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WO1999031267A1 - Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament - Google Patents

Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament Download PDF

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Publication number
WO1999031267A1
WO1999031267A1 PCT/US1998/026894 US9826894W WO9931267A1 WO 1999031267 A1 WO1999031267 A1 WO 1999031267A1 US 9826894 W US9826894 W US 9826894W WO 9931267 A1 WO9931267 A1 WO 9931267A1
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sample
molecular
probes
library
biological
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PCT/US1998/026894
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English (en)
Inventor
Donald L. Heefner
Charles M. Zepp
Yun Gao
Steven W. Jones
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Sepracor Inc.
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Priority to AU19256/99A priority Critical patent/AU1925699A/en
Priority to CA002314422A priority patent/CA2314422A1/fr
Priority to EP98964053A priority patent/EP1049796A1/fr
Priority to CN98813707A priority patent/CN1285001A/zh
Priority to JP2000539165A priority patent/JP2002508507A/ja
Priority to PCT/US1998/026894 priority patent/WO1999031267A1/fr
Publication of WO1999031267A1 publication Critical patent/WO1999031267A1/fr

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    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00576—Chemical means fluorophore
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
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    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to methods for the discovery and identification of novel biological targets of both therapeutic and diagnostic importance, and more particularly, to methods for the simultaneous discovery and identification of novel biological targets and lead structures for drug development.
  • the technologies used to date may provide valuable information regarding targets for therapy or diagnosis, based on the identification of individual differences between normal and abnormal samples.
  • these technologies are not equipped to effectively provide information identifying patterns of differences observed between similar samples. Patterns of differences seen between similar samples can provide valuable information to more fully define particular disease subtypes, and they may aid not only in classifying the subtypes, but also in determining appropriate therapies for a particular subtype.
  • Affinity screening can be accomplished with such known techniques as for example, fluorescence polarization, scintillation proximity assays, enzyme-linked immunosorbent assays, and the like. Recently, a porous silicon biosensor capable of detecting virtually any molecule that binds with high affinity to another molecule has also been disclosed. When the biological receptor is a target of therapeutic importance, library members that exhibit high affinity and specificity for the target can be of value in diagnosis and/or drug development. The advance of combinatorial chemistry has vastly increased the numbers of ligands available for affinity screening. Scintillation proximity assays (SPA) , for example as described in U.S. Patent 4,568,649, are used to test vast chemical libraries for affinity for a known receptor.
  • SPA Scintillation proximity assays
  • the receptor is tagged with a scintillant-loaded bead and screened against radiolabelled ligands in solution. If the labeled ligand has affinity for the receptor and becomes bound, the resulting proximity of the radiolabelled ligand and the scintillant in the beads leads to activation of the scintillant and the emission of light. If the labeled ligand has little or no affinity for the receptor, the radiolabel will not accumulate sufficiently close to the scintillant to allow for energy transfer from radioactive decay and little light emission will be detected.
  • One significant drawback to SPA is the presence of "noise" or background radioactivity in the system caused by the nonspecific adsorption of labeled ligands.
  • the beads are typically incubated with a blocking agent such as albumen, detergent or powdered milk to block sites responsible for such nonspecific adsorption.
  • a blocking agent such as albumen, detergent or powdered milk to block sites responsible for such nonspecific adsorption.
  • Another significant problem with SPA is that the requirement of using radioactive substances, poses a health hazard, such substances are difficult to dispose of, and are expensive to use.
  • a modification of the SPA assay has been used in competitive-type screening procedures where the receptor is immobilized to a scintillant-loaded bead and then placed in a solution containing a radiolabelled substrate for that receptor. Ligand samples are then added to the mixture and any compound that successfully competes with the substrate for the immobilized receptor will reduce the amount of emitted light.
  • the use of SPA in a high throughput screen is described in Wang, P. , Target Identification, Assay Development and High. Throughput Screening in Drug Discovery, in Sino-American Pharmaceutical Professionals Association (SAPA) , The 5th Regional Symposium on Drug Discovery and Development , 1997, Kenilworth, NJ.
  • Fluorescence polarization is another frequently used assay system for identifying compounds that have affinity for a particular receptor.
  • a fluorescent molecule is attached to one end of an oligomer connected to a ligand, the binding of a receptor to the ligand severely limits the rotation of the fluorescent molecule.
  • polarized is passed through a solution containing the fluorophore-tagged oligomer having a receptor bound or absorbed thereto the light that is emitted is also polarized.
  • the resulting change in refractive index causes a shift in the interference pattern that can be detected by a charge-coupled device detector.
  • the biosensor can detect tiny (e.g., femptomolar) concentrations of DNA sequences as well as recognize small organic molecules.
  • the recognition elements of the sensor may be based upon virtually any supramolecular interaction such as, for example, nucleotide hybridization, enzyme- substrate binding, lectin-carbohydrate interactions, antibody-antigen binding, host-guest complexation, and the like.
  • Combinatorial chemistry is capable of creating libraries containing hundreds of thousands of compounds, many of which may be structurally similar. While high throughput screening programs are capable of screening these vast libraries for affinity for known targets, new approaches have been developed that achieve libraries of smaller dimension but which provide maximum chemical diversity. (See . e . g. , Matter . H. Selecting Optimally Diverse Compounds from Structure Databases : A Validation Study of Two-Dimensional and Three- Dimensional Molecular Descriptors , Journal of Medicinal
  • Affinity fingerprinting has previously been used to test a discrete library of small molecules for binding affinities for a defined panel of proteins.
  • the fingerprints obtained by the screen are used to predict the affinity of the individual library members for other proteins or receptors of interest.
  • the fingerprints are compared with fingerprints obtained from other compounds known to react with the protein of interest to predict whether the library compound might similarly react. For example, rather than testing every ligand in a large library for interaction with a known receptor that is associated with a particular pharmacological activity (for example, antihistaminic or anticholinergic activity) , only those ligands having a fingerprint similar to other compounds known to have that activity would be tested. (See, e . g . , Kauvar, L .M . et al . , Predicting ligand binding to proteins by affinity fingerprinting, Chemistry and Biology,
  • the combinatorial screening assays and detection methods of the present invention utilize highly diversified libraries of compounds to interrogate and characterize complex mixtures in order to identify specific molecular differences existing between biological samples, which may serve as targets for diagnosis or development of therapeutics.
  • the invention is based, in part, on the Applicant's design of sensitive, rapid, homogeneous assay systems that permit the evaluation, interrogation, and characterization of samples using complex, highly diversified libraries of molecular probes.
  • the ability to run the high throughput assays in a homogeneous format increases sensitivity of screening.
  • the homogeneous format allows the molecules which interact to maintain their native or active conformations.
  • the homogeneous assay systems of the invention utilize robust detection systems that do not require separation steps for detection of reaction products.
  • fluorescence polarization is used to detect interactions between the sample and library components.
  • the assay can be run in a heterogeneous format in which one of the reaction components (e.g., either the library or the sample components) is releasably linked to a solid support.
  • the reaction products resulting from contacting the sample with the library are released into the liquid phase, and can advantageously be detected using robust detection systems, such as fluorescence polarization.
  • the assays of the invention can be used for diagnostics, drug screening and discovery, target-driven drug discovery, and in the field of proteomics and genomics for the identification of disease markers and drug targets.
  • the highly sensitive combinatorial screening assays and detection methods of the invention utilize ligand/receptor interactions as a discovery tool to detect receptors that exist in complex biological mixtures but that may have been hitherto undetected.
  • the combinatorial screening assays and detection methods of the present invention evaluate the binding interactions of a diversified universe of ligands with the potentially thousands of receptors in a biological sample and identify binding interactions that are unique to or characteristic of the sample, which may be indicative of a certain pathology (including, but not limited to, disease, disorder and infection) .
  • the present invention identifies not only novel receptors, but also characterizes specific differences occurring between normal and diseased 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 such as genomics or differential expression techniques that focus on detecting genotypic changes and detect only DNA or mRNA changes; moreover, proteomics do not detect non-protein ligands, and therefore only relate to proteins encoded for by the particular sequences.
  • the methods of the present invention detect all types of ligand/receptor interactions, whereby the receptor could be proteins, carbohydrates, nucleic acids, or any molecules with a shape that is capable of binding interactions.
  • the present invention provides methods for "fingerprinting" complex biological mixtures, such as for example, blood serum, using diverse libraries of chemical compounds.
  • the specific binding interactions that result between the members of the library and molecules in the biological sample provide a unique fingerprint for the sample.
  • fingerprinting allows for the identification of novel interactions occurring in a particular sample, and when the sample involves a particular pathology, the fingerprint allows for the identification of specific phenotypical differences existing between normal and abnormal, or diseased and undiseased cells and/or tissues of the same type.
  • the molecules involved may be used as lead structures, receptors or targets for diagnostic and/or pharmaceutical deve1opment.
  • the screening assays of the present invention encompass not only the identification of individual novel receptors, but even more importantly, identify a pattern of binding interactions that is characteristic of a particular pathology or class of pathologies. Such patterns of binding affinities provide valuable information useful in more fully and precisely defining and classifying pathology subtypes. Precise diagnostics are important not only to the detection of disease, but also to the management and therapy of disease, all of which in turn allow for predictable clinical outcomes .
  • samples from many different individuals may be collected and tested for interactions with a defined library, and the resulting data used to create a database incorporating all of the binding interactions identified.
  • Data relating to samples obtained from individuals exhibiting a pathology or disease of interest may then be extracted for analysis and compared with the remaining records in the database to identify interactions or patterns of interactions that would be predictive of the pathology or disease state. Interactions and patterns so identified can be used not only to characterize and classify the particular pathology or disease state, but can also be used to format a diagnostic test or to develop a therapy or pharmaceutical.
  • Figures 1-5 are graphic representations of the data compiled in Examples 4 through 7, respectively.
  • Figure 6. The Differential Binding of Probes to Human and Bovine sera. The graph depicts the fluorescence polarization values obtained for human and bovine sera using various probes.
  • Figure 7 The Differential Binding of Probe 18 to the Biological Samples listed in Table 7. The fluorescence polarization values for each sample using Probe 18 are depicted graphically.
  • Figure 8 The Differential Binding of Probe 70 to the Biological Samples listed in Table 7. The fluorescence polarization values for each sample using Probe 70 are depicted graphically.
  • FIG. 9 The Structure and the Differential Binding Pattern of Probe 147.
  • A The structure of Probe 147 is depicted.
  • B The graph depicts the fluorescence polarization values obtained for each sample listed in Table 7 using Probe 147.
  • FIG. 10 The Structure and the Differential Binding Pattern of Probe 129 .
  • A The structure of Probe 129 is depicted.
  • B The graph depicts the fluorescence polarization values obtained for each sample listed in Table 7 using Probe 129.
  • FIG. 11 The Structure and the Differential Binding Pattern of Probe 135.
  • A The structure of Probe 135 is depicted.
  • B The graph depicts the fluorescence polarization values obtained for each sample listed in Table 7 using Probe 135.
  • Heart Infusion medium (BHI) using fluorescein labeled probe 6D were determined.
  • the graph depicts the fluorescence polarization values obtained for each bacterial strain and uninoculated BHI medium after various culture incubation periods.
  • FIG. 14 The Differential Binding of Probes to Normal (Lot HS300) and Diabetic Human Sera.
  • the graph depicts the fluorescence polarization values obtained for normal and diabetic human sera using a variety of probes.
  • FIG. 15 The Differential Binding of Probes to Lipid Depleted Diabetic and Normal (Lot HS300) Human Sera.
  • the graph depicts the fluorescence polarization values obtained for lipid depleted normal and diabetic sera with various probes. 5.
  • the present invention relates to the use of a compound that binds with high affinity to a target molecule as not only a possible lead compound for pharmaceutical development, but also its use as an informational molecule.
