US 20030186454 A1
A method is disclosed for performing drug lead profiling assays for drug discovery utilizing a nanocalorimeter. The method includes depositing not less than one drop containing a profile molecule solution to be screened and not less than one drop containing a drug library compound on a test substrate. After the drops are merged, a determination is made as to whether a reaction has occurred.
1. A method for performing lead profiling assays for drug discovery utilizing a nanocalorimeter, the method comprising:
depositing not less than one drop containing profile molecules to be screened upon a test substrate;
depositing not less than one drop containing a drug library compound upon a test substrate;
merging said not less than one drop containing profile molecules to be screened and said not less than one drop containing a drug library compound; and
detecting whether a reaction has occurred between said not less than one drop containing profile molecules to be screened and said not less than one drop containing a drug library compound.
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 This application is a continuation in part of U.S. application Ser. No. 10/114,611, filed Apr. 1, 2002, the disclosure of which is totally incorporated by reference.
 The following copending application, Attorney Docket Number D/A1578I1, U.S. application Ser. No. XX/XXXXX, filed Nov. 22, 2002, titled “Apparatus and Method for Multiple Target Assay for Drug Discovery”, is assigned to the same assignee of the present application. The entire disclosure of this copending application is totally incorporated herein by reference in its entirety.
 The following U.S. patents are fully incorporated herein by reference: U.S. Pat. No. 5,967,659 (“Ultrasensitive Differential Microcalorimeter with User-selected Gain Setting” to Plotnikov et al.); U.S. Pat. No. 6,079,873 (“Micron-scale Differential Scanning Calorimeter on a Chip” to Cavicchi et al.); U.S. Pat. No. 6,096,559 “Micromechanical Calorimetric Sensor” to Thundat et al.); and U.S. Pat. No. 6,193,413 (“System and Method for an Improved Calorimeter for Determining Thermodynamic Properties of Chemical and Biological Reactions” to Lieberman).
 This invention relates generally to a method for performing assays for drug discovery, and more specifically, to a method for performing lead drug profiling assays.
 In recent years, researchers and companies have turned to combinatorial methods and techniques for synthesizing, discovering and developing new compounds, materials, and chemistries. For example, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. As another example, Symyx Technologies® is applying combinatorial techniques to materials discovery in the life sciences, chemical, and electronics industries. Consequently, there is a need for tools that can measure reactions and interactions of large numbers of small samples in parallel, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable inexpensive measurements and minimize contamination and cross-contamination problems. In addition there has been an explosion in the number of potential drug targets due to the accelerated implementation of genomics technologies and the completion of the Human Genome sequence.
 In some cases, the sample to be studied is precious, and it might not be acceptable to use the relatively large amount of material required by a standard microcalorimeter to perform only one measurement. For example, one may desire to study a natural extract or synthesized compound for biological interactions, but in some cases the available amount of material at concentrations large enough for calorimetry might be no more than a few milliliters. Performing a measurement in standard microcalorimeters, such as those sold, for example, by MicroCal® Inc. (model VP-ITC) or Calorimetry Sciences Corporation® (model CSC-4500), requires about 1 ml of sample, which means that one would possibly be faced with using a majority or all of the precious material for one or a small series of measurements. Tools that enable calorimetric measurements with much smaller sample sizes would be helpful in overcoming this limitation.
 One of the most popular uses of combinatorial techniques to date has been in pharmaceutical research. Pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds which have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks.
 Once a library has been constructed, it must be screened to identify compounds, which possess some kind of biological or pharmacological activity. For example, screening can be done with a specific biological compound, often referred to as a target that participates in a known biological pathway or is involved in some regulatory function. The library compounds that are found to react with the targets are candidates for affecting the biological activity of the target, and hence a candidate for a therapeutic agent.
 Through the years, the pharmaceutical industry has increasingly relied on high throughput screening (HTS) of libraries of chemical compounds to find drug candidates. HTS describes a method where many discrete compounds are tested in parallel so that large numbers of test compounds are screened for biological activity simultaneously or nearly simultaneously. Currently, the most widely established techniques utilize 96-well microtitre plates. In this format, 96 independent tests are performed simultaneously on a single 8 cm×12 cm plastic plate that contains 96 reaction wells. These wells typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers and plate readers are commercially available to fit the 96-well format to a wide range of homogeneous and heterogeneous assays. To achieve faster testing, the industry is evolving to plates that contain 384 and 1536 wells.
