JP2002508507A - A method for simultaneous identification of novel biological targets and drug discovery lead structures - Google Patents

Abstract

(57) SUMMARY The combinatorial screening assays and detection methods of the present invention provide a highly diversified library of compounds that function as fingerprints that allow the identification of particular molecular differences that exist between biological samples. Rally. Certain molecular differences identified by the combinatorial screening assays and detection methods of the invention can be targets for diagnostic and therapeutic drug development. The method of the invention can be used in diagnostics, drug discovery, genomics and proteinology. FIG. 1, which illustrates the method of the invention, illustrates the interaction of the molecular probes described in Table II with human serum samples.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] 1. FIELD OF THE INVENTION The present invention relates to methods for discovering and identifying novel biological targets of both therapeutic and diagnostic importance, and more particularly, to simultaneously identify novel biological targets and drug discovery lead structures. Methods for discovery and identification.

[0002] 2. Background of the Invention Researchers have recently discovered the presence of a unique chromosomal material that circulates in the plasma of patients with catastrophic disease (Umovitz et al., Chronic Disease Syndromes Symposium, Ame
rican Society for Microbiology, May 1997). Similarly, recently, using differential expression techniques, over 500 RNA transcripts have been shown to be expressed at significantly different levels when comparing normal and neoplastic cells (Zhang. L. et al., Gene expression profiles in norm.
al and cancer cells, Science. 276 1268-72, 1997). These findings indicate that there are numerous molecular differences between normal and abnormal cells. In fact, it can be assumed that hundreds or thousands of potential biological receptors are present without being detected by existing techniques.

[0003] The techniques used to date may provide valuable information about targets for treatment or diagnosis based on identifying individual differences between normal and abnormal samples. However, these techniques fail to effectively provide information identifying patterns of differences observed between similar samples. The pattern of differences observed between similar samples can provide valuable information that more completely defines the subtype of a particular disease, as well as the classification of that subtype as well as the determination of the appropriate treatment for that particular subtype Can also help. Clearly, with the wealth of information gained in the discovery of new biological targets, and the unknown number of such targets that remain undetected, the overwhelming need for efficient and effective methods to discover them There is a request.

[0004] It has long been recognized that artificial or naturally occurring chemical libraries can be screened for affinity for known biological receptors such as proteins, enzymes, and the like. Affinity screening can be performed by known techniques, for example, fluorescence polarization, scintillation proximity assay, enzyme-linked immunosorbent assay, and the like. Recently, a porous silicon biosensor has been disclosed that is capable of detecting virtually any molecule that binds with high affinity to another molecule. When the biological receptor is a therapeutically important target, members of the library that exhibit high affinity and specificity for the target are of value in diagnosis and / or drug development. Advances in combinatorial chemistry have vastly increased the number of receptors available for affinity screening.

[0005] The scintillation proximity assay (SPA) is used to investigate vast chemical libraries for affinity to known receptors, for example, as described in US Pat. No. 4,568,649. In a standard SPA assay, receptors are tagged with scintillant load beads and screened in solution for radiolabeled receptors. If the labeled ligand binds with affinity to the receptor, the resulting proximity of the radiolabeled ligand to the scintillant in the bead will lead to activation of the scintillant and light emission. If the labeled ligand has little or no affinity for the receptor, the radiolabel will not accumulate close enough to the scintillant to cause energy transfer due to radioactive decay, and Only light emission will be detected. One obstacle for SPA is caused by non-specific absorption of labeled ligand in the system.
"Noise" or the presence of background radioactivity. For this purpose, the beads are typically incubated with a blocking agent such as egg white, surfactant, milk powder to block sites responsible for such non-specific absorption. Another significant problem with SPA is that the required use of radioactive materials poses a health hazard, and such materials are difficult to dispose of and expensive to use.

A modification of the SPA assay has been used in competitive screening methods in which the receptor is immobilized on scintillant-loaded beads and then placed in a solution containing a radiolabeled substrate for the receptor. Subsequent addition of a sample of the ligand to the mixture will reduce the amount of light emission of any compound that successfully competes with the substrate for the immobilized receptor. The use of SPA in high-throughput screening is described below; Wang, P., Target Idetification, Assay Development and High-Throughput Screening for Drug Discovery
nd High Throughput Screening in Drug Discovery, in Sino-American Pharmac
eutical Professionals Association (SAPA), The 5th Regional Symposium on D
rug Discovery and Development, 1997, Kenilworth, NJ.

[0007] Fluorescence polarization is another commonly used assay system for identifying compounds that have an affinity for a particular receptor. When a fluorescent molecule binds to one of the oligomer termini linked to the ligand, binding of the receptor to the ligand severely limits the rotation of the fluorescent molecule. When polarized light is passed through a solution containing a fluorophore-labeled oligomer to which the receptor is bound or adsorbed, the emitted light is also polarized.

[0008] Another affinity screening technique has recently been described by Lin, V. Motesharei, K., Dancil, K.
, Sailor, M. and Ghadiri, M. et al. (A porous silicon-base
d optical interferometric biosensor, Science 278, 840-43 1997). In essence, this technique involves using an etched silicon wafer to create a porous surface. When light is applied to a porous material, interference fringes are created whose position shifts when the refractive index of the surrounding medium changes. Various molecular recognition elements are attached to the porous surface using well-known chemistry and subsequently reacted with a source of target molecules. As the element under investigation bound to the porous surface binds to the target molecule, the resulting change in refractive index shifts the fringes. This can be detected by a charge pair detector. The biosensor can not only recognize small biomolecules, but also detect DNA sequences at very low concentrations (eg, femtomolar). The recognition element of the sensor may be based on virtually any supramolecular interaction. Such interactions include, for example, nucleotide hybridization, enzyme-substrate binding, lectin-carbohydrate interaction, antigen-antibody binding, host-guest complex formation, and the like.

[0009] Combinatorial chemistry can generate libraries containing hundreds of thousands of compounds. Many of them may be structurally similar. While high-throughput screening programs can screen these vast libraries for affinity to known targets, new methods have been developed to complete libraries that are small but offer the greatest chemical diversity ( Matter. H. Selection of Optimal Diversity Compounds from Structural Database (Sele
cting Optimally Diverse Compounds from Structure Database: 2D and
A Validation Study of Two-Dimensional and T
hree-Dimensional Molecular Descriptors), Journal of Medicinal Chemistry
, 1997, 40: 1219-1229).

Previously, using affinity fingerprinting, discrete libraries of small molecules were investigated for binding affinity to defined protein compartments.
The fingerprints obtained from the screen are used to predict the affinity of individual library members for other proteins or receptors of interest. The fingerprint is compared to a fingerprint obtained from another compound known to react with the protein of interest to predict whether the library compound will react similarly. For example, rather than investigating the interaction of all ligands in a large library with known receptors associated with a particular pharmacological activity (eg, antihistamine activity or hypercholinergic activity), Only ligands with similar fingerprints to other compounds known to have should be investigated (Kauvar, LM et al., Predicting ligand binding to proteins by affinity fingerprinting, Chemistry and Biolog
y, 1995, 2: 107-118; Kauvar. LM, Affinity Fingerprinting, Pharma
ceutical Manufacturing International. 1995 8: 25-28; and Kauvar. LM, Detection of toxic chemicals by pattern recognition in the new frontier of agrochemical immunoassays, D. Kurtz. L. Stanker and JH Skerritt. Editors, 1995.
, AOAC: Washington DC, 305-312).

While the techniques and assays used to date have value with respect to known targets or receptors for therapy or diagnosis, they provide only limited information because they are very specific in nature. For example, differential expression may have different levels of mRNA
It focuses solely on the detection of expression and does not detect the presence of sequence differences. further,
This method does not provide any information about other changes in the biological system, such as post-translational modifications that do not result in a change in expression levels, and in most cases, the response of nearby cells to abnormal cells. No information is provided.

[0012] Accordingly, there is a need for a detection method that is capable of detecting a wide variety of specific differences and changes between biological samples. Such a method should also provide a method to facilitate identification of biological targets and diagnostic and drug discovery lead structures.

[0013] 3. SUMMARY OF THE INVENTION The combinatorial screening assays and detection methods of the present invention utilize a library of highly diversified compounds to interrogate and characterize complex mixtures and to identify specific molecules present between biological samples. It identifies these differences and can serve as a target for diagnostic and therapeutic development.

The present invention is based, in part, on a sensitive, rapid, and homogeneous assay system designed by the applicant. The assay system allows for the evaluation, interrogation and characterization of samples using a complex and highly diversified library of molecular probes. The ability to perform high-throughput assays in a uniform format increases the sensitivity of the screening. In addition, homogeneous forms allow for molecules that interact to maintain their native or active conformation. Further, the homogeneous assay system of the present invention utilizes a crude detection system that does not require a separation step for the detection of reaction products. In a preferred embodiment, fluorescence polarization is used to detect interactions between sample and library components.

[0015] In another embodiment, the assay can be performed in a homogeneous format, wherein one of the reaction components (eg, either a library or a sample component) is releasably linked to a solid support. . The reaction product obtained by contacting the sample with the library is dissociated into a liquid phase, which can be advantageously detected using a crude detection system such as a fluorescence polarization method.

The assays of the present invention can be used in the fields of proteinology and genomics for diagnostics, drug screening and discovery, drug discovery by target, and for identification of disease markers and drug targets.

In another embodiment, the sensitive combinatorial screening assays and detection methods of the invention are discovery tools for detecting receptors present in complex biological mixtures but not previously detected. Utilizes ligand / receptor interaction. The combinatorial screening assays and detection methods of the present invention assess the binding interaction of a diversified population of ligands in a biological sample with thousands of potential receptors and are specific or specific to that sample. Identified binding interactions, which can be revelations of certain pathologies, including but not limited to diseases, disorders, and infections. Thus, the present invention not only identifies novel receptors, but also characterizes the specific differences that occur between normal and abnormal cells and / or tissues of the same type. The screening assays and detection methods of the present invention provide much more information than existing target discovery methods. Existing ones include genomics or differential expression techniques, which only detect changes in genotypes and only detect changes in DNA or mRNA, and proteomics detects non-protein ligands. But not only to the protein encoded by the particular sequence. In contrast, the method of the present invention detects all types of ligand / receptor interactions. Thus, the receptor may be a protein, carbohydrate, nucleic acid, or any molecule having a form that allows for a binding interaction.

According to the method of the present invention, the binding affinities of a diverse library of molecules are evaluated for thousands of potential binding sites present in a complex biological sample, and the sample gives a unique fingerprint to that sample. The pattern of binding affinity shown can be generated.

The present invention provides a method for “fingerprinting” complex biological mixtures, such as serum, using a diverse library of chemical compounds. Specific binding interactions that occur between members of the library and molecules in a biological sample provide a unique fingerprint for the sample. Such fingerprinting allows the identification of new interactions that occur in a particular sample,
If the sample has a particular pathology, the fingerprint will identify the specific phenotypic differences that exist between normal and abnormal, or diseased and healthy, cells and / or tissues of the same type. Enable. Furthermore, once a novel interaction has been identified, the relevant molecule may be used as a lead structure, receptor or target for diagnostic and / or drug development.

The screening assays of the present invention not only involve the identification of individual novel receptors, but more importantly, identify patterns of binding interactions that characterize particular pathologies or classes of pathologies. I do. Such binding affinity patterns provide valuable information for defining and classifying pathological subtypes more completely and accurately. Accurate diagnosis is important not only for disease detection, but also for treatment and treatment of the disease, which in turn all make the clinical prognosis predictable.

Using the principles of the present invention, samples from a number of different individuals are collected, the interactions with defined libraries are investigated, and the results obtained are used to identify all of the identified binding interactions. A database can be created that includes Data on samples from individuals exhibiting the pathology or disease of interest may then be withdrawn for analysis and, compared to the records remaining in the database, interactions or interactions that are predictive of pathology and pathology Can be identified. The interactions and patterns thus identified can be used to characterize and classify a particular pathology or pathology, as well as formalize a diagnostic investigation or develop a therapy or drug. .

[0022] 4. BRIEF DESCRIPTION OF THE DRAWINGS (see below for a description of the drawing) 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of compounds that bind with high affinity to certain target molecules as potential drug discovery compounds as well as information molecules.

While the frequency of a compound in a diverse combinatorial library of chemicals to bind a specific target strongly is relatively low (possibly 0.0001% or less), the frequency of a large number of targets Increases dramatically. For example, if there are 1000 possible target molecules instead of one target molecule, the probability of getting a `` hit '' is 0.
Increase to 1%. Complex biological systems contain thousands of different molecules, many of which can be altered in the case of certain disease states, such as prostate or breast cancer. Thus, the probability of obtaining a high affinity interaction between members in a diversity combinatorial library of organic molecules and the information component of a clinical sample is much higher than the probability of obtaining a "hit" for a single target . If, according to the invention, these interactions are detected and quantified, a fairly complete picture of the sample is formed (depending on the number and diversity of the library components of the system to be evaluated).

The combinatorial screening assays and detection methods of the invention encompass a highly diversified library of compounds that function as fingerprints, allowing identification of specific molecular differences that exist between biological samples. I do. Certain molecular differences identified by the combinatorial screening assays and detection methods of the invention may be targets for diagnostic and therapeutic development. Successful application of the combinatorial screening assays and detection methods of the present invention requires at least three components: (1) a diversity molecule library (probe (s)); (2) a clinical sample Materials (control and test samples); and (3) a sensitive assay for detecting the interaction of members of the library with components of the sample.

Accordingly, one aspect of the present invention is a method for characterizing a condition, comprising: (a) a receptor or target present in a biological sample associated with the condition; And) the identification of a pattern of binding interactions between the library of ligands or probes, wherein the pattern of binding interactions provides a unique fingerprint for the condition. In the present invention, a “receptor” or “target” is a biological molecule that exhibits a binding affinity or interaction with a ligand or probe. Examples of such receptors or targets include, but are not limited to, any biologically active molecule that is differentially expressed or modified and capable of interacting with a probe or ligand, such as, but not limited to, an enzyme or other protein. , Lipids, DNA, nucleic acids such as RNA, carbohydrates, antigens, antibodies, and others. Examples of ligands or probes can be used to interact with or bind to a receptor or target in a given biological sample to measure or indicate the presence of the ligand or probe, Includes any molecule, either natural or synthetic.

In one particular embodiment, the combinatorial screening assays and detection methods of the invention can be used for detecting or characterizing certain conditions. In this embodiment, the condition to be identified or characterized includes, but is not limited to, a transformed phenotype, genetic defect, malignancy, cancer, tumor,
Includes genetic diseases, viral infections, bacterial infections, fungal infections or the presence of parasites.

Vertebrate animals, especially humans, are complex in body structure. Making it difficult to develop accurate diagnostics,
It is this great complexity that has been spent on implementation. For example, plasma contains thousands of proteins, carbohydrates, lipids and nucleic acids. Although any one or many changes or alterations of these components may be highly indicative of a particular disease state or outcome of a particular treatment, due to the great complexity of the system, the precise nature of these changes Detection and analysis are hindered. In one embodiment, the present invention relates to any other clinical assay, including, but not limited to, plasma or serum, tissue homogenate, cerebrospinal fluid, urine, sputum, or substances that can be obtained or prepared in liquid form. Enables the rapid analysis of very complex biological systems, such as biological substances.

One embodiment of the present invention is a medical diagnosis. The fields of diagnosis to which the present invention is applicable include, but are not limited to, early detection of various cancers; detection and identification of infections; detection of toxic side effects of therapeutic agents; and other disease symptoms and conditions. Many physiological changes occur within a complex biological system of an organism in connection with any particular condition. Some changes are a direct consequence of the condition, and others are a consequence of the body's response to the condition. Any or all of these changes should be diagnostic. Unfortunately, for most diseases, no diagnosable changes are known, and prior to the present invention, there was no sensitive and rapid method to detect these diagnosable changes. In some cases, antibody-based diagnostics are used to detect new antigens associated with a particular disease state (eg, PSA antigens associated with prostate cancer), but antibody-based diagnostics first identify the antigen. Relies on identifying. Moreover, the production of antibodies is expensive and time-consuming. In the present invention, the binding interaction of a large diversity library of chemicals to a biological sample, such as plasma from an individual with a particular disease state, is compared to a sample from a normal population and the differences are analyzed. I do. This difference may be qualitative or quantitative, and may be the result of binding to any molecule, for example, proteins, lipids, carbohydrates or nucleic acids, enzymes, antigens, and the like. This method is not biased by the type of molecule that can be detected. As a result of a binding interaction, a single or a small number of binding interactions may be found that diagnose the condition under test, or a unique pattern may be recognizable. In any case, the unique binding interactions identified can be assembled into inexpensive and rapid fluorescence polarization-based assays that can be performed in clinics or beds.

One application of the present invention is in the diagnosis and treatment of various cancers, such as prostate, cervical and ovarian cancer. Currently, diagnosis of prostate cancer relies on antibody-based detection of PSA antigen.
Rely on. Recent evidence from elsewhere indicates that elevated levels of certain cytokines may also be diagnostic for the disease. These two indicators are probably not among the earliest indicators of prostate cancer that actually exist, and the present invention provides a method for the identification of another, yet unknown, diagnostic indicator. Thus, the invention allows the discovery of novel diagnostic markers, for example, by probing plasma samples from prostate cancer individuals and control populations with a library of fluorescent probes. The findings made using this screening assay will in turn lead to the development of therapeutics. Thus, the invention includes the discovery of probes or markers for use in diagnostic assays or kits, as well as their subsequent use in drug discovery.