  • the frequency with which a compound in a diverse combinatorial library of chemicals strongly binds to a particular target is rather low (perhaps 0.0001 percent or less) , but if there are a large number of targets this frequency increases dramatically. For example, if instead of a single target molecule there are 1000 possible target molecules the probability of obtaining a "hit" increases to 0.1 percent.
  • Complex biological systems contain thousands of different molecules many of which may be altered in a particular disease state; for example, prostate or breast cancer.
  • the combinatorial screening assays and detection methods of the present invention encompass highly diversified libraries of compounds which act as fingerprints to allow for the identification of specific molecular differences existing between biological samples.
  • the specific molecular differences identified by the combinatorial screening assay and detection methods of the present invention are potential targets for diagnosis and development of therapeutics.
  • the successful application of the combinatorial screening assays and detection methods of the present invention requires at least three components: (1) a diverse molecular library (probe(s)); (2) a source of clinical samples (control and test sample) ; and (3) a sensitive assay for detecting interactions of members of the library with components of the sample.
  • one aspect of the present invention is a method for characterizing a pathology, said method comprising identifying a pattern of binding interactions between (a) receptors or targets present in biological samples associated with the pathology, and (b) a library of ligands or probes, wherein the pattern of binding interactions provides a unique fingerprint for the pathology.
  • a "receptor" or “target” is a biological molecule which demonstrates a binding affinity or interaction with a ligand or probe.
  • receptors or targets include any biologically active molecule that is differentially expressed or modified and is capable of interacting with a probe or ligand, but are not limited to, proteins including enzymes, lipids, nucleic acids, including DNA, RNA, carbohydrates, antigens, antibodies, etc.
  • ligands or probes include any molecule, either natural or synthetic, which may be used to interact or bind to the receptors or targets to measure or indicate the presence of said ligand or probe in a given biological sample.
  • the combinatorial screening assays and detection methods of the present invention may be used for the detection or characterization of a pathology.
  • the pathology to be identified or characterized may include, but is not limited to, a transformed phenotype, genetic defect, malignancy, cancer, tumor, genetic disorder, viral infection, bacterial infection, fungal infection or the presence of a parasite. Vertebrates, particularly human beings, are physically complex. It is this very complexity that has made accurate diagnostics difficult to develop and time consuming to perform. For example, blood plasma contains thousands of proteins, carbohydrates, lipids and nucleic acids.
  • Changes or alterations in any one or many of these components may be highly diagnostic of a particular disease state or of the outcome of a particular therapy and yet the very complexity of the system prevents accurate detection and analysis of these changes.
  • the present invention allows for the rapid analysis of very complex biological systems such as blood plasma or sera, tissue homogenate, cerebrospinal fluid, urine, sputum or any other clinical material, including but not limited to, those materials that can be obtained or prepared in a fluid form.
  • One embodiment of the present invention is medical diagnostics. Areas of diagnostics where the present invention is applicable include, but are not limited to the early detection of various types of cancer; the detection and identification of infections; the detection of toxic side effects of therapeutics; and other disease states and pathologies. With regard to any given pathology, numerous physiological changes occur within an organism's complex biological system. Some changes are a direct result of the pathology while others result from the body's response to the pathology. Any or all of the changes may be diagnostic. Unfortunately, for most diseases the diagnostic changes are unknown and prior to the present invention there was no sensitive, rapid method for detecting these diagnostic changes.
  • antibody based diagnostics are used to detect a novel antigen associated with a particular pathology (for example, the PSA antigen associated with prostate cancer) , but antibody based diagnostics depend on first identifying the antigen. Also, antibody production is both expensive and time consuming.
  • the binding interactions of large, diverse libraries of chemicals to biological samples, blood plasma, for example, from individuals with a particular pathology are compared with samples from a normal population and the differences are analyzed.
  • the differences may be qualitative or quantitative, and may result from binding to any molecule, such as, for example, proteins, lipids, carbohydrates or nucleic acids, enzymes, antigens, or the like.
  • the method is not biased as to the type of molecule it can detect.
  • the binding interactions may result in the discovery of a single or a small number of binding interactions that are diagnostic for the pathology under study, or alternatively, a unique pattern may be recognizable. Either way the identified unique binding interactions can be assembled into an inexpensive, rapid fluorescence polarization based assay that can be done in the physician's office or at bedside.
  • One application for the present invention is the diagnosis and treatment of various types of cancer, such as, for example, prostate, cervical and ovarian cancers.
  • the present invention enables the discovery of new diagnostic markers by probing, for example, blood plasma samples from individuals with prostate cancer as well as control populations with a library of fluorescent probes. The discoveries made using this screening assay may then result in the development of therapeutics.
  • the present invention includes the discovery of probes or markers for use in diagnostic assays or kits, and their subsequent use in drug discovery.
  • the present invention may also be useful for the detection of diagnostic markers in Pap smear samples.
  • normal and abnormal Pap smear samples obtained from individuals can be screened with a library of labeled probes and those binding interactions that are diagnostic of malignancies or infectious disease can be used in fluorescence polarization-based diagnostics.
  • the discovery of biological molecules associated with a malignancy or infectious disease can then be used to develop therapeutics.
  • the present invention can be used to screen for a biomarker(s) for ovarian cancer, which can be used to develop a diagnostic kit for ovarian cancer.
  • ovarian cancer activating factor (OCAF) has been suggested to serve as a biomarker for ovarian cancer.
  • a serum assay for CH125 a biomarker for ovarian cancer
  • the fluorescence polarization assay, the DNA obstruction assay, and the scintillation proximity assay (SPA) described in the present invention can be used in clinical studies to study known biomarkers for ovarian cancer (OCAF and CA125) and to identify new biomarkers for ovarian cancer.
  • OCAF and CA125 known biomarkers for ovarian cancer
  • SPA scintillation proximity assay
  • the fluorescence polarization values for the interaction of fluorescent compounds with specimens (blood, serum, plasma or other bodily fluids) obtained from patients with ovarian cancer can be compared with the fluorescence polarization values obtained for clinical samples from patients with other types of gynecologic cancer, non-gynecologic cancers, and no cancer.
  • An altered polarization value for specimens from patients with ovarian cancer as compared to the other samples will indicate that an agent binds differentially to a component (s) present in the specimen from ovarian cancer patients.
  • this agent may be a useful biomarker to distinguish ovarian cancer specimens from non-ovarian cancer specimens.
  • a fluorescence polarization value for specimens from patients with ovarian cancer that does not differ significantly from other samples will suggest that the agent is not useful as a biomarker to distinguish ovarian cancer specimens from non-ovarian specimens.
  • Those agents identified that differentially bind to different types of cancers can be used alone or in combination with other agents to detect and diagnose different types of cancers.
  • the identified agents can be used to develop therapeutics for the treatment of cancer.
  • the present invention also provides methods for identifying an early indicator of the onset of diabetes. Current methods for the detection of diabetes are useful only after the disease is fully developed and much damages has already been done. Indeed, diagnosis is done only after gross symptoms are present. In other words, current diagnostic procedures merely confirm that the disease is present.
  • the methods described in the present invention provide methods for identifying agents that are early indicators of diabetes. For example, the glycosylation of blood proteins, a characteristic of. diabetes, creates unique binding sites that can be detected with probes from a tagged library of diverse molecules using the methods described in the present invention. Those probes that are identified to be early indicators of the onset of diabetes can be used in accordance with the invention in a fluorescence polarization- based assay. This assay can be used as a diagnostic in the physician's office or at a diagnostic laboratory.
  • the present invention further provides methods for identifying and distinguishing infectious microorganisms, as well as the diseases that they cause.
  • Infectious microorganisms produce a wide range of mostly uncharacterized proteins, carbohydrates, and other molecules when present in their host.
  • the host organism in response to infection the host organism produces unique proteins, such as, for example, specific antibodies, specific receptors, chemokines, and cytokines. All of these unique molecules are candidates for binding to probes and therefore, represent new diagnostic targets for infectious diseases.
  • the present invention provides methods for identifying binding candidates that differentiate between infectious microorganisms and/or the diseases that they cause. The binding candidiates identified can then be used for diagnosing infectious diseases and may lead to the development of a pharmaceutical therapy.
  • Example 10 describes the discovery of a unique probe from a small library of probes that allows Staphylococcus aureus to be distinguished from methicillin resistant Staphylococcus aureus .
  • the present invention is not only applicable to the detection of infectious bacteria, but also other infectious agents, such as, for example, fungi, parasites, viruses and perhaps, even prions.
  • infectious agents such as, for example, fungi, parasites, viruses and perhaps, even prions.
  • neuraminidase inhibitors have recently been developed that inhibit the Influenza A and B viruses. The effective utilization of these drugs requires that a rapid diagnostic kit be available to identify the presence of the influenza virus.
  • the present invention provides methods for discovering tagged molecules with high avidity for viral neuraminidase or some other viral component. These probes can then be incorporated into a rapid diagnostic test for the presence of the influenza virus.
  • Another aspect of the present invention is a method for identifying biological targets of therapeutic and diagnostic importance and lead structures for drug development, comprising contacting two biological samples with two identical libraries of probes, wherein one biological sample is a control; detecting the binding interaction between each probe and a component of the biological samples; and identifying binding interactions that are characteristic of the second (non-control) biological sample.
  • the ligand or target so identified is a biological target and the probe having affinity for the biological component is a lead structure for drug development.
  • the second biological sample comprises serum from a diseased individual or diseased or otherwise abnormal cells or tissues of the same type as the control.
  • the method is therefore of value in the development of diagnostic tools related to the pathology associated with the cells or tissues being screened.
  • the method is a source of lead structures for the development of a pharmaceutical for use in the treatment of the pathology.
  • the principles of the present invention may be used for the toxicological screening of potential pharmaceuticals.
  • the invention includes methods for determining the toxic properties of a compound as well as methods for determining the presence of toxic compounds in an individual by measuring the biological response to toxic compounds.
  • Many drugs as well as their metabolites have adverse side effects or are toxic.
  • the prediction of toxic properties, or side effects is difficult during the early stages of drug development that occur prior to clinical testing.
  • the presence or absence of toxic properties may be predicted at a much earlier stage in development than is currently possible.
  • liver cell cultures can be treated with known toxins and, over time, samples taken and probed with a diverse library of tagged compounds. Binding interactions that occur in the treated cultures, but not in control may be early markers for cell or tissue damage. These probes can then be tested in animal model systems.
  • the present invention offers methods of detecting early markers that are predictive of impending damages due to drugs of other chemicals.
  • cultured cells or tissues are treated with known toxic or non-toxic compounds, and then extracts are prepared from the cultures and probed with a library of labeled compounds.
  • This probing with the library of labeled compounds provides a "fingerprint" which reflects the responses of the cells or tissues to the toxic or non-toxic compounds. Any changes in the fingerprint patters correlated with toxic properties are analyzed, and optionally further classified. Subsequently, cultured cells or tissues for which a fingerprint pattern has been established as set forth above are treated with a test compound having unknown toxic properties, and extracts are prepared from the treated cultures and probed with the library of labeled compounds. The resulting fingerprint or profile of the cell's or tissue's responses is then compared with the reference profiles generated for that cell or tissue ing the known toxic/non-toxic compounds to predict the nature of the test compound.
  • the principles of the present invention may be used to characterize the function of an expressed protein.
  • Genome sequencing projects including The Human Genome Project, create large databases of gene sequences, which can be cloned and the proteins encoded by these genes can be expressed.
  • knowing the sequence of a gene or expressing a protein from that sequence often does not reveal the function of that protein, its role in disease, or other molecules the expressed protein interacts with in the living cell.