 A variety of measurement approaches has been used to screen combinatorial libraries for lead compounds, one of which is the competitive binding assay. In this assay, a ligand, often the natural ligand in a biological pathway, is identified that will bind well with the target protein molecule. The assay often requires the chemical attachment of a fluorescent molecule to this marker ligand such that the fluorescent molecule does not affect the manner in which the marker ligand reacts with the target protein. Alternatively, the ligand could be radioactively labeled or labeled with a chemiluminescent molecule. To operate an inhibitor assay, the target protein is exposed to a mixture of the test ligands and marker ligands often in microtitre wells. After a time for reaction, the wells are rinsed such that free marker ligand is washed away. In wells where the target protein and the test ligand have reacted, the test ligand has blocked the active site of the target protein so the marker ligand cannot react and is washed away, while in cells where the target protein and test ligand have not reacted, the marker ligand has bound to the target protein and is not washed away. By investigating the wells for the presence of fluorescence after the washing, reactions of test ligands and target proteins can be determined as having occurred in wells where reduced fluorescence is observable relative to control wells to which no test ligands have been added.
 However, the competitive binding assay requires time and expense to develop the labeled reagents and assay. The principal components that need development are discovering a marker ligand and attaching a fluorophore to the marker in a manner that does not affect its reaction with the target protein. Attaching the fluorescent marker can often take 3 months of development or more and cost $250 k or more once the marker ligand is identified. An assay method that avoids such assay development, such as measuring the heat of the reaction with calorimetry, would eliminate this cost and time delay in the discovery process.
 The following disclosures may be relevant and/or helpful in providing an understanding of some aspect of the present invention:
 In Plotnikov et al., U.S. Pat. No. 5,967,659 (“Ultrasensitive Differential Microcalorimeter with User-selected Gain Setting”), a differential calorimeter is disclosed that includes sample and reference cells, a thermal shield surrounding the cells, heating devices thermally coupled to the thermal shield and the cells, a temperature monitoring system, and a control system. The temperature monitoring system monitors the temperature of the shield, cell temperatures, and temperature differentials between the cells and the shield. The control system generates output signals for control of the heating devices, with a gain setting and scan rate selected by means of a user interface. Output control signals are functions of input temperature signals and the user-selected gain setting, as well as functions of input temperature signals and the user-selected scan rate using a mapping function stored in memory.
 In Cavicchi et al., U.S. Pat. No. 6,079,873 (“Micron-scale Differential Scanning Calorimeter on a Chip”), a differential scanning microcalorimeter produced on a silicon chip enables microscopic scanning calorimetry measurements of small samples and thin films. The chip, fabricated using standard CMOS processes, includes a reference zone and a sample zone. The reference and sample zones may be at opposite ends of a suspended platform or may reside on separate platforms. Each zone is heated with an integrated polysilicon heater. A thermopile consisting of a succession of thermocouple junctions generates a voltage representing the temperature difference between the reference and sample zones.
 In Thundat et al., U.S. Pat. No. 6,096,559 (“Micromechanical Calorimetric Sensor”), a calorimeter sensor apparatus utilizes microcantilevered spring elements for detecting thermal changes within a sample containing biomolecules which undergo chemical and biochemical reactions. The spring element includes a bimaterial layer of chemicals on a coated region on at least one surface of the microcantilever. The chemicals generate a differential thermal stress across the surface upon reaction of the chemicals with an analyte or biomolecules within the sample due to the heat of chemical reactions in the sample placed on the coated region. The thermal stress across the spring element surface creates mechanical bending of the microcantilever. The spring element has a low thermal mass to allow detection and measuring of heat transfers associated with chemical and biochemical reactions within a sample place on or near the coated region. Deflections of the cantilever are detected by a variety of detection techniques.
 In Lieberman, U.S. Pat. No. 6,193,413 (“System and Method for an Improved Calorimeter for Determining Thermodynamic Properties of Chemical and Biological Reactions”) a microcalorimeter includes a thin amorphous membrane anchored to a frame within an environmental chamber. Thermometers and heaters are placed on one side of a thermal conduction layer mounted on the central portion of the membrane. Samples are placed on two such membranes; each sample is heated and its individual heat capacity determined. The samples are then mixed by sandwiching the two microcalorimeters together to cause a binding reaction to occur. The amount of heat liberated during the reaction is measured to determine the enthalpy of binding.
