JP2003504641A - Thermochemical sensors and uses thereof - Google Patents

Abstract

(57) Abstract The methods described herein relate the binding event between a test ligand or substrate and a target (eg, a target protein) with the generation of a thermal output. Drugs can be screened using the methods herein.

Description

Detailed Description of the Invention

RELATED APPLICATION This application claims the benefit of Provisional Application No. 60 / 144,570, filed July 19, 1999, which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to the use of thermochemical sensors (eg, calorimeters) for screening ligands or substrates.

BACKGROUND OF THE INVENTION Random screening of natural product and synthetic chemical databases (eg, testing for activity) is a powerful method for discovering compounds that interact with target biomolecules of interest. Identifying substances that interact with target biomolecules is often a crucial step and is generally a necessary condition for drug discovery. Substances that interact with biomolecules are called ligands. Ligands are classified into two main categories: bound ligands that bind to and form stable complexes with target biomolecules; and substrates that interact with target biomolecules and are chemically modified. You can Biomolecule targeting ligands can be identified by their ability to physically associate with a target biomolecule. Binding steps and substrate turnover are generally examined by assays that reveal whether complex formation or a chemical reaction has taken place. These assays are often dependent, for example, on changes in the spectroscopic or chromatographic properties of the complex or ligand of interest.

Large-scale screening assays can be limited in their utility in many ways. For example, a large number of putative ligands searched for a particular target protein is a representative example of a difficult task, especially when the function of the target is unknown.
In particular, enzymatic assays require knowledge of the target function in order to select the appropriate substrate that can be screened. Screening for compounds that interact with targets of unknown function has gained importance in recent years as a result of efforts to sequence the genomes of various organisms. For example, in the field of microbiology, at least 16
The complete genomes of species bacteria and fungi have been sequenced. These sequence data have led to the identification of a number of new protein targets that could be new antibacterial agents. A particularly attractive target for antimicrobial discovery is one that is essential for bacterial growth but has no counterpart in eukaryotes. Many of these potential targets are of unknown function, making them difficult to effectively target new drug discovery programs that rely on large-scale screening assays.

In addition to the limitations associated with assays for targets of unknown function, many screening assays measure compound-target interactions to limit the range of compounds that can be tested. For example, many proteins have multiple functions, but most assays are able to detect only one of these functions. Such assays limit the range of compounds that can be tested. Additional limitations to currently available screening assays include (i) lengthy and / or expensive separation steps often required in heterogeneous assays; (ii) colored compounds or suspensions. Or optical detection problems associated with assays that are unable to effectively detect the activity of cloudy extracts; (iii
) If the assay is not a high throughput assay, the tedious and cumbersome test of a small subset of potential compounds; (iv) the cost and difficulty of the assay required to use cell lines and / or animals. (V) that information on the strength of the interaction between the test compound and the target cannot be obtained by the most used assay method at present.

[0006] Therefore, there is a need for a relatively inexpensive, large-scale assay that can detect the interaction between a target and a test compound without significantly limiting the range of detectable target function.

SUMMARY OF THE INVENTION The methods described herein include the interaction (eg, binding) of a test compound (eg, a test ligand or test substrate) with a target (eg, a target protein or nucleic acid).
Is associated with a change in heat. The heat output is detected by a calorimeter. The methods of the present invention allow interactions to be analyzed without imposing severe restrictions on the type, extent or specific identity of the target activity. As an example, identifying an interactor (eg, a substrate) to a target that has unknown, poorly characterized, or simply deduced, or roughly stated activity by the method of the present invention. You can The method of the present invention detects, for example, when the target is an enzyme, a change in heat generated upon conversion of one or more test substrates into one or more products, or when the target and interacting substances are If it is a ligand and the corresponding ligand,
Detect the change in heat generated when they are bonded. The absorption or release of heat is a universal property of chemical reactions and, therefore, the legal authority of the methods of the present invention imposes unduly limiting hypotheses about the nature of the target or the interaction of the target with other molecules. Beyond the legal authority of how to stand. Some aspects of the invention do not require a hypothesis about the nature of the target or the interaction of the target with its interactors (eg, natural ligands, substrates, or binding partners). Other methods of the invention incorporate knowledge of the target (and / or interactant), or a hypothesis about the target (and / or interactant), as a guide for selecting potential interactants. . For example, aspects of the invention use genomic or other bioinformatics analysis of the target to optimize and prioritize testing of the target for interactant selection.

Accordingly, in one aspect, the invention features a method of analyzing a target (eg, protein). (While the method is described with respect to proteins, other target molecules (eg, other macromolecules, such as nucleic acids) can be analyzed by the methods described herein.) The method comprises the following steps. Including: (1) selectively assigning a putative function to a target (eg, protein).
The function of inference can be assigned by any means, eg, identification of features that the target has (or optionally does not have). Exemplary features include structural features (eg, in the case of a protein, a preselected level of sequence identity with another protein); retention of sequences or motifs, or protein folds; target and another molecule (eg, 3D structural similarity to proteins of known function); promoter structures or other 3 ', 5'or other regulatory structures; chromosomal location or other genetic characteristics (eg suppressors, auxotrophs, Permeation enzyme, drug resistance,
Drug sensitivity, or other similar activity or property); expression profile (eg, tissue specificity, disease or disorder specific expression, transient expression pattern); source (
For example, the species from which the target was obtained. Upon identifying features that the target and a molecule of known function (eg, protein) share (or optionally do not share), the activity of the molecule of known function (eg, protein) can be assigned to the target.
For example, the putative function can be determined, for example, by comparing the sequence of a protein or a nucleic acid encoding the protein with a reference sequence (e.g., the target protein has a preselected sequence (e.g., a preselected motif, e.g., A consensus sequence)), or by comparing a protein with another protein having a known three-dimensional structure (eg, by checking for protein folding), or a crystal of the target protein. Can be assigned by comparing the three-dimensional structure of a protein with the three-dimensional structure of a crystal of another protein of known function; Providing a library of ligands (preferably the library is It is known to contain at least one, and more preferably multiple members, each member being known to interact with a protein having an assigned putative function .. As an example, a library may contain multiple protease substrates. The putative function assignment can optimize the screening strategy, eg, by aiding selection of a particular interactant library); (3) reaction mixture containing the target (eg, protein). (4) contacting a target (eg, protein) with a member of the library; (5) assessing changes in the heat output of the reaction mixture; (6) selectively obtaining the resulting heat. By comparing the value of the change in the target value (eg, protein) with a library that is a ligand or ligand of the target (eg, protein) Analyzing the target (eg, protein) by identifying members. (In this method as well as all other methods described herein, the designation of letters or numbers to the steps of the method is for the convenience of the reader only and unless otherwise necessary. Does not mean that they must be done in the order listed.)

In a preferred embodiment, the target (eg, protein) is produced by a pathogen, including a bacterium, protozoa, virus (eg, phage), or fungus, eg, a prokaryotic or eukaryotic pathogen. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii, Bacillus subtilis, Treponema pallidum,
Borrelia burgdorferi, Helicobacter pylori, Archaeoglobus fulg
idus), Methanobacterium thermo, E. coli (Es
cherichia coli), Mycoplasma pneumoniae,
Synechocystis sp., Methanococcus janas (Methanoc)
occus jannaschii), Saccharomyces cerevisiae
), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia prow
azekii), Pyrococcus abyssii, Bacillus
) Sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitides strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg, protein) is an organelle of an organism (
(Eg, mitochondria).

In a preferred embodiment, the target (eg, protein) has no known activity (eg, enzymatic activity) or has an activity that is difficult to measure. In a preferred embodiment, the protein has a known first activity and is tested against a library containing interactants that interact with the protein by a second activity (eg, an unknown activity).

In a preferred embodiment, the target is a naturally occurring protein or fragment thereof;
A protein of unknown function and / or structure; a protein of unknown ligand, substrate, or other interacting molecule. In other embodiments, the protein has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid molecule, such as DNA or RNA (eg,
Structural RNA, eg ribozymes).

In a preferred embodiment, multiple library members are tested simultaneously, eg, in the same reaction mixture, which can increase the throughput of the method of the invention. Multiple library members (eg, those that produce positive results) can be divided into smaller groups and these smaller groups can be tested. Multiple library members or small groups (eg, those that give positive results)
From, one or more library members can be individually tested.

In a preferred embodiment, the method is performed under different conditions (eg, different salt concentrations,
At different pH, or in the presence of different cofactors), in one or more steps (eg,
Further comprising repeating one or both of steps (4) and (5)).

In a preferred embodiment, the method uses at least one step (eg, steps (3)-(6)) using a second or subsequent member of the library.
It further includes repeating. In a preferred embodiment, multiple library members (eg, candidate substances or test ligands) are tested. In a preferred embodiment, the plurality of library members has at least 10, 10 ² , 10 ³ , 10 ⁴
Number, 10 ^5, 10 ^6, contains 10 ^7, or 10 ⁸ compounds. In a preferred embodiment, at least 10, 10 ² , 10 ³ , 10 ⁴ , 1 of the plurality of library members
0 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ share structural or functional features.

In a preferred embodiment, the library contains a plurality of members with common characteristics. For example, all of the members are enzyme cofactors; substrates (eg, biosynthetic or digestive enzymes) (eg, protease substrates) (including carbohydrates, nucleosides / nucleotides, amino acids, lipids); vitamins; hormones; nucleic acids For example, a DNA molecule; or a natural product (for example, a natural product derived from a bacterium). The library can include any metabolite, precursor, or intermediate of the aforementioned members.

In a preferred embodiment, the library is a substrate library; a cofactor library; a carbohydrate biosynthesis and / or degradation library; a purine and pyrimidine biosynthesis and / or degradation library; an amino acid biosynthesis and / or degradation library. Larry; lipid biosynthesis and / or degradation library; vitamin and / or hormone library; nucleic acid (eg, DNA) library; or natural product library (eg, natural product library from bacteria).

In a preferred embodiment, library members are chemical species that have the potential to interact with a target (eg, target protein). Preferably, the library member is a candidate substrate or test ligand.

In a preferred embodiment, the library members (potential interactors or candidate interactors) are enzyme substrates, metabolites, cofactors, natural products (eg natural products of bacterial origin), carbohydrates, Sugars, nucleic acids (eg nucleoside or nucleotide precursors, double stranded DNA (ds) or single stranded (ss) DNA molecules, circular nucleic acids, supercoiled nucleic acids), amino acids (eg D or L amino acids or precursors thereof) ), Vitamins, hormones, lipids, small organic molecules, metals, peptides, proteins, lipids, glycoproteins, glycolipids, transition state analogs, and combinations thereof.

In a preferred embodiment, the method further comprises testing the protein against at least one member of the second library.

[0023]   In a preferred embodiment, two or more libraries are tested simultaneously.
It As an example, target the first library (eg, cofactor library)
Each member (or some members) of the second library (
For example, each member (or some members) of a potential substrate library
-) Can be tested against. Therefore, the first library contains 50 members.
Bar (1st₁, First₂, ... 1st₅₀) And the second library has 50 members (
No. 2₁,No. 2₂,...No. 2₅₀In case of two libraries with ...)
Or multiple new combinations (eg, (first₁,No. 2₁), (First₁,No. 2₂) ... (first ₁ ,No. 2₅₀)))).

In a preferred embodiment, the library members are members of a combinatorial library.

In a preferred embodiment, the target interacts with the test compound (eg, binds to the test compound and preferably modifies the test compound). As used herein, "modifying" includes causing or abolishing binding (eg, non-covalent or covalent binding) at a test compound or target. Modifications include cleavage, degradation, hydrolysis, changes in phosphorylated label level, ligation, synthesis, and similar reactions. Modifications can include changes in activity (eg, enzymatic activity), physical changes in phase, changes in aggregation or polymerization.

In a preferred embodiment, the method analyzes the structure or function of the target (eg,
Analyzing the physical properties of the target; analyzing the in vitro or in vivo activity of the target; eg, analyzing the target sequence (eg, amino acid sequence or nucleotide sequence) for the presence of conserved amino acid domains), and thereby the structure of the target Or further includes predicting function. In a preferred embodiment, analysis of target structure or function is performed prior to contacting the target with the library.

In a preferred embodiment, the method selects library members (eg, candidate substrates or test ligands) based on their interaction with the target and confirms that the candidate substrate or test ligand is the substrate or ligand, respectively. Further including:

In a preferred embodiment, the method selects library members based on their interaction with a target, contacting the library members with cells (eg, cultured cells) or animals, and selectively It further includes ascertaining whether the member affects cells or animals.

In a preferred embodiment, the method selects interacting agents (eg, library members) based on their interaction with the target, purifies the library (eg, candidate substrate or test ligand), and Crystallize the member (eg, candidate substrate or test ligand) and the physical properties (eg, molecular weight, isoelectric point, sequence (if relevant)) of the library member (eg, candidate substrate or test ligand)
, Or a crystal structure). The library member may be crystallized alone or complexed with the target.

In a preferred embodiment, the method uses the library members selected to interact with the target, and selects agents that modulate the interaction of the target with the selected library member (eg, selection). Binding to a selected library member or by interacting with a selected library member).

In a preferred embodiment, the method selects an interacting agent (eg, a library member) based on its interaction with a target and characterizes the selected library member (eg, a candidate substrate or test ligand). (Eg optimizing affinity for target, varying molecular weight (eg decreasing molecular weight), or varying solubility (eg increasing)). Optimization can be performed using one or more of the known methods disclosed herein.

[0032]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises determining a physical constant of protein-library member interaction (eg, k _cat , K _M , or K _D ).

In a preferred embodiment, the method may include using a binding reaction (eg, a surrogate ligand) as described elsewhere herein.

In another aspect, the invention features a method of purifying or isolating an interactant (or target) from a mixture. (In the embodiments described below, the target is used as an assay reagent to purify or isolate the interactant (eg, substrate or corresponding ligand), but the interactant (eg, substrate) is used as the assay reagent. Similar methods of isolating or purifying a target are also within the scope of the invention.) An interactant is, for example, a ligand, a receptor, a corresponding ligand, a cofactor, or a ligand that interacts with the target (eg, a protein). It can be a substrate. The mixture can be a biological sample (eg, whole blood), a cell homogenate or lysate, a tissue sample, a biological fluid sample consisting of many components. (The method will be described with respect to proteins,
Other molecules, referred to herein as targets (eg, other macromolecules such as nucleic acids), can be analyzed by the methods described herein. ) The method comprises the following steps: (1) providing a mixture; (2) including a first fraction and a second fraction (eg, a soluble fraction and a membrane fraction) Dividing into a plurality of fractions; (3) contacting the target with a first fraction to form a first reaction mixture; (4) assessing the change in heat output of the first reaction mixture Step; (5) selectively comparing the obtained heat change value with a predetermined value; (6) contacting the target with a second fraction to form a second reaction mixture (7) evaluating the change in the heat output of the second reaction mixture; (8) selectively comparing the obtained value of the change in heat with a predetermined value; (9) eg Evaluation is performed by comparing the change in heat of one reaction with the change in heat of the second reaction, and fractions are selected for interaction (eg, target (eg, protein target). A) a ligand or substrate) of)).

In a preferred embodiment, the target (eg protein) is produced by a pathogen (eg prokaryotic or eukaryotic pathogen) including bacteria, protozoa, viruses (eg phage), or fungi. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii,
Bacillus subtilis, Treponema pallidum
, Borrelia burgdorferi, Helicobacter pylori, Alcaeoglobus fu
lgidus), Methanobacterium thermo, E. coli (
Escherichia coli), Mycoplasma pneumoniae
, Synechocystis sp., Methanococcus janas (Methan
ococcus jannaschii), Saccharomyces cerevisia
e), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia priwatzekii
owazekii), Pyrococcus abyssii, Bacillus
s) sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitidis strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg protein) is the organelle (
(Eg, mitochondria).

In a preferred embodiment, the target (eg protein) has no known activity (eg enzymatic activity) or has an activity which is difficult to measure.

In a preferred embodiment, the target (eg, protein) is a naturally occurring protein or fragment thereof; a protein of unknown function and / or structure; a protein of unknown ligand, substrate, or other interacting molecule. . In other embodiments, the target (eg, protein) has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid molecule, such as DNA or RNA (eg,
Structural RNA, eg ribozymes).