  • the present invention is three-dimensional (3-D) and except where the target molecule is very small (less than a few thousand daltons) a binding event gives a clearly discernable signal.
  • PAGE is designed to detect proteins whereas the present invention is designed to detect a binding interaction between a labeled probe and any biomolecule larger than a few thousand daltons.
  • running and analyzing 2-D gels is time consuming, labor intensive and very expensive.
  • the methods of the present invention are on the other hand, rapid and very inexpensive. In any direct comparisons, the methods of the present invention to outcomes predictions is preferable to either Proteomics or Genomics.
  • the present invention provides a means to clarify the biological function of an expressed protein.
  • expressed proteins are probed with tagged libraries of small organic molecules some number of probes can be found that bind to the binding sites of these proteins. These probes themselves are potential inhibitors of these expressed prot ⁇ ins. These probes can be added to cell culture or other systems to determine their biological effects and thus be used to define the activity of this protein.
  • the tagged probes can serve as an artificial substrate for competitive assays with unlabeled molecules in order to discover inhibitors of even stronger avidity. These unlabeled molecules can then be tested in model systems to determine the effect of these agonists or antagonists on cellular function. In this manner, the function of a previously unknown or uncharacterized expressed protein can be determined using the technology described by the present invention.
  • the present invention provide methods for rapidly unraveling the function of these proteins and at the same time discovering leads for developing new pharmaceuticals.
  • the invention includes methods for discovering potential inhibitors of, or affinity ligands useful for the purification or analysis of proteins for which no enzymatic or structural function has yet been ascribed.
  • protein targets include those identified by genomic approaches.
  • the present invention also provides methods, which can be utilized to stratify populations for any purpose. For example, this technology may be used to stratify groups for clinical trials. Moreover, the present invention allows mass screening for the early detection of pathologies or for analysis of the distribution patterns of various disease inexpensively. Once informative probes have been developed, the cost of screening is very low. Screening requires only an inexpensive fluorescence polarization instrument and the probes. The probes are not only inexpensive to manufacture, but only nanomoles are required for an assay.
  • “fingerprints” are established when biological samples, such as blood, sera, body fluids, such as urine, cerebrospinal fluid, amniotic fluid, saliva, mucous, tissue samples, cells, viruses, microorganisms, or organic molecules including RNA, DNA, peptide and proteins, and small organic molecules are exposed to a battery of known reagents to generate a panel of values which reflect a pattern of binding interactions.
  • the diverse ligand library comprises the battery of known reagents or probes, which interact or bind to receptors or targets, to measure or indicate the presence of the receptors or targets in a given biological sample.
  • the ligands or probes of the present invention include, but are limited to, any biological molecule, either natural or synthetic, and may include, but are not limited to, nucleic acids, including DNA or RNA, small organic molecules, peptides, glycoproteins, proteins, polysaccharides, saccharides or inorganic molecules.
  • ligand or probe libraries may be used to generate differential binding patterns to accurately distinguish between biological samples.
  • diverse ligand libraries encompass known molecules which when exposed to a biological sample generate a panel of values which reflect a pattern of binding interactions.
  • the fingerprints of the present invention are used to distinguish between samples, for example to identify or distinguish a particular microorganism, such as a particular strain of virus, bacteria, parasite or fungus
  • the diverse library of ligands should include ligands or probes known to specifically or non-specifically interact with a component of the microorganism.
  • the biological sample to be tested may be exposed to a library of ligands or probes composed of molecules which are in part, randomly selected, thus allowing for the identification of unique binding interactions which may then be identified by known methods.
  • a receptor that is found to exist in only the test sample, or in a different concentration in the test sample may serve as a potential target for diagnosis or treatment of the particular pathology associated with the test sample.
  • the ligand found to have high affinity and specificity for the receptor provides a lead structure for drug development.
  • a ligand having affinity for a receptor existing within the sample will bind to the molecule, forming a ligand/receptor conjugate that can be identified using various assay techniques.
  • Ligand/receptor interactions occurring in a normal or control sample can be compared with those occurring in a second sample that is comprised of abnormal cells or tissues of the same type as the control to identify specific differences between the two.
  • the probes/ligands that are used in the assay systems described can be labeled, tagged, or conjugated such that a detectable signal is generated when a component of a biological sample binds to a probe.
  • the probes/ligands may be labeled with labels known in the art, including but not limited to radioisotopes, fluorescent molecules, chemiluminescent compounds, and bioluminescent compounds.
  • the molecules are preferably labeled with a radioisotope, including but not limited to 32 P, 35 S, 125 I, or 131 l.
  • the radioactive isotope can be detected by a gamma counter or a scintillation counter.
  • Probes/ligands may also be labeled with a fluorescent molecule such as fluorescein (FL) , rhodamines, 4-4-difluoro- 5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid
  • the binding interaction between a probe and a component of a biological sample may also be detected by ELISA (enzyme linked immunosorbent assay) .
  • the probe can be labeled or conjugated to such molecules as biotin, streptavidin or digoxigenin. Probes labeled with these molecules can be detected using enzyme conjugated antibodies specific for the label. Alternatively, the probe can be labeled with an antibody, which may or may not be conjugated to an enzyme. An antibody not conjugated to an enzyme can be detected by a secondary antibody that is conjugated to an enzyme.
  • the enzyme conjugated antibody will react with an appropriate substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label an antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.
  • the methods of the present invention may use ligand libraries synthesized according to any techniques known to those skilled in the art. Preferably, they are made using conventional solution phase reactions or solid phase synthetic techniques. Organic molecules of interests, such as biologically active compounds containing primary or secondary amine group, or hydroxyl groups, or thiol groups, or aldehydes or ketones, or carboxylic acids, can be labeled directly with suitable fluorescent molecules (dyes) in solution to give the corresponding fluorescent-labeled ligands. These methods and dyes are described in Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals, 6 th Ed., 1996.
  • the solution phase syntheses are carried out in a suitable polar organic solvent or solvent mixture such as DMF, DMSO, THF using a slightly excess of dyes to ensure complete labeling.
  • a suitable polar organic solvent or solvent mixture such as DMF, DMSO, THF
  • the resulting fluorescent-labeled ligands are purified by standard techniques in organic synthesis such as liquid-liquid extractions using acid or base, crystallizations and chromatography (thin-layer or column) .
  • Alternative purification methods such as liquid-solid phase extractions using polymer-bound scavengers to removal the unreacted dyes followed by simple filtrations can also be used as described in the following examples (See, Obrecht,
  • the libraries of this invention are made using conventional solid phase techniques. See, e .g. , Bodanszky, Principles of
  • a fluorescent dye is covalently attached to a solid support.
  • the immobilized dye is reacted with a compound or mixture of compounds to form the desired mixture of ligands.
  • the present invention encompasses assays using libraries adhered to the solid supports upon which they were made, or adhered to different solid supports. It is preferred, however, that the mixture of ligands be cleaved from the support in a third step. This optional third step is included in the preferred embodiment of the synthetic method of this invention shown in Scheme II:
  • ⁇ A>, ⁇ B>, ⁇ C>, ⁇ D> and ⁇ E> represent reaction conditions suitable for the formation of the desired products or intermediates represented by Formulas (b) - (g) , and brackets (i.e., [ ]) represent optional parallel or sequential reactions, reactants, and/or products.
  • brackets i.e., [ ]
  • D is a fluorescent moiety
  • X and Y are functional groups independently selected from the group consisting of halogens, alcohols, nitros, thiols, ethers, esters, carboxylic acids, -halo carboxylic acid derivatives, amines, amides, and protected and unprotected derivatives thereof.
  • dye molecules of Formula (a) include, but are not limited to: fluorescein derivatives such as dichlorotriazylaminofluorescein (DTAF) , dichlorosulfofluorescein (DCSF) , and nitrofluorescein; tryptophan derivatives; coumarin derivatives; napthyl derivatives; bipyridine (bpy) derivatives; tripyridine derivatives; cyanines; rhodamines and organometallic complexes such as Ru(bpy) 3 and derivatives thereof.
  • fluorescein derivatives such as dichlorotriazylaminofluorescein (DTAF) , dichlorosulfofluorescein (DCSF) , and nitrofluorescein
  • tryptophan derivatives such as dichlorotriazylaminofluorescein (DTAF) , dichlorosulfofluorescein (DCSF) , and nitrofluorescein
  • tryptophan derivatives such as
  • dye molecules depends on a number of factors including, for example, size, solubility, immunity to degradation under solid phase reaction conditions, absorption and emission wavelengths, quantum yield, and quantum yield and emission wavelength sensitivity to the surrounding chemical environment. Many of these factors, and the synthesis of these and other suitable compounds, are readily determined from the literature. See, e.g., Haugland, R.P.,
  • a reactive substrate of Formula (b) is selected:
  • Resin represents any solid support suitable for solid phase synthesis; L is a linker attached to the solid support; and E is a leaving group bound to L.
  • Suitable solid supports include, for example, polystyrene-divinylbenzene (PS-DVB) copolymer and polyethylene glycol-PEG-PS-DVB copolymer. Wang (polymer-bound 4-benzyloxybenzyl alcohol) and Rink resins, with and without suitable linkers attached, are available from Aldrich Chemical Co., Milwaukee, WI ; Novabiochem, San Diego, CA; and Advanced Chemtech, Louisville, KY.
  • a linker L-E is selected so that its bond 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, esters, aldehydes, ketones, carboxylic acids, nitros, amines, amides, silanes, and protected and unprotected derivatives thereof.
  • the attachment of such linkers to solid supports may be accomplished by methods well known to those skilled in the art. See, e.g., Bunin, B.A. , The Combinatorial Index,
  • L-E is selected so that it will form a covalent bond with the fluorescent moiety D of the dye molecule of Formula (a) under reaction conditions ⁇ A> to yield an immobilized dye of Formula (c) :
  • Suitable reaction conditions ⁇ A> which depend upon Resin, L, E and X, are well known to, or easily determined by, those skilled in the art. Generally they include the use of a solvent that causes the resin to swell and react with X. Suitable solvents include, for example, dimethylformamide (DMF) , l-methyl-2-pyrrolidinone (NMP) , tetrahydrofuran (THF) , CH 2 C1 2 , and mixtures thereof.
  • DMF dimethylformamide
  • NMP l-methyl-2-pyrrolidinone
  • THF tetrahydrofuran
  • the reaction conditions ⁇ A> also may include a base such as diisopropylethlamine (DIPEA) , triethylamine, dimethylaminopyridine (DMAP) , or N- methylmorphaline (NMM) , to neutralize the acid generated during the reaction.
  • DIPEA diisopropylethlamine
  • DMAP dimethylaminopyridine
  • NMM N- methylmorphaline
  • the immobilized dye of Formula (c) serves as a foundation upon which the ligands of the library (represented by Formula (g) in Scheme II) are formed. If the reactive moiety Y is protected, however, it must be deprotected prior to additional reactions. This optional deprotection to form the deprotected moiety Y' is performed under reaction conditions represented by ⁇ B> in Scheme II. These conditions, which vary depending upon the protecting group, are well known to those skilled in the art. See, Greene, T.W. and Wuts, P.G.M. , Protective Groups in Organic Chemistry
  • the immobilized dye is then reacted under reaction conditions ⁇ C> with a compound of formula E-iR-iG-- . to yield a compound of Formula (d) :
  • E-, and G x may be the same or different, E x is a leaving group or protecting group, G : is either the terminal end of Ri or a leaving group or protecting group, and R ⁇ represents any chemical fragment which comprises at least one protected or unprotected reactive moiety that enables the addition of R ⁇ to the fluorescent moiety D under suitable catalytic and/or deprotecting conditions.
  • Suitable reactive moieties include, but are not limited to, halogens, thiols, alcohols, ethers, esters, aldehydes, ketones, carboxylic acids, nitros, amines, amides, silanes, and protected and unprotected derivatives thereof.