The present invention is also useful for detecting a diagnostic marker in a Pap smear sample. According to the present invention, normal and abnormal Pap smear samples obtained from an individual are screened with a library of labeling probes and their binding interactions that are diagnostic of malignancy or infectious disease are used for fluorescence polarization-based diagnosis. Can be. In addition, the discovery of biological molecules associated with malignancies or infections can be used in subsequent therapeutic development.

The present invention can be used to screen for biomarker (s) for ovarian cancer, which can be used to develop diagnostic kits for ovarian cancer. Recently, it was suggested that ovarian cancer activator (OCAF) acts as a biomarker for ovarian cancer. Currently, for the detection of ovarian cancer, CH125, one of the ovarian cancer biomarkers,
Serum assay has been used. According to the present invention, in clinical studies, known biomarkers for ovarian cancer (OCAP and CA125) using the fluorescence polarization assay, DNA obstruction assay and scintillation proximity assay (SPA) described in the present invention To identify new biomarkers for ovarian cancer. These assays are simple, fast, and easy to screen clinical samples with labeled probes or labeled probe libraries.
Provides a sensitive and accurate method.

For example, the fluorescence polarization value relating to the interaction between a fluorescent compound and a specimen (blood, serum, plasma or other body fluid) obtained from an ovarian cancer patient can be used to calculate the fluorescence polarization value of other types of gynecological cancer, non-gynecological cancer and It can be compared to the fluorescence polarization values obtained for clinical samples from non-cancer patients. An altered polarization value for a sample from an ovarian cancer patient relative to another sample means that one agent will differentially bind to the component (s) present in the sample from the ovarian cancer patient. Therefore, the agent can be a useful biomarker to distinguish ovarian cancer samples from non-ovarian cancer samples. For samples from ovarian cancer patients, the fluorescence polarization values that are not significantly different from other samples are:
This suggests that the drug is not useful as a biomarker for distinguishing ovarian cancer samples from non-ovarian cancer samples. Agents identified as differentially binding to other types of cancer can be used alone or in combination with other agents to detect and diagnose other types of cancer. In addition, the identified agents can be used to develop therapeutics for the treatment of cancer.

The present invention also provides a method for identifying an early indicator of the onset of diabetes. Current methods for the detection of diabetes are only useful after the disease has fully developed and many disorders have already occurred. In fact, diagnosis is not made in the absence of gross symptoms. In other words, current diagnostic procedures simply confirm that diabetes is present.
The method described in the present invention provides a method for identifying an agent that is an early indicator of diabetes. For example, glycosylation of blood proteins, one of the hallmarks of diabetes, creates unique binding sites, which are labeled using a method described in the present invention, to a diverse library of labeled molecules. Can be detected by a probe from Probes identified as being an early indicator of the onset of diabetes can be used in accordance with the present invention in a fluorescence polarization-based assay. This assay can be used as a diagnostic method in a clinic or diagnostic laboratory.

[0034] The present invention further provides methods for identifying and identifying infectious microorganisms and diseases caused by them. Infectious microorganisms, when present in a host, produce a wide variety, mostly uncharacterized proteins, carbohydrates and other molecules. Also, in response to infection, the host organism produces unique proteins, such as specific antibodies, specific receptors, chemokines and cytokines. All of these unique molecules are candidates for binding to the probe, thus indicating new diagnostic targets for infectious diseases. The present invention provides a method for identifying a binding candidate substance that distinguishes an infectious microorganism and / or a disease caused thereby. The identified binding candidates may then be used for the diagnosis of infectious diseases, leading to the development of therapeutics with drugs. Example 10 includes mesitillin-resistant Stap
The discovery of a unique probe from a small probe library that can distinguish Staphylococcus aureus from hylococcus aureus is described.

The present invention is applicable to the detection of not only infectious bacteria, but also other infectious agents, such as fungi, parasites, viruses, and possibly prions. For example, neuraminidase inhibitors have recently been developed to inhibit influenza A and B viruses. For effective use of these agents, rapid diagnostic kits need to be available to identify the presence of the influenza virus. The present invention provides methods for discovering labeled tagged molecules that have high avidity for viral neuraminidase or some other viral component. These probes can then be incorporated into rapid diagnostic tests for the presence of influenza virus.

The above considerations confirm that the use of the assay system described in the present invention to test known drugs for the ability to differentially bind to clinical samples and to identify novel drugs. It demonstrates the advantages. The identified agent can be used alone or in combination with another agent to detect and diagnose the condition or disease. After identification using the methods of the present invention, these agents are used as diagnostics (eg, in a diagnostic kit) that can be used to rapidly and inexpensively detect and diagnose various conditions or diseases. Can be used.

Another aspect of the invention is to contact two biological samples with two identical libraries of probes, provided that the first biological sample is a control;
Therapeutic and diagnostic, including detecting the binding interaction between each probe and a component of the biological sample; and identifying the binding interaction characteristic of the second (non-control) biological sample A method for identifying biologically important biological targets and drug discovery lead structures. The ligand or target thus identified is a biological target, and the probe having an affinity for this biological component is a lead structure for drug discovery.

In one preferred embodiment, the second biological sample comprises serum from an individual with a disease, or cells or tissues with the same type of disease or other abnormalities as a control. Since diagnosis is typically based on the presence of a detectable biological target, the method is therefore important to develop a diagnostic tool depending on the condition associated with the cell or tissue being screened. In addition, each time a new receptor or target is discovered, compounds with affinity for that receptor are also discovered, making this method a starting point for drug discovery lead structures used to treat the condition. Become.

In yet another embodiment, the principles of the present invention can be used for toxicological screening of pharmaceutically possible substances. The present invention includes methods for determining the toxicity of a compound, as well as determining the presence of a toxic compound in an individual, by measuring the biological response to the toxic compound. Many drugs and their metabolites have adverse side effects or are toxic. There is a strong demand for earlier indicators of toxicity that can predict damage than merely confirming that damage has occurred. Currently, early in drug development, prior to clinical trials, it is difficult to predict emerging toxicities or side effects. By utilizing the principles of the present invention, the presence or absence of toxicity can be predicted at a much earlier stage of development than is currently possible. Initial studies to detect these early warning signals can be achieved by studying cell cultures, for example, utilizing liver cells. In this approach, a sample can be treated with a known toxicant, after which the sample can be removed and probed with a diversity library of tagged compounds. Binding interactions that occur in the treated culture but not in the control can be an early marker of cell or tissue damage. These probes are then tested in an animal model system. Thus, the present invention provides a method of detecting early markers that can predict imminent damage by drugs or other chemicals.

According to this aspect of the invention, the cultured cells or tissues are treated with a known toxic or non-toxic compound, after which an extract is prepared from the culture and probed with a library of labeled compounds. Probes in the library of labeled compounds provide a "fingerprint" that reflects the response of the cell or tissue to this toxic or non-toxic compound. Any changes in the fingerprint pattern that correlate with toxicity are analyzed and possibly further classified. Subsequently, the cultured cells or tissues whose fingerprint pattern has been determined as described above are treated with a test compound of unknown toxicity, an extract is prepared from the treated culture, and probed with a library of labeled compounds. The fingerprint or profile of the generated cell or tissue response is then compared to a reference profile generated for the cell or tissue using known toxic / non-toxic compounds to predict the nature of the test compound.

In yet another embodiment, the principles of the present invention can be used to characterize the function of an expressed protein. Genome sequencing projects, including the Human Genome Project, have created large databases of gene sequences that can be cloned to express the proteins encoded by these genes. Unfortunately, knowing the sequence of a gene or expressing a protein from that sequence does not reveal the function of the protein in living cells, its role in disease, or another molecule that interacts with the expressed protein Often.

Recognizing the shortcomings of genomics in providing this information, some companies have identified
"Proteinology" techniques have been adopted. In proteinology, two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is used to separate proteins according to charge and mass. Next, we compare the generated protein patterns and try to stratify the patient population according to a unique pattern. This method uses genotype (what it looks like) versus phenotype (what it looks like)
Is advantageous in that It is well known that 2-D electrophoresis cannot detect or analyze large numbers of proteins in very complex samples. The method of the present invention does not have this problem, since binding a single labeled probe to a small, medium or large target gives one distinct and identifiable signal. Furthermore, the present invention is three-dimensional (3-D) and the target molecules are very small (2000-3000).
Otherwise, the binding event gives a clearly discernable signal. Also, while the invention is designed to detect binding interactions between labeled probes and biomolecules greater than 2000-3000 daltons, PAGE is designed to detect proteins. Finally, manipulation and analysis of 2-D gels is time consuming, labor intensive, and very expensive. On the other hand, the method of the present invention is quick and very inexpensive. In any direct comparison, the method of the invention for predicting results is superior to either proteinology or genomics.

The present invention provides a means for elucidating the biological function of an expressed protein. When the expressed proteins are probed with a library of tagged small organic molecules, several probes can be found that bind to the binding sites of these proteins. These probes themselves may be inhibitors of these expressed proteins. These probes can be added to cell cultures or other systems and used to determine their biological effects and thereby determine the activity of the protein. Alternatively, tagged probes can be utilized as artificial substrates for competition assays with unlabeled molecules in order to search for stronger binding inhibitors. These unlabeled molecules can then be tested in model systems to determine the effect of these agonists or antagonists on cell function. Using the technique described in the present invention,
The function of the previously unknown or uncharacterized expressed protein can be determined.

Currently, genes are being cloned and expressed in large amounts from genomes that are undergoing sequencing of organisms from bacteria to humans. The present invention provides a method for rapidly elucidating the functions of these proteins and at the same time searching for new drug lead substances. Moreover, the present invention encompasses methods for searching for potential affinity ligands or inhibitors thereof that are useful for the purification or analysis of proteins whose enzymatic or structural function is not yet known. . Such protein targets include those identified by genomic techniques.

The present invention also provides methods that can be used for stratification of populations for any purpose. For example, this technique can be used to stratify groups for clinical trials. Furthermore, the present invention enables inexpensive mass screening for early detection of disease states or analysis of distribution patterns of various diseases. Once an informative probe has been developed, the cost of screening is very low. Screening requires no more than inexpensive fluorescent polarizers and probes. Probes are not only inexpensive to manufacture, but also require only a few nanomoles for one assay.

Numerous other aspects, aspects and advantages of the present invention will be readily apparent from the following detailed description of the preferred embodiments and the appended claims. However, it is to be understood that this disclosure is to be considered as illustrative of the principles of the present invention, and is not intended to limit the invention to the embodiments disclosed herein.

5.1 Probe Diversity Ligand Library and Detection Thereof According to one embodiment of the present invention, blood, serum, urine, cerebrospinal fluid, amniotic fluid, saliva,
Exposure of bodily fluids such as mucus, tissue samples, biological samples such as cells, viruses, microorganisms, or organic molecules, including RNA, DNA, peptides and proteins, and small organic molecules to a group of known reagents A "fingerprint" is established when a numerical panel is created that reflects the pattern of action. According to the present invention, a diversity ligand library comprises a group of known reagents or probes, which interact with or bind to a receptor or target to bind this receptor in a given biological sample. Or measure or indicate the presence of the target. A ligand or probe of the invention includes, but is not limited to, any biological molecule, either natural or synthetic, including, but not limited to, DNA or R
Includes nucleic acids containing NA, small organic molecules, peptides, glycoproteins, proteins, polysaccharides, saccharides or inorganic molecules.

In accordance with the present invention, a library of ligands or probes can be used to create discriminatory binding patterns to accurately distinguish between biological samples. In one preferred embodiment of the invention, the diversity ligand library includes known molecules that, when exposed to a biological sample, produce a numerical panel that reflects a pattern of binding interactions. When using the present invention to distinguish between samples, for example to identify or identify a particular microorganism, such as a particular strain of virus, bacterium, parasite or fungus, a ligand diversity library contains the microorganism's A fingerprint of the ligand or probe that is known to interact specifically or non-specifically with the component should be included.

[0049] In yet another embodiment of the invention, where the receptor or target is unknown, the biological sample to be tested is exposed to a library of ligands or probes composed of partially randomly selected molecules. However, this allows a unique binding interaction to be identified, which can then be identified in a known manner. Receptors found to be present only in the test sample or at different concentrations in the test sample may be possible targets for the diagnosis or treatment of a particular condition associated with the test sample. Moreover, ligands found to have high affinity and specificity for the receptor provide lead structures for drug discovery.

When a biological sample is contacted with a characteristic source of ligand, a ligand having an affinity for one of the receptors present in the sample binds to this molecule to form a ligand / receptor complex And various assay techniques can be used to identify it. A ligand / receptor interaction that occurs in a normal or control sample may be detected by a second cell composed of abnormal cells or tissues of the same type as the control.
Specific differences between these two can be identified as compared to those occurring in the sample. The probe / ligand used in the described assay system may be labeled, tagged, or complexed such that one component of the biological sample produces a detectable signal when bound to the probe. Can be. Probes / ligands can be labeled with labels known in the art and include, but are not limited to, radioisotopes, fluorescent molecules, chemiluminescent compounds, and bioluminescent compounds.

For use in a scintillation proximity assay, the molecule is preferably labeled with a radioisotope including, but not limited to, ³² P, ³⁵ S, ¹²⁵ I or ¹³¹ I. This radioisotope can be detected by a gamma counter or a scintillation counter.

The probes / ligands were fluorescein (FL), rhodamine, 4-4-difluoro-5
, 7-Dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BO or B
ODIPY), isothiocyanate, cyanine or other fluorescent dyes. The interaction of the fluorescently labeled probe / ligand with the components of the biological sample can be detected by a spectrofluorometer or, preferably, by analyzing the fluorescence polarization of the mixture.

[0053] The binding interaction between the probe and the components of the biological sample can also be detected by ELISA (enzyme-linked immunosorbent assay). The probe can be labeled or conjugated with a molecule such as biotin, streptavidin or digoxigenin. Probes labeled with these molecules can be detected using antibodies specific for this label conjugated to the enzyme. Alternatively, the probe can be labeled with an antibody. This antibody may or may not be complexed with the enzyme. Antibodies not complexed with the enzyme can be detected by a second antibody complexed with the enzyme. Antibodies conjugated with this enzyme, for example, spectrophotometry,
React with an appropriate substrate in such a way as to produce a chemical moiety that can be detected by fluorescence measurement or visible means. Enzymes that can be used to detectably label the antibody include, but are not limited to: malate dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast Alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
β-galactosidase, ribonuclease, urease, catalase, glucose
-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

In the method of the present invention, a ligand library synthesized according to any technique known to those skilled in the art can also be used. Preferably, they are performed using conventional solution phase reactions or solid phase synthesis techniques. Target organic molecules, primary or secondary amine groups, hydroxyl groups, thiol groups, aldehydes or ketones, or biologically active compounds containing carboxylic acids, etc. directly in solution with suitable fluorescent molecules (dyes) Thus, a corresponding fluorescently labeled ligand can be prepared. These methods and dyes are described in Haugland, RP Handbook of Fluorescent Probes and Re
^{search Chemicals, 6 th Ed.,} 1996, which is incorporated herein by reference. In a preferred embodiment of the invention, the liquid phase is used in a suitable polar organic solvent or solvent mixture, DMF, DMSO, THF, etc., using a slight excess of dye to ensure complete labeling. Perform synthesis. The resulting fluorescently labeled ligand is purified by standard techniques in organic synthesis, such as liquid-liquid extraction using acids or bases, crystallization and (thin layer or column) chromatography. As described in the examples below, other purification methods, such as liquid-solid phase extraction using a polymer-bound scavenger to remove unreacted dye, followed by simple filtration, etc. Can also be used (O
brecht, D. and Villalgordo, JM, Solid-Supported Combinatorial and Paral
lel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon, 1998
, Chapter 3, see).

[0055] Scheme I. Solution-Phase Reaction (Product Purified by Scavenger Resin) In one embodiment of the present invention, the libraries of the present invention are made using conventional solid-phase techniques. For example, Bodanszky, Principles of Peptide Synthesis (Springe
r-Verlag: 1984); Bodanszky et al., The Practice of Peptide Synthesis (Springer-
Verlag: 1984); Barany and Merrifield, The Peptides: Analysis, Synthesis a
nd Biology Vol. 2, Chapter 1 (Academic Press: 1980); Atherton et al., Bioorg. Chem.
See Vol. 8 (1979). This is because solid phase synthesis has several advantages over traditional synthesis methods. For example, a large excess of reagents or starting materials can be used to drive the individual reactions completely, and purification by simple filtration and washing because the product is attached to a solid support. And that it can be isolated. In addition, many types of side reactions are prevented because the sites of the material bound to the resin are relatively isolated.