In a preferred embodiment, the method is performed under different conditions (eg different salt concentrations,
At different pHs, or in the presence of cofactors, one or more steps (eg step (4
) And (5) one or both) is further included.

In a preferred embodiment, the target interacts with (eg, binds to, and preferably modifies the interactant) the interactant. As used herein, "modifying" includes causing or abolishing binding (eg, non-covalent or covalent binding) at a test compound or target. Modifications include cleavage, degradation, hydrolysis, changes in phosphorylated label level, ligation, synthesis, and similar reactions. Modifications can include changes in activity (eg, enzymatic activity), physical changes in phase, changes in aggregation or polymerization.

In a preferred embodiment, the method further comprises analyzing the structure or function of the interactant (eg, analyzing the physical properties of the interactant).

In a preferred embodiment, the method further comprises, for example, selecting an interacting agent based on its interaction with the target and confirming that the interacting agent is, for example, a substrate or a ligand.

In a preferred embodiment, the method comprises contacting a purified or isolated interactant with a cell (eg, a cell in culture) or an animal, wherein the purified or isolated interactant is selectively It further includes ascertaining whether it affects the cell or animal.

In a preferred embodiment, the method selects an interactant (eg, a library member) based on its interaction with a target, purifies the purified or isolated interactant, and purifies or isolates it. The method further comprises crystallizing the interacted substance, and evaluating the physical properties of the purified or isolated interactant.

In a preferred embodiment, the method optimizes the properties of the purified or isolated interactant (eg, optimizes affinity for target, alters molecular weight (eg, reduces molecular weight). ), Or changing (eg, increasing) the solubility). Optimization can be performed using one or more of the known methods disclosed herein.

[0049]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises determining the physical constant (eg, k _cat , K _M , or K _D ) of the interaction of the target with the purified or isolated interactant.

In a preferred embodiment, the method may include using a binding reaction (eg, a surrogate ligand) as described elsewhere herein.

In another aspect, the invention features a method of analyzing a target (eg, discovering an interactor (eg, a protein substrate or ligand)). The method comprises the following steps: (a) providing a reaction mixture containing the target; (b) contacting the target with a candidate interactor (eg, a candidate substrate or test ligand); (c) Evaluating the heat change of the reaction mixture; (d) optionally comparing the resulting heat change value with a predetermined value, thereby analyzing the target (eg, discovering the target substrate). Stages. Although much of the discussion below is directed to proteins and their interactors (eg, substrates or corresponding ligands), it will be appreciated that the method is applicable to other targets and other interactants.

In a preferred embodiment, the interactant (eg, substrate or ligand) is identified by a change in the reaction mixture's heat (eg, a change above a predetermined value).

In a preferred embodiment, multiple candidate interactors (eg, candidate substrates or test ligands) (eg, a library of candidate interactors) are tested. In a preferred embodiment, the plurality of candidate substrates or test ligand (e.g., a library of candidate substrates or test ligand), at least 10, 10 ^2, 10 ^3, 10 ^4, 10 ^5, 10 ^6, 10 ⁷ or 10 ⁸ candidate substrates or test ligands are included. Thus, in a preferred embodiment, the method comprises the steps of: (a) providing a reaction mixture comprising a first interactor of a plurality of candidate interactors and a target; (b) a first Interacting the interacting substance with a target molecule; (c) measuring a change in heat in the reaction mixture; and (d) optionally each remaining interacting substance of the plurality of candidate interacting substances. Steps (a), (b), and (c) for testing multiple interactants to confirm that one or more of the interactants interacts with the target. Stages.

In a preferred embodiment, the target (eg, protein) is produced by a pathogen (eg, prokaryotic or eukaryotic pathogen), including bacteria, protozoa, viruses (eg, phage), or fungi. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii,
Bacillus subtilis, Treponema pallidum
, Borrelia burgdorferi, Helicobacter pylori, Alcaeoglobus fu
lgidus), Methanobacterium thermo, E. coli (
Escherichia coli), Mycoplasma pneumoniae
, Synechocystis sp., Methanococcus janas (Methan
ococcus jannaschii), Saccharomyces cerevisia
e), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia priwatzekii
owazekii), Pyrococcus abyssii, Bacillus
s) sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitidis strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg, protein) is an organelle of an organism (
(Eg, mitochondria).

In a preferred embodiment, the target has no known activity (eg enzymatic activity) or has an activity that is difficult to measure. In a preferred embodiment, the target has a known first activity and is tested against a library containing interactors that interact with the target by a second activity (eg, an unknown activity).

In a preferred embodiment, the target is a naturally occurring protein or fragment thereof;
A protein of unknown function and / or structure; a protein of unknown ligand, substrate, or other interacting molecule. In other embodiments, the target (eg, protein) has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid molecule, such as DNA or RNA (eg,
Structural RNA, eg ribozymes).

In a preferred embodiment, multiple candidate interactors (eg, library members) are tested simultaneously, eg, in the same reaction mixture, which can increase the throughput of the methods of the invention. Multiple library members (for example,
Those that produce a positive result) can be divided into smaller groups and these smaller groups can be tested. One or more library members can be individually tested from multiple library members or small groups (eg, those that give a positive result).

In a preferred embodiment, the method is performed under different conditions (eg different salt concentrations,
Further, repeating one or more steps, at different pHs, or in the presence of different cofactors).

In a preferred embodiment, the method further comprises repeating at least one step using a second member of the candidate interactor library or a subsequent member. In a preferred embodiment, multiple candidate interactors (eg, library members) are tested. In preferred embodiments, the plurality of candidate interactors (eg, library members) has at least 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ^Eight compounds are included. In a preferred embodiment, at least 10, 10 ² , 10 ³ , 10 ⁴ , 1 of library members
0 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ share structural or functional features.

In a preferred embodiment, the candidate interactor library comprises a plurality of members with common characteristics. For example, all of the members are enzyme cofactors; substrates (eg, biosynthetic or digestive enzymes) (eg, protease substrates) (including carbohydrates, nucleosides / nucleotides, amino acids, lipids); vitamins; hormones; nucleic acids For example, a DNA molecule; or a natural product (for example, a natural product derived from a bacterium). The library includes any metabolites, precursors, of any of the above members,
Or an intermediate may be included.

In a preferred embodiment, the candidate interactor library is a substrate library; a cofactor library; a carbohydrate biosynthesis and / or degradation library; a purine and pyrimidine biosynthesis and / or degradation library; an amino acid biosynthesis and / Or degradation libraries; lipid biosynthesis and / or degradation libraries; vitamin and / or hormone libraries; nucleic acid (eg, DNA) libraries; or natural product libraries (eg, natural product libraries of bacterial origin) is there.

In a preferred embodiment, the candidate interactor is a chemical species that has the potential to interact with the target (eg, target protein). Preferably, the candidate interactor is a candidate substrate or test ligand.

In a preferred embodiment, the candidate interactor is an enzyme substrate, metabolite, cofactor,
Natural products (eg, natural products from bacteria), carbohydrates, polysaccharides, nucleic acids (eg, nucleoside or nucleotide precursors, ds or ss DNA molecules, circular nucleic acids, supercoiled nucleic acids), amino acids (eg, D amino acids or L amino acids) Or its precursor),
Vitamins, hormones, lipids, small organic molecules, metals, peptides, proteins, lipids,
It is selected from the group consisting of glycoproteins, glycolipids, transition state analogues, and combinations thereof.

In a preferred embodiment, the method further comprises testing the candidate interactor against at least one member of the second library.

In a preferred embodiment, two or more candidate interactor libraries are tested simultaneously. As an example, the target is tested against each member (or some members) of a first library (eg, cofactor library) and a second library (eg, live of potential substrates) is tested. Each member (or some members) of the rally can be tested. Therefore, the first library has 50 members (1st, _1st , _2nd , ... 1st ₅₀ ) and the second library has 50 members (2nd, _1st , _2nd). , 2 ... ₅₀ , the target in the case of two libraries,
All or multiple new combinations (eg (1 _{1 1} , 2 ₁ ), (1 ₁ , 2 ₂ ) ..
( _1st , 2nd _50th, etc.)).

In a preferred embodiment, the members of the candidate interactor library are members of the combinatorial library.

In a preferred embodiment, the target interacts with a candidate interactor (eg,
Bind to and preferably modify the candidate interactor). As used herein, "modifying" includes causing binding (eg, non-covalent or covalent) or destroying binding at a candidate interactor or target. Modifications include cleavage, degradation, hydrolysis, changes in phosphorylated label level, ligation, synthesis, and similar reactions. Modifications can include changes in activity (eg, enzymatic activity), physical changes in phase, changes in aggregation or polymerization.

In a preferred embodiment, the method analyzes the structure or function of the target (eg,
Analyzing the physical properties of the target; analyzing the in vitro or in vivo activity of the target; eg, analyzing the target sequence (eg, amino acid sequence or nucleotide sequence) for the presence of conserved amino acid domains), and thereby the structure of the target Or further includes predicting function. In a preferred embodiment, an analysis of target structure or function is performed prior to contacting the target with the candidate interactor.

In a preferred embodiment, the method selects candidate interactors (eg, library members) based on their interaction with the target, and the candidate interactors that interact with the target are, eg, each of the target's It further comprises confirming that it is a substrate or a ligand.

In a preferred embodiment, the method selects candidate interactors (eg, library members) based on their interaction with the target and contacts the library members with cells (eg, cultured cells) or animals. And optionally verifying whether the library member affects cells or animals.

In a preferred embodiment, the method selects candidate interactors (eg, library members) based on their interaction with the target, purifies the library (eg, candidate substrate or test ligand), and Crystallize a rally member (eg, candidate substrate or test ligand) and the physical properties (eg, molecular weight, isoelectric point, sequence (if relevant), or crystal structure of a library member (eg, candidate substrate or test ligand). ) Is further included.

In a preferred embodiment, the method uses the library members selected to interact with the target and selects agents that modulate the interaction of the target with the selected library member (eg, selection). Binding to a selected library member or by interacting with a selected library member).

In a preferred embodiment, the method selects candidate interactors (eg, library members) based on their interaction with the target, and selects the selected library members (eg, candidate substrates or test ligands). It further includes optimizing properties (eg, optimizing affinity for the target, changing molecular weight (eg, decreasing molecular weight), or changing solubility (eg, increasing)). Optimization can be performed using one or more of the known methods disclosed herein.

[0078]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises determining a physical constant (eg, k _cat , K _M , or K _D ) for the interaction of the protein with the library member.

In a preferred embodiment, the method may include using a binding reaction (eg, a surrogate ligand) as described elsewhere herein.

Embodiments of the method include binding reactions (eg, surrogate ligands) as described herein.
May be used. Thus, in a preferred embodiment, one or more steps (eg, step (b)) includes the inclusion of a surrogate ligand and a signaling entity, interacting the surrogate ligand (eg, a substituted surrogate ligand) with the signaling entity. It further includes that.

In another aspect, the invention features a method of modifying (eg, optimizing) the structure of a compound. The optimized parameter can be, for example, the ability of the compound to interact with the target (eg, the ability to bind to the target or modify the target). The method comprises the steps of: (a) preparing a target; (b) testing by a process involving, for example, producing or breaking a bond (eg, covalent or non-covalent). Modifying the structure of the compound to obtain a modified compound; (c) contacting the target molecule with the modified compound to obtain a reaction mixture; (d) altering the heat associated with the reaction mixture. Evaluating; (e) optionally comparing the value measured in (d) with a predetermined value, thereby obtaining a modified compound.

In a preferred embodiment, the method comprises the following steps: (a) preparing a target; (b) contacting the target molecule with a test compound to obtain a reaction mixture; (c) reaction Measuring the change in heat associated with the mixture; (d) optionally comparing the value measured in (c) with a default value, and if the value and the default value exhibit a predetermined relationship ( (E.g., if the value is equal to or less than the default value), (e) For example, the structure of the test compound by causing a bond (eg, covalent or non-covalent bond) or breaking the bond. To obtain a compound and thereby a modified compound. In a preferred embodiment, the method comprises repeating one or more of the following steps: (f) contacting the target molecule with a modified test compound to obtain a reaction mixture; (g) in the reaction mixture. Measuring the associated change in heat; (h) optionally comparing the value measured in (g) with a default value, and if the value and the default value exhibit a default relationship (eg, If the value is equal to or less than the default value), (i) a test compound modified by, for example, causing a bond (eg, covalent or non-covalent bond) or breaking the bond. Modifying the structure of to obtain a compound.

In a preferred embodiment, the target (eg, protein) is produced by a pathogen, including a bacterium, protozoa, virus (eg, phage), or fungus, eg, a prokaryotic or eukaryotic pathogen. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii, Bacillus subtilis, Treponema pallidum,
Borrelia burgdorferi, Helicobacter pylori, Archaeoglobus fulg
idus), Methanobacterium thermo, E. coli (Es
cherichia coli), Mycoplasma pneumoniae,
Synechocystis sp., Methanococcus janas (Methanoc)
occus jannaschii), Saccharomyces cerevisiae
), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia prow
azekii), Pyrococcus abyssii, Bacillus
) Sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitides strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg, protein) is an organelle of an organism (
(Eg, mitochondria).

In a preferred embodiment, the target (eg protein) has no known activity (eg enzymatic activity) or has an activity which is difficult to measure. In a preferred embodiment, the target (eg, protein) has a known first activity and is second to the library containing interactants that interact with the protein.
The protein is tested for its activity (eg, unknown activity).

In a preferred embodiment, the target is a naturally occurring protein or fragment thereof, a protein having an unknown function and / or structure, a ligand, a substrate, or a protein in which other molecules with which it interacts are unknown, etc. . In other embodiments, the target (eg, protein) has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid such as, for example, DNA or RNA (eg structured RNA (eg ribozyme)).

In a preferred embodiment, the test compound (potential interactor or candidate interactor) is a molecular species that is likely to interact with the target (eg, target protein). Preferably, the test compound is a candidate substrate or test ligand.

In a preferred embodiment, the test compound is one of the members in a library containing multiple members with common properties. For example, all of the plurality of members are enzyme cofactors, or carbohydrates, nucleosides / nucleotides,
Substrates of, for example, biosynthetic or degradative enzymes, including amino acids and lipids (eg,
It is a substrate for proteolytic enzymes), or is a vitamin, hormone, nucleic acid (eg, DNA molecule), or natural product (eg, natural product derived from bacteria). The library includes metabolites, precursors, or intermediates of the above members.

In a preferred embodiment, the test compound is one of the members of the library selected from the group consisting of: Substrate libraries, cofactor libraries, carbohydrate biosynthesis and / or degradation libraries, purine and pyrimidine biosynthesis and / or degradation libraries, amino acid biosynthesis and / or degradation libraries, lipid biosynthesis And / or degradation libraries, vitamin and / or hormone libraries, nucleic acid (eg DNA) libraries, or natural product libraries (eg bacterial derived natural product libraries).

In a preferred embodiment, the test compound is one of the members of the library selected from the group consisting of: That is, enzyme substrates, metabolites, cofactors, natural products (for example, natural products derived from bacteria), carbohydrates, polysaccharides, nucleic acids (for example,
Nucleoside or nucleotide precursors, double-stranded or single-stranded DNA molecules, circular nucleic acids, supercoiled nucleic acids, etc.), amino acids (eg D- or L-amino acids or their precursors), vitamins, hormones, lipids, organic compounds Molecules, metals, peptides, proteins, lipids, glycoproteins, glycolipids, transition state analogs, and combinations thereof.

In a preferred embodiment, the test compound is a member of a combinatorial library.

In a preferred embodiment, the target interacts with, eg, binds to, and preferably modifies, the test compound. As used herein, “modifying” includes producing or cleaving a bond (eg, non-covalent bond or covalent bond) in a test compound or target. "Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A “modification” may be a change in activity (eg enzymatic activity), a physical change in phase, a change in association or polymerization.

In a preferred embodiment, the method further comprises the following steps. That is, the step of analyzing the structure or function of the test compound or modified test compound, for example, analyzing the physical properties of the test compound or modified test compound, or analyzing the in vitro or in vivo activity of the test compound or modified test compound. Including.

In a preferred embodiment, the method further comprises the following steps. That is, a test compound or modified test compound is selected, contacted with a cell (for example, a cultured cell or animal), and whether the test compound or modified test compound selectively exerts an effect on the cell or animal is determined. Including the step of examining.