  • Suitable reaction conditions ⁇ C> include those which have been developed for solid phase combinatorial chemistry. See, e.g., Brown, R. , Contemporary Organic Synthesis , 216 (1997); Felder, E.R., and Poppinger, D., Adv . Drug Res . , 30:111 (1997); Balkenhohl, F. , et al . , Angew . Chem . Int . Ed . Engl . 35:2288 (1996); Hermkens, P.H.H., et al . , Tetrahedron 52:4527 (1996); Hermkens, P.H.H. , et al .
  • Exemplary addition reactions include that of a primary amine with an aldehyde to form an imine, which in turn can react with a variety of different moieties including, for example, ⁇ -lactams, pyrrolidines, thiozolidinones, and amides. Acid groups are equally flexible, and be used, for example, with aldehydes, amines and isonitriles under Ugi multicomponent condensation conditions to form either small amides or heterocyclic compounds.
  • the immobilized dye molecules of Formula (c) may also be reacted with a mixture of compounds, each of which is different but is of the general formula " + - +
  • n-1 subsequent addition reactions may be performed under reaction conditions that are collectively referred to in Scheme II by ⁇ D>, wherein n represents the number of moieties bound to the fluorescent moiety D, and is an integer of preferably less than about 100.
  • each of these subsequent addition reactions may employ both single compounds or mixtures of compounds of formulas E k R k G k , wherein k is an integer between 2 and n-1, R k is the k th moeity bound to the immobilized fluorescent moiety D (via the k-1 moieties already bound to D) , E k and G k are the same or different, E k is a leaving group or protecting group, G k is the terminal end of R k or a leaving group or protecting group, and R k represents any chemical fragment which comprises at least one reactive moiety that enables the addition of R k to the immobilized compound (s) .
  • Suitable reaction conditions ⁇ C> include the use of catalysts, deprotectants, and the like which facilitate the addition of R k to the immobilized fluorescent compounds.
  • Resin-L-D- (R 1 R 2 R 3 -R n ) + Resin-L-D- (R 1 R 2 R 3 —R n ) / + Resin-L-D- + - + Resin-L-D- (R 1 R 2 R 3 -R n ) m , wherein m has a maximum value 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.
  • G n is omitted from Formula (f) because the terminal end of the ligand (e.g., R n ) undergoes no further addition reactions.
  • Formula (g) encompasses all possible compounds and mixtures of compounds produced by the reactions indicated in Scheme II.
  • Suitable cleavage conditions ⁇ E> are known to those skilled in the art, and depend upon the bond between Resin and L. Cleavage may be accomplished under acidic or basic conditions, or may be photoinduced. Many suitable cleavage methods have been reported in the literature. For example, some cleavage reactions accomplished by treating the modified resin of Formula (f) with trifluoroacetic acid (TFA) in ethylene chloride are shown in Scheme III:
  • (j) is a Wang resin derivative;
  • (k) is a Wang carbamate resin derivative; (1) is a Wang amino acid resin derivative;
  • (m) is a Rink resin derivative;
  • (n) is a Rink amino acid resin derivative; and
  • L x represents any side chain or spacer stable under solid phase reaction conditions. Examples of suitable side chains include, but are not limited to, substituted and unsubstituted alkyl, aryl and aralkyl groups.
  • the solvent is preferably removed to isolate the fluorescent library.
  • the library may then be dissolved in a solvent such as dimethylsulfoxide (DMSO) suitable for use in the assays of this invention.
  • DMSO dimethylsulfoxide
  • Schemes IV - VIII Specific 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 mixtures. It is to be understood, however, that each of the individual reactions shown represents the possibility of numerous parallel reactions.
  • a particular simplified embodiment of the general approach of Scheme II is shown in Scheme IV:
  • L- L is any moiety that does not sterically hinder or otherwise inhibit the coupling reaction under the reactions conditions shown; P represents the end of ⁇ after it has been cleaved from the solid support; and R ⁇ and R 2 are the same or different and may be any moieties desired to provide a library with preferred structural and reactive characteristics.
  • suitable moieties include, but are not limited to, substituted and unsubstituted alkyl, aryl and aralkyl.
  • DTAF is immobilized upon a solid support using diamino carbamate Wang resin or amino acid Rink resin or diamino/amino alcohol trityl/chlorotrityl resin to give monochlorotriazylaminofluorescein resin.
  • This reaction is preferably conducted at ambient temperature.
  • DTAF is dissolved in a suitable solvent such as DMF, NMP, THF, methylene chloride, or mixtures thereof with between about 0.5 to about 3 equivalents of a base such as DIPEA, triethylamine, DMAP, or NMM.
  • DCSF is attached to a solid support via substitution of a chlorine atom by a secondary alkyl amine, and preferably a cyclic secondary amine, bound to a resin.
  • L thus forms part of a cyclic dia ine.
  • Suitable cyclic diamines include, for example, piperazine, homopiperazine, 4,4'- trimethylenedipiperidine, and derivatives and isomers thereof.
  • R ⁇ also forms part of a cyclic diamine, although HNRiNH may be replaced by any compound having suitable reactive groups.
  • R 2 and R 3 represent any moieties suitable for incorporation within the fluorescent ligands of the libraries of this invention and include, for example, the side chains of natural amino acids; substituted and unsubstituted alkyl, aryl and aralkyl; and the like.
  • an N-Fmoc protected amino acid is attached to the fluorescent resin by the reaction of the free amino group of the fluorescent compounds with the Fmoc amino acid under standard amide formation conditions (i.e.,
  • fluorescent-labeled ligand libraries are also made by general solid-phase synthesis techniques (Obrecht, D. and Villalgordo, J.M. , Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon, 1998; and Bunin, B.A., The Combinatorial Index, Academic Press, 1998) .
  • the desired compounds are synthesized on the solid supports according the methods described in the literature. Before cleavage from the solid supports, the compounds on the solid supports are treated with suitable dyes to give the fluorescent-labeled ligands on resins. The ligands are then cleaved from the resins to give the fluorescent-labeled ligands.
  • the ligands are synthesized in a linear fashion by reaction of a solid supported reactive block with different reactive blocks step by step.
  • the dye is then added in the last step before cleavage to give the labeled ligands as shown in Scheme VIII.
  • fluorescent-labeled N-hydroxyquinzolinones are prepared.
  • Quinzalinones are one of the most common bioactive nitrogen containing heterocycles (See, Sinha, S. and Srivastava, M. in Progress in Drug Research, 1994, vol. 43, 143-238). They display a broad spectrum of biological and pharmacological activities in human and animals. They have been used as anticonvulsant, antibacterial and antidiabetic agents. Therefore, fluorescent-labeled quinzolines would be useful for diagnostic applications and for drug discovery.
  • the ligands are prepared in a convergent manner using multicomponent condensation reactions such as the Ugi condensation (Tempest, P.A. , et al. Angew .
  • the methods of the present invention may be utilized to identify specific phenotypical differences which exist between normal or abnormal, healthy or diseased, nontransformed or transformed, noninfected or infected cells and/or tissues.
  • the methods of the present invention may 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, whereby the receptor could be proteins, carbohydrates, nucleic acids, or any molecules with a shape that is capable of interacting with a probe or ligand.
  • biological receptor As used herein, “biological receptor”, “receptor”, “biological target”, “target” and “component of a biological sample” are intended to include any biological molecule, binding surface, binding site or the like, that is for example, differentially expressed, or is differentially modified in the test sample, e.g., the diseased or otherwise abnormal cells and/or tissues. Any one of these receptors may serve as a target for diagnosis and/or development of potential therapeutics.
  • one aspect of the present invention is a method for characterizing a pathology, said method comprising identifying a pattern of binding interactions between receptors or targets present in biological samples associated with the pathology, and a library of ligands or probes, wherein the pattern of binding interactions provides a unique fingerprint for the pathology.
  • a "receptor” or “target” is a biological molecule which demonstrates a binding affinity or interaction with a library of ligands or probes in the test sample which may or may not differ between the test sample and the control sample.
  • 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, etc.
  • the biological samples utilized in the method can be any sample that is a source of biological molecules, including but not limited to, biological materials such as body fluids, such as blood plasma, blood sera, urine, cerebrospinal fluid, amniotic fluid, saliva, mucous, tissue samples, cell extracts, products from in vitro transcription and translation systems (obtained, for example by the method of U.S. Patent 5,654,150 issued to King et ai . ) , and the like.
  • extracts derived from or fluids containing pathogenic organisms such as bacteria, yeast, fungi, viruses, protozoa, and the like may also be used.
  • ligands exhibiting high affinity and specificity for a protein or other receptor in the pathogen may reveal new targets and can be tested for inhibitory effect against the pathogen.
  • biological samples which may be screened in accordance with the methods of the present invention may be obtained from a wide variety of sources.
  • biological samples or mixtures may be obtained from patients and include bodily fluids, blood, serum, mucous, including oral, rectal or intestinal mucosa, urine, feces, etc.
  • biological samples may include tissue samples, biopsy tissue, cell samples, including bone marrow cells, lymphocytes, immune cells, mucousal cells obtained from oral, rectal or intestinal mucousal linings, etc.
  • the biological samples or mixtures may encompass cell lysates or portions thereof, carbohydrates including lectins; proteins including glycoproteins, cell surface receptors, peptides; nucleic acids including DNA or RNA etc.
  • the biological sample may be or may be derived from a virus, bacteria, microorganism or parasite or fluids containing such biological samples, e.g. , testing water supplies for microorganism content.
  • the biological samples of the present invention may be obtained from individuals inflicted with a disease, disorder, or pathology infected with a virus, bacteria or other microorganism.
  • the biological samples may be generated by exposing a tissue, cells in culture, cell extracts etc. to a toxin or pathogenic agent, or by genetically engineering the genome of a cell in culture to encode a mutation or protein or peptide known to be associated with any given pathology or disorder.
  • Collections of biological materials as sources for clinical samples may be obtained from hospitals or national research facilities.
  • the third element of the invention the sensitive assay systems, must be capable of detecting binding interactions occurring in only microliters of sample and in addition, must be capable of detecting binding interactions of less than optimal affinity.
  • the assay systems of the present invention eliminate or greatly reduce nonspecific binding of ligands to receptors present in the sample.
  • an aspect of the assays of the present invention is to provide a means for detecting the interaction between the probe or ligand and the receptor, target or active site.
  • homogeneous assays are utilized for the detection of receptors or targets present in a biological sample.
  • the assays comprise contacting a biological sample with a library of diverse probes in a microtiter dish or other well-type device and detecting any binding of individual members of the library of probes to components in the system.
  • the binding pattern of members of the library of probes with the sample provides a molecular fingerprint of the sample.
  • the binding of a member of the library to a component of the sample is detected by fluorescence polarization.
  • the assay for detecting receptors or targets present in a biological sample encompasses attaching the probes and ligands of the library to a structure which contains a site cleavable by a specific catalyst, the cleavage of the structure or scaffold provides the signal or the means for detecting the interaction between the probe and the receptor.
  • the members of the library of ligands or probes are attached to scaffolds having a site cleavable by a specific catalyst and a marker on each side of the site, to provide ligand/scaffold constructs; the ligand/scaffold constructs are contacted with a biological sample, wherein a ligand having affinity for a receptor present in the sample binds with the receptor and blocks the cleavable site of the scaffold.
  • the scaffolds that do not include a bound receptor are cleaved and the intact scaffolds are identified.
  • a preferred embodiment of the assay 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 opposite side of the scaffold.
  • a marker such as biotin
  • a second marker such as digoxigenin
  • the scaffolds used in the assay may be comprised of DNA, RNA, peptides, peptoids, oligosaccharides or any hydrophobic or hydrophilic, synthetic or natural polymer including block copolymers.