The solid-phase synthesis methods which have been found to be particularly suitable for the synthesis of the libraries according to the invention are described below. The method can rapidly and effectively form a diversity library of fluorescently labeled ligands. This method involves two general steps, first, covalently attaching a fluorescent dye to a solid support. . In a second step, this may be repeated as many times as necessary, but the immobilized dye is reacted with the compound or mixture of compounds to form the desired mixture of ligands. The invention encompasses assays that use libraries attached to a solid support as made, or to another solid support. However, it is preferred in the third step to cleave the mixture of ligands from the support. A preferred embodiment of the synthetic method of the present invention shown in Scheme II includes this third optional step.

[0057] Scheme II In Scheme II, <A>, <B>, <C>, <D> and <E> represent a reaction suitable for forming a desired product or an intermediate represented by formula (b)-(g). Conditions are indicated, and brackets (ie, []) denote any parallel or sequential reactions, reactants, and / or products.

According to Scheme II, a dye molecule of formula (a) is selected: XDY formula (a) where D is a fluorescent moiety and X and Y are halogen, alcohol, nitro, thiol, ether, ester Carboxylic acid, α-halocarboxylic acid derivative, amine,
Functional groups independently selected from the group consisting of amides and their protected and unprotected derivatives. Examples of dye molecules of formula (a) include: fluorescein derivatives such as dichlorotriazylaminofluoroscein (DTAF), dichlorosulfofluorescein
(DCSF), and nitrofluorescein; tryptophan derivatives; coumarin derivatives;
Naphthyl derivatives; bipyridine (bpy) derivatives; tripyridine derivatives; cyanines; rhodamines and organometallic complexes, such as, but not limited to, Ru (bpy) ₃ and their derivatives. The choice of dye molecule includes, for example, size, solubility, insensitivity to degradation under solid-state reaction conditions, absorption and emission wavelengths, quantum yield,
And depends on several factors, including the sensitivity of the quantum yield and emission wavelength to the surrounding chemical environment. Many of these factors, as well as the synthesis of these and other suitable compounds, are readily determined from the literature. For example, Haugland, RP, Handbook of Fl
See uorescent Probes and Research Chemicals (6th ed .; 1996).

Also according to Scheme II, a reactive substrate of formula (b) is selected: Resin-LE Formula (b) where resin represents any solid support suitable for solid phase synthesis; Is a linker attached to the solid support; E is a leaving group attached to L. Suitable solid supports include, for example, polystyrene-divinylbenzene (PS-DVB) copolymer and polyethylene glycol-PEG-PS-DVB copolymer. Wang resin (polymer-bound 4-benzyloxybenzyl alcohol) and Rink resin (with and without a suitable linker attached) are available from Aldrich Chemical Co., Milwaukee, WI; Novabiochem, San Diego, CA; and Advanced Chemteck. Available from Lewisville, KY.

The linker LE is selected such that its binding to the solid support is readily cleaved under the reaction conditions represented by <E> in Scheme II. Suitable linkers are known to those skilled in the art and include, for example, halogens, thiols, alcohols, ethers,
Includes esters, aldehydes, ketones, carboxylic acids, nitros, amines, amides, silanes and their protected and unprotected derivatives. Attachment of such a linker to a solid support can be achieved by methods known to those skilled in the art. For example, Bu
nin, BA, The Combinatorial Index, Academic Press, 1998.

In addition to the above criteria, the linker LE forms a covalent bond with the fluorescent moiety D of the dye molecule of formula (a) under the reaction conditions <A> to yield an immobilized dye of formula (c) The resin-LDY formula (c) The appropriate reaction conditions <A> (which depends on the resin, L, E and X) are known to the person skilled in the art or readily Can be determined. Generally, these are
Includes the use of a solvent that causes the resin to swell and react with X. Suitable solvents include, for example,
Dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP), tetrahydrofuran run _{(THF), CH 2 Cl 2} , and includes mixtures thereof. The reaction condition <A> may also include a base such as diisopropylethylamine (DIPEA), triethylamine, dimethylaminopyridine (DMAP), or N-methylmorphine (NMM) to neutralize the acid generated during the reaction. May be included.

The immobilized dye of formula (c) serves as a basis on which the ligands of the library (represented by formula (g) in Scheme II) are formed. However, if the reactive moiety Y is protected, it must be deprotected before further reaction. Any deprotection reaction to form the deprotected moiety Y 'is performed under the reaction conditions represented by <B> in Scheme II. These conditions (which vary with the protecting group) are known to those skilled in the art. See Greene, TW and Wuts, PGM, Protective Groups in Organic Chemistry (2nd ed .; 1991).

The immobilized dye is then reacted with a compound of formula E ₁ R ₁ G ₁ under reaction conditions <C> to form a compound of formula
Obtain a compound of (d): resin-LDR ₁ -G ₁ formula (d) where E ₁ and G ₁ may be the same or different, and E ₁ is a leaving or protecting group , G ₁ is a terminator of R ₁ or is a leaving or protecting group, and R ₁ is at least _one that enables the addition of R ₁ to fluorescent moiety D under suitable catalytic and / or deprotecting conditions. Represents any chemical fragment that contains one protected or unprotected reactive moiety. Suitable reactive moieties include, but are not limited to, halogen, thiol, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, amide, silane, and protected and unprotected derivatives thereof. Not something. Suitable reaction conditions <C> include those developed for solid phase combinatorial chemistry. For example, Brown, R., Contemporary Organic Synthesis, 216 (1997);
der, ER, and Poppinger, D., Adv. Drug Res., 30: 111 (1997); Balkenhohl,
F., et al., Angew. Chem. Int. Ed. Engl. 35: 2288 (1996); Hermkens, PHH.
., et al., Tetrahedron 52: 4527 (1996); Hermkens, PHH, et al., Tetrahe.
dron 53: 5643 (1997); Thompson, LA, et al., Chem. Rev. 96: 555 (1996); and Chem. Rev. 97 (2) (1997). Exemplary addition reactions include reacting a primary amine with an aldehyde to form an imine, which in turn reacts with a variety of different moieties including, for example, β-lactam, pyrrolidine, thiozolidinone, and amide. Can be done. Similarly, the acid groups are also flexible, and can be used, for example, with aldehydes, amines and isonitriles under Ugi multicomponent condensation conditions to form either small amides or heterocyclic compounds.

As shown by Scheme II, the immobilized dye molecules of formula (c) are each a different compound, but a mixture of compounds having the general formula E ₁ R ₁ G ₁ , ie, E ₁ R ₁ G _{1 + (E 1 R 1 G} 1) '+ (E 1 R 1 G 1) "+ ··· + (E 1 R 1 G 1) i ( where, i is the number of compounds in the mixture, (Preferably an integer less than or equal to about 50), in which case a mixture of compounds of formula (d) results, each having a different R ₁ G ₁ fragment; resin -LDR ₁ G ₁ + resin _{-LD- (R 1 G 1) '} + resin _{-LD- (R 1 G 1) "} + ··· + resin ^{_{-LD- (R 1 G 1) i}} . However, Rukoto a compound of formula (c) is only react with one of the compounds the formula E ₁ R ₁ G ₁ is preferred.

[0065] Since the compounds of many pharmacologically active containing reactive moieties, such as amines and carboxylic acids, the present invention contemplates that such compounds are encompassed by the formula E ₁ R ₁ G ₁ And in such cases, further reactions may or may not be desirable. However, when R ₁ fragment of compounds of formula (d) (1 or more) is a reactive moiety, collectively mentioned reaction conditions a n-1 times of the subsequent addition reactions in Scheme II by <D> Where n represents the number of reactive moieties attached to the fluorescent moiety D, and is preferably an integer of about 100 or less.

As above, each of these subsequent addition reactions can employ both a single compound or a mixture of compounds of the formula E _k R _k G _k , where k is from 2 to n−1 Where R _k is the k-th moiety that binds to the immobilized fluorescent moiety D (via k-1 moieties already attached to D), and E _k and G _k are the same Or is different, E _k is a leaving or protecting group, G _k is a terminator of R _k , or is a leaving or protecting group, and R _k is an immobilized compound (one or more At least one that allows the addition of R _k to
Represents any chemical fragment that contains two reactive moieties. Suitable reaction conditions <C> include the use of catalysts, deprotecting agents, etc. that promote the addition of R _k to the immobilized fluorescent compound.

Upon completion of the reaction described above, the immobilized compound of formula (f): resin-LDR _n a mixture of the immobilized compound of formula (f) or (f); ie, resin-LD- (R ₁ R ₂ R ₃・ ・ ・ R _n ) + Resin-L
-D- (R ₁ R ₂ R ₃ ... R _n ) '+ Resin-LD- (R ₁ R ₂ R ₃ ... R _n ) "+ ... + Resin-LD- (R ₁ R ₂ R ₃ ... R _n ) ^m, where m has a maximum of about i * n when i is equal to the number of compounds in the E _k R _k G _k mixture having the largest number of compounds. any are formed. for simplicity, omitted end of ligands (e.g., R _n) a G _n since it is not subject to a further addition reaction from formula (f).

In the final step of Scheme II, the dye-ligand compound is cleaved from the solid support under reaction conditions <E> to obtain a library of compounds of formula (g): PDR _n formula (g) where It will be understood that formula (g) encompasses all possible compounds and mixtures of compounds that result from the reactions depicted in Scheme II. Suitable cleavage conditions <E> are known to those skilled in the art and depend on the bond between the resin and L. Cleavage can be achieved under acidic or basic conditions, or can be photoinduced.
Many suitable cleavage methods have been reported in the literature. For example, some of the cleavage reactions achieved by treating a modified resin of formula (f) with trifluoroacetic acid (TFA) in methylene chloride are shown in Scheme III: Scheme III where (j) is a Wang resin derivative; (k) is a Wang carbamate resin derivative; (l) is a Wang amino acid resin derivative; (m) is a Rink resin derivative; (n) is a Rink amino acid resin derivative; o) is a trityl or chlorotritylamine (ie, X = NH 2) or alcohol (ie, X = O) resin derivative; L ₁ represents any side chain or spacer that is stable under solid state reaction conditions. Examples of suitable side chains include, but are not limited to, substituted and unsubstituted alkyl, aryl and aralkyl groups.

After cleavage, the fluorescent library is isolated, preferably by removing the solvent. Next, the library is dissolved in a solvent suitable for use in the assays of the present invention, for example, dimethyl sulfoxide (DMSO).

Certain embodiments of the method of Scheme II are shown in Schemes IV-VIII. For clarity, these schemes do not show the reaction and formation of the mixture. However, it will be appreciated that each of the individual reactions shown represents a number of parallel reaction possibilities.

A specific simplified embodiment of the general approach of Scheme II is shown in Scheme IV.

[0072] Scheme IV wherein L ₁ is any moiety that does not sterically hinder or otherwise inhibit the binding reaction under the reaction conditions indicated; P is a solid support It represents an end portion of the L ₁ after being cleaved from; R ₁ and R ₂ may be any group desired to provide a library having a different or the same, and preferred structure and reactivity. Examples of suitable groups include, but are not limited to, substituted or unsubstituted alkyl, aryl, and aralkyl.

According to Scheme IV, using a diamino carbamate Wang resin or an amino acid Rink resin or a diamino / amino alcohol trityl / chlorotrityl resin,
Is immobilized on a solid support to obtain a monochlorotriazylaminofluorescein resin. This reaction is preferably performed at ambient temperature. DTAF is combined with about 0.5 to about 3 equivalents of a base such as DIPEA, triethylamine, DMAP or NMM in a suitable solvent such as DM
Dissolve in F, NMP, THF, methylene chloride, or a mixture thereof. For example, DMF or NM
Replacement of the remaining chlorine on the triazine ring with an excess of the symmetric diamine (preferably about 2 to about 6 equivalents) at ambient temperature in P creates new reactive groups for further synthesis. After this process has been repeated the desired number of times with the desired number of different reactants, the resulting fluorescent compound or mixture of compounds is cleaved from the reactive support.

Another embodiment of the general approach of Scheme II is shown in Scheme V: Scheme V wherein DCSF is attached to a solid support by replacing the chlorine atom with a secondary alkylamine, preferably a cyclic secondary amine, which is attached to the resin. Thus, L ₁ forms part of a cyclic diamine. Suitable cyclic diamines include, for example, piperazine, homopiperazine, 4,4'-trimethylene dipiperidine, and derivatives and isomers thereof. As shown, HNR ₁ NH can be replaced with any compound having a suitable reactive group, but R ₁ also forms part of the cyclic diamine. R ₂ and R ₃ represent any moiety suitable for incorporation into the fluorescent ligands of the libraries of the invention, including, for example, the side chains of natural amino acids; substituted and unsubstituted alkyl, aryl and aralkyl, and the like. It is.

As shown in Scheme IV, standard amide formation conditions (ie, PyBrOP / DMAP /
Reacting the free amino group of the fluorescent compound with an Fmoc amino acid under DMF)
-Fmoc protected amino acid is bound to the fluorescent resin. After removal of the Fmoc group by piperidine in DMF, the new amino group provided by the amino acid can be derivatized with, for example, an acid chloride, chloroformate or isocyanate to give various fluorescently labeled compounds. Non-limiting examples of suitable isocyanide compounds are shown in Scheme VI: Scheme VI As will be readily apparent to those skilled in the art, reactive moieties such as amino acids, acid chlorides, chloroformates, and many other moieties including isocyanates (ie, R ₄ , R ₅ ,...) -, it is possible to form a ligand bound to DCSF with R _n). Likewise, if the reaction conditions are appropriately changed, the chemical fragment R ₂ and R ₃ groups in Scheme IV is bound, it is replaced with others that enable the growth of chains attached to the dye molecule Can be.

The final embodiment of the general approach of Scheme II is shown in Scheme VII: Scheme VII wherein DTAF is coupled to a Rink amino acid resin and subsequently derivatized by the methods described above. L ₁ , R ₁ and R ₂ are defined as above.

In addition to the above method, a fluorescently labeled ligand library is also prepared by a general solid-phase synthesis technique (Obrecht, D. and Villalgordo, JM, Solid-Supported Com.
binatorial and Parallel Synthesis of Small-Molecular-Weight Compound Lib
raries, Pergamon, 1998; and Bunin, BA, The Combinatorial Index, Acade
mic Press, 1998). In a preferred embodiment of the invention, the desired compound is synthesized on a solid support according to methods described in the literature. Prior to cleavage from the solid support, the compound on the solid support is treated with a suitable dye to obtain a fluorescently labeled ligand on the resin. Next, these ligands are cleaved from the resin to obtain fluorescently labeled ligands.

In one approach, these ligands are synthesized in a linear fashion by reacting the solid phase supported reactive blocks stepwise with different reactive blocks. Next, in the final step, a dye was added prior to cleavage, resulting in Scheme VI
The labeled ligand shown in II is obtained. In this scheme, fluorescently labeled N-hydroxyquinazolinone is preferred. Quinazolinones are one of the most common biologically active nitrogen-containing heterocycles (Sinha, S. and Srivastava, M. in Progress in Drug Res.
earch, 1994, vol. 43, 143-238). They exhibit a wide range of biological and pharmacological activities in humans and animals. These have been used as anticonvulsants, antibacterials and antidiabetic agents. Therefore, fluorescently labeled quinazoline is useful for diagnostic applications and drug discovery.

[0079] Scheme VIII In another approach, centralization is performed using a multi-component condensation reaction such as Ugi condensation (Tempest, PA, et al. Angew. Chem. Int. Ed. Engl. 1996, vol. 35, 640-642). These ligands are prepared by a standard method. Using this approach, the amine component is immobilized on a solid support as a Rink amine resin, and aldehydes, Fmoc protected amino acids, and isocyanides are swelled with a mixture of MeOH / DCM (1: 2 v / v). Add in excess (Scheme IX). Numerous ligands can be synthesized by using different aldehydes, acids and isocyanides in combination.

[0080] Scheme IX 5.2 Biological Samples The present invention provides a method for "fingerprinting" complex biological mixtures or samples obtained from a wide range of sources. The method of the invention is for identifying specific phenotypic differences that exist between normal or abnormal, healthy or diseased, untransformed or transformed, uninfected or infected cells and / or tissues. Can be used for The methods of the present invention can also be applied to identify or distinguish between species of microorganisms, viruses, bacteria, fungi or parasites.

The methods of the present invention detect all types of ligand / receptor interactions, where the receptor is a protein, carbohydrate, nucleic acid, or any form that is capable of interacting with a probe or ligand. Molecule. As used herein, "biological receptor", "receptor", "biological target", "target" and "component of a biological sample" are any biological molecule, binding surface, It is intended to include a binding site or the like, eg, differentially expressed or differentially modified in a test sample, eg, a diseased or otherwise abnormal cell and / or tissue. Any one of these receptors can be a target for diagnosis and / or development of potential therapeutics.

Accordingly, one aspect of the present invention is a method of characterizing a pathology, the method comprising the steps of identifying a receptor or target, and a ligand or probe, present in a biological sample in connection with a pathology. Identifying a pattern of binding interactions with the library, wherein the pattern of binding interactions provides a fingerprint specific to the pathology.

According to the present invention, a “receptor” or “target” binds to a library of probes or ligands in a test sample, which may or may not differ between the test and control samples. A biological molecule that exhibits affinity or interaction. Examples of such receptors or targets include, but are not limited to, proteins including enzymes, antigens, antibodies, lipids, nucleic acids including DNA and RNA, carbohydrates including lectins, cell surface proteins or receptors, and the like. Including.