 Briefly stated, and in accordance with one aspect of the present invention, there is disclosed a method for performing drug lead profiling assays for drug discovery utilizing a nanocalorimeter. The method includes depositing not less than one drop containing a target solution to be screened and not less than one drop containing a drug library compound on a test substrate. After the drops are merged, a determination is made as to whether a reaction has occurred.
 The foregoing and other features of the instant invention will be apparent and easily understood from a further reading of the specification, claims and by reference to the accompanying drawings in which:
FIG. 1 is a flow chart illustrating a prior art method for performing drug lead profiling; and
FIG. 2 is an embodiment of the method for performing lead profiling assays in accordance with the present invention.
 As used herein, the term “ligand” refers to an agent that binds a target molecule. According to the present invention, a ligand is not limited to an agent that binds a recognized functional region of the target protein e.g. the active site of an enzyme, the antigen-combining site of an antibody, the hormone-binding site of a receptor, a cofactor-binding site, and the like. In practicing the present invention, a ligand can also be an agent that binds any surface or conformational domains of the target protein. Therefore, the ligands of the present invention encompass agents that in and of themselves may have no apparent or known biological function, beyond their ability to bind to the target protein in the manner described above.
 As used herein, the term “test ligand” refers to an agent, comprising a compound, molecule or complex, which is being tested for its ability to bind to a target molecule. Test ligands can be virtually any agent, including without limitation metals, peptides, proteins, lipids, polysaccharides, nucleic acids, small organic molecules, and combinations thereof. Complex mixtures of substances such as natural product extracts, which may include more than one test ligand, can also be tested, and the component that binds the target molecule can be purified from the mixture in a subsequent step.
 As used herein, the term “target protein” refers to a peptide, protein or protein complex for which identification of a ligand or binding partner is desired. Target proteins include without limitation peptides or proteins known or believed to be involved in the etiology of a given disease, condition or pathophysiological state, or in the regulation of physiological function. Target proteins may be derived from any living organism, such as a vertebrate, particularly a mammal and even more particularly a human. For use in the present invention, it is not necessary that the protein's biochemical function be specifically identified. Target proteins include without limitation receptors, enzymes, oncogene products, tumor suppressor gene products, vital proteins, and transcription factors, either in purified form or as part of a complex mixture of proteins and other compounds. Furthermore, target proteins may comprise wild type proteins, or, alternatively, mutant or variant proteins, including those with altered stability, activity, or other variant properties, or hybrid proteins to which foreign amino acid sequences, e.g. sequences that facilitate purification, have been added.
 As used herein, “test combination” refers to the combination of a test ligand and a target protein. “Control combination” refers to the target protein in the absence of a test ligand.
 As used herein, “screening” refers to the testing of a multiplicity of molecules or compounds for their ability to bind to a target molecule.
 As used herein, the term “lead molecule” refers to a molecule or compound, from a combinatorial library or other collection, which displays relatively high affinity for a target molecule. High affinity is detected by this invention through the heat released in a chemical reaction. The terms “lead compound” and “lead molecule” are synonymous.
 As used herein, the term “target molecule” encompasses peptides, proteins, nucleic-acids, protein-nucleic acid complexes and other receptors. The term encompasses both enzymes and proteins which are not enzymes. The term encompasses monomeric and multimeric proteins. Multimeric proteins may be homomeric or heteromeric. The term encompasses nucleic acids comprising at least two nucleotides, such as oligonucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded. 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 target molecule also encompasses portions of peptides, secondary, tertiary, or quaternary structure through folding, with substituents including, but not limited to, cofactors, coenzymes, prosthetic groups, lipids, oligosaccharides, or phosphate groups.
 As used herein, the term “molecule” refers to the compound, which is tested for binding affinity for the target molecule. This term encompasses chemical compounds of any structure, including, but not limited to nucleic acids and peptides. More specifically, the term “molecule” encompasses compounds in a compound or a combinatorial library. The terms “molecule” and “ligand” are synonymous.
 As used herein, the term “thermal change” encompasses the release of energy in the form of heat or the absorption of energy in the form of heat.
 As used herein, the term “contacting a target molecule” refers broadly to placing the target molecule in solution with the molecule to be screened for binding. Less broadly, contacting refers to the turning, swirling, shaking or vibrating 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 for binding. Mixing can be accomplished, for example, by repeated uptake and discharge through a pipette tip or by deposition by robotic means. Preferably, contacting refers to the equilibration of binding between the target molecule and the molecule to be tested for binding.