In a preferred embodiment, the method further comprises the following steps. That is, (for example, based on the interaction with the target) selecting a test compound or modified test compound, purifying the test compound or modified test compound, crystallizing the test compound or modified test compound, the test compound or The step of assessing the physical properties of the modified test compound (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure) is included.

In a preferred embodiment, the method further comprises the following steps. That is, the step of purifying the test compound or modified test compound (eg, based on the interaction with the target), optimizing the properties of the test compound or modified test compound (eg, optimizing the affinity for the target, the molecular weight Altering (eg, reducing molecular weight), changing solubility (eg, increasing solubility). Optimization can be performed using known methods or the methods disclosed herein.

[0100]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises the step of determining a physical constant (eg, k _cat , K _M , or K _D ) for the interaction between the target and the test compound or modified test compound.

In a preferred embodiment, the method can include the use of a binding reaction (eg, a surrogate ligand as described elsewhere herein).

In another aspect, the invention features a method of comparing two interactants (eg, ligands) (eg, comparing the original ligand structure and the modified structure).
The interacting substances can be compared with respect to, for example, the ability to interact with the target, for example, the ability to bind to the target or the ability to modify the target. The method comprises the steps of: (a) providing a target, (b) contacting the target with a first interacting agent (eg, a first ligand) and providing a reaction mixture, c) measuring a change in heat in the reaction mixture, (d) providing a modified interactant (eg, a modified ligand, ie, a change in one or more sites in the ligand molecule), e) contacting the target with a modified interactor (eg, a modified ligand) to provide a reaction mixture, (f) measuring the change in heat in the reaction mixture in (e), and (g) (c). And comparing the measurements in (f), thereby comparing the two interactants or ligands (eg, the original structure and the modified structure).

In a preferred embodiment, these steps may be performed in any order. For example, it is possible to first carry out the steps (a to c) and then the steps (d to f), or in another preferred embodiment, the steps (a to c) and (d to f). ) Stage,
It is also possible to carry out at exactly the same time or partly at the same time.

In a preferred embodiment, the target (eg, protein) is produced by a pathogen, including a bacterium, protozoa, virus (eg, phage), or fungus, eg, a prokaryotic or eukaryotic pathogen. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii, Bacillus subtilis, Treponema pallidum,
Borrelia burgdorferi, Helicobacter pylori, Archaeoglobus fulg
idus), Methanobacterium thermo, E. coli (Es
cherichia coli), Mycoplasma pneumoniae,
Synechocystis sp., Methanococcus janas (Methanoc)
occus jannaschii), Saccharomyces cerevisiae
), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia prow
azekii), Pyrococcus abyssii, Bacillus
) Sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitides strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg, protein) is an organelle of an organism (
(Eg, mitochondria).

In a preferred embodiment, the target (eg, protein) has no known activity (eg, enzymatic activity) or has an activity that is difficult to measure.

In a preferred embodiment, the target is a naturally occurring protein or fragment thereof, a protein having an unknown function and / or structure, a ligand, a substrate, or a protein in which other molecules with which it interacts are unknown, etc. .. In other embodiments, the target (eg, protein) has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid such as, for example, DNA or RNA (eg structured RNA (eg ribozyme)).

In a preferred embodiment, the method further comprises repeating one or more steps with different conditions (eg different salt concentrations, different pH, or in the presence of different cofactors).

In a preferred embodiment, the target interacts with, eg, binds, the interacting agent,
It is preferably modified. As used herein, “modifying” includes producing or cleaving a bond (eg, non-covalent bond or covalent bond) in a test compound or target. "Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A “modification” may be a change in activity (eg enzymatic activity), a physical change in phase, a change in association or polymerization.

In a preferred embodiment, the method further comprises the following steps. That is, the step of analyzing the structure or function of the interacting substance, for example, analyzing the physical property of the interacting substance, and analyzing the in vitro or in vivo activity of the interacting substance.

In a preferred embodiment, the method further comprises the following steps. That is, selecting an interacting agent (eg, based on interaction with a target), contacting it with a cell (eg, a cultured cell or animal), and selectively allowing the interacting agent to interact with the cell or animal. It includes the step of checking whether or not the effect is exhibited.

In a preferred embodiment, the method further comprises the following steps. That is, the interacting agent is selected (eg, based on the interaction with the target), and the interacting agent and the physical properties of the interacting agent (eg, molecular weight, isoelectric point, sequence (if applicable), crystal, etc.) Structure).

In a preferred embodiment, the method further comprises the following steps. That is, for example, the interacting substance is purified based on the interaction with the target, and the properties of the interacting substance are optimized (eg, the affinity for the target is optimized, the molecular weight is changed (
For example, reducing molecular weight) and changing solubility (eg, increasing solubility)). Optimization can be performed using known methods or the methods disclosed herein.

[0117]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises the step of determining a physical constant (eg, k _cat , K _M , or K _D ) for the interaction between the protein and the interactant.

In a preferred embodiment, the method can include the use of a binding reaction (eg, a surrogate ligand described elsewhere herein).

In another aspect, the invention features a method of comparing a test molecule with a modification product thereof (eg, comparing an original structure and a structure after modification). The method comprises the steps of: (a) providing a test molecule, (b) allowing the test molecule to interact with a second molecule (eg, binding to a ligand), or the first of the test molecules. A step of interacting a part with a second part of the test molecule, (c) a step of measuring a change in heat associated with the interaction, (d) a modified test molecule having a change at one or more positions in the molecule Providing, (e) allowing the modified test molecule to interact with a second molecule (eg, binding to a ligand)
Or, interacting a first portion of the test molecule with a second portion of the test molecule,
(F) measuring the heat change associated with the interaction of (e), and comparing the measurements of (g), (c) and (f), thereby comparing the test molecule with its modification product.

In a preferred embodiment, these steps may be performed in any order. For example, it is possible to first carry out the steps (a to c) and then the steps (d to f), or to carry out the steps (a to c) and (d to f) at the same time or It is also possible to carry out partially simultaneously.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a modifying molecule that interacts with the target, and in a second test, that the modifying molecule interacts (eg, binds) with the target (eg, in the absence of a surrogate modified molecule) Including the step of confirming.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a modifying molecule that interacts with the target, and contacting the modifying molecule with the target in vitro (eg, in the absence of a surrogate modifying molecule) allows the modifying molecule to interact with the target. Confirming (eg, binding).

In a preferred embodiment, the method further comprises the following steps. That is, selecting a modifying molecule that interacts with a target, and contacting the ligand with a cell (for example, a cultured cell, or an animal), and examining whether or not the ligand selectively exerts an effect on the cell or the animal. Including stages.

In a preferred embodiment, the method further comprises the following steps. That is, it includes the steps of purifying the test ligand, crystallizing the test ligand, and evaluating the physical properties of the test ligand (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure).

In a preferred embodiment, the method further comprises the following steps. That is, a step of optimizing the properties of the selected test ligand (eg, optimizing affinity for a target, changing molecular weight (eg, reducing molecular weight), changing solubility (eg, increasing solubility)). Including. Optimization can be performed using known methods or the methods disclosed herein.

[0127]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In another aspect, the invention features a method of analyzing a compound (eg, protein or nucleic acid, eg, structured RNA, or other target).
The method comprises the following steps: (a) providing a reaction mixture containing the target, (b) interacting or in the absence of an acting molecule (eg, interacting with a ligand (or library member)). Does not occur, or in the absence of a ligand (or library member)), including a structural change in the target, (c) measuring the change in heat that accompanies the structural change, (d) A step involving conformational change of the target in the presence of the interaction or the acting molecule (eg, the interaction with the ligand (or library member) occurs, or in the presence of the ligand (or library member)), (E) measuring the change in heat that accompanies the structural change, and (g) comparing the value obtained in (c) with the value obtained in (e), This is the step of analyzing the target.

In a preferred embodiment, a denaturing agent (eg, guanidine hydrochloride, urea, or similar agent) is added to the reaction mixture.

[0130]   In a preferred embodiment, the measurement is carried out using a microcalorimeter.

In a preferred embodiment, the method further comprises the following steps. That is, (a) providing a reaction mixture containing a target, (b) in the absence of an interaction or an acting molecule (eg, no interaction with a ligand, or in the absence of a ligand), the reaction mixture Heating (c), for example, measuring the change in heat that accompanies the structural change to obtain an apparent specific heat output, (d) in the presence of an interaction or an acting molecule (eg, The interaction with the ligand takes place, or in the presence of the ligand), the step of heating the reaction mixture, (e) the change in heat that accompanies the structural change, for example to obtain an apparent specific heat output. Measuring and comparing the values obtained in (g) (c) with the values obtained in (e), thereby analyzing the target, eg, for examining the binding of the ligand to the target.

In a preferred embodiment, the target is a protein or polypeptide, a nucleic acid (eg RNA). This may be purified, partially purified, or crude.

In a preferred embodiment, a denaturing agent (eg, guanidine hydrochloride, urea, or similar agent) is added to the reaction mixture.

In a preferred embodiment, one or more conditions (eg, denaturant) are provided such that the presence of a ligand that binds to the relatively more compact folded state results in a relatively large change in heat. And target concentration, or temperature). This is done, for example, by allowing the target molecule to assume a folded state.

[0135]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In a preferred embodiment, the target (eg, protein) is produced by a pathogen, including a bacterium, protozoa, virus (eg, phage), or fungus, eg, a prokaryotic or eukaryotic pathogen. For example, the protein can be a protein produced by any of the following species: Aquifex aeolicus, Pyrococcus horikoshii, Bacillus subtilis, Treponema pallidum,
Borrelia burgdorferi, Helicobacter pylori, Archaeoglobus fulg
idus), Methanobacterium thermo, E. coli (Es
cherichia coli), Mycoplasma pneumoniae,
Synechocystis sp., Methanococcus janas (Methanoc)
occus jannaschii), Saccharomyces cerevisiae
), Mycoplasma genitalium, Haemophilus influenzae, Rickettsia prow
azekii), Pyrococcus abyssii, Bacillus
) Sp., Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aeruginosa
erophilum), Pyrococcus furiosus, Mycobacterium tuberculosis (Myc)
obacterium tuberculosis), Neisseria gonorhea,
Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi (Bo)
rellia burgdorferi), Caulobacter crescentu
s), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enter)
ococcus faecalis), Legionella pneumophila,
Mycobacterium avium, Mycobacteriu
m tuberculosis), Methanococcus jannaschii
, Neisseria meningitides, Pseudomonas putida, Porphyromonas gingivalis (Porphyro)
monas gingivalis), Salmonella typhimurium
, Shewanella putrefaciens, Streptococcus pneumoniae, Vibrio cholerae, Clostridium acetobutylicum, Campylobacter jejunin, Halobacterium Institute), Listeria monocytogenes, Mycobacterium tubercul
osis Sanger), Mycoplasma mycoides, Neisseria meningitides strain, Streptomyces
Streptomyces coelicolor, Actinobacillus actinomyce, Chlamydia trachomatis
matis), Halobacterium sp., Mycoplasma capricolum, Neisseria gonorrhea
), Pseudomonas aeruginosa, Aspergillus nidulans (Asper)
gillus nidulans), Candida albicans, Leishmania major, Neurospora crassa, Pneumocystis carinii, Plasmodium falciparum (Plasm)
odium falciparum), Saccharomyces cerevisiae
), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
brucei), Abelson murine leukemia virus, adeno-associated virus
Dengue virus 2 or 3, type 1, type 2, or type 3, hepatitis A virus, hepatitis GB
Virus B, human T lymphotropic virus type 1 or 2, human T lymphotropic virus type I, human adenovirus type 12 or 2, human herpesvirus 1-4, human immunodeficiency virus 1-2 Type, human parainfluenza virus 3, human respiratory system inclusion virus, infectious hematopoietic necrosis virus, influenza A virus,
Influenza B virus, influenza C virus, and measles virus. Further examples of species producing targets tested using the methods of the invention are described below.

In a preferred embodiment, the target (eg protein) is eukaryotic (eg
Unicellular or multicellular organisms). Examples of such eukaryotes include Arabidopsis thaliana M, and the Brugia mala.
yi), nematode (Caenorhabditis elegans), Drosophila
melanogaster), Shistosoma mansoni, Shistosoma
Examples include japonicum (Shistosoma japonicum), and mammals (eg, humans). Preferably the target is produced by humans.

In a preferred embodiment, the target (eg, protein) is an organelle of an organism (
(Eg, mitochondria).

In a preferred embodiment, the target (eg, protein) has no known activity (eg, enzymatic activity) or has an activity that is difficult to measure. In a preferred embodiment, the target (eg, protein) has a known first activity and is second to the library containing interactants that interact with the protein.
The protein is tested for its activity (eg, unknown activity).

In a preferred embodiment, the target is a naturally occurring protein or fragment thereof, a protein with unknown function and / or structure, a ligand, a substrate, or a protein with which other molecules with which it interacts are unknown, etc. . In other embodiments, the target (eg, protein) has at least one enzymatic activity.

In a preferred embodiment, the target is a nucleic acid such as, for example, DNA or RNA (eg, structured RNA (eg, ribozyme)).

In a preferred embodiment, multiple members of the library are tested simultaneously. For example, if it is possible to increase the throughput of the process, the same reaction mixture is used. If the result is positive, multiple members of the library can be divided into smaller groups and each group tested. If the result is positive, one or more of the members in the library or one or more members from the grouped subgroups can be tested individually.

In a preferred embodiment, the method further comprises repeating one or more steps under different conditions (eg different salt concentrations, different pH, or in the presence of different cofactors).

In a preferred embodiment, the method further comprises repeating the first member then the second member of the library, or the next member or other members. In a preferred embodiment, testing is performed on multiple members of the library (eg, candidate substrates, or test ligands). In a preferred embodiment, the plurality of said members of the library are
It comprises at least 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ compounds. In a preferred embodiment, at least 10, 10 ² , 10 ³ constituting the library,
10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ members have the same structural and functional characteristics.

In a preferred embodiment, the library comprises multiple members with common characteristics. For example, all of the plurality of members are enzyme cofactors or include carbohydrates, nucleosides / nucleotides, amino acids, lipids, eg, biosynthetic or degradative enzyme substrates (eg, proteolytic enzyme substrates). ,
Alternatively, it is a vitamin, hormone, nucleic acid (eg, DNA molecule), or natural product (eg, natural product of bacterial origin). The library includes metabolites, precursors, or intermediates of the above members.

In a preferred embodiment, the library is a substrate library, a cofactor library, a carbohydrate biosynthesis and / or degradation library, a purine and / or purine and / or degradation library.
Alternatively, a pyrimidine biosynthesis and / or degradation library, an amino acid biosynthesis and / or degradation library, a lipid biosynthesis and / or degradation library, a vitamin and / or hormone library, a nucleic acid (eg, DNA
) Library or natural product library (for example, natural product library derived from bacteria).

In a preferred embodiment, members of the library (potential or candidate interactors) are molecular species that are likely to interact with the target (eg, target protein). Preferably, the members of the library are candidate substrates or test ligands.

In a preferred embodiment, the members of the library are selected from the group consisting of: That is, enzyme substrates, metabolites, cofactors, natural products (eg, natural products from bacteria), carbohydrates, polysaccharides, nucleic acids (eg, nucleoside or nucleotide precursors, double-stranded or single-stranded DNA molecules, circular nucleic acids , Supercoiled nucleic acids, etc.), amino acids (eg D- or L-amino acids or their precursors), vitamins, hormones, lipids, small organic molecules, metals, peptides, proteins, lipids, glycoproteins, glycolipids, transition states Analogs, and combinations thereof.

In a preferred embodiment, the method further comprises testing the protein against at least one member of the second library.

[0150]   In a preferred embodiment, two or more types of libraries are
To test. As an example, target the first library (eg, cofactor library
Second library, tested against each (or more) members of
Each (or more) member of (eg, potential substrate library)
Test against. Thus, the first library has 50 members (first₁,
First₂, ... 1st₅₀) And the second library has 50 members (second₁,No. 2₂,.
..No. 2₅₀When using a library with ...
Or multiple new combinations (eg (first₁,No. 2₁), (First₁,No. 2₂) ... (first ₁ ,No. 2₅₀)))).

In a preferred embodiment, the members of the library are members of a combinatorial library.