The biological samples utilized in this method include, but are not limited to, plasma, serum, urine, cerebrospinal fluid, amniotic fluid, saliva, bodily fluids such as mucus, tissue samples, cell extracts,
Products from in vitro transcription and translation systems (see, for example, US Patent No.
5,654,150) and any other sample that is a source of biological molecules. In addition, extracts or liquids thereof containing pathogenic organisms such as bacteria, yeasts, fungi, viruses, protozoa, etc. can be used. In these examples, ligands that exhibit high affinity and specificity for proteins or other receptors in the pathogen are:
It can be a new target and its inhibitory effect on pathogens can be tested.

[0085] Biological samples that can be screened by the methods of the present invention include:
Can be obtained from a wide range of sources. By way of example, and not limitation, a biological sample or mixture can be obtained from a patient, including bodily fluids, blood, serum, oral cavity, mucus, including rectal or intestinal mucus, urine, feces, and the like. . further,
Biological samples can include tissue samples, biopsy tissue, bone marrow cells, lymphocytes, immune cells, cell samples including mucosal cells obtained from oral, rectal or intestinal mucus lining, and the like. In yet other embodiments, the biological sample or mixture comprises cell lysates or portions thereof, carbohydrates including lectins; proteins including glycoproteins, cell surface receptors, peptides; nucleic acids including DNA or RNA, etc. Can be. In still other embodiments, the biological sample is a virus, bacterium, microorganism or parasite, or a liquid containing such biological samples, if example embodiment, even in the test water feed microbial content Or may be derived from them.

A biological sample of the present invention can be obtained from an individual suffering from a disease, disorder or pathology infected with a virus, bacterium or other microorganism. In still other embodiments, the biological sample is obtained by exposing the tissue, cells in the medium, cell extracts, etc. to toxins or pathogens, or altering the genome of the cells in the medium to a given pathology or disorder. Can be made by genetic manipulation to encode a mutation or protein or peptide known to be associated with

A collection of biological material as the source of a clinical sample can be obtained from a hospital or a national laboratory.

5.3 Assays for Detection of Ligand / Receptor Interactions Clinical substances are often the third element of this invention, recognizing that only a limited supply is available for a given pathology. A sensitive assay system
The say system must be capable of detecting binding interactions that occur in only a few microliters of the sample, and must be capable of detecting binding interactions that are weaker than optimal affinity. The assay system of the present invention eliminates or significantly reduces non-specific binding of the ligand to the receptor present in the sample.

Thus, one aspect of the assay of the present invention is to provide a means for detecting the interaction between a probe or ligand and a receptor, target or active site. One such embodiment utilizes a homogeneous assay for the detection of a receptor or target present in a biological sample. The assay contacts a biological sample with a library of diverse probes in a microtiter dish or other well-type device and detects binding of individual members of the probe library to components in the system. To do. The binding pattern between the members of the probe library and the sample gives the molecular fingerprint of the sample. In a preferred embodiment, binding between a member of the library and a component of the sample is detected by fluorescence polarization.

In another embodiment, assays for detecting receptors or targets present in a biological sample bind probes and ligands of the library to a structure having a site cleavable by a specific catalyst. And the cleavage of the structure or scaffold provides a signal or means for detecting the interaction between the probe and the receptor. More specifically, members of a library of ligands or probes are attached to a scaffold having a site cleavable by specific catalysis and markers on both sides of the site to provide a ligand / scaffold construct. Contacting the ligand / scaffold construct with a biological sample, ligands having an affinity for the receptor present in the sample bind to the receptor and block the cleavable site of the scaffold. When the ligand / scaffold construct is contacted with the specific catalyst, the scaffold without the bound receptor is cleaved and an intact scaffold is identified.

A preferred embodiment of the assay of the present invention further comprises attaching a marker, such as biotin, to the same side of the scaffold as the cleavable site, and attaching a second marker, such as digoxigenin, to the scaffold. Bonding to the opposite side. The scaffold is immobilized on the surface, and after addition of the catalyst, the mixture is washed to remove the portion of the cleaved scaffold containing the second marker, and the intact scaffold is identified by the presence of the second marker.

[0092] The scaffold used in the assay may comprise any hydrophobic or hydrophilic, synthetic or natural polymer, including DNA, RNA, peptides, peptoids, oligosaccharides or block copolymers. In a preferred embodiment, the scaffold comprises a first
, A second complementary oligonucleotide having a ligand attached thereto, and a double-stranded DNA construct comprising one restriction enzyme cleavage site. In this embodiment, the specific catalyst is a restriction enzyme specific for a restriction enzyme cleavage site. The first marker can be, for example, biotin, in which case the ligand structure can be immobilized on a streptavidin or avidin-coated surface. Digoxigenin is a preferred second marker, in which case anti-digoxigenin peroxidase is used to indicate the presence of intact scaffolds.

Another assay for detecting the presence of a receptor in a biological sample involves binding a member of a ligand library to a scaffold and binding the scaffold to a scaffold-specific binding agent and a biological sample. And detecting the interaction between the ligand and the receptor present in the biological sample. The scaffold used in this assay can comprise any hydrophobic or hydrophilic, synthetic or natural polymer, including DNA, RNA, peptides, peptoids, oligosaccharides or block copolymers. In a preferred embodiment, the scaffold-specific binding agent does not bind to the region of the scaffold where the interaction between the ligand and the receptor occurs. Scaffold specific binding agents include drugs, peptides, glycoproteins,
It can be a protein, polysaccharide, sugar, or inorganic molecule. In other embodiments, the scaffold comprises a marker that is blocked when the scaffold-specific agent binds to the scaffold, and access to the marker is due to the absence of the scaffold-specific binding agent and ligand / receptor interaction. Indicates the presence of In a preferred embodiment, the scaffold comprises double-stranded DNA and the marker is a biotinylated nucleotide that is detected by streptavidin or avidin when a receptor / ligand interaction occurs.

In yet another embodiment, the principles of the present invention may be used for toxicological screening of potential pharmaceuticals. Currently, it is difficult to predict toxicity or side effects that occur early in drug development and before clinical trials. By utilizing the principles of the present invention, the presence or absence of toxicity can be predicted at a much earlier stage of development than is currently possible.

According to this aspect of the invention, the cultured cells or tissues are treated with a known toxic or non-toxic compound, after which extracts are prepared from the culture and probed with a library of labeled compounds. . Probing with this library of labeled compounds gives a "fingerprint" pattern that reflects the response of the cells or tissues to toxic or non-toxic compounds. Any changes in the fingerprint pattern related to the toxic properties were indicated, presumably statistical analysis or artificial intelligence routines (
Neural networks). Subsequently, the cultured cells or tissues with the fingerprint pattern set as described above are treated with a test compound having unknown toxicity, an extract is prepared from the treated culture, and a library of labeled compounds is used. Probe. The fingerprint or profile obtained from the response of the cell or tissue is then compared to a reference profile generated for the cell or tissue using a known toxic / non-toxic compound to predict the nature of the test compound. .

In accordance with the present invention, Sections 5.3.1 through 5.3.3 provide a method for detecting a difference in the binding affinity of a probe to a biological sample and generating a fingerprint or pattern of binding affinity. Describes assays that can be used.

The results obtained using the present invention help to distinguish between two sample populations (eg, normal and diseased) and can be expected to fit into two categories. In the first category, the population can be distinguished with one or two probes, and the probe response will be easily distinguishable between the two groups ("shining pebbles"). In the second category, subtle differences in binding to the two sample populations can be identified by using a larger number of probes with a small difference in binding to the sample populations. The combination of these probes (diagnostic clusters) provides the power to statistically analyze reliable discrimination in unknown samples.
cluster)). Larger numbers of probes are expected to provide a more useful analytical system, as variations in individual differences do not significantly affect the overall binding pattern. In systems where only one or two probes are available, individual differences in the probes make diagnosis more difficult.

Analysis of multiple probe bindings on multiple samples requires the use of sophisticated computer algorithms. Two types of data analysis can be performed. The first analysis type focuses on determining which probes are useful for identifying members of two sample populations. Algorithms for this analysis type come from the field of statistical analysis (discriminate function analysis) or from the field of artificial intelligence (supervised back-propagation neural networks (supe
rvised back propagation neural network)). A second type of analysis that can be performed is to classify probe binding data based on the results of the probes and then ask if there is any similarity in the history (eg, medical) of the category based on each probe. including. Algorithms for these analysis types are based on statistical analysis (principal component analysis) and artificial intelligence (unsupervised, Kohonen neural networks (unsuper
vised, Kohonen neural network)). Data analysis programs or packages with the above functions are commercially available. For example, the commercial program SPSS (S
PSS, Inc., Chicago, Illinois) and SAS (SAS Institute, Inc., Cary, NC)
Provides discriminant function analysis and principal component analysis. Neuroshell 2 (Word Systems Gro
up, Inc., Frederick, MD) or several packages, such as Stuttgart neural network simulation (The University of Stuttgart, Stuttgart, Germany), provide backpropagation and Kohonen neural networks.

5.3.1 Fluorescence Polarization Assay In the present invention, fluorescence polarization can be used to identify binding interactions between receptors and ligands present in a biological sample.

[0101] Fluorescence polarization assays are designed to measure the binding of fluorescently labeled compounds to unlabeled biomolecules. Assays based on fluorescence polarization can be used to detect the interaction of fluorescently labeled compounds with biomolecules with molecular weights up to approximately 10,000. The types of fluorescently labeled compounds that can be used include, but are not limited to, small organic molecules, peptides, small proteins, nucleic acids, lipids and polysaccharides. When excited by plane-polarized light, fluorescent molecules will emit light in a fixed plane only when they do not rotate during excitation and emission. The degree of depolarization of the emitted light depends on the amount of rotation of the molecule, which will depend on the size of the molecule. Between excitation and emission, small molecules rotate more than large molecules. The optimal conditions for this assay are when the labeled compound is much smaller than the unlabeled molecule of the binding partner. Unbound small fluorescently labeled compounds rotate rapidly and the emitted light is depolarized. The interaction between the biomolecule and the fluorescently labeled compound increases the effective size of the fluorescently labeled compound, thus reducing the rotation of the compound and leaving the emitted light polarized. The intensity of the emitted polarization can be measured by inserting a movable polarization filter in front of the detector. Intensity is measured in planes offset by 90 °, often in parallel and perpendicular. In some instruments, the excitation filter is movable and the emission filter is fixed. In certain other instruments, the parallel and perpendicular intensities are measured simultaneously via optical fibers. Pan Ve
ra, BMG Lab Technologies, and LJL Biosystems provide market research grade fluorescence polarizers, and Abott provides clinical research instrumentation. The fluorescence polarization value is determined by the following equation: Polarization = (Vertical Intensity-Parallel Intensity) / (Vertical Intensity + Parallel Intensity) Fluorescence polarization values are often expressed in millipolarization units (mP), divided by 1000. .

In one form of such an assay, a fluorescent molecule, such as fluorescein, is labeled
Attached to oligomers of sufficient length to cause a "propeller effect". Thereafter, a short linker is attached to the fluorescent molecule and the end of the oligomer opposite the fluorescent molecule is attached to the wall of a microtiter dish or other well-type device suitable for use in fluorescence polarization. Then, the free end of the short linker is
Derivatize with the ligand to be screened. The identity of the ligand bound in each well is well known. Thereafter, a source of receptor was added to each well,
Incubate. Thereafter, the fluorescent molecules are excited using the polarized light, and the degree of polarization of the emitted polarized light is measured. Receptors that bind the ligand will reduce the free rotation of the fluorescent molecule and an increase in polarization will be detected. On the other hand, if no receptor binds to the ligand, the fluorescent molecule will remain free to rotate and the emitted light will be less and will not be polarized. To obtain an approximation of the affinity of any bound receptor for the ligand, the extract is removed from the wells, fresh buffer is added, and the polarization of the light emitted from each well is measured. Receptors with low affinity will dissociate from the bound receptor and the emitted light will be less polarized. Repeating this process will identify the highest affinity ligand / receptor binding interaction.

5.3.2 Scintillation Proximity Assay (SPA) In a typical SPA assay, the receptor is bound to scintillant load beads, and screening is performed directly or competitively with the ligand in solution. I do. However, it is also possible to reverse this procedure, so that the ligand is bound to the beads and the receptor is in solution. This variant can be particularly adapted to combinatorial chemistry, because in many synthesis schemes, library synthesis is performed on beads, and then saturates the beads with scintillant when needed in SPA assays. Because you can do it. Further, one or more ligands can be synthesized on one bead. In such systems, beads loaded with scintillant and coated with ligand are immersed in a liquid phase containing the radiolabeled reactant. If the labeled reactant has an affinity for the tagged ligand and the two bind, the proximity of the resulting radiolabeled reactant to the scintillant of the beads activates the scintillant and emits light. If the labeled reactant has little or no affinity, the radiolabel will not accumulate close enough to the scintillant to cause radioactive decay following energy transfer. Since SPA does not require a washing step, detection of relatively low affinity ligand / receptor binding interactions is possible.

5.3.3 DNA Restriction Cleavage Site Interference Assay The principle of this system is that the presence or absence of restriction enzyme activity on a DNA construct synthesized to include a single restriction site and a ligand-reporter system. Based on location. When the construct is contacted with a biological mixture, the ligand / receptor interaction will block access of the restriction enzyme to the restriction enzyme cleavage site and prevent hydrolysis of the construct at that site. Constructs that remain intact can be isolated and ligand / receptor interactions identified.

A description of the DNA restriction enzyme cleavage site assay system is provided herein. A single-stranded DNA oligonucleotide containing biotin at the 5 'end and digoxigenin at the 3' end was annealed to the complementary oligonucleotide, and the annealed double-stranded oligonucleotide was placed with a single restriction endonuclease cleavage site at the center. Contained. The complementary oligonucleotide was modified to contain a linker with a terminal amino group. In this case, the position of the amino group between the 5th A and the 6th T from the 5 'end does not interfere with the restriction enzyme activity for the double-stranded oligonucleotide.

The resulting construct (where the sequence: (Sequence number ) Is the restriction enzyme cleavage site): (Sequence number The amino groups were derivatized with ligands from various chemical libraries to create the constructs shown below: (Sequence number The derivatization of amino groups can be performed during the synthesis of single-stranded oligomers, while still attached to CPG beads, after which the oligomers are released from the beads using alkaline cleavage, followed by complementary biotin -Can be annealed to digoxigenin oligomers. As an alternative to attaching the ligand, a derivatized base can be incorporated during the synthesis of the complementary oligonucleotide.

When incubated with a restriction enzyme specific for a restriction enzyme cleavage site by methods known in the art, the derivatized construct hydrolyzes at the restriction enzyme cleavage site to provide the following two parts: (Sequence number and If the hydrolysis mixture is reacted with a streptavidin or avidin-coated surface, the biotin-labeled moiety will be fixed and the digoxigenin-labeled moiety will be eliminated by washing. Reaction of the immobilized mixture with the anti-digoxigenin peroxidase antibody will give a negative result since hydrolysis by restriction enzymes and subsequent washing will remove the digoxigenin-labeled moiety from the mixture.

When an intact ligand-derivatized construct, which functions as a probe to detect potential receptors in a sample, is mixed with a clinical sample, the ligand captures a receptor with high affinity as shown below. Will: (Sequence number After the incubation, the reaction mixture is diluted with a suitable buffer,
Treat with restriction enzymes, then incubate with streptavidin coated surface to fix biotin molecules. If a biological molecule with a high affinity for the ligand on the construct is present, it will block access of the enzyme to the restriction enzyme cleavage site and prevent hydrolysis of the DNA scaffold, thereby rendering the intact two It will result in immobilization of the strand oligonucleotide. Alternatively, if the biological molecule does not bind to the ligand of the construct, the restriction enzyme will not be blocked and the DN
Hydrolysis of the A scaffold will be possible and will result in immobilization of the truncated construct without the digoxigenin labeling moiety. A standard enzyme-linked immunosorbent assay (ELISA) using a digoxigenin peroxidase antibody can be used to detect the presence of digoxigenin on streptavidin. The positive assay is
Indicates that the restriction enzyme was blocked, which is an indication that the molecule was attached to the linker. Thus, this technique can be used to detect interactions between any receptor and any ligand with sufficient affinity and size to block access of the restriction enzyme to the restriction enzyme cleavage site. .

In a modification of the assay described above, as shown below, certain “gaps” (
(A deletion of a base) is inserted into one strand of the double-stranded sequence and a ligand is bound next to the gap: (Sequence number ) Treatment of this construct with an endonuclease such as mung bean nuclease or nuclease S1 will result in hydrolysis of the construct in the gap region. Reaction of the hydrolysis mixture with immobilized avidin or streptavidin and subsequent washing will remove the digoxigenin-containing portion of the construct. As described above, incubation of this construct with a receptor with high affinity for the ligand prior to nuclease addition,
It will prevent hydrolysis of the construct in the gap region and result in a strong positive response during the assay for the presence of digoxigenin.