 As used herein, the term “biochemical conditions” encompasses any component, thermodynamic property, or kinetic property of a physical, chemical, or biochemical reaction. Specifically, the term refers to conditions of temperature, pressure, protein concentration, pH, ionic strength, salt concentration, time, electric current, potential difference, and concentrations of cofactor, coenzyme, oxidizing agents, reducing agents, detergents, metal ion, ligands, buffer components, co-solvents including DMSO (dimethyl sulfoxide), glycerol, and related compounds, enhancers, and inhibitors.
 As used here in the term “lead profiling assay” encompasses the testing for reaction of a compound or a series of compounds with known binding activity towards a desired target against a plurality of other targets so as to determine a reactivity profile for said compound of compounds.
 The present invention encompasses nanocalorimeters and nanocalorimeter arrays that enable measurement of enthalpic changes, such as enthalpic changes arising from reactions, phase changes, changes in molecular conformation, and the like. Furthermore, the present invention encompasses combinatorial methods and high-throughput screening methods that use nanocalorimeters in the study, discovery, and development of new compounds, materials, chemistries, and chemical processes, as well as high-throughput monitoring of compounds or materials, or high-throughput monitoring of the processes used to synthesize or modify compounds or materials. The present invention also relates to compounds or materials identified by the above methods and their therapeutic uses (for diagnostic, preventive or treatment purposes), uses in purification and separation methods, and uses related to their novel physical or chemical properties. For the purposes herein, a nanocalorimeter refers to a device capable of measuring heats of reaction in the range of nanocalories.
 As an example, the present invention encompasses high-throughput screening methods for identifying a ligand that binds a target protein. If the target protein to which the test ligand binds is associated with or causative of a disease or condition, the ligand may be useful for diagnosing, preventing or treating the disease or condition. A ligand identified by the present method can also be one that is used in a purification or separation method, such as a method that results in purification or separation of the target protein from a mixture. The present invention also relates to ligands identified by the present method and their therapeutic uses (for diagnostic, preventive or treatment purposes) and uses in purification and separation methods.
 In practicing the present invention, the test ligand is combined with a target molecule, and the mixture is maintained under appropriate conditions and for a sufficient time to allow binding of the test ligand to the target molecule. Experimental conditions are determined empirically for each target molecule. When testing multiple test ligands, incubation conditions are usually chosen so that most ligand:target molecule interactions would be expected to proceed to completion. In high-throughput screening applications, the test ligand is usually present in molar excess relative to the target molecule. The target molecule can be in a soluble form, or, alternatively, can be bound to a solid phase matrix. The matrix may comprise without limitation beads, membrane filters, plastic surfaces, or other suitable solid supports.
 Binding to a given target is a prerequisite for pharmaceuticals intended to modify directly the action of that target. Thus, if a test ligand is shown, through use of the present method, to bind a target that reflects or affects the etiology of a condition, it may indicate the potential ability of the test ligand to alter target function and to be an effective pharmaceutical or lead compound for the development of such a pharmaceutical. Alternatively, the ligand may serve as the basis for the construction of hybrid compounds containing an additional component that has the potential to alter the target's function. For example, a known compound that inhibits the activity of a family of related enzymes may be rendered specific to one member of the family by conjugation of the known compound to a ligand, identified by the methods of the present invention, that binds specifically to that member at a different site than that recognized by the known compound.
 The fact that the present method is based on physicochemical properties common to most targets gives it widespread application. The present invention can be applied to large-scale systematic high-throughput procedures that allow a cost-effective screening of many thousands of test ligands. Once a ligand has been identified by the methods of the present invention, it can be further analyzed in more detail using known methods specific to the particular target used. Also, the ligand can be tested for its ability to influence, either positively or negatively, a known biological activity of the target.
 In the drug discovery process, a drug target protein is screened for reactivity against a large number (500,000) of compounds from a drug library of compounds. Often it is desirable to screen several different drug target proteins against the same library of proteins if the target proteins are thought to have a similar function such as the enzymatic function of a kinase.
