to receive a first heat conducting container for containing the first plurality of samples.

180. The apparatus according to claim 170, characterized in that the first heat conducting container comprises: a microtitre plate.

181. The apparatus according to claim 170, characterized in that it further comprises a computer program product containing a useful computing means having the logic control mode in the medium, the logical control comprises: means controlling the temperature to cause the computer system controls the temperature controller; medium that controls the light source to cause the computer system to energize the light source; Y medium that receives the fluorescence emission to cause the computation system to receive fluorescence emissions from the fluorescence emission detector.

182. The apparatus according to claim 170, characterized in that the means registering the thermal unrolling results comprises: means that registers the results of thermal denaturation to cause the computer system to record the thermal denaturation results of one or more samples.

183. The apparatus according to claim 191, characterized in that the means for generating the thermal curve comprises: means of generation of the thermal denaturation curve to cause the computation system to generate the thermal denaturation curves from the thermal denaturation results.

184. The apparatus according to claim 170, characterized in that the means for comparing the thermal curve comprises: comparison means of the thermal denaturation curve to cause the computer system to compare the thermal denaturation curves.

185. The apparatus according to claim 170, characterized in that the means for comparing the curve comprises: means of comparison of the midpoint of temperature to cause the computation system to compare the mean points of thermal unwinding temperature of two or more of the samples.

186. The apparatus according to claim 185, characterized in that the means of comparison of the temperature midpoint omprende: means of comparison of the midpoint of the denaturing temperature to cause the computation system to compare the mean points of thermal denaturation temperature of two or more of the samples.

187. The apparatus according to claim 170, characterized in that the light source comprises a tungsten-halogen lamp.

188. The apparatus according to claim 170, characterized in that the light source comprises a xenon arc lamp.

189. The apparatus according to claim 170, characterized in that the light source comprises a mercury lamp.

190. The apparatus according to claim 170, characterized in that the light source comprises a laser.

191. The apparatus according to claim 170, characterized in that the light source comprises an optical fiber cable.

192. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a fluorescence plate reader.

193. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a charging device coupled.

194. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a fiber optic probe.

195. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a photomultiplier tube.

196. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a fluorescence search.

197. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a fluorescence imaging camera.

198. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a fluorescence polarization detector.

199. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a charging device coupled to the fluorescence imaging camera.

200. The apparatus according to claim 170, characterized in that the fluorescence emission detector comprises: a diode array.

201. The apparatus according to claim 170, characterized in that it further comprises: a temperature detector coupled to the temperature controller.

202. The apparatus according to claim 217, characterized in that the temperature detector comprises: an infrared temperature detector.

203. The apparatus according to claim 217, characterized in that the temperature detector is arranged adjacent to the fluorescence emission detector.

204. An apparatus for detecting fluorescence emissions of a plurality of heated samples, characterized in that it comprises: a plurality of heat conducting blocks; a temperature controller coupled to the plurality of heat conducting blocks; a light source arranged adjacent to the plurality of heat conducting blocks; a fluorescence emission detector arranged adjacent to the plurality of heat conducting blocks; Y a positioning system coupled between the plurality of heat conducting blocks and the fluorescence emission detector.

205. A method for detecting fluorescence emissions of a plurality of heated samples, characterized in that it comprises the steps of (1) aligning a fluorescence emission detector with a first heat conducting block having a first plurality of samples disposed therein; (2) heat the first block; (3) shining an exciting light towards the first block; (4) detect fluorescence emissions of the first plurality of samples; and (5) recording the thermal unwinding results of the first plurality of samples.

206. The method according to claim 205, characterized in that it also comprises the step of: (6) aligning the fluorescence emission detector with a second heat conducting block having a second plurality disposed therein; (7) heating the second block; (8) shining an exciting light towards the first block; (9) detect fluorescence emissions of the second plurality of samples; Y (10) recording the results of thermal unwinding of the first plurality of samples.

207. The apparatus according to claim 170, characterized in that the temperature controller comprises: a step-up temperature controller.

208. The apparatus according to claim 170, characterized in that it further comprises: a positioning system coupled between the first heat conducting block and the fluorescence emission detector.

209. The apparatus according to claim 208, characterized in that the positioning system comprises: a moveable platform configured to receive the first heat conductor block.

210. The apparatus according to claim 209, characterized in that the positioning system further comprises: a transposable armature of the detector coupled to the fluorescence emission detector.

211. The apparatus according to claim 209, characterized in that it also comprises: a second heat conductor block disposed on the movable platform, the second heat conductor block is configured to receive a second plurality of samples.

212. The apparatus according to claim 211, characterized in that the temperature controller comprises: first and second temperature controllers independently controlling the respective temperatures of the first and second heat conducting blocks.

213. The apparatus according to claim 208, characterized in that the positioning system comprises: a rotating platform configured to receive the first heat conducting block.

214. The apparatus according to claim 213, characterized in that the positioning system further comprises: a transposable armature of the detector coupled to the fluorescence emission detector.

215. The apparatus according to claim 213, characterized in that it further comprises: a second heat conducting block disposed on the turntable, the second heat conducting block is configured to receive a second plurality of samples.

216. The apparatus according to claim 215, characterized in that the temperature controller comprises: first and second temperature controllers independently controlling the respective temperatures of the first and second heat conducting blocks.

217. The apparatus according to claim 208, characterized in that the logic controller further comprises: means of positioning control to cause the computer system to control the positioning system.

218. The method according to claim 205, characterized in that step (4) comprises the step of: (a) detect the fluorescence emissions of one of the samples at a time.

219. The method according to claim 205, characterized in that step (4) comprises the step of: (a) detecting the fluorescence emissions of multiple samples at the same time.

220. The method according to claim 205, characterized in that step (4) comprises the step of: (a) detect the fluorescence emissions of all the samples at the same time. SUMMARY OF THE INVENTION The present invention is a method for ordering the affinity of each of a multiplicity of different molecules of a target molecule that is capable of being denatured due to a thermal change. The method comprises contacting the target molecule with a molecule of the multiplicity of different molecules in each of a multiplicity of vessels, simultaneously heating the multiplicity of vessels, measuring in each of the vessels a physical change associated with the thermal denaturation of the white molecule that results from the heating in each of the containers, generating a curve of thermal denaturation for the target molecule as a function of the temperature for each of the containers and determining a midpoint of temperature (TJ of it, comparing the Tm of each of the thermal denaturation curves with the Tm of a denaturation curve obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules, and ordering the affinities of the multiplicity of different molecules according to the change in Tm of each of the thermal denaturation curves.The present invention also provides a testing apparatus that includes a means for adjusting the temperature for simultaneously heating a plurality of samples, and a means for receives spectral emission from the samples while the samples are being heated In additional aspects of the invention, the receiving means may be configured to receive the fluorescent emission, ultraviolet light, and visible light.The receiving means may be configured to receive the emission of the spectrum in a variety of ways, eg, one sample at a time, simultaneously give you more than one sample, or simultaneously of all the samples. The means for adjusting the temperature can be configured with a temperature controller to change the temperature according to a predetermined profile.