In a further variation of the DNA restriction enzyme cleavage site assay, a DNA scaffold is blocked by a bond cleavable by a particular enzyme or other mechanism, and the cleavage is blocked by the generation of a ligand / receptor interaction near the binding site. Backbone (bac) consisting of a peptide or peptoid or any polymer with a central bond
kbone). For example, commercially available poly (glycine) or poly (alanine) backbones into which chains containing phenylalanine residues and adjacent ligands have been inserted by standard peptide synthesis techniques can be utilized. afterwards,
Enzymes such as chymotrypsin A (EC 3.4.21.1) can be used in an analogous manner to the nucleases described above. In addition, the assay can be modified to a SPA assay using radioactive bases or ligands for the synthesis of the portion of the construct opposite biotin and binding streptavidin to the scintillant-loaded beads.

Although any of the assays described herein are suitable for use in the present invention, this system provides radiolabeled nucleotides to adapt the assay to the SPA format, which offers several advantages: It may be located downstream of the restriction sites, but the system does not require radioactivity; the system detects the presence of a supersensitive (single intact DNA backbone) by using a PCR technique in the detection system. The system allows the entire assay to be performed in a streptavidin-coated microtiter dish; and the system uses a DNA analog with a nucleotide analog unless the restriction site is changed. The strand can be protected against nuclease activity in the sample.

In the present invention, a film containing a specific reactive group (for example, Pall Corpo)
ration, East Hills, NY) can be placed in a well device such as a 96 well dot blot apparatus. Thereafter, the specific functional group added to the library synthesis is combined with the specific reactive group on the membrane in the device, and the compound library can be synthesized in the device. For example, if the library compound synthesized contains amino groups, the membrane will contain active carboxylic acid groups and both groups will be linked by amide bonds. Excess groups on the membrane will be blocked with a drastic blocking agent. Thereafter, the ligand binding membrane can be reacted with a biological sample that has been previously derivatized with, for example, biotin or radioactivity. afterwards,
A suitable assay can be used to detect the ligand / receptor interaction.

5.3.4 Equilibrium Perturbation Assay The DNA restriction site assay described above can be characterized as an equilibrium perturbation assay, and the preferred approach is to equilibrate the ligand / molecular interaction DNA binding protein with its cognate sequence. Monitoring the impact on the environment. For example, the viral DNA binding protein UL9 can be used to screen for compounds that bind to a selected DNA test sequence located downstream of its cognate UL9 sequence. The assay is based on the ability of the test sequence to disrupt the equilibrium of the binding protein and prevent the binding protein from binding to the cognate sequence. The assay is described in US Pat. No. 5,306,619, which is incorporated herein by reference.

In accordance with the principles of the present invention, test sequences located downstream of the UL9 recognition site may serve as a backbone for a ligand library rather than as a target for DNA binding proteins in US Pat. No. 5,306,619. it can. A scaffold construct can be formed such that a particular nucleotide base comprises, for example, a UL9 cognate sequence tagged with a marker such as biotin; and an oligonucleotide derivatized with a digoxigenin-terminated ligand. . The presence of biotin in the cognate sequence does not interfere with the binding of UL9 to the cognate sequence, and once UL9 binds to the biotinylated sequence, the biotin molecule can be blocked from immobilization on, for example, avidin or streptavidin. Are known. UL9 can be reacted with the construct and incubated until equilibrium is established between UL9 and the biotinylated sequence. Thereafter, when the biological sample is introduced into the reaction mixture, any receptors present in the sample that have an affinity for the ligand on the scaffold test sequence will bind to the ligand and bind between the binding protein and the biotinylated sequence. It can disrupt the equilibrium and cause a change in the amount of free biotin available for binding to avidin or streptavidin.

In a variation of this assay, a test sequence other than the second DNA sequence can be synthesized using the screening sequence prior to binding of the compound. Any scaffold capable of binding the ligand can be used. Alternatively, the ligand can be covalently linked directly to the screening sequence without the need for additional linkers or scaffolds.

[0115] 6. EXAMPLES The following examples are provided so that the invention described herein may be more fully understood. These examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

Example 1 Scintillation Proximity Assay is a Sensitive Method for Detecting the Interaction between a Ligand and a Receptor The following example demonstrates the sensitivity of a scintillation proximity assay (SPA). This example demonstrates that 2,5-diphenyloxazole (PPO) impregnated p-aminophenyl-β-D-
The interaction between thiogalactosidase agarose beads and β-galactosidase is evaluated using SPA.

E. coli transformed with the β-galactosidase expression vector was incubated at 37 ° C. with H _Two ³⁵ SO_FourGrow in M9CA medium with or without (0.1 mCi / mL medium).
β-galactosidase expression was induced by treating E. coli cultures with 1 mM IPTG for 3 hours. Subsequently, E. coli is pelleted by centrifugation and resuspended in lysis buffer.
Then, the cells were thawed and freeze-thawed for 5 cycles to be thawed. Bacterial debris is removed by centrifugation,
β-galactosidase was isolated from the supernatant using ammonium sulfate precipitation, after which 80% purification was obtained by affinity chromatography.

Unlabeled or labeled β-galactosidase and 2,5-diphenyloxazole (PPO) impregnated p-aminophenyl-β-D-thiogalactosidase agarose beads (prepared by the Bertoglio-Matte method of US Pat. No. 4,568,649) ) Were evaluated. PPO-impregnated p-aminophenyl-β-D-thiogalactosidase agarose beads were washed overnight with PBS (8.1 mm Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 137 mM NaCl, 2.7 mM KCl, 0.5 mM MgC
Incubation in a solution of 10% powdered milk in l ₂ , 0.9 mM CaCl ₂ ) blocked sites involved in non-specific binding. Alternatively, the beads can be blocked with an agent, such as albumin, a detergent, or an extract from a control solution. Subsequently, 20 μL of PPO-impregnated p-aminophenyl-β-D-thiogalactosidase agarose beads were added to ³⁵ S-β-galactosidase (as counted in the scintillation cocktail,
2.1 × 10 ⁵ cpm) and either 200 μL of PBS or 200 μL of 1 mg / mL unlabeled β-galactosidase. In other experiments, ³⁵ S - labeled Escherichia coli (E. coli) extract or H ₂ ³⁵ SO ₄ equal cpm of ^{(6.5l~7.7 X 10 3 cpm),} PPO containing immersion p- aminophenyl-beta-D -Mix with 20 μL of thiogalactosidoagarose beads. The solution was mixed for 10 minutes before being transferred to a scintillation vial containing 1 mL of PBS containing 0.2% casein. The interaction between beads and β- galactosidase or H ₂ ³⁵ SO _4, and analyzed to assess the radioactivity with a Beckman LS 6500 scintillation counter.

In the sample containing ³⁵ S-β-galactosidase and PPO impregnated p-aminophenyl-β-D-thiogalactosidase agarose beads, 487 +/− 52 counts / min (cpm; n = 3) were detected. In contrast, both labeled and unlabeled β-galactosidase and PPO-impregnated p-aminophenyl-β-D-thiogalactosidase agarose beads detected 341 +/− 16 cpm (n = 3). These results demonstrate that unlabeled β-galactosidase can competitively displace labeled β-galactosidase bound to beads. Furthermore, these results indicate that SPA can be used to detect mM interactions. The released aminophenylthiogalactoside was confirmed to have a Ki of only 1.9 mM. Furthermore, the results show that SPA can be used to detect interactions between receptors and ligands that have low affinity for each other. In this example, the bead affinity for β-galactosidase was reduced almost 2.3-fold by using PPO.

6.5-7.7 × 10 ³ cpm of ³⁵ S-labeled E. coli extract and PPO impregnated p-aminophenyl-
About 3000 cpm was detected in the sample containing β-D-thiogalactosidoagarose beads. In contrast, in samples containing 7.7 X 10 ³ cpm and PPO impregnated p- Aminofeni Le-beta-D-thiogalactoside agarose beads H ₂ ³⁵ SO _4, about 670 cp
m was detected. This result indicates that the background is very low due to the interaction between the bead and H ₂ ³⁵ SO _4. Thus, the signal detected in the sample containing the ³⁵ S-labeled E. coli extract is primarily due to the interaction between the beads and the labeled E. coli protein. The high cpm detected for the interaction between the beads and the ³⁵ S-labeled E. coli extract indicates that in the absence of a purification step, background radioactivity masks the adsorption of specific β-galactosidase to SPA beads Suggests.

This example demonstrates that the scintillation proximity assay (SPA) is a sensitive assay for detecting interactions at the mM level between ligand and receptor. In addition, the SPA is useful for detecting interactions between low affinity receptors and ligands.

[0122] Example 2: DNA interfering assays are highly sensitive method To evaluate the interaction between the receptor and ligand. The following example demonstrates the sensitivity of a DNA interference assay to assess the interaction between receptor and ligand.

The following two oligomers were synthesized by Clontech, Palo Alto, CA: (SEQ ID NO: ) I 5'BTT-TTT-TTT-TTT-TAT-ATA-GGA-TCC-TAT-ATA-TTT-TTT-TTT-TTT-TTD-3 '(B = biotin, D = digoxigenin); ) II 5′AAA-AA-AAA-AAA-AAT-ATA-TAG-GAT-CCA-AAT-TAA-AAA-AAA-AAA-A-3 ′; It was designed to form an enzyme cleavage site. The oligomer was prepared in an annealing buffer (10 mM Tris HCl [pH 7.8] 0.1 M NaCl and 1.0 mM EDTA) at a ratio of II: I of 10: 1.
C., 1 hour, then 57.degree. C., 3 hours, followed by storage and annealing at -20.degree. Serially dilute 32.8 pmol duplicate samples of the annealed oligomers into annealing buffer and transfer one of each duplicate dilution overnight.
Instructions (New England BioLabs, Beverly, MA) at 37 ° C with 40 units of restriction enzyme
). The remaining dilution was processed the same except that the restriction enzyme was omitted from the incubation mixture. After an overnight incubation, the samples were added to a streptavidin-coated microtiter dish, and HRP-derivatized D anti-digoxigenin antibody and ABTS substrate were used to prepare instructions (Boehri
nger Manheim GmbH, Manheim, GER). Thereafter, the absorbance of the sample was read at 405 nm.

The enzyme-treated sample showed no absorption on background (no oligomer in the incubation mixture), whereas the untreated sample showed clearly detectable absorption at oligomer concentrations as low as 3 pmol. Thus, this system is capable of detecting pmol levels of substrate with essentially no background.

[0125] Example 3: ability to cleave the DNA scaffold restriction enzymes, this embodiment is influenced by the molecular interactions between Sukaho field of other drugs, molecular mutual away from the restriction enzyme cleavage sites Explain that action (ligand / receptor interaction) affects enzyme activity.

To immobilize DNA oligomers on the well walls of a 96-well microtiter plate, 65.6 μl of annealed DNA labeled with biotin at one end and digoxigenin at the other end and having a centrally located restriction enzyme cleavage site. Pmol samples were added to streptavidin coated wells. Control wells without DNA were also provided (Table 1, lane 1). Thereafter, the four wells were added to the conjugate buffer (
In Boehringer Mannheim, anti-digoxigenin peroxidase (anti-dig-PO
D) Antibody treatment. Three wells were treated with conjugate buffer without antibody. After a one hour incubation, the wells were washed and three of the wells were treated with 120 units of restriction enzyme at 37 ° C. for one hour (Table 1, lanes 3, 5, and 7).
). Other wells were treated with the same solution without restriction enzymes. The wells were then washed again and assayed for intact (unhydrolyzed) oligomers by standard ELISA. The complete, annealed DNA in the sample was indicated by Vmax in mOD / mm.

The results of this experiment are summarized in Table 1 below. In the absence of DNA, the background Vmax value of the reaction was 0.89. Absence of restriction enzymes and no pretreatment (Table 1,
In lane 2), the Vmax value of the complete, unhydrolyzed annealed DNA was 30.96. In contrast, when the immobilized DNA was treated with the restriction enzyme without pretreatment, the Vmax value was 2.52 (Table 1, lane 3). A decrease in the Vmax value indicates that the restriction enzyme hydrolyzed the immobilized oligomer.

Pretreatment of the immobilized oligomers with ant-dig-POD did not affect the assay for intact oligomers (Table 1, lanes 4, 6, and 8), and
Values of 34.96, 45.33 and 53.71 were obtained. However, pretreatment with anti-dig-POD does affect restriction enzyme activity (Table 1, lanes 5 and 7). This is because pretreatment of the oligomer before addition of the restriction enzyme reduces hydrolysis of the oligomer. The pretreated sample was later subjected to restriction enzymes (Table 1, lanes 5 and 7)
Oligomers that had Vmax values of 15.17 and 14.64, respectively, but were not pretreated but were subjected to restriction enzymes had a Vmax of only 2.52. Therefore, pretreatment of the oligomer with anti-dig-POD before addition of the restriction enzyme reduces the activity of the restriction enzyme by 17-fold, and the interaction between anti-dig-POD, digoxigenin-labeled DNA and oligomer is limited by the restriction enzyme. Affects the ability to cleave oligomers of These results demonstrate that the addition of an agent that interacts with the oligomer to the reaction mixture affects the ability of the restriction enzyme to cleave the oligomer.

[0129]

[Table 1] This example demonstrates that the “receptor” that interacts with the ligand that binds to the DNA backbone is DNA
Demonstrates that it can have a strong effect on restriction enzyme cleavage of the main chain

Examples 4-8 demonstrate the sensitivity of a fluorescence polarization assay and the differential binding patterns of biological samples to various probes. The molecular probes used in these examples are listed in Table II.

[0131] Example 4: The following example is a method having sensitivity to detect the interaction between the fluorescence polarization assay probe with a biological sample, of various probes, the biological sample It demonstrates that the ability to bind a component depends on the concentration of the component in the sample. Further, this example demonstrates the differential binding of the probe to components of a biological sample.

A series of dilutions of pooled human serum (Sigma Lot # 116H4661) in PBS (pH = 7.4) was used to give a total protein concentration of 65.6 mg / mL, 13.1 mg / mL, 2.6 mg / mL, 0.52
It was adjusted to be mg / mL and 0.1 mg / mL. Thereafter, 150 μL of PBS and 1.0 μL of a DMSO solution containing probe # 5 (Table II) at a concentration of 1.0 × 10 ⁻³ mg / mL were added to wells A1-A6 of a 96-well microtiter plate. Subsequently, a 10 μL sample of human serum solution containing 65.6 mg / mL, 13.1 mg / mL, 2.6 mg / mL, 0.52 mg / mL, and 0.1 mg / mL of total protein was added to wells A1, A2, A3, and Added to A4 and A5 respectively. No human serum solution was added to well A6. Wells B1-B6 were prepared as described above and were duplicates of the samples tested in wells A1-A6.

As before, 150 μL of PBS and 1.0 μL of a DMSO solution containing probe # 18 (Table II) at a concentration of 1.0 × 10 ⁻³ mg / mL were added to wells C1-C6. Then, a 10 μL sample of a human serum solution containing 65.6 mg / mL, 13.1 mg / mL, 2.6 mg / mL, 0.52 mg / mL, and 0.1 mg / mL of the total protein was added to wells C1, C2, C3, and Added to C4 and C5 respectively. No human serum solution was added to well C6. Wells D1-D6 were prepared as described above and were duplicates of the samples tested in wells C1-C6.

Probe # 20-24 (Table II) was treated as described above. The 96-well microtiter plate was then read on a Fluorolite FPM-2 fluorescence polarization microtiter system, and the resulting polarization (mP) data was averaged for duplicate runs and plotted against total protein. The results shown in FIG. 1 show that the binding of the various probes to human serum components varies considerably. In addition, the concentration of components present in human serum affects mP values (a measure of probe binding).

[0135] Example 5: Dose Response This embodiment for the probe # 25 pooled and streptavidin -HRP conjugate to human serum, it is possible to distinguish between biological sample with a probe Prove that. Further, this example demonstrates that the ability of a probe to bind to a component of a biological sample varies with the concentration of the component. To determine whether strong binding interactions with the probe can be observed in the presence of serum proteins, the dose response of probe # 25 (Table II) to pooled human serum and to streptavidin-HRP conjugate was determined. Examined. A series of dilutions of the pooled human serum (Sigma Lot # 116H4661) in PBS (pH = 7.4) was performed at a total protein concentration of 65.6 mg / mL, 13.1 mg / mL, 2.6 mg / mL, 0.52 mg / mL, and 0.1
It was adjusted to be mg / mL. Then, add 150 μL of PBS and probe # 25 to 1.0 X 1
The 1.0μL of DMSO solution containing a concentration of 0 ^-3 mg / mL, were added to the wells A1-A6 in 96-well microtiter tarp rate. Subsequently, 65.6 mg / mL of total protein, 13.1 mg / m
10 μL samples of human serum solution containing L, 2.6 mg / mL, 0.52 mg / mL, and 0.1 mg / mL were added to wells A1, A2, A3, A4 and A5, respectively. No human serum solution was added to well A6. Wells B1-B6 and C1-C6 were prepared as described above and were duplicates of the samples tested in wells A1-A6.