In a preferred embodiment, the target interacts with, eg, binds to, and preferably modifies members of the library. As used herein, “modifying” includes producing or cleaving a bond (eg, non-covalent bond or covalent bond) in a test compound or target. "Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A “modification” may be a change in activity (eg enzymatic activity), a physical change in phase, a change in association or polymerization.

In a preferred embodiment, the method further comprises the following steps. That is, the step of analyzing the structure or function of the members of the library, eg, analyzing the physical properties of the target, analyzing the in vitro or in vivo activity of the members of the library.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a member of the library (eg, selecting a candidate substrate or test ligand based on interaction with the target) and confirming that the candidate substrate or test ligand is the substrate or ligand, respectively. Including.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a member of the library based on its interaction with a target, contacting it with a cell (eg, a cultured cell or animal), and selectively allowing the member of the library to effect the cell or animal. Including whether to show or not.

In a preferred embodiment, the method further comprises the following steps. That is, an interactant (eg, a member of a library) based on its interaction with a target
, Purifying the library (eg, candidate substrate or test ligand), crystallizing a library member (eg, candidate substrate or test ligand), library member (eg, candidate substrate) Or the physical properties of the test ligand (eg, molecular weight, isoelectric point, sequence (if applicable),
Crystal structure) evaluation step.

In a preferred embodiment, the method further comprises utilizing members of the library selected to interact with the target. The purpose of its use is to identify agents that modulate the interaction between the target and the member selected from the library, for example by binding or interacting with the member selected from the library. .

In a preferred embodiment, the method further comprises the following steps. That is, an interactant (eg, a member of a library) based on its interaction with a target
, Purifying the library (eg, candidate substrate or test ligand), and optimizing the properties of the members of the library (eg, candidate substrate or test ligand) (eg, affinity for target) Optimize, change molecular weight (eg reduce molecular weight), change solubility (eg
Increase solubility))). Optimization can be performed using known methods or the methods disclosed herein.

[0159]   In a preferred embodiment, the change in heat output is measured with a microcalorimeter.

In a preferred embodiment, the method further comprises the step of determining a physical constant (eg, k _cat , K _M , or K _D ) for the interaction between the protein and members of the library.

In another aspect, the invention features a method of analyzing an interactant (eg, a substrate) (eg, discovering a target molecule that modifies the substrate). The method comprises the steps of: providing a reaction mixture that includes an interactant (eg, substrate), contacting the interactant with a candidate target, assessing a change in heat in the reaction mixture, Optionally, including the step of comparing the obtained value of the change in heat with a predetermined value, thereby analyzing the interactant (eg, discovering the target of the interactant).

In a preferred embodiment, the change in heat in the reaction mixture (eg, a change value greater than a predetermined value) identifies the interactant.

In a preferred embodiment, multiple candidate targets are tested. In preferred embodiments, the plurality of candidate targets is at least 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or
Contains 10 ⁸ candidate targets.

In a preferred embodiment, the target interacts with, eg, binds to, and preferably modifies a substrate. “Modifying” includes generating or cleaving a bond (eg, noncovalent bond or covalent bond) in a surrogate ligand (or a substance that generates a signal). "Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A "modification" may be a physical change in phase, an association or a change in polymerization.

In a preferred embodiment, the method further comprises the following steps. That is, it includes the steps of selecting a candidate target and confirming that the candidate target has modified the target.

In a preferred embodiment, the method further comprises the following steps. That is, the steps include selecting a candidate target, contacting the candidate target with a cell (eg, cultured cell, or animal), and selectively examining whether the candidate target has an effect on the cell or animal.

In a preferred embodiment, the method further comprises the following steps. That is, it includes the steps of purifying the candidate target, crystallizing the candidate target, and assessing the physical properties of the candidate target (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure).

In a preferred embodiment, the method further comprises the following steps. That is, optimizing the properties of the selected candidate target (eg, optimizing affinity for substrate,
Changing the molecular weight (eg decreasing the molecular weight), changing the solubility (eg increasing the solubility). Optimization can be performed using known methods or the methods disclosed herein.

[0169]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In another aspect, the invention features a method of analyzing a target (eg, analyzing the interaction between a target and a second substance). The method comprises the following steps: providing a reaction mixture containing a target, interacting (where the interaction is associated with a binding interaction (eg, a binding reaction) (eg, of a surrogate ligand)). Dissociation, or conformational change)), the product of the binding reaction to a second reaction (eg cleavage or decomposition of the surrogate ligand).
And the step of measuring the change in heat in the second reaction. This allows analysis of target interactions.

In a preferred embodiment, the coupling reaction or the second reaction can involve a phase change.

In a preferred embodiment, another reaction or reactions can be inserted between the coupling reaction and the second reaction.

In a preferred embodiment, the interaction of the target is, for example, the interaction of the target with another molecule (eg, a ligand or a solute molecule), or a first portion of the target and a second portion of the target. Interactions (eg, autophosphorylation, or target structure (
For example, changes in target secondary structure, tertiary structure, quaternary structure)).

In a preferred embodiment, the reaction mixture is not transparent, the reaction mixture is colored, or the reaction mixture is cloudy and the reaction mixture is a substance that inhibits fluorescence or colorimetric detection. , Or the reaction mixture is not a pure solution and contains, for example, a product other than the target. In a preferred embodiment, the reaction mixture comprises a substance that inhibits radiometric analysis, a substance that inhibits spectroscopic analysis (eg, NMR analysis).

[0175]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In another aspect, the invention features a method of analyzing a test ligand, a target, or an interaction between the two. The method comprises the steps of: providing a reaction mixture comprising a surrogate ligand and a target, contacting the reaction mixture with a surrogate ligand and a signal generator, wherein:
The signal generator is allowed to co-exist with the surrogate ligand under conditions capable of interacting with the surrogate ligand (eg, dissociating the surrogate ligand from the target by binding the test ligand to the target), and changing the heat in the reaction mixture. Including the step of measuring. This allows analysis of the test ligand, the target, or the interaction between the two.

In a preferred embodiment, the surrogate ligand exhibits negative ectopic binding with respect to the test ligand capable of binding the target (ie, dissociation upon binding of the test ligand to the target). This is the preferred embodiment and is the subject of the description herein. However, the present invention also includes a mode in which the surrogate ligand binds to the target depending on the test ligand, thereby decreasing the concentration of the surrogate ligand and decreasing the surrogate ligand interacting with the signal generating substance.

[0178]   In a preferred embodiment, the interaction between the signal generator and the surrogate ligand is
Between signal generator and free surrogate ligand (relative to bound target)
Occurs more easily at. As an example, signal generators and (target binding
Interactions between free surrogate ligands (for
Between the free surrogate ligands (relative to those bound to
10 times, 10²Double, 10³Double, 10^FourDouble, 10^FiveDouble, 10⁶Double, 10⁷Double or 10⁸Double, more waking
It is easy to get. However, the present invention provides a method for
Surrogate ligation that interacts with the signal generator and the target (as opposed to free)
It also includes a mode that is more likely to occur between hands. As an example, the signal product
The interaction between the quality and the surrogate ligand bound to the target is dependent on the signal generator and surrogate ligand.
The interaction between the probe and the signal generator and the target (relative to the free one).
At least 2 times, 5 times, 10 times, 10²Double, 10³Double, 10^FourDouble, 10^FiveDouble, 10 ⁶ Double, 10⁷Double or 10⁸Twice more likely to happen.

In a preferred embodiment, the surrogate ligand is an ion, eg a proton. In a preferred embodiment, the signal generating substance is a buffer molecule, eg, a buffer molecule having a relatively high heat of ionization (eg, Tris-HCl).

In a preferred embodiment, the surrogate ligand is an agent that modulates (eg, increases or decreases) the activity of the signal generator. As an example, the surrogate ligand may be a metal ion that activates (or inhibits) the enzyme that is the signal generator.

In a preferred embodiment, the signal generator interacts with (eg, binds to) a surrogate ligand (eg, free surrogate ligand), and preferably modifies it. "
"Modify" means to bind (in a surrogate ligand (or signal generator))
For example, generating or cleaving (non-covalent bond or covalent bond). "
"Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A "modification" may be a physical change in phase, an association or a change in polymerization.

In a preferred embodiment, the target is a protein or polypeptide, the surrogate ligand is a nucleic acid, and the signal generator is an enzyme (eg, a nuclease) that cleaves a nucleic acid bond.

In a preferred embodiment, the method further comprises the following steps. That is, it includes the step of selecting a ligand that interacts with the target, and the step of confirming that the ligand interacts with, for example, binds to the target in the second test (for example, in the absence of surrogate ligand).

In a preferred embodiment, the method further comprises the following steps. That is, selecting a test ligand that interacts with the target, and contacting the ligand with the target in vitro (eg, in the absence of a surrogate ligand) causes the test ligand to interact with the target, For example, the step of confirming that the binding is included.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a ligand that interacts with the target, and
Contacting with cultured cells or animals), and optionally determining whether or not the ligand has an effect on the cells or animals.

In a preferred embodiment, the method further comprises the following steps. That is, the steps of purifying the test ligand, crystallizing the test ligand, and evaluating the physical properties of the test ligand (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure). Including.

In a preferred embodiment, the method further comprises the following steps. That is, a step of optimizing properties of the selected test ligand (eg, optimizing affinity for a target, changing molecular weight (eg, reducing molecular weight), changing solubility (eg, increasing solubility)) including. Optimization can be performed using known methods or the methods disclosed herein.

In a preferred embodiment, the surrogate ligand (eg, certain surrogate ligands,
For example, the nucleic acid) is amplified before it interacts with the signal generating substance, for example by PCR or, more preferably, using a method of amplification under isothermal conditions.

[0189]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In a preferred embodiment, the signal generator interacts directly with the surrogate ligand. In another aspect, they interact indirectly. For example, it interacts with amplification products generated from surrogate ligands. Alternatively, it acts on the product of the reaction between the surrogate ligand and another substance.

In another aspect, the invention features a method of analyzing a target, a test ligand, or an interaction between them. The method comprises the steps of: providing a reaction mixture comprising a surrogate ligand and the target, contacting the reaction mixture with a test ligand and a signal generator, where:
The signal generator measures the change in heat in the surrogate ligand (eg, the test ligand that dissociates from the target due to binding of the test ligand) under conditions that allow it to interact with the free surrogate ligand, and the reaction mixture. Including stages. This allows analysis of the test ligand, the target, or the interaction between the two.

In a preferred embodiment, the surrogate ligand exhibits negative ectopic binding with respect to the test ligand capable of binding the target (ie, dissociation upon binding of the test ligand to the target). This is the preferred embodiment and is the subject of the description herein. However, the present invention also includes a mode in which the surrogate ligand binds to the target depending on the test ligand, thereby reducing the concentration of the free surrogate ligand and decreasing the surrogate ligand interacting with the signal generating substance.

In a preferred embodiment, the interaction between the signal generator and the surrogate ligand is
It is more likely in the case between the signal generator and the free surrogate ligand (relative to the target bound). As an example, the interaction between the signal generator and the free surrogate ligand (relative to the target bound) is at least 2-fold between the signal generator and the free surrogate ligand (relative to the target bound), 5 times, 10
Double, 10 ² times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, 10 ⁶ times, 10 ⁷ times, or 10 ⁸ times more likely to occur. However, the present invention also includes embodiments in which the interaction between the signal generator and the surrogate ligand is more likely to occur between the signal generator and the surrogate ligand bound to the target (as opposed to free). As an example, the interaction between the signal generator and the surrogate ligand bound to the target is the interaction between the signal generator and the surrogate ligand between the signal generator and the surrogate ligand bound to the target (relative to the free one), at least 2-fold, 5-fold, 10-fold, 10 ^twice, 10 ^three times, 10 ⁴ times, 10 ⁵ times, 10 ⁶
Double, 10 ⁷ times, or 10 ⁸ times more likely to occur.

In a preferred embodiment, the signal generator interacts with, eg, binds, preferably modifies, the free surrogate ligand. “Modifying” includes producing or cleaving a bond (eg, non-covalent bond or covalent bond) in a surrogate ligand (or signal generator). "Modification" includes cleavage, degradation, hydrolysis, changes in the degree of phosphorylation labeling, ligation, synthesis, and similar reactions. A "modification" may be a physical change in phase, an association or a change in polymerization.

[0195]   In a preferred embodiment, the signal generator is a degrading enzyme.

In a preferred embodiment, the surrogate ligand is a nucleic acid and the signal generator is an enzyme that modifies the nucleic acid or an enzyme that uses the nucleic acid as a substrate or template. For example, a signal generator may be an enzyme (eg, nuclease, (eg, DNAase, (eg, endonuclease or exonuclease)), polymerase (eg, DN).
A polymerase),)). Alternatively, the signal generating agent modifies the protein (eg, in the surrogate ligand (or the agent itself) by creating or cleaving covalent or non-covalent bonds (eg, cleaving peptide bonds (eg, cleaving peptide bonds)). Is a protease))), the surrogate ligand comprises a peptide bond (eg, a protein).

In a preferred embodiment, the method further comprises the following steps. That is, selecting a ligand that interacts with the target, and confirming that the test ligand interacts with, eg, binds to, the target in a second test (eg, in the absence of a surrogate ligand) .

In a preferred embodiment, the method further comprises the following steps. That is, selecting a test ligand that interacts with the target, and contacting the test ligand with the target in vitro (eg, in the absence of a surrogate ligand) causes the ligand to interact with the target, For example, the step of confirming that the binding is included.

In a preferred embodiment, the method further comprises the following steps. That is, the step of selecting a test ligand that interacts with the target, and
For example, the step of contacting with a cultured cell or animal), and the step of selectively examining whether or not the test ligand has an effect on the cell or animal.

In a preferred embodiment, the method further comprises the following steps. That is, the steps of purifying the test ligand, crystallizing the test ligand, and evaluating the physical properties of the test ligand (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure). Including.

In a preferred embodiment, the method further comprises the following steps. That is, a step of optimizing properties of the selected test ligand (eg, optimizing affinity for a target, changing molecular weight (eg, reducing molecular weight), changing solubility (eg, increasing solubility)) including. Optimization can be performed using known methods or the methods disclosed herein.

In a preferred embodiment, the reaction mixture is not transparent, the reaction mixture is colored, or the reaction mixture is cloudy and the reaction mixture is a substance that inhibits fluorescence or colorimetric detection. , Or the reaction mixture is not a pure solution and contains, for example, products other than the target. In a preferred embodiment, the reaction mixture comprises a substance that inhibits radiometric analysis, a substance that inhibits spectroscopic analysis (eg, NMR analysis).

In a preferred embodiment, the surrogate ligand (eg, nucleic acid) is amplified prior to interacting with the signal generating agent, eg, by PCR, or, more preferably, using methods that amplify under isothermal conditions. To do.

[0204]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

In a preferred embodiment, the signal generator interacts directly with the surrogate ligand. In another aspect, they interact indirectly. For example, it interacts with amplification products generated from surrogate ligands. Alternatively, it acts on the product of the reaction between the surrogate ligand and another substance.

In another aspect, the invention features a method of analyzing a target, a test ligand, or an interaction between them. The method comprises the steps of: providing a reaction mixture comprising a surrogate ligand that is a nucleic acid and the target, the reaction mixture comprising a test ligand and a signal generator (wherein the signal generator is in the surrogate ligand, Contacting with a bond (eg, a molecule that produces or cleaves a bond (eg, covalent or non-covalent)), where the signal generator is a surrogate ligand (eg, a surrogate ligand) under conditions capable of interacting with a free surrogate ligand. , Test ligand that dissociates from the target due to binding of the test ligand)))
, And measuring the change in heat in the reaction mixture. This allows analysis of the test ligand, the target, or the interaction between the two.

[0207] In a preferred embodiment, the surrogate ligand exhibits negative ectopic binding with respect to the test ligand capable of binding the target (ie, dissociation upon binding of the test ligand to the target).

In a preferred embodiment, the interaction between the signal generator and the surrogate ligand is
It is more likely in the case between the signal generator and the free surrogate ligand (relative to the target bound). As an example, the interaction between the signal generator and the free surrogate ligand (relative to the target bound) is at least 2-fold between the signal generator and the free surrogate ligand (relative to the target bound), 5 times, 10
Double, 10 ² times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, 10 ⁶ times, 10 ⁷ times, or 10 ⁸ times more likely to occur.