  • the scaffolds are double-stranded DNA, constructs comprised of a first oligonucleotide, a second complementary oligonucleotide having a ligand attached thereto; and a single restriction site.
  • the specific catalyst is the restriction enzyme specific for the restriction site.
  • the first marker can be, for example, biotin, whereby the ligand structures can be immobilized on a streptavidin or avidin coated surface.
  • Digoxigenin is a preferred second marker, whereby anti-digoxigenin-peroxidase is used to indicate the presence of intact scaffolds.
  • Another assay for detecting the presence of receptors in a biological sample consists of attaching members of a library of ligands to a scaffold, contacting the scaffold with a scaffold-specific binding agent and a biological sample, and detecting the interaction between a ligand and a receptor present in a biological sample.
  • the scaffold used in this assay may be comprised of DNA, RNA, peptides, peptoids, oligosaccharides or any hydrophobic or hydrophilic, synthetic or natural polymer including block copolymers.
  • the scaffold-specific binding agent does not bind to the region of the scaffold where the interaction between a ligand and a receptor occurs.
  • the scaffold-specific binding agent may be a drug, peptide, glycoprotein, protein, polysaccharide, saccharide, or inorganic molecule.
  • the scaffold includes a marker that is blocked when the scaffold-specific agent binds to the scaffold such that access to the marker indicates the absence of the scaffold-specific binding agent and the presence of the ligand/receptor interaction.
  • the scaffold is comprised of double- stranded DNA and the marker is a biotinylated nucleotide, which is detected with streptavidin or avidin when a receptor/ligand interaction occurs.
  • the principles of the present invention may be used for the toxicological screening of potential pharmaceuticals.
  • the prediction of toxic properties, or side effects is difficult during the early stages of drug development that occur prior to clinical testing.
  • the presence or absence of toxic properties may be predicted at a much earlier stage in development than is currently possible.
  • cultured cells or tissues are treated with known toxic or non-toxic compounds, and then extracts are prepared from the cultures and probed with a library of labeled compounds.
  • This probing with the library of labeled compounds provides a "fingerprint" patterns which reflects the responses of the cells or tissues to the toxic or non-toxic compounds. Any changes in the fingerprint patterns correlated with toxic properties are noted, and perhaps further classified using statistical analysis or analysis by artificial intelligence routines (neural networks) .
  • cultured cells or tissues for which a fingerprint pattern has been established as set forth above are treated with a test compound having unknown toxic properties, and extracts are prepared from the treated cultures and probed with the library of labeled compounds. The resulting fingerprint or profile of the cell's or tissue's responses is then compared with the reference profiles generated for that cell or tissue using the known toxic/non-toxic compounds to predict the nature of the test compound.
  • sections 5.3.1 through 5.3.3 describe the assays that may be utilized to detect the differences in binding affinities of probes to biological samples such that a fingerprint or pattern of binding affinities is generated.
  • the results obtained with the present invention serve to differentiate two sample populations (e.g., normal and diseased) and can be expected to fit into two categories.
  • one or two probes can serve to differentiate the populations and the probe response will be easily distinguishable between the two groups ("shining pebbles") .
  • the second category subtle difference in binding to two sample populations can be differentiated using a greater number of probes, which have a small difference in binding to the sample populations.
  • the combination of these probes (a diagnostic cluster) will be necessary to provide the statistical power for a reliable differentiation in unknown samples.
  • a larger number of probes is expected to result in a more useful analytical system since the overall binding pattern will not be severely affected by fluctuations due to individual variation. In a system where only one or two probes are used, individual variation in the probes might make diagnosis more difficult.
  • the first type of analysis focuses on determining which probes are useful for distinguishing members of two sample populations. Algorithms for this type of analysis are available from either the statistical analysis field (discriminate function analysis) or from the field of artificial intelligence (supervised, back- propagation neural networks) .
  • the second type of analysis that can be performed involves categorizing the probe binding data based upon the probe results and then asking if there are any similarities in the histories (e.g., medical) in the respective probe-based categories.
  • Algorithms exist in statistical analysis (principal component analysis) and in artificial intelligence (unsupervised, Kohonen neural networks) for these types of analysis.
  • Data analysis programs or packages are available commercially which include the above functions.
  • SPSS SPSS, Inc., Chicago, Illinois
  • SAS SAS Institute, Inc., Cary, NC
  • Neuroshell 2 Word Systems Group, Inc. , Frederick, MD
  • Stuttgart Neural Network Simulation The University of Stuttgart, Stuttgart, Germany
  • a fluorescence polarization assay can be utilized to identify binding interactions between a ligand and a receptor present in a biological sample.
  • a fluorescence polarization assay is designed to measure the binding of a fluorescent-labeled compound to an unlabeled biomolecule.
  • a fluorescence polarization-based assay can utilize fluorescence labeled compounds up to a molecular weight around 10,000 to detect interactions with biomolecules.
  • the type of fluorescent labeled compounds that can be used include, but are not limited to, small organic molecules, peptides, small proteins, nucleic acids, lipids, and polysaccharides. Fluorescent molecules when excited with plan polarized light, will emit light in a fixed plane only if they do not rotate during the period between excitation and emission. The extent of depolarization of the emitted light will depend upon the amount of rotation of the molecules, which is dependent upon the size of the molecule.
  • the intensity of emitted, polarized light can be measured by inserting a moveable polarizing filter in front of the detector. The intensities are measured in planes 90° apart and are many times designated the horizontal and vertical intensities. In some instruments the excitation filter is moveable while the emission filter is fixed. In certain other machines the horizontal and vertical intensities are measured simultaneously via fiber optics.
  • Fluorescence polarization values are most often divided by 1000 and expressed as millipolarization units (mP) .
  • a fluorescent molecule such as fluorescein
  • an oligomer of sufficient length that the "propeller effect" comes into play.
  • a short linker is then attached to the fluorescent molecule and the end of the oligomer opposite the fluorescent molecule is attached to the walls of a microtiter dish or other well-type device suitable for use in fluorescence polarization.
  • the free end of the short linker is then derivatized with ligands to be screened.
  • the identity of the attached ligands in each well is known.
  • a source of receptors is then added to each well and incubated. Polarized light is then used to excite the fluorescent molecule, and the polarity of emitted polarized light is determined.
  • Receptors that bind to the ligand will reduce the free rotation of the fluorescent molecule and increases in polarization will be detected. On the other hand if no receptor binds to the ligand, the fluorescent molecule remains freely rotating and the emitted light will be less is not polarized. To obtain an approximation of the affinity of any bound receptor for the ligands, the extracts are removed from the wells, fresh buffer added and the polarization of light emitted from each well determined. Those receptors with low affinity will dissociate from the receptor and the polarization of emitted light will decrease. Repeating this process will identify ligand/receptor binding interactions of the highest affinity.
  • SPA SCINTILLATION PROXIMITY ASSAY
  • the receptor is bound to a scintillant loaded bead and screening is done directly or competitively against ligands in solution. It is possible, however, to reverse this arrangement and have the ligand attached to the bead and have the receptor in solution. This variation is especially compatible with combinatorial chemistry because in many synthetic schemes, the library synthesis is done on beads, which may subsequently be impregnated with scintillant as needed by an SPA assay system. In addition, more than one ligand can be synthesized on a bead.
  • beads loaded with scintillant and coated with a ligand are immersed in a fluid phase containing radiolabelled reactant. If the labeled reactant has affinity for a tagged ligand and the two become bound, the resulting proximity of the radiolabelled reactant and the scintillant in the beads leads to activation of the scintillant and the emission of light. If the labeled reactant has little or no affinity for a ligand, the radiolabel will not accumulate sufficiently close to the scintillant to allow for energy transfer following radioactive decay. Because SPA does not require a washing step, it allows for the detection of relatively low affinity ligand/receptor binding interactions.
  • DNA RESTRICTION SITE OBSTRUCTION ASSAY The principles of this system are based upon the presence or absence of restriction enzyme activity upon a DNA construct that has been synthesized to include a single restriction site and a ligand-reporter system. When the construct is contacted with a biological mixture, ligand/receptor interactions will block access of the restriction site by the restriction enzyme and prevent hydrolysis of the construct at the site. Constructs that remain intact can be isolated and the ligand/receptor interaction identified.
  • a description of a DNA restriction site assay system is provided herein.
  • oligonucleotide 5 is annealed to a complementary oligonucleotide such that the annealed, double stranded oligonucleotide contains a single centrally located restriction endonuclease site.
  • the complementary oligonucleotide is modified to contain a linker having a terminal amino group. The location of the amino acids
  • amino group is derivatized with ligands of a diverse chemical library to form the construct shown below:
  • the derivatization of the amino group can be accomplished during synthesis of the single-stranded oligomer while it is still attached to the CPG bead, and alkaline cleavage then used to release the oligomer from the bead where it can then be annealed to the complementary biotin- digoxigenin oligomer.
  • derivatized bases may be incorporated during the synthesis of the complementary oligonucleotide.
  • Reaction of the hydrolyzed mixture with a streptavidin or avidin coated surface will result in the immobilization of the biotin labeled section and the digoxigenin labeled section will be eliminated by washing.
  • Reaction of the immobilized mixture with anti-digoxigenin-peroxidase antibody will provide a negative result because the digoxigenin labeled section will have been eliminated from the mixture by hydrolysis by the restriction enzyme and subsequent washing.
  • the ligand When the intact ligand-derivatized construct functioning as a probe for detecting potential receptors within the sample, is mixed with a clinical sample, the ligand will capture any receptor for which is has high affinity for as shown below:
  • the reaction mixture is diluted with an appropriate buffer and treated with restriction enzyme and then incubated with a streptavidin coated surface to immobilize the biotin molecules.
  • a biological molecule with high affinity to the ligand on a construct is present, the molecule will block access of the enzyme to the restriction site and prevent hydrolysis of the DNA scaffold, thereby resulting in the immobilization of the intact double-stranded oligonucleotide.
  • the restriction enzyme will not be blocked, allowing hydrolysis of the DNA scaffold as seen above, and resulting in the immobilization of a truncated construct, free of the digoxigenin-labeled portion.
  • a standard enzyme- linked immunosorbent assay (ELISA) with digoxigenin- peroxidase antibody can be used to detect the presence of digoxigenin on the streptavidin surface.
  • a positive assay indicates that the restriction enzyme was blocked and this is an indication that a molecule was bound to the linker.
  • this approach can be used to detect the interaction between any ligand and any receptor having sufficient affinity and size to block access of a restriction enzyme to its restriction site.
  • a "gap" deletion of a base
  • a ligand is attached next to the gap as shown below:
  • this construct Treatment of this construct with an endonuclease such as mung bean nuclease or nuclease SI will result in hydrolysis of the construct at the gap region. Reaction of the hydrolyzed mixture with immobilized avidin or streptavidin and subsequent washing will eliminate the digoxigenin containing sections of the construct. As described above, incubation of this construct with receptors having high affinity for the ligand prior to addition of a nuclease will prevent hydrolysis of the construct at the gap region and a strong positive response will be obtained during an assay for the presence of digoxigenin.
  • an endonuclease such as mung bean nuclease or nuclease SI
  • the DNA scaffold can be replaced by a backbone comprised of peptides or peptoids or any polymer with a centrally located bond that can be cleaved by a particular enzyme or other mechanism wherein cleaving can be blocked by occurrence of a ligand/receptor interaction near the site of the bond.
  • a backbone comprised of peptides or peptoids or any polymer with a centrally located bond that can be cleaved by a particular enzyme or other mechanism wherein cleaving can be blocked by occurrence of a ligand/receptor interaction near the site of the bond.