A series of dilutions of streptavidin-HRP conjugate (Sigma 59420) in PBS (pH = 7.4) were prepared at a total protein concentration of 1.0 mg / mL, 0.1 mg / mL, 0.01 mg / mL,
It was adjusted to be mg / mL, 0.001 mg / mL, 0.0001 mg / mL, and 0.00001 mg / mL.
Then, DM containing 150 μL of PBS and probe # 25 at a concentration of 1.0 × 10 ⁻³ mg / mL.
1.0 μL of the SO solution was added to wells A1-A7 of another 96-well microtiter plate. Subsequently, a 10 μL sample of human serum solution containing 1.0 mg / mL, 0.1 mg / mL, 0.01 mg / mL, 0.001 mg / mL, 0.0001 mg / mL, and 0.00001 mg / mL of total protein was added to well A1- Each was added to A6. No human serum solution was added to well A7. Wells B1-B7 were prepared as described above and were duplicates of the samples tested in wells A1-A7. The fluorescence polarization of the samples in both 96-well microtiter plates was determined or measured on a Fluorolite FPM-2 fluorescence polarization microtiter system. The resulting polarization (mP) data was averaged for duplicate runs and plotted against the log of total protein.

The data shown in FIG. 2 shows that probe # 25 binds very weakly to serum proteins, and under experimental conditions, approximately 100 μg of serum protein is required before binding to probe is observed. Explain that there is. In contrast, the binding of probe # 25 to the streptavidin-HRP conjugate was complete in the presence of 1.0 μg of the streptavidin-HRP conjugate. Therefore, probe # 25 has a higher affinity for streptavidin-HRP conjugate than for human serum.

[0138] Example 6: pooled detected following experiment streptavidin A avidin -HRP conjugate using probe # 25 in human and bovine serum, the high affinity for mixed probes in biological samples Demonstrates that low-concentration components can be detected.

The size of the tightly bound protein (streptavidin-HRP conjugate)
The amount should be pooled to determine if it can be detected in the presence of serum proteins.
Streptavidin-HRP conjugate was added to the human serum and the response to probe # 25 (Table II) was examined. First, add 150 μL of PBS and probe # 25 to 1.0 X 10 ^-3 10 μL of a DMSO solution containing a concentration of mg / mL was added to wells A1-A3 of a 96-well microtiter plate. Thereafter, human blood with a total protein concentration of 13.1 mg / mL was
10 μL sample of the clear solution (Sigma pool serum Lot # 116H4661 diluted 1: 5 in PBS
Was added to all three wells. Therefore, the human serum in each well
The total amount was 131 μg. Three wells contain serum protein 1 for probe # 25.
This is a triplicate measurement for 31 μg binding.

Next, DM containing 150 μL of PBS and probe # 25 at a concentration of 1.0 × 10 ⁻³ mg / mL.
1.0 μL of the SO solution was added to wells B1-B3 of a 96-well microtiter plate. Thereafter, a 10 μL sample of human serum solution containing 13.1 mg / mL of total protein (obtained by diluting Sigma pool serum Lot # 116H4661 1: 5 in PBS) and 0.1 mg / mL of total protein were obtained. A 10 μL sample of the containing streptavidin-HRP conjugate solution was added to wells B1-B3. The total amount of human serum protein in each well was 131 μg, and the total amount of streptavidin-HRP conjugate protein was 1 μg.
0.0 μg. Wells B1, B2, and B3 are triplicate measurements of the binding of probe # 25 to 1.0 μg of streptavidin-HRP conjugate protein in the presence of 131 μg of serum protein.

The fluorescence polarization for the sample in the microtiter plate was determined using the Fluorolite FPM
Read with a -2 fluorescence polarization microtiter system. Polarization (mP) data 3
The measurements were averaged and a bar chart was generated to show the polarization (m) that occurred in the presence of the streptavidin-HRP conjugate and serum protein compared to serum protein alone.
P) showed a change. The results shown in FIG. 3 illustrate that the binding of probe # 25 to 1.0 μg of streptavidin-HRP conjugate protein is easily detected in the presence of 131 μg of serum protein. These results indicate that the fluorescence polarization assay
This shows that the method is sensitive enough to detect binding between the probe and sample components present at a concentration of μg / mL.

Example 7: Construction of a serum "fingerprint" with probe # 1-25, both with and without streptavidin-HRP conjugate. It demonstrates that the presence of an amount can alter the fingerprint of a biological sample.

First, the probe concentrations of 150 μL of PBS and probes 1-12 were each 1 × 10 ^−3.
1.0 μL of a mg / mL DMSO solution was added to wells A1-A1 of a 96-well microtiter plate.
Added to 2. This allows probes # 1, # 2, # 3, # 4, # 5, # 6, # 7, #
1.0 μL of a 1 × 10 ⁻³ mg / mL solution of 8, # 9, # 10, # 11, and # 12 (Table II), and
Wells A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A12 containing 150 μL of PBS were obtained. Wells B1-B12 were treated in a similar manner to create an overlap for probes 1-12.

Next, the probe concentrations of 150 μl of PBS and probes 13-24 were each 1 × 10 ⁻³ m
Add 1.0 μL of the g / mL DMSO solution to wells C1-C12 of a 96-well microtiter plate.
Was added. This allows probes # 13, # 14, # 15, # 16, # 17, # 18,
1.0 μL of a 1 × 10 ⁻³ mg / mL solution of # 19, # 20, # 21, # 22, # 23, and # 24 (Table II),
And wells C1, C2, C3, C4, C5, C6, C7, C8, C9, C containing 150 μL of PBS
10, C11 and C12 were obtained. Wells D1-D12 were treated in a similar manner and probe C1
Duplicate for 3-C24. Then, 1 × 10 ⁻³ mg / m
1.0 μL of LDMSO solution was added and added to wells E1 and E2. E1 and E2 were duplicates of probe # 25. Subsequently, pooled human serum (Sigma Lot # 116H4661)
10 μL of the 1:10 dilution was added to wells A1-A12, B1-B12, C1-C12, D1-D12, and E1-E2 in a 96-well plate. Total protein content in a 10 μL serum aliquot was 65.6 μg.

The fluorescence polarization of each well of the 96-well plate was then read on a Fluorolite FPM-2 fluorescence polarization microtiter system and the resulting polarization (mP) values were plotted on a bar graph to provide a “fingerprint” of the serum sample. Indicated. FIG. 4 shows the results.

Next, 10 μL of a streptavidin-HRP conjugate solution containing 0.1 mg / mL of total protein was added to each well. Total streptavidin-HRP conjugate protein added to each well was 1.0 μg. Then, remove the plate from Flu
The resulting polarization (mP) values were read on an orolite FPM-2 fluorescence polarization microtiter system and plotted on the bar graph shown in FIG. This figure represents the "fingerprint" of a serum sample to which streptavidin-HRP conjugate was added. This result allows detection of the presence of small amounts of streptavidin-HRP conjugate protein in serum samples, indicating that it alters the fingerprint observed for serum. These results suggest that the fluorescence polarization assay may be used to screen for probes that can differentiate between normal and abnormal components of a biological sample.

Examples 8-9 demonstrate that a library of probes can be screened using a fluorescence polarization assay for probes that distinguish complex mixtures from each other.

Example 8: Identification of probes that bind differentially to human and bovine serum 4,4-Difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene as described in Table II The interaction between 3-proponic acid (BO) labeled compound 1-19 (Molecular Probes, Eugene, OR) and fetal calf serum or human serum was quantified by fluorescence polarization.
Since the amount of protein in the sample affects the binding to the probe, the total amount of protein in human and fetal calf serum used in this experiment was quantified. The human serum sample (Sigma Lot # 116H4661) contained 65.6 mg / mL total protein and fetal calf serum (Sigma Lot # 96H4615) contained 47.3 mg / mL total protein. To minimize the effect of total protein concentration, experiments were performed with 10 μL of fetal calf serum and 47.3 / 65.6 × 10 μL = 7.2 μL of human serum.

A1-A10, B1-B10, C1-C9, D1-D9, E1-E10 in 96-well microtiter plates
, F1-F10, G1-G9, and H1-H9, 150 μL of PBS was added. A1-A10, B1-B10, C
To 1-C9, D1-D9, E1-E10, and F1-F10, 1.0 μL of a solution of compound 1-10 in dimethylsulfoxide (DMSO) having a compound concentration of 1 × 10 ⁻³ mg / mL was added. In this way, wells A1, B1, E1 and F1 each contained 150 μL of PBS plus 1 × 1 of compound # 1.
Containing 1.0μL of 0 ^-3 mg / mL DMSO solution. Similarly, wells A2, B2, E2 and F2 contain 1.0 μL of a 1 × 10 ⁻³ mg / mL DMSO solution of compound # 2 in addition to 150 μL of PBS,
And well 3-10 was the same. To wells C1-C9, D1-D9, G1-G9 and H1-H9, 1.0 μL of a DMSO solution having a compound concentration of compound # 11-19 of 1 × 10 ⁻³ mg / mL was added. Thus, wells C1, D1, G1, and H1 each contain 150 μL of PBS plus 1.0 μL of a 1 × 10 ⁻³ mg / mL DMSO solution of compound # 12, and wells 13-19 also. It was similar. The plate was then read on a Fluorolite FPM-2 fluorescence polarization microtiter system using a 485 nm excitation filter and a 530 nm emission filter. The polarization (mP) and intensity data obtained represent a blank reading.

Next, wells A1-A10, B1-B10, C1-C9, D1 containing PBS and the above-described probe were used.
To -D9, 7.2 μL of human serum (Sigma Lot # 116H4661) was added. Thus, in human serum, the series A1-A10 and B1-B10 are duplications of compounds 1-10 and the series C1
-C9 and D1-D9 were duplicates of compound 11-19. Similarly, wells E1-E10, F1-F10, G1-G19, and H1-H9 containing PBS and the above probe were added to fetal bovine serum (S
igma Lot # 96H4615) was added. Thus, in fetal calf serum, lines E1-E10 and F1-F10 were duplications of compound 1-10, and lines G1-G9 and H1-H9 were duplications of compound 11-19. The plate was then read on a Fluorolite FPM-2 fluorescence polarization microtiter system as described above. The obtained polarization (mP) and intensity data are shown in FIG. The mP values for each probe were plotted as a bar graph. For each probe, the first two bars represent values for human serum duplicates and the next two bars represent values for fetal calf serum duplicates. This mP data represents the degree of polarization from tag emission. A high reading indicates that the tagged compound bound to the macromolecule, and a low reading indicates that the tagged compound bound to a lesser extent and weakly. As can be seen, compounds # 2 and # 18 bound macromolecules to a greater extent in human serum than in fetal calf serum.

[0151] Example 9: The following examples identify a variety of mammalian species biological sample with differentially bind probe is a biological sample, are fabricated at the time of screening with a probe library Demonstrates that species can be distinguished from each other based on differential binding patterns, such as distinguishing species from species and diseased from unaffected.

1 μL of each of the samples shown in Table 3 was combined with fluorescently tagged molecules in a 96-well microtiter plate and the fluorescence polarization was measured using a Fluorolite FPM-2 fluorescence polarization microtiter system. . The following fluorescently labeled probes were used in the fluorescence polarization assay: final concentration of about 0.18 nM BODIPY-labeled myo-inositol-1-phosphate (probe 18; molecular probe), 10.5 nM final concentration of fluorescein-labeled phenytoin (probe) 70; Sigma), fluorescein-labeled probe 129 (Sepracor) at a final concentration of 0.096 μg / ml, fluorescein-labeled probe 135 (Sepracor) at a final concentration of 0.078 μg / ml, and fluorescein-labeled probe at a final concentration of 0.09 μg / ml. 147 (Sepracor). Fluorescence polarization values for the interaction between each probe and plasma or cell extracts were measured.

[0153]

[Table 2] Based on the fluorescence polarization values, BODIPY-labeled myo-inositol-1-phosphate (Probe 18) showed differential binding to samples from various species (FIG. 7). For example, using probe 18, the fluorescence polarization values for pooled human plasma were almost three times higher than those for pooled calf serum or pooled fetal calf serum. However, little difference was observed between the fluorescence polarization values for samples from the same species. For example, using probe 18, pooled WK
No significant differences in fluorescence polarization values between Y rat plasma and pooled SHR rat plasma were detected. These results indicate that probes such as myo-inositol-1-phosphate can be used to gain information about heterologous samples.

Fluorescence polarization values for fluoresceinated phenytoin (probe 70) indicate that it is also a useful probe to differentiate plasma from various species (FIG. 8). For example, the fluorescence polarization values obtained for human plasma pooled with probe 70 were much higher compared to the other samples. In addition, significant differences in fluorescence polarization values were also detected between pooled calf serum and pooled fetal calf serum. These results suggest that probes such as fluoresceinated phenytoin are useful for distinguishing not only samples from different species but also samples from different growth stages of the same species.

[0155] Probes with a similar structure may also be useful for distinguishing samples from different species. Similar probes 147, 129, and 135 (FIGS. 9A, 10A, and 11A), synthesized by solid phase chemistry and constructed on a fluorescein backbone, were derived from the six primates described in Table 3. Provide sufficient information for the classification of blood samples. Probes 147 and 129 showed higher polarization values after incubation in primate (human and non-human) plasma than in non-primate plasma (FIGS. 9B and 10B). However, probe 129 showed a higher polarization signal when incubated with monkey-derived plasma than with human-derived plasma (FIG. 10B). The fluorescence polarization values observed with probe 135 were higher when incubated with African green monkey plasma samples than with other monkey or human-derived plasma samples (FIG. 11B). Thus, the information can be used to compile the differential binding patterns of probes 129, 135, and 147 to a primate plasma sample and to accurately identify the samples from one another.

[0156] Example 10: Staphylococcus aureus identification following experiments methicillin-resistant Staphylococcus aureus or we identify probes, Staphylococcus aureus (. S aureus) , methicillin resistant Staphylo
Performed to identify probes that can be used to identify coccus aureus (MRSA), and E. coli.

Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA
), And E. coli in brain heart infusion medium (BHI, Difc).
o) Inoculated in 25 ml or 35 ml volumes. The culture is _treated with an OD _{600 nm} value of 0 for E. coli.
Incubation was carried out in a shaker at 37 ° C. until .344, 0.41 for SA and 0.367 for MRSA. The differences between the OD _{600 nm} values of the above cultures are shown in the table. At this point, a 5 μl sample of the culture was diluted in 100 μl of phosphate buffered saline (PBS) + 0.1% bovine gamma globulin BGG in a 96-well black, fluorescence polarized microtiter dish. Subsequently, 5 μl of the diluted fluorescently labeled probe from the library with a final concentration of about 10 nM
Is added to the cell suspension and the plates are incubated at 37 ° C. for 30 minutes or for the indicated culture period,
Incubated. After incubation, the amount of fluorescence polarization was measured for each sample using a Fluorolite FPM-2 fluorescence polarization microtiter system. Duplicate measurements of fluorescence polarization were performed for each probe.

Fluorescently labeled probes used in the fluorescence polarization assay were purchased from external suppliers or synthesized at Sepracor. Table 4 lists the name of the probe, the fluorescent label used, the source of the probe, and the probe number.

[0159]

[Table 3] Based on the fluorescence polarization values, the four probes (11A, 10B, 6D, and 10E)
It showed differential binding to the Staphylococcus strain compared to medium (uninoculated) or E. coli samples (Table 5). The fluorescence polarization assay was repeated using probes 10B, 10E, and 6D to see if it could be used to distinguish SA from MRSA. Probes 10B and 10E were used for methicillin-resistant Staphylococcus aureus (M
(RSA) did not show differential binding to Staphylococcus aureus (SA). Using probes 10B or 10E, no significant differences were found in the fluorescence polarization values for SA, MRSA, E. coli, and uninoculated BHI medium samples (Table 6). Thus, probes 10B and 10E are not useful in differentiating SA from MRSA, E. coli, and inoculated BHI media.

[0160]

[Table 4]

[Table 5] Results from a fluorescence polarization assay using probe 6D show that this probe
Demonstrated to be useful for differentiating from SA, E. coli, and uninoculated BHI media (
Table 7; FIG. 12). Staphylococcus aureus (SA) is a methicillin-resistant Staphylococcu
s aureus (MRSA), uninoculated BHI medium, and higher fluorescence polarization values compared to E. coli. This difference in fluorescence polarization values was only detected when bacterial samples were obtained from cultures incubated for more than 5 hours (Table 7, FIG. 12). SA samples are only available for MRS when the culture incubation time is 7.5 or 24 hours.
As compared to the A sample, it showed a higher fluorescence polarization value. Fluorescence polarization values obtained in uninoculated BHI medium were only available when the culture incubation time was 7.5 or 24 hours.
It was lower than the fluorescence polarization obtained for A and MRSA. In contrast, E. coli samples always showed the lowest fluorescence polarization values. Thus, the ability of probe 6D to bind the components of the bacterial sample changed with increasing incubation time of the bacterial culture.

The level of fluorescence polarization obtained with probe 6D increases over time for SA samples, but decreases for other samples. For example, the fluorescence polarization value for E. coli is 211.8 for samples obtained from cultures incubated for 1 hour.
From +/- 8.8 mP, 1 for samples from cultures incubated for 24 hours
73.4 +/- 3.1 mP (Table 7). In contrast, the fluorescence polarization value of SA increased from 223.3 +/- 5.1 mP for samples from cultures incubated for 1 hour to 279.0 +/- 3.3 mP for samples from cultures incubated for 24 hours. (Table 7). These results suggest that probe 6D preferentially binds to an unknown component of SA and that increasing the culture incubation time increases the expression of this component. Furthermore, this result indicates that probe 6D can be used to distinguish SA from MRSA.