[0209]   In a preferred embodiment, the signal generator is a degrading enzyme.

In a preferred embodiment, the signal generating substance is an enzyme that modifies a nucleic acid or an enzyme that uses a nucleic acid as a substrate or template. For example, the signal-generating substance is, for example, a nuclease (eg, endonuclease or exonuclease),
An enzyme such as a polymerase (eg, DNA polymerase).

In a preferred embodiment, the method further comprises the following steps. That is, a step of selecting a test ligand that interacts with the target, and a step of confirming that the test ligand interacts with the target in the second test (for example, a condition in which a surrogate ligand does not coexist), for example, binding is performed. Including.

In a preferred embodiment, the method further comprises the following steps. That is, selecting a test ligand that interacts with the target, and contacting the test ligand with the target in vitro (eg, in the absence of a surrogate ligand) causes the ligand to interact with the target, For example, the step of confirming that the binding is included.

In a preferred embodiment, the method further comprises the following steps. That is, the step of selecting a test ligand that interacts with the target, and
For example, the step of contacting with a cultured cell or animal) and the step of selectively examining whether or not the ligand has an effect on the cell or animal are included.

[0214] In a preferred embodiment, the method further comprises the following steps. That is, the steps of purifying the test ligand, crystallizing the test ligand, and evaluating the physical properties of the test ligand (eg, molecular weight, isoelectric point, sequence (if applicable), crystal structure). Including.

In a preferred embodiment, the method further comprises the following steps. That is, a step of optimizing properties of the selected test ligand (eg, optimizing affinity for a target, changing molecular weight (eg, reducing molecular weight), changing solubility (eg, increasing solubility)) including. Optimization can be performed using known methods or the methods disclosed herein.

In a preferred embodiment, the surrogate ligand is amplified prior to interacting with the signal generating substance, eg by PCR or, more preferably, using methods that amplify under isothermal conditions.

[0217]   In a preferred embodiment, the change in heat is measured with a microcalorimeter.

[0218] In a preferred embodiment, the signal generator interacts directly with the surrogate ligand. In another aspect, they interact indirectly. For example, it interacts with amplification products generated from surrogate ligands. Alternatively, it acts on the product of the reaction between the surrogate ligand and another substance.

In another aspect, the invention features a library of candidate interactors, eg, a candidate substrate or test ligand library described herein. The method includes the following steps. Preferably, the library comprises at least one member known to interact with the target.

In a preferred embodiment, the library is a substrate library, a cofactor library, a carbohydrate biosynthesis and / or degradation library, a purine and / or purine and / or degradation library.
Alternatively, a pyrimidine biosynthesis and / or degradation library, an amino acid biosynthesis and / or degradation library, a lipid biosynthesis and / or degradation library, a vitamin and / or hormone library, a nucleic acid (eg, DNA
) Library or natural product library (for example, natural product library derived from bacteria).

In a preferred embodiment, the members of the library are molecular species capable of interacting with the target, eg proteins. Preferably, the members of the library are candidate substrates or test ligands.

In a preferred embodiment, the test compound is one of the members of the library selected from the group consisting of: That is, enzyme substrates, metabolites, cofactors, natural products (for example, natural products derived from bacteria), carbohydrates, polysaccharides, nucleic acids (for example,
Nucleoside or nucleotide precursors, double-stranded or single-stranded DNA molecules, circular nucleic acids, supercoiled nucleic acids, etc.), amino acids (eg D- or L-amino acids or their precursors), vitamins, hormones, lipids, organic compounds Molecules, metals, peptides, proteins, lipids, glycoproteins, glycolipids, transition state analogs, and combinations thereof.

[0223] In a preferred embodiment, the library comprises multiple members having common characteristics. For example, all of the plurality of members are cofactors for the enzyme or include carbohydrates, nucleosides / nucleotides, amino acids, fats, etc. A vitamin, hormone, nucleic acid (eg, DNA molecule), or natural product (
For example, natural products derived from bacteria). The library includes metabolites, precursors, or intermediates of the above members. The library can also include a combination of members with different properties. For example, the cofactor library can be combined with a biosynthetic or degrading enzyme substrate library.

In a preferred embodiment, the library is at least 10, 10 ² , 10 ³ , 10 ^4.
, 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ compounds.

[0225]   In a preferred embodiment, the libraries have the same structural or functional characteristics.
Have at least 10, 10²,Ten³,Ten^Four,Ten^Five,Ten⁶,Ten⁷, Or 10⁸Pieces of la
Includes Ibrary members. In another embodiment, the library is structurally
Alternatively, it may include a combination of members that share functional characteristics. For example,
The library has at least 10,10 identical structural or functional features. ² ,Ten³,Ten^Four,Ten^Five,Ten⁶,Ten⁷, Or 10⁸Different library members
At least 10,10 with structural or functional characteristics²,Ten³,Ten^Four,Ten^Five,Ten⁶,Ten⁷ , Or 10⁸It may contain up to 8 library members.

In a preferred embodiment, two or more libraries are tested simultaneously against a single target. As an example, the target is tested against a member of each (or more) of the first library, eg, the cofactor library, and the second library, eg, each (or more) of the potential substrate libraries. Test against members of. Thus, the first library has 50 members ( ₁
The first _2, has a ~ first _50), the second library 50 kinds of members (second _1, second _2,
~ 2 ₅₀ ), the target may be all combinations or multiple novel combinations, such as (1 ₁ and 2 ₁ ), (1 ₂ 2 _{2 2} ) (No. 1 ₅₀ ,
The test will be conducted for the second ₅₀ ) etc.

Other features and advantages of the invention will be apparent from the description and claims that follow.

DETAILED DESCRIPTION Heat absorption or release is a universal property of chemical reactions. The methods described herein are directed to test compounds, or targets of interactants (eg, test ligands or candidate substrates) (eg, target macromolecules such as target proteins or nucleic acids).
It relates interactions with (eg, bonds) to changes in heat. The heat output is detected by a calorimeter. This allows analysis of interactions without imposing explicit restrictions on the type, extent, or special nature of the target's activity. By way of example, it may be possible to identify interacting substances, for example substances, with targets whose activity is unknown, poorly characterized, or only putative or roughly described. it can. The method of the invention occurs, for example, in the conversion of a test substance to a product when the target is an enzyme, or in binding when the target and interacting substances are ligands and counter-ligands. Detect changes in heat. In some embodiments of the invention, no assumptions are made about the nature of the target and its interaction with its interacting substances, such as, for example, natural product ligands, substances or binding partners. As another method of the present invention, a target (
(And / or interacting substances) are incorporated to guide in the selection of potential interacting substances. As an aspect of the present invention,
For example, in order to optimize and / or prioritize the selection of interactants for testing the target against the interactant, use of the target's genome, or other bioinformatic analysis.

Also included within the scope of the invention are candidate libraries of interactions, eg, libraries of candidate substrates or test ligands as described herein.
Constructs such as the methods and libraries of the present application can be used for diagnostic tests, research purposes, or for screening for agents such as drugs. Substances such as agents identified using the methods described herein are also within the scope of the invention.

[0230]   Certain terms are initially defined so that the invention may be more readily understood.

As used herein, “test compound”, also referred to as “interacting substance”
Are those types that have the potential to interact with a target, such as a target macromolecule (eg, protein or nucleic acid). The test compound can also be a candidate substrate or test ligand. Test compounds include, without limitation, small organic molecules, metals, peptides, proteins, lipids, glycoproteins, glycolipids, carbohydrates, polysaccharides, nucleic acids (eg nucleoside or nucleotide precursors, double-stranded DNA or single-stranded DNA molecules. , Cyclic nucleic acids, supercoiled nucleic acids), amino acids (eg D-amino acids or L-amino acids or their precursors), vitamins, hormones, enzyme substrates, metabolites, transition state analogues, cofactors, natural products (eg, Any substance, including natural products of bacterial origin), and combinations thereof. The library may contain a plurality of test compounds.

[0232] As used herein, a "mixture" or "reaction mixture" may be a complex combination of substances, such as suspensions, extracts of natural products, cell homogenates, cell lysates. , Or impure samples such as cell extracts, whole cells, reconstituted systems, biochemical mixtures, biological samples, tissue samples, biological fluids,
Alternatively, it may be a substance such as a stained solution, which may contain one or more test compounds.

As used herein, a “candidate substrate” is a substance that, when acted upon, produces a different chemical entity. Typical candidate substrates include, among others, enzyme substrates, metabolites, cofactors (eg, group transfer and energy coupled molecules), natural products such as those from bacteria, carbohydrates, polysaccharides such as, for example: Included are amino acids, vitamins, hormones, lipids such as nucleoside or nucleotide precursors, double-stranded DNA or single-stranded DNA molecules, eg D-amino acids or L-amino acids, or their precursors.

As used herein, a “test ligand” is one of a combinatorial library of drug candidates that is a library of compounds, such as fungal or fermentative products. It is derived from a library of natural compounds and a library of organic synthesis. In a preferred embodiment, the ligand is predetermined in, for example, a cDNA library, a specifically expressed cDNA library, a genomic library, a library created by expression characterization, or a predetermined tissue. Selected at the time of development, or in a predetermined disease or in the absence of the disease, by enriched species-expressed libraries, such as hybridization of ordered probes to a two-dimensional array. And a polypeptide expressed from a nucleic acid from a population of nucleic acids, such as a large number of nucleic acids, such as a library produced after that treatment of cells or organisms that have been treated with a drug or the like.

As used herein, a “library” is a collection of substances that may potentially interact with a target. Preferably, the library comprises at least one known to interact with the target.

As used herein, a “substrate library” is a target, eg, a protein, for which the function is unknown (herein “unknown”).
(Also referred to as) is a collection of compounds that can be screened for possible interactions such as enzyme activity. Interactions, such as enzyme activity, will result in changes in the heat output detected by the calorimeter.

As used herein, the term “target” refers to any molecule of interest. In a preferred embodiment, the target is a protein or polypeptide, such as a protein of natural origin or a fragment thereof, a protein of unknown function,
There are proteins for which the ligand, substrate, or other interacting molecule is unknown. The target may be a nucleic acid, eg DNA or RNA (
For example, structural RNAs such as ribozymes). Targets include molecules of known or unknown structure or function (eg, peptides, proteins or nucleic acids).
In a preferred embodiment, the target is a protein having no catalytic activity, a known catalytic activity, or a catalytic activity that is difficult to measure. Targets can be carbohydrates, polysaccharides, and glycoproteins, among others.

[0238]   As used herein, the term "surrogate ligand" refers to, for example, a target.
Refers to factors that interact with (eg, bind to) targets such as proteins. Surrogate
A ligand may be naturally associated with its target, or
You don't have to. In a preferred embodiment, the surrogate ligand is at least 10^-1 M ^-1 ,Ten^-2 M^-1,Ten^-Four M^-1,Ten^-6 M^-1,Ten^-8 M^-1,Ten^-Ten M^-1,Ten^-12 M^-1,Ten^-15 
M^-1,Ten^-20 M^-1,Ten^{-twenty five} M^-1,Ten^-30 M^-1K for the target_DWith. Preferably,
The surrogate ligand fulfills the following characteristics: (1) The surrogate ligand relates to the test ligand
Indicates a heterootropic linkage (ie, it
Must be displaced upon binding of the test ligand (negative heterotropic linkage
Binding) or binding to its target is possible with respect to the test ligand (positive binding).
Teloscopic linkage). ), And (2) its "free" or substituted
The surrogate ligand as a switch to generate an amplified signal
work. For example, the substituted surrogate ligand acts as a substrate for the enzyme. Preferably
Is a surrogate ligand with any surface or internal sequence or conformational region of the target.
Can interact (bind). In another embodiment, the surrogate ligand is the target
Can be catalytically altered or altered the functional activity of the target
You can Examples of natural surrogate ligands are anions, cations, protons, water
, Other solution phase components are also found in relation to the target. Non-natural surrogate Riga
Examples of proteins include synthetic proteins, peptides and nucleic acid sequences (eg DNA
Or RNA molecule).

The surrogate ligands of the invention interact with recognized functional regions of the target protein, such as, for example, enzyme active sites, antibody antigen binding sites, receptor hormone binding sites, cofactor binding sites ( For example, it is not limited to factors that bind).

In a preferred embodiment, the surrogate ligand is a nucleic acid molecule (also referred to herein as “surrogate nucleic acid ligand”). As used herein, "nucleic acid molecule"
The term refers to DNA, RNA, single or double strands thereof, and any chemical modifications. Typical modifications include, but are not limited to, those that provide the nucleic acid with other chemical groups that incorporate additional charge, polarizability, hydrogen bonding and electrostatic interactions. Such modifications include 2'-sugar modification, 5-position pyrimidine modification, 8
Position 3'and 5'modifications such as purine modification, exocyclic amine modification, 4-thiouridine substitution, 5-bromo-uracil or 5-iodo-uracil substitution, backbone modification, methylation, capping , But not limited to, base pair combinations such as the isobases isocytidine and isoguanidine.

As used herein, the term “surrogate nucleic acid ligand” refers to 2 to 40
Up to nucleotides, preferably from 10 to 30 nucleotides, more preferably
It includes nucleic acid molecules containing from 15 to 25 nucleotides, most preferably 20 nucleotides. Therefore, in a preferred embodiment, the surrogate ligand is an oligonucleotide. In one embodiment, surrogate nucleic acid ligands are identified using the SELEX process, as described in detail below; Gold et al., (1995) Annu. Rev.
Biochem. 64: 763-797, "Diversity of Olig
nucleotide functions); US Pat. No. 5,270,163, "Methods for Identifying Nucleic Acid Ligands", December 1993.
14-day registration, Gold et al., US Pat. No. 5,567,588, "Systemic Evolution of Ligands by E".
xponential Enrichment Solutions Selex) '', registered October 22, 1996, Gold (G
old) et al., U.S. Pat. No. 5,763,177, "Tissue systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution Selex (Systemic Evolution of Liga).
nds by Exponential Enrichment: Photoselection of Nucleic Acid Ligands an
d Solutions Selex), registered June 9, 1998, Gold et al .; US Pat. No. 5,87.
4,219, "Methods for detecting target compounds in substances using nucleic acid ligands (Meth
od for Detecting a Target Compound in a Substance Using a Nucleic Acid L
igand) ”, registered February 23, 1999, Drolet et al .; all of these contents are expressly incorporated herein by reference.

As used herein, the term "signal generator" is an entity that interacts with a surrogate ligand in a non-isothermal process, preferably an exothermic process. Preferably, the signal generator amplifies the signal generated by the test ligand. For example, the signal generator may interact with the surrogate ligand in a form that produces a signal, eg, heat output (eg,
Binding) and, preferably, the surrogate ligand may be modified. Typical signal generators are enzymes that undergo exothermic or endothermic reactions with surrogate ligands.

Preferably, the signal generator interacts more readily with the free surrogate ligand as opposed to the surrogate ligand bound to the target. In a particular embodiment, the signal generator modifies the free (as opposed to bound to the target) surrogate ligand by, for example, forming or cleaving covalent or non-covalent bonds. For example, the modification step may involve cleavage, degradation, phosphorylation, polymerization, or any other event that gives rise to a signal, such as a heat signal. The signal generator may be a degrading enzyme (eg, nuclease or protease).
Alternatively, the signal generator may be a polymerizing enzyme such as a polymerase. By way of example, in those embodiments, when the free surrogate ligand is a DNA molecule, the signal generator is staphylococcal nuclease (SNase), Serratia marcescens nuclease (SNase), bovine pancreatic nuclease (DNase I). ), Or a nuclease such as human (type IV) nuclease. Alternatively, the signal generator is, for example,
It may be a polymerase such as Taq polymerase. In those embodiments, when the free surrogate ligand is an RNA molecule, the signal generator may be a ribonuclease (eg RNAse). In those embodiments, the signal generator may be a protease when the free surrogate ligand is a protein or peptide. Exemplary proteases include, but are not limited to, trypsin, chymotrypsin, V8 protease, elastase, carboxypeptidase, proteinase K, thermolysin, papain and subtilisin. In embodiments where the surrogate ligand is a metal ion, an enzyme that requires a metal for activation can be used as a signal generator.