  • a commercially available poly (glycine) or poly (alanine) backbone into which has been inserted by standard techniques of peptide synthesis, a string containing a phenylalanine residue and a nearby ligand.
  • An enzyme such as chymotrypsin A 4 (EC 3.4.21.1) can then be used in analogous fashion to the nuclease described above.
  • the assay could be modified to an SPA assay by using radioactive bases or ligands in the synthesis of the section of the construct opposite the biotin and attaching the streptavidin to scintillant loaded beads. While any of the assays described herein are suitable for use in the present invention, this system provides several advantages such as, it does not require radioactivity, although radiolabelled nucleotides may be placed downstream of the restriction site to adapt the assay to an SPA format; the system may be made ultra sensitive (detecting the presence of a single intact DNA backbone) by using PCR methodology for the detection system; the entire assay can be carried out in streptavidin coated microtiter dishes; and nucleotide analogs may be used to protect the DNA backbone against nuclease activity in the sample, as long as the restriction site is not altered.
  • a membrane (available e.g., from Pall Corporation, East Hills, NY) containing particular reactive groups, may be placed into a well device, such as a
  • a library of compounds can then synthesize in the device, wherein a particular functional group added in the library synthesis couples with the particular reactive group on the membrane.
  • a particular functional group added in the library synthesis couples with the particular reactive group on the membrane.
  • the membrane will contain activate carboxylic acid groups and the groups will couple by amide bonding. Any excess groups on the membrane will be blocked with an appropriate blocking reagent. Then the ligand-bound membrane can be reacted with a biological sample that has previously been derivatized, for example with biotin or radioactivity.
  • An appropriate assay can then be used to detect ligand/receptor interactions.
  • the viral DNA binding protein UL9 may be used in a screen for compounds that bind to selected DNA test sequences that are placed downstream of the cognate UL9 sequence.
  • the assay is based upon the test sequences' ability to disturb the equilibrium of the binding protein that the binding protein does not bind to its cognate sequence.
  • the assay as described in U.S. Patent No. 5,306,619, which is incorporated herein by reference.
  • test sequence located downstream of the UL9 recognition site may serve as the backbone for the library of ligands, rather than as a target for DNA binding proteins as in U.S. Patent No. 5,306,619.
  • a scaffold construct can be formed that is comprised of the UL9 cognate sequence wherein a specific nucleotide base has been tagged with, for example, a marker such as biotin; and a ligand-derivatized oligonucleotide terminating in digoxigenin.
  • a biological sample can then be introduced into the reaction mixture whereby any receptors present in the sample that have affinity for the ligand on the scaffold test sequence will bind to the ligand, disturbing the equilibrium between the binding protein and the biotinylated sequence, resulting in a change in the amount of free biotin available for binding with avidin or streptavidin.
  • a test sequence other than a second DNA sequence may be synthesized with the screening sequence prior to attachment of the compounds. Any scaffold that allows for the attachment of the ligands may be used. Alternatively, the ligands may be covalently attached directly to the screening sequence without the need of an additional linker or scaffold.
  • SPA scintillation proximity assay
  • E. coli transformed with a ⁇ -galactosidase expression vector were grown at 37°C in M9CA medium supplemented with or without H 2 35 S0 4 (0.1 mCi/mL of culture media).
  • ⁇ -galactosidase expression was induced by treating the E. coli cultures with
  • the PPO-impregnated p-aminophenyl ⁇ -D- thiogalactoside agarose beads were incubated overnight in a solution of 10% powdered milk in PBS (8.1 mmNa 2 HP0 4 , 1.5mM KH 2 P0 4 , 137mM NaCl, 2.7mM KC1, 0.5 mM MgCl 2 , 0.9 mM CaCl 2 ) to block any sites responsible for non-specific binding.
  • the beads may be blocked with agents such as albumin, detergents, or even an extract from the control solution.
  • E. coli extracts or H 2 35 S0 4 were mixed with 20 ⁇ L of PPO- impregnated p-aminophenyl ⁇ -D-thiogalactoside agarose beads.
  • D-thiogalactoside agarose beads approximately 3000 cpm were detected. In contrast, only about 670 cpm were detected in samples containing 7.7 X 10 5 cpm of H 2 35 S0 4 and PPO-impregnated p-aminophenyl ⁇ -D-thiogalactoside agarose beads. These results indicate that the background due to the interaction between the beads and H 2 35 S0 4 is very low. Therefore, the signal detected in the sample containing 35 S-labeled E. coli extracts is primarily due to the interaction between the beads and labeled E. coli protein. The high cpm detected for the interaction between the beads and the 35 S-labeled E .
  • the following example demonstrates the sensitivity of the DNA obstruction assay for assessing the interaction between receptors and ligands.
  • the oligomers were designed such that a centrally located restriction site was formed upon annealing.
  • the oligomers were annealed using a 10:1 ratio of II: I by incubating the mixture in annealing buffer (10 mM Tris «Cl [pH 7.8] 0.1 M NaCl and 1.0 mM EDTA) at 65°C for 1 hour, then 57°C for 3 hours followed by storage at -20°C.
  • Duplicate samples of 32.8 pmol of annealed oligomer were serially diluted in annealing buffer and one of each duplicate dilution was hydrolyzed overnight with 40 units of restriction enzyme at 37°C as per instructions (New England BioLabs, Beverly, MA) .
  • This example illustrates that a molecular interaction (ligand/receptor interaction) at a distance from a restriction site influences restriction enzyme activity.
  • Examples 4-8 demonstrate the sensitivity of the fluorescence polarization assay and the differential binding pattern of biological samples to various probes.
  • the molecular probes used in these examples are listed in Table II.
  • ASSAY IS A SENSITIVE METHOD FOR DETECTING AN INTERACTION BETWEEN A PROBE AND A BIOLOGICAL SAMPLE
  • the following example demonstrates that the ability of various probes to bind to a component (s) of a biological sample varies with the concentration of the component (s) in the sample. Furthermore, this example demonstrates the differential binding of probes to a component (s) of a biological sample.
  • Probes # 20-24 were similarly treated as described above.
  • the 96-well microtiter plates were then read in 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 as depicted in Figure 1 demonstrate that the binding of the various probes to a component (s) of human serum varies considerably. Furthermore, the concentration of the component (s) present in the human serum affects mP values (a measure of probe binding) .
  • probes may be used to differentiate between biological samples. Furthermore, this example demonstrates that the ability of a probe to bind to a component (s) of a biological samples varies as the concentration of the component varies.
  • the dose response of probe #25 (Table II) to pooled human serum and to streptavidin-HRP conjugate was determined.
  • Wells B1-B7 were prepared as above to serve as a duplicate for the samples tested in wells A1-A7.
  • the fluorescence polarization of the samples in both 96-wcll microtiter plates were then determined or measured with a Fluorolite FPM-2 fluorescence polarization microtiter system.
  • probe #25 has a higher affinity for the streptavidin-HRP conjugate than for human serum.
  • the fluorescence polarization for samples in the microtiter plate was read in a Fluorolite FPM-2 fluorescence polarization microtiter system.
  • the resulting polarization (mP) data were averaged for the three determinations and a bar graph was constructed to show the change in polarization (mP) that occurred in the presence of the streptavidin-HRP conjugate and serum proteins as compared to the serum proteins alone.
  • the results as shown in Figure 3 illustrate that the binding of the probe #25 to 1.0 ⁇ g of streptavidin- HRP conjugate protein is easily detected in the presence of 131 ⁇ g of serum protein.
  • Wells BI-B12 were treated in the same manner to serve as a duplicate for probes 1-12.
  • El and E2 served as duplicates for probe #25. Subsequently, 10 ⁇ L of a 1:10 dilution of pooled human serum (Sigma Lot #116H4661) was added to wells A1-A12, B1-B12 , C1-C12 , D1-D12, and E1-E2 in the 96-well plate. The total protein content in the 10 ⁇ L serum aliquot was 65.6 ⁇ g.
  • each well of the 96- well plate was then read in a Fluorolite FPM-2 fluorescence polarization microtiter system and the resulting polarization (mP) values were plotted in a bar graph to show a "fingerprint" of the serum sample.
  • the results are shown in Figure 4.
  • each well was spiked with 10 ⁇ L of a streptavidin- HRP conjugate solution containing 0.1 mg/mL of total protein.
  • the total streptavidin-HRP conjugate protein added to each well was 1.0 ⁇ g.
  • Examples 8-9 demonstrate that the fluorescence polarization assay can be utilized to screen libraries of probes for those that distinguish complex mixtures from each other.
  • series A1-A10 and B1-B10 were duplicates for compounds 1-10 and series Cl- C9 and D1-D9 were duplicates for compounds 11-19 in human serum.
  • E1-E10, F1-F10, Gl -G19, and Hl- H9 which contained PBS and a probe as described above, lO ⁇ L of fetal bovine serum (Sigma Lot #96H4615) was added.
  • series E1-E10 and F1-F10 are duplicates for compounds 1-10 and series G1-G9 and H1-H9 were duplicates for compounds 11- 19 in fetal bovine serum.
  • the plate was then read in a
  • Fluorolite FPM-2 fluorescence polarization microtiter system as described above.
  • the resulting polarization (mP) and intensity data is depicted in Figure 6.
  • the mP value for each probe is plotted as a bar graph.
  • the first two bars represent the values for the human serum duplicates and the last two bars represent the values for the bovine serum duplicates for each probe.
  • This mP data represents the extent of polarization from the tag emission. A high reading indicates that the tagged compound was bound to a macrom-olecule and a low reading indicates that the tagged compound was less bound to a lesser extent.
  • compounds #2 and #18 are bound to a macromolecule to a greater extent in human serum than in fetal bovine serum.
  • EXAMPLE 9 THE IDENTIFICATION OF PROBES WHICH DIFFERENTIALLY BIND TO BIOLOGICAL SAMPLES FROM DIFFERENT MAMMALIAN SPECIES
  • 5 samples can be distinguished from one another, including distinguishing species from species and diseased from undiseased, based on the differential binding pattern generated when they are screened with a library of probes. l ⁇ L of each of the samples listed in Table 3 were
  • BODIPY labeled myo-inositol-1-phosphate demonstrated differential binding for samples from different species ( Figure 7) .
  • the fluorescence polarization value for pooled human plasma was almost 3 -fold higher than the
  • Probes with similar structure may also be useful in distinguishing samples from different species. Probes 147, 129, and 135, which are similar in structure ( Figures 9A,
  • probe 129 exhibited a higher polarization signal when incubated with blood plasma from monkeys than with blood plasma from humans ( Figure 10B) .
  • the fluorescence polarization value observed for probe 135 was higher when it was incubated with a sample of African green monkey plasma than with the plasma samples from other monkeys or humans ( Figure 11B) . Therefore, the differential binding pattern of probes 129, 135, and 147 to primate blood plasma samples can be complied so that the information may be used to accurately distinguish the samples from one another.
  • Staphylococcus aureus (SA) , methicillin resistant Staphylococcus aureus (MRSA) , and Escherichia coli (E. coli ) were inoculated into 25 ml or 35ml volumes of Brain Heart Infusion medium (BHI, Difco) .
  • BHI, Difco Brain Heart Infusion medium
  • the cultures were incubated in a 37°C shaker until the O.D. 600nm values were 0.344 for E. coli , 0.411 for SA and 0.367 for MRSA. Any differences in the O.D. 500nm values of the cultures from those above are indicated in the tables.
  • the fluorescently labeled probes used in the fluorescence polarization assays were purchased from outside sources or synthesized at Sepracor. Table 4 lists the probe name, the fluorescent label utilized, the source of the probes, and the probe number. Table 4. Library of Fluorescently Tagged Probes.