This example demonstrates that this assay system is useful for screening probes that differentiate bacterial strains. In this example, one probe (6D) from a small library of 80 fluorescently labeled probes was found to be useful for identifying the three bacterial strains used in the above experiments.

[0163]

[Table 6] Example 11: Ability of probe 6D to distinguish Staphylococcus aureus from other bacterial strains To determine whether probe 6D can be used to distinguish Staphylococcus aureus (SA) from other bacterial strains The following experiment was performed.

A volume of 35 ml of Brain Heart Infusion medium (BHI, Difco) was inoculated with 12 cultures with 5 different bacterial strains. Although the identity of each culture was unknown to the experimenter, four of the cultures contained Staphylococcus aureus (SA), two contained methicillin-resistant Staphylococcus aureus (MRSA), and two contained quinoline-resistant Staphylococcus aureus (QRSA), and two were found to contain vancomycin resistant Enterococcus faecium (VREF). Cultures were incubated overnight on a 37 ° C shaker. The next day, seven 5 μl samples were taken from each of the twelve cultures and 10 μl in a 96-well black fluorescent polarization microtiter dish.
Diluted in 0 μl phosphate buffered saline (PBS) + 0.1% BGG. Subsequently, 5 μl of about 10 n
M probe 6D fluorescein-tagged Sep0119288 was added to the cell suspension and plates were incubated at 37 ° C for 30 minutes or for the indicated incubation period. After the incubation period, the amount of fluorescence polarization was measured for each sample using the Fluorolite FPM-2 fluorescence polarization microtiter system. The standard deviation was determined by averaging the remaining five values, excluding the high and low polarization readings from seven samples from each of the twelve unidentified cultures.

Table 8 shows the measured fluorescence polarization values for samples of bacterial cultures containing bacterial cells.
It was shown to. The results of the fluorescence polarization assay for the 12 cultures were grouped based on their fluorescence polarization values. FIG. 13 shows their groupings. Each group corresponded to one bacterial strain (Table 9). The SA culture showed the highest fluorescence polarization value and the E. coli culture showed the lowest fluorescence polarization value (FIG. 13). Thus, using this single fluorescent probe, 12 unknown cultures could be accurately grouped into 5 strains, distinguishing SA from other microorganisms used in the study. .

[0166]

[Table 7]

[Table 8] This example demonstrates that the present invention provides a method for distinguishing between bacterial strains. Furthermore, this example shows that the technology of the present invention can be used to detect and diagnose infectious microorganisms.

Example 12: Synthesis of Ligand Library Procedure 1: Liquid Phase Synthesis of Fluorescently Labeled Amines Primary and secondary amines at ambient temperature in the presence of dimethylaminopyridine (DMAP) dissolved in DMF or DMSO. Individual treatment with FITC (1.2 eq) (if amine salt was used, 1 eq of triethylamine was added to liberate the amine). After about 24 hours, 2 equivalents of amine scavenging resin (tris- (2-aminoethyl) -amine polystyrene
HL resin) was added to the reaction mixture. After 24-48 hours, the mixture was filtered to remove the resin and the filtrate containing the purified fluorescently labeled ligand was further diluted with DMSO to the desired screening concentration. Table 10 lists the amines fluorescently labeled by the liquid phase synthesis of Scheme I below.

[0168]

[Table 9] Procedure 2: Synthesis of amine carbamate one (Wang) resin One resin (25 g, 20 mmol, 0.8 mmol / g loading) and carbonyldiimidazole (CDI) (20 g, 120 g) dissolved in anhydrous THF (180 ml). (mmol) at room temperature for 2 days. The mixture is filtered and the resin is washed thoroughly with DMF, THF and CH ₂ Cl ₂ and dried to give imidazole carbonyl one resin (27 g).

Next, imidazole carbonylone resin (5 g, 4 mmol, 0.8 mmol / g loading) was added to piperazine (1.7 g, 20 mmol), homopiperazine (2.0 g, 20 mmol) or 4,4′-trimethylene. Treat with dipiperidine (4.2 g, 20 mmol) in a separate reaction tube in THF / DMF (1: 1, 40 ml) at room temperature for 17 hours. The resin was then added to DMF, THF and CH _Two Cl_TwoWash thoroughly with and dry under reduced pressure to obtain the corresponding amine carbamate one resin
Respectively.

Procedure 3: Synthesis of DTAF on One Resin via Amine Carbamate Linker Amine carbamate one resin (1.0 equivalent) dissolved in DMF and DTF hydrochloride (2.5 equivalents)
Is shaken for 12-17 hours at room temperature in the presence of diisopropylethylamine (DIPEA) (4.0 eq). The resin is filtered and washed thoroughly with DMF, THF and then CH ₂ Cl ₂ and dried under reduced pressure to give monochlorotriazyl fluorescein on one resin.

Procedure 4: Synthesis of DT AF on trityl resin or chlorotrityl resin via amine linker Aminotrityl resin or amino 2-chlorotrityl resin dissolved in DMF (ml)
(Novabiochem) (0.12 mmol, 1.0 eq) and DTAF (0.3 mmol, 2.5 eq) were added to DIPEA (
(0.48 mmol, 4.0 equiv) at room temperature for 24-40 hours. The resin is washed with DMF, THF and then with DCM and dried under reduced pressure to give monochlorotriazylfluorescein on trityl resin.

Procedure 5: Synthesis of DTAF on Rink resin Rink amide resin (1 g, 0.8 mmol, 0.8 mmol / g loading) was treated with a 30% solution of piperidine in DMF (5 ml) with 2%. Time treatment, the resulting link amine resin is DMF, then D
Wash with CM and dry. The rinkamine resin (0.8 mmol) is then shaken for 24 hours with DTAF (2.5 eq, 2 mmol) and DIPEA (4.0 eq, 3.2 mmol) dissolved in 5 ml of DMF. The resin is washed with DMF / THF, then DCM, and dried under reduced pressure to yield monochlorotriazyl fluorescein on Link resin.

Similarly, Fmoc-amino acids on link resin (prepared from link amine resin and Fmoc-amino acids under standard peptide synthesis conditions, eg, DIC / DMAP / DCM), are treated with piperidine / DMF. To remove the Fmoc group. The amino acid link resin is then treated for 24 hours at room temperature with 2.5 equivalents of DTAF and 4.0 equivalents of DIPEA dissolved in DMF. After washing and drying, monochlorotriazyl fluorescein is obtained on the link resin.

Procedure 6: Reaction of Fluorescent Linker with Diamine Monochlorotriazyl fluorescein on a solid support such as one resin or trityl resin is dissolved in 4.0 equivalents of symmetric diamine in DMF at room temperature for 24 to 40 hours. To process. The resin is then washed with DMF / MeOH, then with DCM and dried under reduced pressure to yield aminofluorescein on a solid support.

Procedure 7: Preparation of a Library of Fluorescently Labeled Urea Compounds Three amino acid linked resins (ie, L ₁ in Scheme I is CH ₃ , isobutyl, benzyl) (500 mg, 0.4 mmol, 0.8 respectively) (mmol / g loading) are each treated with DTAF (2.5 eq) in a separation tube as described in Procedure 5 above to give three different monochlorofluorescein derivatives on the resin. Then, these resins (250 m each)
g, 0.2 mmol) were individually treated with piperazine (4.0 eq each) and 4,4′-trimethylene dipiperidine (4.0 eq each) as in Procedure 6 to give the six aminofluorescein-labeled resins. obtain. Then, six aminofluorescein-labeled resins (
Each of 25 mg, 0.02 mmol) is reacted individually with 10 isocyanates (3.0 equivalents each) dissolved in THF in the same manner to obtain 60 fluorescently labeled urea compounds on the resin. The resin is then cleaved with 30% TFA / DCM and dried under reduced pressure to obtain 60 individually labeled fluorescent urea compounds (confirmed by HPLC / MS analysis), Dissolve in 1 ml DMSO for screening.

Procedure 8: Coupling of DCSF to One Resin or Trityl Resin via Amine Linker Amine carbamate one resin dissolved in 50 ml of anhydrous DMF (4 mmol, 0.8 mmol /
g) and DCSF (2.0 eq, 8 mmol) in the presence of DIPEA (2.0 eq, 8 mmol) at room temperature for 2
Shake for ~ 3 days. The resin is then washed with DMF, MeOH, then DCM, and dried to give monochlorosulfofluorescein on a one-piece resin.

In a similar manner, piperazine trityl resin is treated with DCSF in the presence of DIPEA dissolved in DMF to give monochlorosulfofluorescein on trityl resin.

Procedure 9: Reaction of Monochlorosulfofluorescein with Cyclic Diamine Fluorescein (1.33 mmol, 1.0 equiv.) On one resin obtained from procedure 8 was added to 10 ml of DMF with piperazine (4.0 mmol, 3.0 equiv.) Treat with cyclic diamine for 24 hours. The resin is then washed with DMF / MeOH, then DCM, and dried under reduced pressure to give aminosulfofluorescein on One Resin.

Procedure 10: Preparation of a Library Labeled with Sulfofluorescein Aminosulfofluorescein one resin containing different diamine linkers (eg, a combination of piperazine, homopiperazine and 4,4′-trimethylene dipiperidine) was added to DMF. Different organic acids (10 equivalents) in a similar manner for 24-48 hours at room temperature in the presence of coupling reagents such as diisopropylcarbodiimide (DIC) (10 equivalents) and DMAP (5.0 equivalents) dissolved in / DCM To process. After complete acylation of the amine (and then HPLC by cleaving a small amount of the resin), the resin was DMF /
Wash with MeOH then with DCM and dry under reduced pressure. Next, after cleavage from the resin using 30% TFA / DCM, drying is performed under reduced pressure to obtain a desired fluorescently labeled compound.

Procedure 11: Synthesis of Fluorescently Labeled Quinazolinone In Step 1, O-hydroxylamine (see: Floyd, CD et al., Tetrahedro)
n Lett. 1996, vol. 37, 8045) bound resin (2.0 g, 1.6 mmol, 0.8 mm)
ol / g load) in the presence of DMAP (0.8 mmol, 0.5 equiv.), isatoic anhydride (6.4 mmol, 4.0 equiv.) dissolved in 25 ml of DMF with shaking at 65-70 ° C. for 26 h. Process with. The resulting resin mixture is then filtered, washed thoroughly with DMF, MeOH and DCM, and dried to give N-hydroxyamide on the resin.

In Step 2, Fmoc dissolved in dimethylacetamide (DMAC) solvent (0.6 ml) was used.
A commercially available product consisting of a protected α-amino acid (0.2 mmol, 5.0 equiv), coupling reagent PyBrOP (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, 0.2 mmol, 5.0 equiv) and DMAP (0.2 mmol, 5.0 equiv) In the first group, N-hydroxyamide resin (0.04 ml, 50 mg, 0.8 mg) was shaken at 60-65 ° C. for 20-24 hours.
mmol / g load). Filter the resin and wash thoroughly with DMF, MeOH and DCM,
Dry to obtain quinazolinone on resin. The resin is then suspended in 30% piperidine in DMF (0.6 ml) to remove the Fmoc protecting group from the amine groups and to obtain the free aminoquinazolinone resin after filtration and washing for subsequent reaction block attachment.

In step 3, the Fmoc-protected α-amino acid (0.2 mmol, 5.0 equiv.) Dissolved in DMF (0.6 ml), coupling reagent DIC (diisopropylcarbodiimide, 0.2 mmol
, 5.0 eq.), HOBt (N-hydroxybenzotriazole, 0.2 mmol, 5.0 eq.) And DMAP (0.04 mmol, 1.0 eq.) For 20-24 hours at room temperature,
Treat aminoquinazolinone on resin (50 mg, 0.04 mmol). The resin is then filtered, washed thoroughly with DMF, MeOH and DCM and dried. The resin is then suspended in 30% piperidine dissolved in DMF (0.6 ml) to remove the Fmoc protecting group from the amine groups and to obtain a free amino resin after filtration and washing for dye attachment.

In step 4, DTAF.HCl dye (dichlorotriazylaminofluorescein monohydrochloride, 0.08 mmol, 2.0 equiv.) And DIPEA (diisopropylethylamine, 0.16 mmol, 4.0 equiv.) Dissolved in DMF (0.6 ml). Treat the amine resin (0.04 mmol) with shaking at room temperature for 20-24 hours. The resin is then filtered, washed thoroughly with DMF, MeOH and DCM, and dried to give quinazolinone fluorescently labeled on the resin.

In step 5, the fluorescently labeled resin (0.04 mmol) is treated with 30% trifluoroacetic acid (TFA) dissolved in DCM (1.0 ml) while shaking at room temperature for 1-2 hours. The mixture is filtered and the resin is rinsed with 0.5 ml of DCM. The combined filtrates are collected and evaporated to dryness under reduced pressure to give the desired fluorescently labeled quinazolinone ligand, which is dissolved in DMSO for screening.

By performing the above reaction in a similar manner using different isatoic anhydrides and Fmoc-protected amino acids, the above-mentioned library of fluorescently labeled quinazolinone ligands is prepared.

Procedure 12: Synthesis of Ligand Using UGI-Coupling on Solid Phase Linkamine resin (1.0 equivalent) (Commercially available Linkamine resin was dissolved in DMF
Prepared by treatment with 5% piperidine) in MeOH / DCM (1: 2, v / v). Add aldehyde (10 eq) and Fmoc protected amino acid (10 eq). The mixture is shaken at room temperature for 1-2 hours. Thereafter, isocyanide (10 equivalents) is added. The mixture is shaken at room temperature for 20-24 hours. The resin is then filtered and DMF, M
Wash thoroughly with eOH and DCM and dry. The resin is then treated with 25% piperidine dissolved in DMF to yield a resin containing free amine groups. The resin is then treated with 2.0-3.0 equivalents of DTAF and 4-6 equivalents of DIPEA dissolved in DMF, and after filtering and washing as usual, a fluorescently labeled ligand on the resin is obtained. Then TF
After cleavage from the resin using A / DCM and drying, the desired fluorescently labeled ligand is obtained.

Example 13: Use of Fluorescence Polarization to Differentiate and Identify Human Blood Serum Samples The following example illustrates the invention for distinguishing and / or identifying two or more human blood serum samples. This is an example of the use of. For example, normal and abnormal sera can be distinguished from each other based on different binding patterns that occur when screening a sample using a library of probes. Furthermore, the invention can be used to distinguish diabetic sera from normal sera or otherwise non-diabetic sera using a solution of a suitable probe. The following discussion illustrates the general procedure and is used to distinguish between human serum samples.

Fluorescein-labeled probes obtained from plates from three different libraries (XN1192-54, XN1043-58, GY1175-96 and Plate 1) were purchased from Western States P
Bound to serum samples purchased from lasma Company (Fallbrook, CA). The serum samples were obtained from diabetic sera pooled from three diabetic patients (confirmed diabetic by reference experiments) and serum pooled from healthy individuals (lot number HS300; SeraCare, Oceanside, California). It was something.

First, 2.5 μl of each fluorescein-labeled probe (1-10 nM) was bound to 95 μl of buffer (PBS + 0.03% lithium dodecyl sulfate) and 5 μl of serum in a 96-well plate. The mixture was then incubated at 37 ° C. for 30 minutes. Serum samples were centrifuged at 20,000 × g for 3.5 hours as indicated, and 5 μl of the lower layer, the pale yellow serum layer, was assayed for probe binding. Fluorolite F
Fluorescence polarization was measured for each probe of each sample using a PM-2 fluorescence polarization microtiter system. Each probe was tested in triplicate using each sample, and the obtained fluorescence polarization values (MP) were averaged.

In a primary screen using 80 fluorescein-labeled probes, the probes showed significant differences in the fluorescence polarization values obtained for the two serum samples. These probes were screened again in triplicate under the same conditions. The probes listed in Table 11 and shown in Table 12 were used for normal pooled sera (lot number).
HS300) and the diabetic serum showed significant differences in the fluorescence polarization values obtained (FIG. 14). Fluorescence polarization values obtained for 10 of the 12 probes listed in Table 11 were higher for diabetic sera than for normal sera (lot number HS300). This result indicates that probes 58A2, 58A3, 58A6, 58B6, 58B7, 58B11, 58H8, 96G3, 54G3 and P1B4 bind preferentially to any components present in diabetic serum.

[0191]

[Table 10] One possible reason for the difference in fluorescence polarization values obtained for normal (Lot No. HS300) and diabetic serum samples is that the lipid content in diabetic serum is higher than in normal serum. To determine whether the lipid content of diabetic serum affected the resulting fluorescence polarization values, lipids were depleted from serum samples by centrifugation and using the same probes listed in Table 11. The fluorescence polarization was measured. The fluorescence polarization values obtained with the probe are listed in Table 12 and are illustrated in FIG. Probe 58A2, 58A3, 58A6, 58
The magnitude of the difference between the fluorescence polarization values for diabetic and normal sera (lot number HS300) using B6, 58B7, 58B11, 58H8, 96G3, 54G3 and P1B4 is greater when serum is depleted of lipids. Seems to grow. Polarization values are higher for diabetic sera depleted than for diabetic sera without lipid depletion.
These results indicate that the lipid content of diabetic sera was lower than that of normal serum (lot number HS300) when diabetic sera were probed 58A2, 58A3, 58A6, 58B6, 58B7, 58B11, 5B.
It shows that the reason for preferentially binding to 8H8, 96G3, 54G3 and P1B4 is not explained. In addition, these results indicate that the lipid component present in diabetic serum was probed against another component present in diabetic serum, probes 58A2, 58A3, 58A6,
It suggests reducing the binding affinity of 58B6, 58B7, 58B11, 58H8, 96G3, 54G3 and P1B4.