 Turning to FIG. 1, there is shown an embodiment of a conventional drug screening process, a competitive screen, using a fluorescent assay. In a competitive screen, the reaction of the drug library compound with the target protein prevents the reaction of a second known reactive compound that contains a detectable label such as, in the case of a fluorescent assay, a fluorescent label. The level of reactivity of the drug library compound is inferred by detecting the fluorescence coming from the bound labeled compound. Several types of fluorescent assays are currently utilized in the art, but the two most practiced are fluorescence intensity and fluorescence polarization.
 In a fluorescence intensity assay, a labeled ligand 1130 at a low concentration and one or more drug library compounds 1150 at a higher concentration (5 μM) are mixed with a target protein 1110. The labeled ligand 1130 is known to react strongly with the target protein 1110 and is often the natural ligand. The receptor of the target protein 1110 is immobilized to the container and incubated with the mixture of labeled ligand and drug library compounds at step 1140. The label on the ligand in this example is a molecule that fluoresces in a particular way when stimulated by light such as a laser or an ultraviolet light source. Radioactive compounds can also be used as labels. Following the incubation step 1152, the free ligand is removed by washing at step 1155 and the amount of bound, labeled ligand is measured by detecting the amount and nature of the light emitted from the fluorescent label attached to ligand 1130 at step 1160. If the fluorescence is reduced, then a reaction with a drug library compound has occurred, as shown at step 1180. The reaction will reduce the amount of labeled ligand that reacts by a predicted amount ranging from 20% to 50% or more. If the fluorescence is not reduced, then a reaction has not occurred, as shown at step 1170, since the labeled ligand reaction is not inhibited. The variation in the amount of fluorescent light for uninhibited binding is approximately 10%, resulting in an acceptable signal to noise ratio.
 For the fluorescence polarization approach, the labeled ligand is incubated with the receptor and the drug library compounds but the receptor does not need to be immobilized. Here the assay relies on the observation that fluorescence from the labeled ligand bound to the receptor is substantially more polarized than the fluorescence from an unbound labeled ligand. Again in this approach, the signal produced is maximum when no the drug library compounds bind to the target receptor and is reduced by binding. In both straight fluorescence and fluorescence polarization, reaction of the target protein and a labeled ligand is required for the assay.
 For competitive assays, a compound is required that strongly interacts with the protein target. This compound is often the ligand that reacts with the protein target in nature. In the fluorescent assays described here, this ligand must have an attached molecule that serves as a fluorescent label. The label must be attached in a manner that does not affect the ligand reaction to the protein target. Developing a competitive assay by creating such a labeled ligand is a costly, time-consuming effort.
 Lead profiling identifies and prioritizes a portfolio of compounds that have the best chance of success in clinical development. Assays can range from relatively simple ligand-receptor interaction to cell-based assays using genetically modified cells with multiple endpoints or reporter genes. The choice for a specific assay is determined by factors such as the desired target profile, sensitivity, robustness, and ease of automation. Drug leads are screened against other naturally occurring proteins to determine the level of interaction with these other targets. To become a drug lead, the protein must not only interact with the desired target but must not interact with other possibly similar proteins. Leads that do not affect proteins other than their target protein are more likely to not be toxic. Alternatively, a specific profile of interaction with more than one target may be desired, and specific other targets definitely excluded. Testing for interactions with these other proteins using existing technology requires a unique assay for each protein. In many cases, screening against other proteins can require many different assay formats and reagent development and consequently be costly and time-consuming. For competitive assays this requires a high affinity interaction between the protein and a labeled ligand.
 However, with the device taught herein, there is no need to develop a specific assay for each target, since a direct measurement of the heat of interaction is made utilizing calorimetry. Turning now to FIG. 2, there is shown an embodiment of the nanocalorimeter as used for a lead profiling assay. In FIG. 12, a target substance to be screened, for example a protein, 1210 is combined at step 1220 with one or more drug library compounds 1230, which had reacted with an original target protein. For example, with a kinase inhibitor, the drug lead is tested for reaction with other kinases as well as other key proteins to test activity level. The enthalpy of this reaction is measured at step 1240. The enthalpy of this reaction is then compared with the enthalpy for a reaction with no inhibitor. If there is a reaction, it is identified at step 1260; if a reaction is not present, it is identified at step 1250.
 While the present invention has been illustrated and described with reference to specific embodiments, further modification and improvements will occur to those skilled in the art. It is to be understood, therefore, that this invention is not limited to the particular forms illustrated and that it is intended in the appended claims to embrace all alternatives, modifications, and variations which do not depart from the spirit and scope of this invention.