  • SA methicillin resistant Staphylococcus aureus
  • probes 10B and 10E are not useful for differentiating SA from MRSA, E . coli , and uninoculated BHI medium.
  • the level of fluorescence polarization obtained with probe 6D increased over time for SA samples, but decreased with the other samples.
  • the fluorescence polarization value for E. coli decreased from 211.8 +/- 8.8 mP for samples obtained from cultures incubated, for 1 hour to 173.4 +/- 3.1 P for samples obtained from cultures incubated for 24 hours (Table 7) .
  • the fluorescence polarization value of SA increased from 223.3 +/- 5.1 mP for samples obtained from cultures incubated 1 hour to 279.0 -»/- 3.3 mP for samples obtained from cultures incubated 24 hours (Table 7) .
  • This example demonstrates that this assay system is useful for screening for probes which differentiate bacterial strains.
  • one probe (6D) from a small library of 80 fluorescently labeled probes was found useful for distinguishing the three strains of bacteria used in the experiments described above.
  • EXAMPLE 11 THE ABILITY OF PROBE 6D TO DISTINGUISH STAPHYLOCOCCUS AUREUS FROM OTHER BACTERIAL STRAINS
  • Sep0119288 was added to the cell suspensions and the plates were incubated for 30 minutes at 37°C or the indicated incubation period. Following the incubation period, the amount of fluorescence polarization was determined for each sample using the Fluorolite FPM-2 fluorescence polarization microtiter system. The high and low values of the polarization readings for the seven samples from each of the twelve unidentified cultures were discarded, and the remaining five values were averaged and the standard deviations determined.
  • the fluorescence polarization values determined for bacterial culture samples containing bacterial cells are shown in Table 8.
  • the results from the fluorescence polarization assays for the twelve cultures were grouped based upon their fluorescence polarization values. These groupings are shown in Figure 13. Each grouping corresponded to a bacterial strain (Table 9) .
  • SA cultures had the highest fluorescence polarization values and the E . coli cultures had the lowest fluorescence polarization values ( Figure 13) . Therefore, with this single fluorescent probe twelve unknown cultures could be accurately grouped into five strains and SA could be distinguished from the other organisms used in the stud .
  • This example demonstrates that the present invention provides a method of distinguishing between bacterial strains. Furthermore, that this example indicates that the technology of the present invention can be used to detect and diagnose infectious microorganisms.
  • PROCEDURE 1 SOLUTION PHASE SYNTHESIS OF FLUORESCENTLY LABELED AMINES
  • PROCEDURE 2 SYNTHESIS OF AMINE CARBAMATE WANG RESIN
  • the imidazole carbonyl Wang resin (5 g, 4 mmol, 0.8 mmol/g loading) is then treated with piperazine (1.7 g, 20 mmol), homopiperazine (2.0 g, 20 mmol) or 4,4'- trimethylenedipiperidine (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 is then thoroughly washed with DMF, THF, and CH 2 C1 2 and dried under vacuum to give the corresponding amine carbamate Wang resin, respectively.
  • PROCEDURE 3 SYNTHESIS OF DTAF ON WANG RESIN
  • PROCEDURE 4 SYNTHESIS OF DTAF ON TRITYL OR
  • PROCEDURE 5 SYNTHESIS OF DTAF ON RINK RESIN
  • Rink amide resin (1 g, 0.8 mmol, 0.8 mmol/g loading) is treated with a 30% solution of piperidine in DMF (5 ml) for 2 hours and the resulting Rink amine resin is washed with DMF following DCM and dried.
  • the Rink amine resin (0.8 mmol) is then shaken with DTAF (2.5 equivalents, 2 mmol) and DIPEA (4.0 equivalents, 3.2 mmol) in 5 ml of DMF for 24 hours.
  • the resin is washed with DMF/THF and then DCM and dried under vacuum to give the monochlorotriazylfluorescein on Rink resin.
  • Fmoc-amino acid on Rink resin (prepared from Rink amine resin and Fmoc-amino acid under standard peptide synthesis conditions, e . g . , DIC/DMAP/DCM) is treated with piperidine in DMF to remove the Fmoc group.
  • the amino acid Rink resin is then treated with 2.5 equivalents of DTAF and 4.0 equivalents of DIPEA in DMF for 24 hours at room temperature. After washing and drying, the monochlorotriazylfluorescein on Rink resin is obtained.
  • PROCEDURE 6 REACTION OF FLUORESCENT
  • Monochlorotriazylfluorescein on a solid support such as Wang resin or trityl resin is treated with 4.0 equivalents of a symmetrical diamine in DMF at room temperature for 24-40 hours. The resin is then washed with DMF/MeOH followed by DCM and dried under vacuum to give an amino fluorescein on solid support.
  • PROCEDURE 7 REPARATION OF A LIBRARY OF
  • PROCEDURE 9 REACTION OF MONOCHLOROSULFOFLUORESCEIN
  • PROCEDURE 10 PREPARATION OF
  • the amino sulfofluorescein Wang resins containing different diamine linkers e.g., a combination of piperazine, homopiperazine and 4 , 4 ' -trimethylenedipiperidine
  • a coupling agent such as diisopropylcarbodiimide (DIC) (10 equivalents) and DMAP (5.0 equivalents) in DMF/DCM at room temperature for 24-48 hours.
  • DIC diisopropylcarbodiimide
  • DMAP 5.0 equivalents
  • PROCEDURE 11 SYNTHESIS OF FLUORESCENT
  • Step 2 the N-hydroxyamide resin 0.04 mmol, 50 mg, 0.8 mmol/g loading) is treated with commercially available first group of Fmoc-protected -amino acid (0.2 mmol, 5.0 equivalent) , coupling agent PyBrOP (Bromo-tris-pyrrolidino- phosphonium hexafluorophosphate, 0.2 mmol, 5.0 equivalent) and DMAP (0.2 mmol, 5.0 equivalent) in dimethyl acetamide (DMAC) solvent (0.6 ml) at 60-65°C with shaking for 20-24 hours.
  • DMAC dimethyl acetamide
  • the resin is filtered and washed with DMF, MeOH and DCM thoroughly and dried to give the quinazolinone on resin.
  • the resin is then suspended in 30% piperidine in DMF (0.6 ml) to remove the F oc-protecting group from the amine group to give the free amino quinazolinone resin after filtration and washing for attachment of next reactive
  • Step 3 the amino quinazolinone on resin (50 mg, 0.04 mmol) is. treated with second group of Fmoc-protected amino acid (0.2 mmol, 5.0 equivalent), coupling agent DIC (diisopropylcarbodiimide, 0.2 mmol, 5.0 equivalent), HOBt(N- hydroxybenzotriazole, 0.2 mmol, 5.0 equivalent) and DMAP (0.04 mmol, 1.0 equivalent) in DMF (0.6 ml) at room temperature for 20-24 hours. The resin is then filtered and washed thoroughly with DMF, MeOH and DCM and dried. The resin is then suspended in 30% piperidine in DMF (0.6 ml) to remove the Fmoc-protecting group from the amine group to give the free amino resin after filtration and washing for attachment of the dye.
  • DMF 0.6 ml
  • Step 4 the amine resin (0.04 mmol) is treated with DTAF»HC1 dye (Dichlorotriazylamino fluorescein monohydrochloride, 0.08 mmol, 2.0 equivalent) and DIPEA (diisopropylethylamine, 0.16 mmol, 4.0 equivalents) in DMF (0.6 ml) at room temperature with shaking for 20-24 hours.
  • DTAF Dichlorotriazylamino fluorescein monohydrochloride, 0.08 mmol, 2.0 equivalent
  • DIPEA diisopropylethylamine, 0.16 mmol, 4.0 equivalents
  • Step 5 the fluorescent-labeled resin (0.04 mmol) is treated with 30% Trifluoracetic acid (TFA) in DCM (1.0 ml) at room temperature with shaking for 1-2 hours. This mixture is filtered and the resin is rinsed with 0.5 ml of DCM. The combined filtrate is collected and evaporated under vacuum to dryness to give the desired fluorescent-labeled quinazolinone ligand which may be dissolved in DMSO for screenings.
  • TFA Trifluoracetic acid
  • a library of the above fluorescent-labeled quinazolinone ligands are made by performing the above reactions in a parallel manner using different isatoic anhydrides and Fmoc- protected amino acids.
  • PROCEDURE 12 SYNTHESIS OF LIGANDS USING
  • Rink amine resin (1.0 equivalents) (Prepared by treatment of commercial Rink amine resin with 25% piperidine in DMF) is swelled in MeOH/DCM (1:2, v/v) .
  • the aldehyde (10 equivalent) and Fmoc-protected amino acid (10 equivalent) are added.
  • the mixture is shaken at room temperature for 1-2 hours.
  • the isocyanide (10 equivalent) is then added.
  • the mixture is shaken at room temperature for 20-24 hours.
  • the resin is then filtered and washed thoroughly with DMF, MeOH and DCM and dried.
  • the resin is then treated with 25% piperidine in DMF to give the resin containing a free amine group.
  • the resin is then treated with 2.0-3.0 equivalent of DTAF and 4-6 equivalents of DIPEA in DMF to give the fluorescent-labeled ligand on resin after filtration and washing as usual.
  • the desired fluorescent-labeled ligand is then obtained after cleavage from the resin with TFA/DCM and drying.
  • the following example illustrates the use of the present invention to distinguish and/or identify two or more human blood sera samples.
  • normal sera and abnormal sera can be distinguished from each other based upon the differential binding pattern generated when the samples are screened with a library of probes.
  • the invention may be used, upon solution of the appropriate probes, to distinguish diabetic sera from normal or otherwise non-diabetic sera.
  • the discussion below illustrates the general procedures, to be employed to distinguish between human sera samples.
  • Fluorescein labeled probes from three different library plates (XN1192-54, XN1043-58, GY1175-96 and Plate 1) are combined with sera samples purchased from Western States Plasma Company (Fallbrook, CA) .
  • the sera samples consisted of diabetic sera pooled from three diabetic individuals (confirmed to be diabetic by reference laboratories) and pooled sera from normal individuals (Lot HS300; SeraCare, Oceanside, California) .
  • each fluorescein labeled probe (1-10 nM) was combined with 95 ⁇ l of buffer (PBS + 0.03% lithium dodecysulfate) and 5 ⁇ l of sera in 96-well plates. Then the mixtures were incubated at 37°C for 30 minutes. The sera samples, where indicated, were centrifuged at 20,000 xg for 3.5 hours and 5 ⁇ l of the lower, straw colored sera layer was assayed for probe binding. The fluorescence polarization for each probe with each sample was determined by utilizing the Fluorolite FPM-2 fluorescence polarization microtiter system. Each probe was tested with each sample in triplicate and the resulting fluorescence polarization values (MP) were averaged.
  • MP fluorescence polarization values
  • probes showed a significant difference in the fluorescence polarization values obtained for the two sera samples. These probes were screened again in triplicate under the same conditions.
  • the probes listed in Table 11 and depicted in Table 12 showed a significant difference in the fluorescence polarization values obtained for normal pooled sera (Lot HS300) and diabetic sera ( Figure 14) .
  • the fluorescence polarization values obtained for 10 of the 12 probes listed in Table 11 were higher for diabetic sera than for normal sera (Lot HS300) .
  • the results suggest that probes 58A2,
  • 58A3, 58A6, 58B6, 58B7, 58B11, 58H8, 96G3, 54G3 , and P1B4 are preferentially binding to some component (s) present in diabetic sera.
  • lipid component (s) present in the diabetic sera reduces the binding affinity of probes 58A2, 58A3 , 58A6, 58B6, 58B7, 58B11, 58H8, 96G3, 54G3 , and P1B4 for another component (s) present in diabetic sera.