In another embodiment, the signal generator may be a solution (eg, buffer solution) that amplifies the generation of that molecule when the test ligand binds to the target (eg, target protein). For example, when many target proteins bind to the test ligand, they release or bind a large number of protons. These release or absorption events are referred to as "linkage" events. The linkage process can be amplified by introducing into the solution a buffer molecule with a high heat of ionization, such as Tris-HCl. In yet another aspect, the signal generator may be a change in phase, or an aggregation or polymerization of substances.

As used herein, the interaction of a first molecule with a second molecule results in a change in the association of two molecules, a change in the binding of two molecules, or a modification of one or both of the molecules. Changes may be included, such as increases or decreases. As used herein, modification includes forming or cleaving a bond, eg, a non-covalent bond or a covalent bond. It includes resolution, degradation, hydrolysis, changes in phosphorylation levels, labeling, ligation, synthesis, and similar reactions. Modifications may include physical changes in phase, aggregation or changes in polymerization.

In the case of interaction with the signal generator, the modification is not isothermal and is preferably exothermic.

As used herein, the term "analyzing a ligand" determines whether that ligand binds to a target; one of assessing the affinity of a test ligand for a target, or You may include a plurality.

Target Generation The target used in the methods of the invention may be any molecule of interest. Preferably, the target is a protein or polypeptide (also referred to herein as a "target protein"), eg, a naturally occurring protein or fragment thereof; a protein of unknown function; a ligand, substrate, or other thereof. There are proteins whose interacting molecules are unknown. Typical target proteins include, without limitation, receptors, enzymes, products of oncogenes, products of tumor suppressor genes, transcription factors, and infectious proteins (eg, infectious organisms such as viruses, parasites). , Proteins derived from bacterial, and / or fungal proteins). In addition, the target protein may be a wild-type protein or a mutant or variant protein, including those with altered stability, activity or various other properties, or a foreign amino acid sequence, such as a sequence that facilitates purification. Hybrid proteins (eg glutathione S-transferase (GST))
Part of)) may be included.

The target protein may be either in pure or impure form (eg, as part of a complex mixture of proteins and other compounds, as described herein). In particular aspects, the target protein may be a recombinant protein or a biochemical isolate. For example, the target can be any protein encoded by a gene isolated from prokaryotes or eukaryotes. The isolated gene is cloned into an expression vector and under the conditions that allow expression of the cloned gene by performing standard molecular biology techniques,
It can be introduced into a suitable host cell (Ausubel, F. et al., Edited, "Current Protocols in Molecular Biology", 1999.
, J. Wiley: New York; Sambrook, J., Fritsh, EF and Maniatis, T., "Molecular Cloning: A Laboratory Manual",
Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Pres
S., Cold Spring Harbor, NY, 1989). The vector may be, for example, a plasmid or a viral vector, among others. Preferably the vector is
For example, the gene encoding the target protein is modified by linking it to the appropriate regulatory sequences so that proper expression of the target protein is obtained. Examples of regulatory sequences include promoters, enhancers and other expression control elements (eg, polyadenylation signals) (eg, Goeddel; (1990) “Gene Expression Technology: Methods in 185. Enzym
ology 185) ", Academic Press, San Diego, CA).

Targets may be obtained from prokaryotes or eukaryotes, such as microorganisms (eg bacteria, viruses, parasites), vertebrates or invertebrates (eg mammals (eg humans)).

Typical prokaryotes include: Aquifex aeolyx (A
quifex aeolicus), Pyrococcus horikoshii, Bacillus subtilis, Treponema pallidum, Borrelia burgdorferi, Helicobacter pylori
Helicobacter pylori), Archaeoglobus fulgid
us), Methanobacterium thermo, Escherichia coli (Esch
erichia coli), Mycoplasma pneumoniae, Synechocystis sp. PCC6803, Methanococcus janas (Metha)
nococcus jannaschii), Mycoplasma genitaliu
m), Haemophilus influenzae, Rickettsia prowazekii, Pyrococcus abyssii,
Bacillus sp. C-125, Pseudomonas aeruginosa, Ureaplasma urealyticum, Pyrobaculum aerophilum, Pyrococcus fu
riosus), Mycobacterium tuberculosis H37Rv, Mycobacteri
um tuberculosis) CSU93, Neisseria gonorhea, Neisseria meningiditis, Streptococcus pyogenes, Borrelia burgdorferi
llia burgdorferi), Caulobacter crescentus
), Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis (Enteroc)
occus faecalis), Legionella pneumophila, Mycobacterium avium, Mycobacterium
tuberculosis), Methanococcus jannaschii, Neisseria meningitides, Pseudomonas putida, Porphyromonas
gingivalis), Salmonella typhimurium, Shewanella putrefaciens, Streptococcus pneumoniae (Strep)
tococcus pneumoniae), Thermotoga maritime (Thermotoga maritime), Treponema denticola, Thiobacillus ferroxidans (Thiobacillus ferroxidans), cholera fungus (Vibrio cholerae), Clostridium acetobutyricum (Clostridium acetobutylicum), Clostridium acetobutylicum (Clostridium acetobutylicum) (Mycobacterium leprae)
, Pseudomonas aeruginosa, Staphylococcus aureu
s), Bacillus species, Bartonella henselae
, Bordetella pertussis, Campylobacter jejuni
bacter jejuni), Francisella tularensis, Halobacterium salinarium
Institute), Listeria monocytogenes, Mycobacterium tuberculosis Sanger, Mycoplasma mycoides, Neisseria
ningitides strain, Streptomyces coelicolor
), Rash typhus rickettsia (Rickettsia prowazekii), Sulfolobus solfataricus (Sulfolobus solfataricus), Synechocystis (Synechocystis) PC
C6803, Thermoplasma acidophilum, Yersinia pestis, Xylella fastidiosa, Actinobacillus actinomyce, Chlamydia
Chlamydia trachomatis, Halobacterium
Species NRC-1, Mycoplasma capricolum, Neisseria gonorrhea, Pseudomonas aeruginosa, Pyrococcus furiosus, Pyrobaculum aerophilhocapsula
sulatus), Rhodobacter sphaeroides, Streptococcus pyogenes, Ureaplasma urealyticum, Crenarchaeum symbiosum, Pasteurella pasteurella maltosida.
), Ehrlichia species (HGE agent), Haemophilus ducreyii
), And Streptomyces hygroscopius
).

Typical eukaryotes include: Aspergillus nidulans (Asp
ergillus nidulans), Candida albicans, Leishmania major, Neurospora crassa,
Pneumocystis carinii, Plasmodium falciparum (Pla
smodium falciparum), Saccharomyces cerevisia
e), Schizosaccharomyces pombe, Trypanosoma cruzi, Trypanosoma brucei
ma brucei), Tetrahymena species, Cryptosporidium parvum, Arabidopsis thaliana M
, Malayan filamentous worm (Brugia malayi), nematode (Caenorhabditis elegans), Drosophila melanogaster, cystsoma mansoni
soma mansoni), Shistosoma japonicum, and mammals (eg, humans).

Targets can be produced by eukaryotic organelles. For example,
The target may be a mitochondrial enzyme. Examples of organisms whose organelle genome is known include: Chlorarachnion,
Guillardia theta, Cyanophora paradoxa
nophora paradoxa), Epifagus virginiana (Epifagus virginiana), Euglena gracilis, Guillardia seta (Guillard)
ia theta), Marchantia polymorpha, Nicotiana tabacum, Odontella sinensis
ensis), Oryza sativa (Oryza sativa), Porphyr (Porphyr)
a purpurea), Pinus thunbergiana, Acanthamoeba castellanii, Allomyces macrogynus, Bos Taurus, Cafeteria roenbergensis, Chrysodeymus cinou Chrysodidymus synuroideus), Condras crispa (Chondrus c
rispus), Chlamydomonas reinhardtii
, Drosophila melanogaster, Drosophila yakuba, Equus asinus, Homo sapiens, Mus mascaras (Mus musculus), Ochronas Danica (Opuronas pular), Prototheca wickerhamii, Reclinomonas Americana, Saccharomyces Saccharomyces
cerevisiae), Schizosaccharomyces pombe
, Tetrahymena pyriformis and Xenopus laevis.

Targets can also be produced by phage and are not limited and include: Acholeplasma bacteriophage, Accholeplasma phage / virus, bacteriophage bIL67, bacteriophage Cp-1.
, Bacteriophage G4, bacteriophage HP1, bacteriophage IKe, bacteriophage λ, bacteriophage MS2, bacteriophage PRD1, bacteriophage PZA, bacteriophage T4 and Lactococcus (Lactococcus).
) Bacteriophage C2.

The target may also be a viral protein. Examples of viruses that can generate targets include: Abelson murine leukemia virus, adeno-associated virus 2, adeno-associated virus 3, African swine fever virus, alfalfa mosaic virus, apple ahIorotic leaf virus.
spot virus), Apple stem grooving vir
us) 、 Arabis mosaic virus satellite
), Arctic ground squirrel hepatitis
B virus), Artichoke mottled crinkle v
irus), Autographa californica nuclear polyhedrosis virus (Autographa calif
ornica nuclear polyhedrosis virus), avian carcinoma v
irus), Avian infectious bronchitis viru
s), avian leukemia virus, avian sarcoma virus, BK virus, baboon endogenous virus, baboon endogenous virus (BaEV), bamboo mosaic virus, barley yellow virus, Barmah Forest viru.
s,), Bean golden mosaic virus, Beet curly top virus, Beet curly top virus, Beetle yellow virus, Black beetle virus, Silkworm nuclear polyhedrosis virus (Bombyx m)
orl nuclear polyhedrosis virus), border disease virus, borna virus,
Bovine immunodeficiency virus, bovine leukemia virus, bovine viral diarrhea virus, Brome mosaic virus, Cacao swollen shoot virus, goat arthritis-encephalitis virus (Caprine)
arthritis-encephalitis virus), Cardamine chlorotic fleck virus, Carrot mottle virus A (Carrot mot)
tle virus A), Cassava common mosaic v
irus), Cassava latent virus, Cassava vein mosaic virus, Cauliflower mosaic virus, Chicken anemia virus, Chloris streathe mosaic virus (Chloris st virus)
riate mosaic virus, Citrus tristeza virus
), Clover yellow mosaic virus, coconut leaf rot virus (Coconut foli
ar decay virus), Commelina yellow mottle viru
s), cucumber green mottle mosaic virus, cucumber mosaic virus, cucumber necrosis virus, dengue virus (Dengu
e virus) 3, dengue virus type 1, dengue virus type 2, digitaria streak virus, duck hepatitis B virus, ebola virus (constructed), eggplant mosaic virus, encephalomyocarditis virus, equine infectious anemia virus, cat Immunodeficiency virus,
Foxtail mosaic virus, Friend murine leukemia virus, Friend spleen focus-forming virus, Fujinami sarcoma virus, ground squirrel hepatitis virus, hepatitis A virus, B Hepatitis virus, C
Hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, hepatitis GB virus B, heron hepatitis B virus, swine fever virus, human T cell leukemia virus type 1, human T cell leukemia virus type 2 , Human adenovirus type 12, human adenovirus type 2, human foamy virus, human herpesvirus 1, human herpesvirus 3, human herpesvirus 4, human immunodeficiency virus type 1, human immunodeficiency virus type 2 , Human parainfluenza virus 3
, Human RS virus, infectious hematopoietic necrosis virus, influenza A virus, influenza B virus, influenza C virus, JC virus, Japanese encephalitis virus, Jembrana disease virus, Kennedya yellow mosaic virus , Lactate dehydrogenase-elevating virus, Leishmania RNA virus, Leishmania RNA virus 1, Lucerne transient streak virus, Corn streak virus , Marburg virus, Mason-Pfizer monkey virus, measles virus, Melon necrotic spot virus, mouse minute virus (Mic
e minute virus), Molluscum contagiosum vir subtype 1
us subtype l), Moloney mouse sarcoma virus, mouse breast cancer virus, mouse leukemia virus, mouse osteosarcoma virus, mouse sarcoma virus, mushroom bacilliform virus, narcissus mosaic virus (Na
rcissus mosaic virus), O'nyong-nyong virus,
Odontoglossum ringspot virus, olive latent virus 1, Ononis yellow mosaic virus, sheep lung adenocarcinoma virus, millet streak virus, papaya mosaic virus, papaya ring spot virus, pea larval brown virus, pea species Mosaic virus, peanut white streak virus, peanut striped virus, peanut dwarf virus, Pepper huasteco virus, pepper mottle virus, Plum pox virus, polyoma virus strain a2 (Polyomavirus strai
a2), polyoma virus strain a3, potato leaf curl virus, potato mop-top virus, potato virus A, potato virus M, potato virus X, potato virus Y, Punta Toro virus (Punta Toro). virus), Rabbit hemorrhagic virus, Rabies virus, Rice tungro spherical virus, Rice yellow spotted virus, Ross River virus, Rous sarcoma virus, Rubella virus, Saccharomyces cerevisiae virus La (Sacchar
omyces cerevisiae virus La), Saguaro cactus v
irus), Satellite tobacco necrosis
virus), Sendai virus, Sendai virus (Simi
an foamy virus), simian immunodeficiency virus, sarcoma virus, simian virus 40
, Sindbis virus, Sindbis-like virus, Sonchus yellow net virus, Southern bean mosaic virus, Soybean chlorophyll virus
tic mottle virus), Spiroplasma virus, Strawberry vein banding virus, Sulfolobus virus-like particle ssvI, Swine vesicular disease virus , Taylor's encephalomyelitis virus, Tick-borne encephalitis virus, Tobacco etch virus, Tobacco mild green mosaic virus, Tobacco moss virus
aic virus), Tobacco necrosis virus, Tobacco vein mottling virus, Tomato bushy stunt virus, Tomato golden mosaic virus, Tomato leaf winding virus , Tomato yellow leaf curl virus, turnip vein-clearing virus,
Turnip yellow mosaic virus, vaccinia virus, smallpox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, Visna virus,
West Nile virus, Woodchuck hepatitis B virus, Woodchuck hepatitis virus, Y73 sarcoma virus and yellow fever virus.

Bioinformatics Analysis of Targets In a preferred embodiment, hypothetical or putative functions are preferably assigned prior to studying the target with a test compound. Several bioinformatics techniques can be used in assigning virtual functions to targets (eg proteins). By identifying features of known function that the target and the molecule (eg, protein) share (or, in some cases, do not share) with the molecule (
The corresponding activity of the protein (for example) or any activity can be assigned to the target. Examples of methods currently used to predict protein function include sequence-based searching, folding recognition methods (threading).
Algorithms and neural networks), homology modeling,
And structure-based analysis.

For example, using FASTA, BLAST, and Smith-Watermann algorithms, one-to-one with any sequence for any known function.
Can be compared (Shpaer, EG et al. (1996) Genomics 38 (2)). Two approaches can be used to identify conserved regions of proteins (eg, sequence motifs with predictable function). One is PROSITE [http: //expasy.hcuge.ch
/sprot/prosite.html] and the other is BLOCKS [http: //www.blocks.fhcrc.
org / blocks /]. The PROSITE analysis is based on the matching pattern of amino acid residues when using a consensus sequence or motif. The second approach, BLOCKS, is not just a consensus sequence, but a highly sensitive selection of unrelated sequences by matching sequences against multiple complete, gap-free sequence alignments of conserved regions. it can.

The target amino acid sequence also includes the class, structure, topology (folding family) and homology superfamily (Class, Architecture, Topology (fold fam
ily), and Homologous superfamily) (CATH) database [http: //www.bioc
[hem.ucl.ac.uk/bsm/cath] can also be used to analyze the presence or absence of protein folding. At present, more than 670 different types of protein folds are included in this database. The predictive ability of such structural motifs based on the primary sequence can be improved by the threading method (that is, by adapting the amino acid sequence of the target protein to a known three-dimensional protein structure).
ant, SH et al. (1993) Proteins 16: 92-112). It is also possible to predict protein folding using neural networks (Bohr, H et al.
90) FEBS Lett 261, 43-46).

Homologous to another protein of known function (at least 30
Another method of predicting the functional correctness of target proteins (35% identical) is homology modeling. Homology modeling (Johnson, MS et al. (1994) Crit. Rev.
Biochem. Mol Biol 29: 1-68) compares the amino acid sequence of a protein of interest with another related protein of known three-dimensional structure using a computational algorithm.