[0192]

[Table 11] For probes 54C6 and 54E8, differences in fluorescence polarization values between lipid-depleted diabetic serum and lipid-depleted normal serum (lot number HS300) are observed between lipid-containing sera Not much different than the difference. However, the effect of removing lipid components from serum dramatically affects the difference in fluorescence polarization values obtained for normal (lot number HS300) and diabetic sera using probes 54G3 and P1B4. In the presence of lipids, the fluorescence polarization value for diabetic serum is normal serum (
Significantly higher for these two probes than for lot number HS300). However, in lipid-depleted serum, the fluorescence polarization values for normal (lot number HS300) and diabetic sera using probes 54G3 and P1B4 are nearly identical. These results suggest that probes 54G3 and P1B4 bind lipid-soluble ligands in diabetic serum. The effect of lipid depletion on probe binding suggests that the probe binds at least three or more different targets.

In summary, the above results demonstrate that human serum samples can be distinguished from each other using the fluorescence polarization-based assay described in the present invention.
In addition, the method used in this example can be used to identify probes that distinguish between normal and diabetic sera.

[0194]

[Table 12]

[Table 13] The invention is not to be limited in scope by the specific embodiments described,
The embodiments are intended only as exemplifications of individual aspects of the invention.
Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. It is intended that the scope of the appended claims include such modifications.

[0195] All references mentioned herein are hereby incorporated by reference in their entirety for all purposes.

[Brief description of the drawings]

FIG. 1 is a graph showing data accumulated in Examples 4 to 7.

FIG. 2 is a graph showing data accumulated in Examples 4 to 7;

FIG. 3 is a graph showing data accumulated in Examples 4 to 7;

FIG. 4 is a graph showing data accumulated in Examples 4 to 7.

FIG. 5 is a graph showing data accumulated in Examples 4 to 7;

FIG. 6. Differential binding of probes to human and bovine serum. The graph shows the fluorescence polarization values obtained for human and bovine serum using various probes.

FIG. 7: Differential binding of probe 18 to the biological samples listed in Table 7. The fluorescence polarization value of each sample using probe 18 is shown in the graph.

FIG. 8: Differential binding of probe 70 to the biological samples listed in Table 7. The fluorescence polarization values for each sample using probe 70 are shown in the graph.

FIG. 9: Structure and discriminative binding pattern of probe 147. (A) shows the structure of the probe 147. (B) Graph shows the fluorescence polarization values obtained for each sample listed in Table 7 using probe 147.

FIG. 10: Structure and differential binding pattern of probe 129. (A) shows the structure of probe 129. (B) Graph shows the fluorescence polarization values obtained for each sample listed in Table 7 using probe 129.

FIG. 11: Structure and discriminatory binding pattern of probe 135. (A) shows the structure of probe 135. (B) Graph shows the fluorescence polarization values obtained for each sample listed in Table 7 using probe 135.

FIG. 12: Differential binding of probe D6. Staphylococcus using fluorescently labeled probe 6D
Fluorescence polarization values of culture samples of auureus (SA), methicillin-resistant Staphylococcus auureus (MRSA), E. coli, and non-inoculated brain heart infusion medium (BHI) were measured. The graph shows the fluorescence polarization values obtained for each bacterial strain and uninoculated BHI medium after various culture incubation periods.

FIG. 13: Classification of bacterial cultures based on fluorescence polarization values obtained using probe 6D. The results of the fluorescence polarization assay for the 12 unknown cultures were grouped based on their fluorescence polarization values.

FIG. 14. Differential binding of the probe to normal (lot number HS300) and diabetic human serum. The graph shows the fluorescence polarization values obtained for normal and diabetic human serum using various probes.

FIG. 15. Differential binding of probes to lipid-depleted diabetic and normal (Lot HS300) human serum. The graph shows the fluorescence polarization values obtained for lipid-depleting normal and diabetic sera using various probes.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 21/76 G01N 21/76 33/53 33/53 M 33/566 33/566 // C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Gao, Yun USA 01772 Massachusetts, Southborough, Davis Road 7 (72) Inventor Jones , Steven, W. United States 01752 Broadmeadow, Marlborough, Mass. 169A-8 F-term (reference) 2G054 AA07 AA08 AA10 AB02 AB05 AB07 CA20 CA21 CA22 CA23 CA25 CE02 EA03 EB05 FA28 GA04 GB02 2G059 AA01 AA05 AA06 BB05 CC16 DD17 FF08 FF13 GG04 JJ02 JJ17 JJ19 KK01 4B024 AA11 CA01 HA14 4B063 QA01 QA07 QA18 QA19 QQ06 QR55 QS39 QX02

Claims (57)

[Claims] 1. A method for characterizing a sample containing a mixture of molecular components, comprising: (a) contacting the sample with a library of molecular probes under conditions that allow the molecular probes to bind to their specific binding partners. (B) detecting the binding of individual members of the library to molecular components in the sample using a homogeneous assay system, comprising: Such a method, wherein the binding pattern provides a molecular fingerprint of the sample. 2. The method of claim 1, wherein the members of the library of molecular probes are labeled. 3. The method of claim 2, wherein said label is a phosphor. 4. The method of claim 3, wherein the binding between the members of the library and the components of the sample is detected by fluorescence polarization. 5. The method of claim 2, wherein said molecular probe is cross-linked to a photoactivatable functional group. 6. The method of claim 5, wherein said functional group is activated to form a chemical bond between a probe and a component of said sample. 7. The method of claim 2, wherein said probe is labeled with a scintillant and components of said sample are labeled with a radioisotope. 8. The method of claim 7, wherein the interaction of the probe with a component of the biological sample results in luminescence. 9. The method of claim 1, wherein the members of the library are biomolecules. 10. The biomolecules in the library are glycoproteins, lipoproteins, proteins, polypeptides, peptides, peptoids, amino acids, polysaccharides, oligosaccharides, saccharides, polynucleotides, oligonucleotides, nucleotides,
10. The molecule according to claim 9, which is a nucleoside, DNA or a derivative thereof, RNA or a derivative thereof, lipid, glycolipid, lipopolysaccharide, organic molecule or inorganic molecule. 11. The molecule according to claim 10, wherein the biomolecule in the library is a receptor, a ligand, an antibody, an antigen, an epitope, a growth factor, a cytokine, or a chemokine. 12. The method according to claim 1, wherein the sample is a patient-derived sample or a clinical sample. 13. The method according to claim 13, wherein the sample is a body fluid, urine, lymph, whole blood, serum, plasma,
The method of claim 1 or 4, wherein the method is an extract of a red blood cell, a white blood cell, a tissue, a cell, a cell extract, a product from in vitro transcription or translation, a virus, a microorganism, or a pathogen. 14. The molecular fingerprint of the sample generated using the library of molecular probes is compared with the molecular fingerprint of a negative control sample generated using the library of molecular probes. 13
The described method. 15. The negative control sample is from a normal subject.
The method according to claim 14. 16. The method according to claim 16, wherein a molecular fingerprint of the sample generated using the library of molecular probes is compared with a molecular fingerprint of a positive control sample generated using the library of molecular probes. 13
The described method. 17. The method of claim 1, wherein the positive control sample is from a patient.
6. The method according to 6. 18. The method according to claim 1, wherein the molecular component in the sample is a glycoprotein, a lipoprotein, a protein, a polypeptide, a peptide, a peptoid, or an amino acid. 19. The method according to claim 1, wherein the molecular components in the sample are polynucleotides, oligonucleotides, nucleotides, nucleosides, DNA or derivatives thereof, RNA or derivatives thereof. 20. The method according to claim 1, wherein the molecular component in the sample is a polysaccharide, an oligosaccharide, or a saccharide. 21. The molecular component in the sample is lipid, glycolipid, lipopolysaccharide,
5. The method according to claim 1, which is a lipoprotein. 22. The method according to claim 1, wherein the molecular component in the sample is an organic molecule or an inorganic molecule. 23. The molecular component in the sample is a receptor, ligand, antibody, antigen, epitope, growth factor, cytokine, or chemokine.
Or the method of 4. 24. An assay for characterizing a biological sample, comprising: (a) contacting a library of fluorescently labeled probes with a biological sample; Detecting the binding interaction with the sample. 25. A method for identifying biological samples, comprising: (a) contacting the same library of fluorescently labeled probes with the biological samples; (b) contacting each probe with each biological sample. Detecting the binding interaction with the sample; (c) identifying a pattern of binding interactions that distinguishes the biological sample from each other. 26. A method for identifying therapeutically and diagnostically important biological targets and drug lead structures, comprising: (a) contacting two biological samples with two identical probe libraries. That the first biological sample is a control sample; (b) detecting the binding interaction between each probe and a component of the biological sample using a homogeneous assay system; Identifying a probe / biological component binding interaction characteristic of a second biological sample, wherein the component so identified becomes a biological target and Wherein the probe having a high affinity has a lead structure for drug discovery. 27. An assay method for characterizing a sample containing a mixture of molecular components, comprising: (a) placing a molecular probe on a scaffold having a site cleavable by a particular catalyst and a marker on each side of the site. Binding the members of the library to create a molecular probe / scaffold construct; (b) combining the molecular probe / scaffold construct with the sample;
At this time, a molecular probe having an affinity for a molecular component present in the sample binds to the sample component to form a complex that blocks a cleavable site of the scaffold, (c) Adding a specific catalyst to the mixture to cleave the scaffold free of bound sample components; (d) releasing unbound molecular probes from the scaffold or releasing the complex on the scaffold. Detecting retention, wherein the binding pattern between the members of the library and the sample provides a molecular fingerprint of the sample. 28. The assay of claim 27, wherein said scaffold consists of DNA, peptide, peptoid, or any polymer. 29. A ligand having an affinity for a receptor present in a biological sample binds to said receptor and blocks access of a scaffold-specific agent to said scaffold. The assay described. 30. The scaffold-specific binding agent according to claim 27, wherein the scaffold-specific binding agent is a drug, peptide, oligopeptide, polypeptide, protein, glycoprotein, lipoprotein, saccharide, oligosaccharide, polysaccharide, organic or inorganic molecule. Assay method. 31. The assay of claim 27, wherein the scaffold-specific binding agent does not bind to regions of the scaffold where interaction of the ligand with the receptor occurs. 32. The scaffold comprises a marker that is blocked when a binding protein binds to a screening sequence, wherein access to said marker is absent of a scaffold-specific binding agent and the presence of a ligand / receptor interaction. 28. The assay of claim 27, wherein 33. The ligand binds to a scaffold at the site on the same side as a first marker, and the assay comprises immobilizing the scaffold with the first marker and washing the mixture after addition of a catalyst. 29. The assay of claim 27, further comprising removing an unimmobilized portion of the cleavage scaffold (such a cleavage portion comprising a second marker), wherein an intact scaffold is identified by the presence of the second marker. Law. 34. The assay of claim 27, wherein said scaffold comprises double-stranded DNA and said marker is a biotinylated nucleotide. 35. An assay for identifying biological samples of the same type, comprising: (a) binding members of the same probe library to a scaffold having a cleavage site and markers on both sides of the site. (B) contacting biological samples with the same probe library and the same specific catalyst; (c) detecting the binding interaction between each probe and each biological sample; d) identifying a pattern of binding interactions that distinguishes the biological samples from each other. 36. The method further comprising using a control biological sample, and identifying a probe / biological component binding interaction characteristic of the non-control biological sample. 31. The component becomes a biological target, and the probe having affinity for the biological component becomes a lead structure for drug discovery.
The assay described. 37. A method for synthesizing a library of labeled ligands, comprising: (a) immobilizing a fluorescent dye on a solid support via a cleavable linkage to form a first product mixture. (B) combining the first product mixture with one or more compounds or mixtures of compounds in a second
Reacting at a temperature and for a sufficient time to form a product mixture of (c) cleaving the product mixture from a solid support to form a library of labeled ligands, provided that the fluorescence The dye has a reactive moiety independently selected from the group consisting of halogen, thiol, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, amide, silane, and their protected and unprotected derivatives. The above method, comprising: two. 38. The fluorescent dye, wherein the rhodamine derivative, cyanine derivative, fluorescein derivative, tryptophan derivative, coumarin derivative, naphthyl derivative,
4. A bipyridine derivative, a tripyridine derivative, or an organometallic complex.
7. The method according to 7. 39. The solid support comprises a linker, wherein the linker is halogen, thiol, alcohol, ether, ester, aldehyde, ketone, carboxylic acid, nitro, amine, amide, silane, or protected or unprotected thereof. 38. The method of claim 37, which is a derivative of 40. A method of synthesizing a library of labeled ligands, comprising: (a) immobilizing a compound on a solid support via a cleavable linkage; (b) fluorescently attaching the immobilized compound to a solid support. Binding the dye, (c) cleaving the molecule-fluorescent dye reaction product from the solid support to obtain a library of fluorescently labeled ligands. 41. A method for characterizing the toxicity of a test agent, comprising: (a) contacting two identical probe libraries with two biological samples, provided that the first biological sample is a control Wherein the second biological sample has been exposed to the test agent; (b) detecting the binding interaction between each probe and the biological sample; (c) the second biological sample Identifying a probe / sample binding interaction characteristic of the biological sample. 42. A method for confirming the presence of a toxic substance in a biological sample, comprising: (a) contacting two identical probe libraries with two biological samples, provided that the first (B) detecting the binding interaction between each probe and each biological sample, (c) characterizing the positive control sample, Identifying a probe / toxicant binding interaction in the second biological sample. 43. The method of claim 41 or claim 42, wherein the binding interaction between the probe and a biological sample is detected using a homogeneous assay system. 44. The method of claim 43, wherein said library of probes is fluorescently or chemiluminescently labeled. 45. A method for characterizing a protein component of a sample, comprising using the method according to claim 1 or 26 to molecular fingers of a glycoprotein, lipoprotein, protein, polypeptide, peptide, peptoid or amino acid in the sample. A method comprising creating a print. 46. A method for diagnosing a disease or condition in a patient, comprising: (a) generating a molecular fingerprint of a biological sample from the patient using the method of claim 45; Comparing the molecular fingerprint of the patient sample to the molecular fingerprint of the positive or negative control sample. 47. A method for predicting the efficacy of a drug or treatment in a patient, comprising: (a) creating a molecular fingerprint of a biological sample from the patient using the method of claim 45; Comparing the molecular fingerprint of the patient sample with the molecular fingerprints of (i) patients responding to and not responding to the drug or treatment, or (ii) patients exhibiting an adverse response to the drug or treatment. The above method, comprising: 48. A method of monitoring the efficacy of a treatment in a patient, the method comprising: (a) using the method of claim 45, wherein the biological sample is obtained from the patient before treatment and thereafter periodically. Creating a molecular fingerprint; (b) comparing the molecular fingerprint of the patient sample to the molecular fingerprint of a positive or negative control sample. 49. A method for characterizing a nucleotide component of a sample, comprising:
27. A method comprising using the method of claim 1 or 26 to create a molecular fingerprint of a polynucleotide, oligonucleotide, nucleotide, DNA or derivative thereof, or RNA or derivative thereof in a sample. 50. A method of diagnosing a disease or condition in a patient, comprising: (a) creating a molecular fingerprint of a biological sample from the patient using the method of claim 49; Comparing the molecular fingerprint of the patient sample to the molecular fingerprint of the positive or negative control sample. 51. A method for predicting the efficacy of a drug or treatment in a patient, comprising: (a) creating a molecular fingerprint of a biological sample from the patient using the method of claim 49; Comparing the molecular fingerprint of the patient sample with the molecular fingerprints of (i) patients responding to and not responding to the drug or treatment, or (ii) patients exhibiting an adverse response to the drug or treatment. The above method, comprising: 52. A method of monitoring the efficacy of a treatment in a patient, the method comprising: (a) using the method of claim 49, wherein the biological sample is obtained from the patient before treatment and thereafter periodically. Creating a molecular fingerprint; (b) comparing the molecular fingerprint of the patient sample to the molecular fingerprint of a positive or negative control sample. 53. A kit for characterizing a molecular component of a sample, comprising: (a) a library of molecular probes; and (b) a member of the library to a molecular component in the sample in a homogeneous or liquid phase. A kit for detecting the binding of the kit. 54. The probe library is labeled with a fluorophore,
54. The kit of claim 53, wherein said detection means is a fluorescence polarizer. 55. The kit of claim 54, further comprising reaction vessel means for each member of said library, suitable for performing a homogeneous assay. 56. The kit of claim 54, further comprising a scaffold having releasably linked members of said library. 57. The library of molecular probes is selected to bind to molecular components associated with diabetes, infectious diseases, inflammatory diseases, heart diseases, neoplastic diseases, autoimmune diseases, and disorders of the central nervous system. 55. The kit of claim 54, wherein