  • results described above demonstrate that human sera samples can be distinguished from one another using the fluorescence polarization-based assay described in the present invention. Furthermore, the methods utilized in this example can be used to identify probes that distinguish normal and diabetic sera.
  • C12-MPP bis indolylmaleim.de IS / nyoi ⁇ ositol-l-phosphate

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Abstract

L'invention concerne des techniques de criblage combinatoire et de détection portant sur des banques de composants extrêmement diversifiées qui agissent comme empreintes permettant l'identification des différences moléculaires spécifiques existant entre des échantillons biologiques. Les différences moléculaires spécifiques identifiées par la technique de criblage combinatoire et par les techniques de détection représentent des cibles potentielles pour le diagnostic et la mise au point de traitements. On peut utiliser ces techniques pour établir des diagnostics, découvrir des médicaments ainsi que pour déterminer les génomes et les protéines. La figure (1) qui illustre ces techniques montre l'interaction entre les sondes moléculaires décrites dans la table II et un échantillon de sérum humain.
PCT/US1998/026894 1997-12-18 1998-12-18 Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament WO1999031267A1 (fr)

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AU19256/99A AU1925699A (en) 1997-12-18 1998-12-18 Methods for the simultaneous identification of novel biological targets and leadstructures for drug development
CA002314422A CA2314422A1 (fr) 1997-12-18 1998-12-18 Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament
EP98964053A EP1049796A1 (fr) 1997-12-18 1998-12-18 Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament
CN98813707A CN1285001A (zh) 1997-12-18 1998-12-18 同时鉴定新型生物靶和用于药物开发的引导结构的方法
JP2000539165A JP2002508507A (ja) 1997-12-18 1998-12-18 新規な生物学的標的および創薬リード構造体の同時同定方法
PCT/US1998/026894 WO1999031267A1 (fr) 1997-12-18 1998-12-18 Techniques d'identification simultanee de nouvelles cibles biologiques et structures conductrices pour mise au point d'un medicament

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WO2002037106A3 (fr) * 2000-11-03 2002-11-21 Glycodata Ltd Procedes d'analyse comparative de polymeres a base d'hydrates de carbone et polymeres a base d'hydrates de carbone ainsi identifies
US6573369B2 (en) 1999-05-21 2003-06-03 Bioforce Nanosciences, Inc. Method and apparatus for solid state molecular analysis
WO2003084997A1 (fr) * 2001-04-10 2003-10-16 Transtech Pharma, Inc. Sondes, systemes et procedes pour la decouverte de medicaments
US6635311B1 (en) 1999-01-07 2003-10-21 Northwestern University Methods utilizing scanning probe microscope tips and products therefor or products thereby
WO2003089662A1 (fr) * 2002-04-15 2003-10-30 The Regents Of The University Of California Procede pour obtenir les affinites de liaison a une proteine d'une banque de peptides
WO2003033512A3 (fr) * 2001-10-16 2003-12-24 Procognia Ltd Procede de preparation de banques d'oligosaccharides biologiquement actifs purifies
US6703214B2 (en) * 2000-05-19 2004-03-09 Devgen Nv Lipid uptake assays
US6827979B2 (en) 1999-01-07 2004-12-07 Northwestern University Methods utilizing scanning probe microscope tips and products therefor or produced thereby
US6890919B2 (en) 2001-06-26 2005-05-10 Shitij Kapur Atypical antipsychotic agents having low affinity for the D2 receptor
US6897015B2 (en) 2000-03-07 2005-05-24 Bioforce Nanosciences, Inc. Device and method of use for detection and characterization of pathogens and biological materials
US7008769B2 (en) 2000-08-15 2006-03-07 Bioforce Nanosciences, Inc. Nanoscale molecular arrayer
EP1646371A2 (fr) * 2003-06-26 2006-04-19 Biotron Limited Compositions et methodes antivirales
US7042488B2 (en) 2001-09-27 2006-05-09 Fujinon Corporation Electronic endoscope for highlighting blood vessel
US7056678B1 (en) 2000-05-04 2006-06-06 Procognia Ltd Polysaccharide structure and sequence determination
US7060448B2 (en) 2000-10-10 2006-06-13 Bioforce Nanosciences, Inc. Evaluating binding affinities by force stratification and force panning
US7079955B2 (en) 2000-11-03 2006-07-18 Procognia, Ltd. System and method for integrated analysis of data for characterizing carbohydrate polymers
US7132251B1 (en) 2000-05-04 2006-11-07 Procognia Ltd Method and composition for analyzing a carbohydrate polymer
US7157239B2 (en) * 2001-06-11 2007-01-02 Applied Research Systems Ars Holding N.V. Method and kit for identifying and/or quantifying radiolabeled aminoglycoside binding molecules
US7344832B2 (en) 2003-01-02 2008-03-18 Bioforce Nanosciences, Inc. Method and apparatus for molecular analysis in small sample volumes
US7354721B2 (en) 2000-09-22 2008-04-08 Clontech Laboratories, Inc. Highly sensitive proteomic analysis methods, and kits and systems for practicing the same
US7361310B1 (en) 2001-11-30 2008-04-22 Northwestern University Direct write nanolithographic deposition of nucleic acids from nanoscopic tips
US7407773B2 (en) 2000-11-03 2008-08-05 Procognia, Ltd. Method for characterizing a carbohydrate polymer
US7455979B2 (en) 1999-05-06 2008-11-25 Procognia Ltd. Polysaccharide structure and sequence determination
US7887885B2 (en) 2000-10-20 2011-02-15 Northwestern University Nanolithography methods and products therefor and produced thereby
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US8119357B2 (en) 2003-12-18 2012-02-21 Procognia, Ltd. Method for analyzing a glycomolecule
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US9405885B2 (en) 2002-07-24 2016-08-02 Keddem Bioscience Ltd. Drug discovery method
US20180327424A1 (en) * 2017-05-15 2018-11-15 Indicator Systems International, Inc. Compositions to detect remnant cancer cells
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US10890588B2 (en) 2016-08-02 2021-01-12 Isi Life Sciences, Inc. Compositions and methods for detecting cancer cells in a tissue sample
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US6827979B2 (en) 1999-01-07 2004-12-07 Northwestern University Methods utilizing scanning probe microscope tips and products therefor or produced thereby
US7741061B2 (en) 1999-05-06 2010-06-22 Procognia Ltd. Polysaccharide structure and sequence determination
US7455979B2 (en) 1999-05-06 2008-11-25 Procognia Ltd. Polysaccharide structure and sequence determination
US6998228B2 (en) 1999-05-21 2006-02-14 Bioforce Nanosciences, Inc. Method and apparatus for solid state molecular analysis
US6573369B2 (en) 1999-05-21 2003-06-03 Bioforce Nanosciences, Inc. Method and apparatus for solid state molecular analysis
WO2001036980A3 (fr) * 1999-11-18 2002-03-14 Melacure Therapeutics Ab Procede d'identification du site actif dans une cible biologique
US6897015B2 (en) 2000-03-07 2005-05-24 Bioforce Nanosciences, Inc. Device and method of use for detection and characterization of pathogens and biological materials
GB2360282A (en) * 2000-03-17 2001-09-19 Bioinvent Int Ab Making and using micro-arrays of biological materials
EP2330219A2 (fr) 2000-04-14 2011-06-08 Metabolon, Inc. Procédé de découverte de médicament, traitement de maladie et diagnostic utilisant les métabolomiques
US7132251B1 (en) 2000-05-04 2006-11-07 Procognia Ltd Method and composition for analyzing a carbohydrate polymer
US7056678B1 (en) 2000-05-04 2006-06-06 Procognia Ltd Polysaccharide structure and sequence determination
US6703214B2 (en) * 2000-05-19 2004-03-09 Devgen Nv Lipid uptake assays
US7008769B2 (en) 2000-08-15 2006-03-07 Bioforce Nanosciences, Inc. Nanoscale molecular arrayer
US7354721B2 (en) 2000-09-22 2008-04-08 Clontech Laboratories, Inc. Highly sensitive proteomic analysis methods, and kits and systems for practicing the same
US7723125B2 (en) 2000-09-22 2010-05-25 Clontech Laboratories Inc. Highly sensitive proteomic analysis methods, and kits and systems for practicing the same
US7060448B2 (en) 2000-10-10 2006-06-13 Bioforce Nanosciences, Inc. Evaluating binding affinities by force stratification and force panning
US7887885B2 (en) 2000-10-20 2011-02-15 Northwestern University Nanolithography methods and products therefor and produced thereby
WO2002037106A3 (fr) * 2000-11-03 2002-11-21 Glycodata Ltd Procedes d'analyse comparative de polymeres a base d'hydrates de carbone et polymeres a base d'hydrates de carbone ainsi identifies
US7407773B2 (en) 2000-11-03 2008-08-05 Procognia, Ltd. Method for characterizing a carbohydrate polymer
US7079955B2 (en) 2000-11-03 2006-07-18 Procognia, Ltd. System and method for integrated analysis of data for characterizing carbohydrate polymers
WO2003084997A1 (fr) * 2001-04-10 2003-10-16 Transtech Pharma, Inc. Sondes, systemes et procedes pour la decouverte de medicaments
US7157239B2 (en) * 2001-06-11 2007-01-02 Applied Research Systems Ars Holding N.V. Method and kit for identifying and/or quantifying radiolabeled aminoglycoside binding molecules
US6890919B2 (en) 2001-06-26 2005-05-10 Shitij Kapur Atypical antipsychotic agents having low affinity for the D2 receptor
US7042488B2 (en) 2001-09-27 2006-05-09 Fujinon Corporation Electronic endoscope for highlighting blood vessel
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US7361310B1 (en) 2001-11-30 2008-04-22 Northwestern University Direct write nanolithographic deposition of nucleic acids from nanoscopic tips
US7951334B2 (en) 2001-11-30 2011-05-31 Northwestern University Direct write nanolithographic deposition of nucleic acids from nanoscopic tips
WO2003089662A1 (fr) * 2002-04-15 2003-10-30 The Regents Of The University Of California Procede pour obtenir les affinites de liaison a une proteine d'une banque de peptides
EP2508204A3 (fr) * 2002-06-26 2012-12-12 Ono Pharmaceutical Co., Ltd. Remèdes pour les maladies provoquées par la contraction ou la dilatation vasculaire
US8765783B2 (en) 2002-06-26 2014-07-01 Ono Pharmaceuticals Co., Ltd. Pharmaceutical composition for treatment of disease due to vascular constriction or vasodilation
US9405885B2 (en) 2002-07-24 2016-08-02 Keddem Bioscience Ltd. Drug discovery method
US7344832B2 (en) 2003-01-02 2008-03-18 Bioforce Nanosciences, Inc. Method and apparatus for molecular analysis in small sample volumes
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EP1646371A4 (fr) * 2003-06-26 2010-09-22 Biotron Ltd Compositions et methodes antivirales
US10472332B2 (en) 2003-06-26 2019-11-12 Biotron Limited Antiviral compounds and methods
US11192863B2 (en) 2003-06-26 2021-12-07 Biotron Limited Antiviral compounds and methods
US8119357B2 (en) 2003-12-18 2012-02-21 Procognia, Ltd. Method for analyzing a glycomolecule
US10890588B2 (en) 2016-08-02 2021-01-12 Isi Life Sciences, Inc. Compositions and methods for detecting cancer cells in a tissue sample
US20180327424A1 (en) * 2017-05-15 2018-11-15 Indicator Systems International, Inc. Compositions to detect remnant cancer cells
US10239891B2 (en) * 2017-05-15 2019-03-26 Indicator Systems International, Inc. Compositions to detect remnant cancer cells
US10753942B2 (en) 2017-05-15 2020-08-25 Indicator Systems International, Inc. Methods to detect remnant cancer cells
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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