Further, by determining the function of the protein based on the structure, the biological function of the target can be estimated. In this assay, the crystal structure of the target protein is determined and then its three-dimensional structure is compared to other proteins of known function. If there is a matching portion, the biological function of the target can be estimated based on the known functions of other proteins. With this approach, Methanococcus has recently become
A novel NTPase derived from jannascii has been identified (Hwang, KY et al. (1999) Nat.
Struct. Biol. 6: 691-696).

Preparation of Test Compound The test compound (interacting substance) is a compound, a molecule, or a complex, and is a target (
For example, the interaction force (eg, binding force) with the target protein can be examined. In one embodiment, the test compound is a small organic molecule (eg, a synthetic or naturally occurring non-proteinaceous molecule). The test compound can be designed to interact with the target, and based on the desired activity, a library of various compounds (
For example, it can be selected from a substrate library or a combinatorial library (eg, random screening of drugs based on desired activity (eg, ability to interact with target)).

Libraries The methods of the invention use libraries as a source of candidate interactors (eg, candidate agents whose ability to interact with a target is considered). A library can include multiple members that are structurally or functionally related. But,
The factors that make up the library may be structurally and functionally unrelated.

Libraries that include multiple members that are functionally or structurally related may be particularly useful where the target can be assigned activity or hypothetical activity. For example, when the target is a nuclease, a library of nuclease substrates can be tested against the target. A library of nuclease substrates can include various or hypothetical substrates.

The library can cover a wide range of target “activity”. An example of the library will be described later. The substrate requirements for the newly discovered enzyme can be determined by serially classifying the substrate library. This method will be referred to herein as substrate profiling.

Cofactor Library “Cofactor” libraries include group transfer and energy-coupled molecules, coenzymes (eg, ATP, GTP, TTP, CTP, UTP, NADH, NADPH, NAD, NADP, FAD, FADH. , Phosphoenolpyruvate, coenzyme A, lipoamide, S-adenosylmethionine, thiamine pyrophosphate, biotin, tetrahydrofolate, uridine diphosphate glucose, cytidine diphosphate diacylglycerol, and succinyl-CoA CoA-modified molecule) can be optionally included. These groups of molecules, called cofactors, are involved in a large number of different enzymatic reactions. Examples of cofactor libraries discussed and described in Example 5 below include the following members: ATP, GTP
, CTP, TTP, UTP, NADH, NADPH, NAD, NADP, FAD, flavin, thiamine monophosphate chloride, pyridoxal 5'-phosphate, coenzyme A, and cocarboxylase.

In a preferred embodiment, the library comprises at least 1, 2, 5, or 10 substrates described herein. Often, cofactor libraries are considered along with other libraries. For example, cofactor libraries are considered in combination with carbohydrate libraries (see Example 5, below).

Carbohydrate Metabolism Library A carbohydrate metabolism library (eg, a biosynthetic library and / or a degradation library) can be used to screen for carbohydrate modifying enzymes. This library includes carbohydrates involved in known biochemical pathways, including, for example, long units, short units, and single units of carbohydrates and modified carbohydrates derived from known biochemical pathways such as phosphorylated carbohydrates. Can be included.
This type of library can include carbohydrates or modified carbohydrates that are unknown to be substrates for any enzyme. Examples of carbohydrates used can include glucose, fructose, arabinose, xylose, mannose, galactose, lactose, sucrose, and ribose. Examples of other substrates that can be used in carbohydrate libraries are found in the Metabolic Pathway Chart (Metabolic
Pathway Chart), 1997, 20th Edition (Sigma-Aldrich). In a preferred embodiment, this library comprises at least 1,2,5 described herein.
, Or 10 members. Carbohydrate library is
For example, it can be examined in combination with a cofactor library).

Examples of carbohydrate libraries discussed and described in Example 5 below include the following members for consideration: D-glucose, arabinose, sucrose,
Ribose, lactose, galactose, maltose, and xylose.

Purine and Pyrimidine Metabolism Libraries Purine and pyrimidine metabolism libraries (eg, biosynthetic and / or degradation libraries) can include nucleoside / nucleotide precursors. This library can also be used to screen for enzymes involved in purine and pyrimidine biosynthesis or degradation. Examples of purine and pyrimidine compounds used include glycinamide-ribose-phosphate, urea, formylglycinamide-RP, 5-aminoimidazole carboxylate-RP, inosine-P.
, Formylamide-imidazole-carboxamide-RP. Examples of other substrates that can be used for purine and pyrimidine libraries are described in the Metabolic Pathway Chart, 1997, 20th Edition (Sigma-Aldrich). In a preferred embodiment, this library comprises at least 1, 2, 5, or 10 substrates described herein. Purine and pyrimidine metabolism libraries can be examined in combination with other libraries (eg, cofactor libraries).

Amino Acid Metabolism Library Amino Acid Metabolism Library (eg Biosynthesis and / or Degradation Library)
Can include D- and L-form amino acids, and precursors of amino acids. This can include peptides that have a known protease domain and serve as substrates for any current known protease. This library may also contain some non-enzymatic proteins such as BSA that allow us to explore undiscovered or unclassified proteolytic activity to find new classes of proteases. Examples of amino acids used in the amino acid metabolism library include alanine, aspartic acid, cysteine, histidine, glycine, and isoleucine. Examples of other substrates that can be used in the amino acid metabolism library are:
Metabolic Pathway Chart, 1997, 20th Edition (Sigma-Aldrich
)It is described in. Examples of peptides that can be used as protease substrates include:
It includes an acetyl group-serine-glutamine-asparagine-tyrosine-proline-valine-valine-amide group (Sigma, page 1132 of the 1999 edition catalog, No. A0806), and serine-proline-arginine (Sigma). In a preferred embodiment, the library comprises at least 1, 2, 5, or 10 members described herein. The amino acid metabolism library can be examined in combination with other libraries (for example, cofactor library).

Lipid Metabolism Libraries For lipid metabolism (eg, biosynthesis and / or degradation libraries), fatty acids, fatty acid precursors, steroids, and steroid precursors previously discovered as substrates for known enzymes, and Both undiscovered or unclassified fatty acids and steroids can be included as substrates for the enzyme. This library can be used to screen for enzymes involved in fatty acid metabolism. Examples of substrates used in lipid metabolism libraries include cholesterol, desmosterol, thymosterol, lanosterol, choline, lecithin, cephalin, linoleic acid, cardiolipin, and acetylcholine. Examples of other substrates that can be used in a lipid biosynthesis and degradation library are found in the Metabolic Path Chart.
thway Chart), 1997, 20th Edition (Sigma-Aldrich). In a preferred embodiment, this library comprises at least 1, 2, 5,
Or including 10 members. Lipid metabolism libraries can be examined in combination with other libraries (eg cofactor libraries).

Vitamin and Hormone Libraries This class of libraries can include vitamins and hormones, and their metabolic precursors, for screening enzymes involved in hormone or vitamin synthesis, degradation, or modification. Can be used. Examples of substrates used in vitamin and hormone libraries include retinoic acid, metalrhodopsin, rhodopsin, vitamin K, opsin, and vitamin E. Examples of other substrates that can be used in vitamin and hormone libraries are described in the Metabolic Pathway Chart, 1997, 20th Edition (Sigma-Aldrich). In a preferred embodiment, the library comprises at least 1, 2, 5, or 10 members described herein. Vitamin and hormone libraries can be examined in combination with other libraries (eg cofactor libraries).

DNA Molecule Libraries This class of libraries can be used to screen for DNA modifying enzymes. This can include double-stranded and single-stranded DNA molecules, as well as partially double-stranded DNA molecules, and random sequences of DNA such as calf thymus DNA. This can include covalently bound closed circular DNA, whether in the supercoiled or relaxed state. These DNA molecules have the sigma (S
igma), Amersham, and Biorad. In a preferred embodiment, the library comprises at least 1, 2, 5, or 10 members described herein. The DNA molecule library can be examined in combination with other libraries (for example, cofactor library).

Natural Product Libraries Natural product libraries (eg, natural product libraries from bacteria) include organisms (
Natural products derived from, for example, bacteria) can be included. By using this library, an unknown enzyme activity can be screened from an unknown substance. The substrate requirements for natural product libraries can be determined by deconvolution of natural products by chromatographic methods. In a preferred embodiment, the library comprises at least 1, 2, 5, or 10 members described herein.
Natural product libraries can be examined in combination with other libraries (eg cofactor libraries).

In some embodiments, a natural product (eg, a bacterial-derived natural product), for example, accumulates any metabolite when grown under non-permissive conditions (eg, non-permissive temperature or in the absence of essential nutrients), It is generated in vivo by a mutant organism (eg, a temperature-sensitive bacterial mutant, or an auxotrophy). The accumulated metabolites can then be purified from the organism before studying.

The different substrate libraries can each be incubated as a solution with the target protein / protein group and the cofactor library, respectively, at several different pH values and with a general mixture containing different salts. Enzyme activity can be detected as a change in heat output detected by a calorimeter. This makes it possible to quickly classify a wide range of types of enzyme activities possessed by the novel protein. The exact requirements that the substrate must meet can then be determined by systematically dividing the substrate library. It should be noted that the substrate requirements and solution conditions of the newly discovered enzyme can be optimized and key parameters such as k _cat , k _M , and / or k _D can be determined.

In another embodiment, the test compound is a member of a combinatorial library. Combinatorial libraries can be synthesized by methods well known in the art, such as the paper of EM Gordon et al.
Med. Chem (1994) 37: 1385 to 1401); DeWitt, SH; Czarnik, AW et al. (Acc. Chem. Res. (1996) 29: 114);
Armstrong (Armstrong, RW); Combs (AP); Tempest (
Tempest, PA); Brown, SD; Keating, TA, et al. (Acc. Chem. Res (1996) 29: 123; Elman, JA) (Acc. Chem. Res. (1996) 29: 132; Gordon, EM; Gallop, MA; Patel, DV et al. (Acc. Chem. Res. (1996)
29: 144); Lowe, G. paper (Chem. Soc. Rev. (1995) 309) and Blondelle et al. Paper (Trends Anal. Chem. (1995) 14:83).
As reviewed by Chen et al. (J. Am. Chem. Soc. (1994) 116: 2661), and in US Pat. Nos. 5,359,115, 5,362,899, and 5,288,514;
WO92 / 10092, 93/09668, 91/07087, 93/20242, 94/0
No. 8051.

Libraries of test ligands can be prepared by a variety of methods well known in the art. For example, the "split-pool" method is performed in the following procedure: beads of functionalized polymeric support are placed in multiple reaction vessels; various polymeric supports suitable for solid phase support peptide synthesis are available. Known and partly commercially available (eg M. Bodansky "Principles of P
eptide Synthesis) ", 2nd edition, Springer-Verlag, Berlin (1993)).
A solution of individually activated amino acids is added to each aliquot of the beads and reacted to produce multiple immobilized amino acids in each reaction vessel. Next, the aliquots of derivatized beads are washed, "pooled (ie, recombined)," the pool of beads is further divided, and each aliquot is placed in a separate reaction vessel. Then another activated amino acid is added to each bead aliquot. The cycle of synthesis is repeated until the desired length is reached. The residues added at each synthesis cycle can be randomly selected; or the residues can be selected to provide a "biased" library. A wide variety of peptides, peptidomimetics, or non-peptidic compounds can be easily made in this way.

In another illustrative synthetic method, the “diversomer library” is based on the method of Hobbs DeWitte et al. (Proc Natl Acad Sci US A. 9).
0: 6909 (1993)). Other synthetic methods include Houghten's
The “tea bag” method (see, eg, Houghten et al., Nature 354: 84-86 (1991)) can also be used for the synthesis of compound libraries according to the present invention.

Combinatorial libraries of compounds can be synthesized with a “tag” that marks each member of the library (eg, US Pat. No. 5,565,324 by WC Still et al. 94/08051 and 95/28
See No. 640). Generally, this method is characterized by the use of an inert but easily detectable tag that binds to the solid support or compound. When detecting an active compound (eg, by one of the methods described above), the identity of the compound is determined by the unique tag attached. This tagging method can be used to synthesize large libraries of compounds that can be used for identification at very low levels. Such a tagging method is useful in identifying a compound released from beads.

In a preferred embodiment, the library of test compounds comprises at least 30 compounds, more preferably at least 100 compounds, and even more preferably at least 500 compounds. In a preferred embodiment, the library of test compounds comprises less than 10 ⁹ compounds, more preferably less than 10 ⁸ compounds, and even more preferably less than 10 ⁷ compounds.

Formats The methods described herein can be performed in many physical formats. In a preferred embodiment, the measurement is carried out in an isothermal titration calorimeter. A calorimeter, such as an isothermal titration calorimeter, can include a flow cell. In a preferred embodiment, the target is immobilized on the flow cell. Samples can be introduced into the flow cell from a multi-compartment sample holder, eg, a microtiter plate, eg, a multi-well plate such as a 96-well plate. The sample can be premixed in the compartment of the sample holder.

In another preferred embodiment, a multi-compartment sample holder, eg a microtiter plate, eg a multi-well plate such as a 96-well plate, wherein each fraction contains a thermopyle, each being a calorimeter cell. Can be used. Channels may be included for transporting liquid to the compartment.

In a preferred embodiment, the method can be carried out on a microchip in which the appropriate well channels and other components have been formed, eg by etching or deposition. Liquids can be pumped and moved by electrokinetic methods.

In the methods described herein, the reaction mixture can contain a single target or multiple targets. Similarly, one or more ligands can be added to the reaction mixture. For example, it may be useful to multiplex one or both of these elements to screen multiple species. For example, if a large number of ligands have to be evaluated, the first group of candidates can be pooled, and if one pool shows promising results, the members of the pool are evaluated. Similarly, in the method of evaluating a substrate, candidate substances can be pooled.

For large scale screening, the calorimeter can be combined with high throughput screening formats. For the purposes of high throughput screening, the experimental conditions described above are adjusted to give a threshold ratio of test ligands identified as "positive" compounds or ligands to total compounds screened. Preferably, this threshold is set according to two criteria. First, the number of positive compounds must be manageable in a practical sense. Second, the number of positive compounds must reflect ligands with a recognizable affinity for the target protein. A preferred threshold is obtained when 0.1% to 1% of the total tested ligands are shown to be ligands for a given target.

ITCs are commercially available and routinely used by those skilled in the art. For example, 199
U.S. Patent No. granted to Protnikov (Plotonikov, VV) on September 29, 8
5,873,763; Indyk et al. (1998, Meth. Enzymol. 295: 350-364).
: See Brandts et al. (1990, American Laboratory 30-41). ITC is a two-cell differential instrument. It operates at a fixed temperature with constant stirring of the liquid in the sample. The device measures the heat generated or absorbed as a result of the test ligand binding to the target.

In another embodiment, a differential scanning calorimeter (DSC) can be used to detect heat output. DSCs are commercially available and routinely used by those of ordinary skill in the art. For example, US Pat. No. 5,873,763 to Plotonikov, VV et al.
No .; Freire (1995, Meth. Mol. Biol. 40: 191-218). The differential scanning calorimeter automatically raises or lowers the temperature at a predetermined rate while monitoring the temperature difference between cells. From the temperature difference information, a small difference in heat capacity between the sample and control cells can be determined and attributed to the test substance.

In a preferred embodiment, the reaction mixture is not transparent; the reaction mixture is colored; the reaction mixture is cloudy; the reaction mixture contains substances that interfere with fluorescence or colorimetric detection; the reaction mixture is pure It includes products that are not in solution, eg, other than the target. In a preferred embodiment, the reaction mixture comprises a substance that interferes with radioactivity analysis: a substance that interferes with spectrophotometric analysis, eg, NMR analysis. In a preferred embodiment, a complex mixture of substances is tested, for example an impurity sample such as a suspension, a natural product extract, a cell extract, a biochemical mixture or a colored solution which may contain one or more test compounds. .

Methods Based on Surrogate Ligands Identification of Surrogate Nucleic Acid Ligands A surrogate ligand can be a nucleic acid (eg, an oligonucleotide). The SELEX technique can be used to identify surrogate ligands. SELEX technique (Gold)
(1995), supra) can be used to investigate whether a large number of random sequence oligonucleotides bind with high affinity to a target, eg a target protein. The larger the nucleotide library, the greater the chance of discovering at least one sequence that binds to the target with a dissociation constant in the picomolar to nanomolar range. Preferably, the ligand is about 20 nucleotides in length, longer oligonucleotides probably only bind with a fraction of that length, and some residues, even if the ligand binds to the target. However, they are susceptible to degradation or processing by signal generators. For example, in the case of a surrogate nucleic acid ligand, longer oligonucleotide sequences may undergo background hydrolysis when using DNase.

In one embodiment, the target used in the methods of the invention is a protein (
Target protein). First, target proteins can be identified and purified using standard biochemical techniques such as HPLC and ion exchange or size exclusion chromatography. Preferably, a highly purified target protein sample is obtained. The SELEX method is then used to identify single-stranded oligonucleotide (DNA) ligands that bind to proteins with high affinity (> nM). The first step in this process is 10 ¹⁴ to 10 ¹⁵ single-stranded DNAs with the 5'to 3'end structure shown below.
Generation of a sequence random oligonucleotide library is required. In the formula, Fixed A and Fixed B mean the constant sequences present at the 5'and 3'ends of each member of the library. These constant sequences flank the random sequence, which allows transcription and subsequent pool amplification after each round of the SELEX process (Tuerk and Gold (1990), Science 24.
9: 505-510). Preferably, the random sequence must not exceed 20 nucleotides in length (the larger ligand may use the central region of its residue to bind to the target, so that its ends are Even if it binds to the target protein, it may be exposed to the activity of DNase).

The library is then mixed with the target protein and then partitioned by passage through a nitrocellulose membrane. Those DNA sequences bound to the filter by the protein are then eluted and amplified by the polymerase chain reaction (PCR), followed by transcription of the (currently modified) library in the second round of SELEX. The process is repeated until a ligand is obtained that binds with the desired affinity.

Each round of method results in an average 10-fold enrichment for high affinity ligands (Schneider et al., (1992), J. Mol. Biol. 228: 862-869).
, And to identify ligands that bind with Kd in the nanomolar range, SELEX 10
~ 20 round range normally required (Gold et al., 1995, supra)
It is shown. In the first round, if an even more abundant low affinity ligand is present in the library, it is recommended to use a fairly large amount of protein to ensure that all high affinity ligands are retained. The selectivity of the SELEX process can be increased by using lower amounts of target (eg, target protein) in later rounds if the high affinity ligand is enriched enough to survive competitive binding states. You can This process has been attempted on more than 30 proteins, and in almost all cases, oligonucleotide ligands were found that bind with an affinity of nM or higher (Gold et al., 19
95, above).

Selection of Signal Producer After identifying the surrogate ligand, a suitable signal producer having high specific activity for the surrogate ligand is selected. Preferably, the signal producing substance interacts more easily (eg, binds) with the free surrogate ligand as compared to the surrogate ligand bound to the target. Preferably, such interaction amplifies the ligand and produces, for example, a thermal signal. In certain embodiments, the signal producing agent modifies the free surrogate ligand by, for example, forming or cleaving covalent or non-covalent bonds. For example, the modification step may include cleavage, degradation, phosphorylation, polymerization, and other events that produce a signal, eg, a thermal signal. The signal producing substance can be a degrading enzyme (eg, a nuclease or a protease). Alternatively, the signal producing substance can be a polymerizing enzyme such as a polymerase.

The signal producing substance can be immobilized, eg attached to a solid support, or cross-linked. For example, in those embodiments where the signal producing substance is an enzyme, the enzymes can be cross-linked to form a crystalline enzyme.

For example, in those embodiments where the free surrogate ligand is a DNA molecule, the signal generator can be a nuclease. Examples of nucleases that can be used are staphylococcal nuclease (SNase), Serratia marcescens (
Serratia marcescens) nuclease (SNase), bovine pancreatic nuclease (DN)
These include, but are not limited to, Aase I), or human (type IV) nucleases. The activity may vary by tens of folds with a single nuclease and with a variety of oligonucleotide substrates, but the optimal DNase may be selected. Optimal solution conditions, such as pH, temperature, and solvent conditions, may be selected as well. The activity of specific DNases against specific surrogate ligands has been demonstrated by Friedhoff et al. (1996, Eur. J. Biochem. 241: 572-580) and Friedhoff et al. (1999, FEBS Lett. 443: 209-214). ) Can be used to assay. Some example DNases are listed in the table below along with their activity on specific substrates. The table is by no means complete,
It is not to be construed as limiting the scope of the invention.

[0297]

Table 1 Steady-state parameters for cleavage of various nucleic acid substrates by several nucleases a Staphylococcal nuclease b Bovine pancreatic desoxyribonuclease c Serratia marcescens nuclease (SNase) d Barley nuclease

The signal producing substance can also be a polymerase, such as Tac polymerase. In those embodiments in which the free surrogate ligand is an RNA molecule, the signal generator can be a ribonuclease (eg, RNase).

In other embodiments where the free surrogate ligand (for the target binding ligand) is a protein or peptide, the signal generator can be a protease. Proteases useful for practicing the present invention include trypsin, chymotrypsin, V8
Protease, elastase, carboxypeptidase, proteinase K, thermolysin, and subtilisin (all of which are Sigma Chemical Co., St. Louis,
(Available from Missouri), but is not limited to these. The most important criterion for selecting one or more proteases used to practice the invention is that the protease must be able to digest a particular target protein under the chosen incubation conditions. To avoid "false positive" results caused by test ligands that directly inhibit the protease, one or more proteases, particularly those with different enzymatic mechanisms of action, can be used in simultaneous or parallel assays. In addition, the cofactors required for the activity of the protease are provided in excess to avoid false positive results with test ligands that could sequester these factors.

Calorimetric Ligand Screening Using Surrogate Nucleic Acids The following description illustrates examples using surrogate nucleic acid ligands and target proteins. The experimental conditions described herein are for those skilled in the art to use other surrogate ligands (eg, target ligands). ) And the target can be easily expanded. Briefly, once a surrogate ligand (eg, surrogate nucleic acid ligand) that binds to the target protein with a suitable dissociation constant is identified, a 1: 1 solution of the target protein and surrogate nucleic acid ligand is prepared (concentration is about 10 mM), equilibrate the interior of the microcalorimeter cell for a few minutes with a much lower concentration of signal producing substance. For example, a concentration of about 1 nM of a specific deoxyribonuclease (DNase was chosen because of its high activity against the surrogate nucleic acids identified in the SELEX process. One assumption in the present invention is that the surrogate nucleic acid ligand binds to the target protein. , Is less susceptible to rapid DNase degradation than free surrogate nucleic acid ligands.

More specifically, the target protein, surrogate nucleic acid ligand and specific nuclease are mixed in solution to form a reaction mixture. The target protein and surrogate nucleic acid ligand can both be present at about 1 μM, while the nuclease is present at about 1 nM. The total volume of the sample is about 1 ml. The sample is incubated in a microcalorimeter cell (eg, an isothermal titration calorimeter cell). Enclose the two cells in an insulating container. The vessel is cooled so that thermal energy is needed to maintain the cell and its contents at the experimental temperature. Keep the two cells in thermal equilibrium with each other. Then a small amount (5-25 μl) of the test ligand, probably bound to the target protein, is added to the sample cell, if the test ligand binds to the target protein with significant affinity (compared to the oligonucleotide). It is believed that some fraction of the surrogate ligand is released into solution depending on the extent of each binding constant. This newly released surrogate ligand then begins to be hydrolyzed by the nucleases present in solution, thus producing a heat output (output) greater than the heat output produced by the original competitive binding of the test compound. To occur. The heat output can be recorded for about 1 minute. The total heat output for a given test can be related (eg, proportional) to the ratio of the affinity of the surrogate ligand to the test ligand for the target protein.

In other embodiments, structural changes in the target when the test ligand binds can be measured. Protein targets and structural RNA targets (eg, ribozymes) share one common feature: they change their conformation when denaturant is added to a solution or when heat is applied by increasing the temperature. It is a type that is not compressed very much. The conformational change that a protein or RNA undergoes involves a large change in heat, which may be conveniently detected by a calorimeter. If the test ligand binds to a more compacted form of the target protein or RNA, it can inhibit conformational changes and thereby detect a difference in heat output as compared to a control solution without the test ligand.
A detailed description of this detection is provided in Example 3 and Example 4.

The method can also be used to analyze (eg, identify) substances that bind to the target when the target is present on the cell surface, eg, bacterial cell wall components. Thus, the method can be used to identify substances that interact with (eg, bind to) cell surface molecules.

The interaction of the target with the surrogate ligand, test ligand and / or substrate can occur in vitro (eg, cell-free system) or in vivo (eg, cell, eg, prokaryotic or eukaryotic cell). For example, this interaction can be tested by adding these compounds to cells placed inside the calorimeter, eg living cells.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are given for illustrative purposes and should not be construed as limiting the invention in any way.

Example 1 Screening for Compounds That Can Bind Target Proteins First, single-stranded oligonucleotides of DNA that bind with high affinity to the target protein of interest are identified. For the purposes of this example, the ligand is assumed to be 20 residues in length and Kd = 1 nM. Proteins and oligonucleotide ligands at concentrations of 10 μM each are mixed with each other. Add a small amount (1 nM) of deoxyribonuclease, which is known to have high activity for ligands (total amount of solution 500
μl). The binding reaction between the target protein (P) and the ligand (L) can be described as follows: Kd PL = P + L, and rewritten in the following equation: Kd = [P] [L] / [ PL] (equation 1)

If the concentration of the protein-ligand complex [PL] is known, the amount of free protein and free ligand in solution can be calculated from the following formula [P _τ ] = [P] + [PL] (Equation 2) and [L _τ ] = [L] + [PL] (equation 3) Substituting equations 2 and 3 for equation 1 and solving for [PL] gives a quadratic equation: _{^{[PL] 2 - (L T}} + P T + K d) [PL] + P T L T = 0 This can be solved with respect to [PL]. In this case, both [P _T ] and [L _T ] are 10 μM
, Kd = 1 nM and [PL] equals 0.99 μM, which means that 1% of the ligand is free in solution (from equation 3). This small fraction will begin to hydrolyze when the protein ligand solution is combined with DNase (resulting in some baseline heat output: 70 μcal / sec × 10 sec ⁻¹ = 700 μcal / sec).

After equilibrating an initial solution of target protein, ligand and nuclease in the sample cell, the test compound is added at a final concentration of 10 μM. For the purposes of this example, assuming that it has the same Kd for the target as the original oligonucleotide, the test compound will eventually compete with half the DNA and be degraded by DNase. The total amount of DNA in the cell is about 5 × 10 ⁻⁹ mol. When this half is completely hydrolyzed, this results in (20) (2.5 × 10 ^-9 ) = 5 × 10 ^-8 mol of phosphodiester bonds being cleaved and total heat output (70 kcal / mol) (5 × 10 ^-8 mol) (10 seconds ^-1 ) = 35,00
It is meant to produce 0 μcal / sec. This is 50 times greater than the baseline heat output from the original 1% free ligand.

Example 2 Calorimetric Detection of Enzyme Turnover and Inhibition The measurement of activity of enzymatic reactions is directly proportional to the differential output in the calorimetric cell due to the catalyzed conversion of the substrate to the product. The detection signal (force) of the enzymatic reaction, when monitored by a calorimeter, is one of the heat of reaction, as shown by the following equation:
Equivalent to substrate turnover per second: ΔP = ΔH × V = ΔH [-dS / dt] where S is substrate concentration and V is enzyme rate.

To measure a small change of 5%, the required change in calorimeter output is 0.5 μc
Will not exceed al / sec. This change in output is due to the typical heat of reaction of the substrate being 10 kcal.
Substrate turnover of 5 × 10 ^-11 mol / sec, assuming / mol, would be the cause. Since enzyme turnover numbers range from 10 to 10,000 / sec, it appears that the amount required to perform each drug screening assay is as low as picomolar or fentomolar of enzyme in the calorimeter.

In order to optimize the calorimetric assay to ensure maximal signal change, it is helpful to consider the following. Simple expression equation for the relationship of the rate of the enzyme to the concentration of substrate and inhibitor: V = Vmax {1 + K m / S [1 + I / Ki]} in ^-1 formulas, respectively S and I, substrate and inhibitor Where K _i is the dissociation constant of the inhibitor and K _m is the Michaelis-Menten parameter of the substrate. _Km values are mainly 10 ^-1 to 10 ^-6 M
Changes in the range of. Enzyme activity is most sensitive to changes in the low concentration of inhibitor if the substrate concentration is equal to K _m. When the inhibitor is added to the enzyme solution at a substrate concentration K _{m at} a concentration equal to K _i , the enzyme rate (and thus output) is reduced by 34%.

Example 3: Detection of test ligand by measuring heat of conformational change of the target using an isothermal calorimeter After purification of the target of interest (eg target protein or structural RNA), guanidine hydrochloride Alternatively, a denaturing buffer solution such as urea is introduced into the calorimetric reaction cell. The syringe is then filled with protein to a specific concentration (~ 100 μM). About 1
0 μl (containing 1 nmol of target) is introduced into the calorimetric cell. For a typical protein, when the conformation of the protein changes to a less compact state, 100 microcalories can be detected after subtracting the thermal effect of denaturant dilution.
00 kcal / mol is absorbed. The thermal effect of denaturant dilution will be small, since 10 μl of titrant is added to 1000 μl of solution. However, the actual value of this dilution effect can be determined by adding 10 μl of buffer to the denaturant solution without adding protein.

To detect whether the test ligand binds to the protein, the above experiment can be repeated so that the test ligand is introduced at equal concentrations in the denaturant solution and the protein solution. If the test ligand binds to the compressed state of the protein, when injected into the calorimeter, a small amount of the protein will be converted to less compressed state and the total heat output will change. The injection process can be easily automated by coupling the flow injection system to the titer plate so that each sample is introduced into the same large amount of denaturant.

Example 4: Detection of test ligands by measuring the heat of conformational change of the target using a differential scanning calorimeter Differential scanning calorimetry with a buffer containing purified target (eg, target protein of interest) Introduce into the reaction cell. The temperature of the solution is raised and heat is applied. Heat is released when the conformation of the protein changes to a less compacted form. Integrating the apparent specific heat capacity curve gives the apparent specific heat output due to changes in the three-dimensional structure. The experiment is repeated with a fresh solution of protein with the addition of some test ligands. If the test ligand binds to the protein, the apparent specific heat output obtained by integrating the apparent specific heat capacity curve at the same two temperature points as in the previous experiment will be reduced. This difference serves as a convenient signal to indicate which ligand binds to the protein.

Example 5: Experiments investigating the profile of a hexokinase substrate This example demonstrates the monitoring of heat release rate due to phosphorylation of glucose by hexokinase using isothermal titration calorimetry (ITC).

Materials and Methods: F-300 type hexokinase from baker's yeast was purchased from Sigma and used without further purification. The ATP and carbohydrate and coenzyme kits used to probe the substrate profile were also obtained from Sigma. All reagents are 100 mM HEPES (pH
8.0), 10 mM MgCl ₂ , 10 mM KCl, and 1 mM ATP (reaction buffer). To make multiple combinations of carbohydrate and coenzyme mixtures (see flow chart below), each individual component was adjusted so that final dilution with reaction buffer yielded 1 mM carbohydrate and 0.5 mM coenzyme. Solution at 2 mM and 1 respectively
Adjusted to mM. Hexokinase was prepared as 50 units / ml stock in reaction buffer.

Enthalpies of reactions reflecting enzyme turnover were obtained from thermograms obtained with a VP-ITC microcalorimeter (Microcalink, Northampton, Mass.). The VP-ITC instrument directly measures the heat released or absorbed in a liquid sample as a result of injecting the correct amount of reactant into a thermal equilibrium reaction cell. The volume of the reaction cell is about 1.7 ml and is enclosed in the same control cell in an insulating inner shield inside the insulating outer shield. After the device is fully assembled with the rotating syringe, it is brought to the desired experimental temperature. When the cell reaches thermal equilibrium, the temperature difference between the control and sample cells is measured. The differential force (DP) between the control cell and the sample cell
Signal) calibration is obtained electrically by applying a known amount of force through a resistive heating element on the cell. The injection causes a chemical release (heat release) or heat absorption (endotherm) in the sample cell, which causes a negative change in the DP signal in the case of an endothermic reaction and a positive change in the DP signal in the case of an endothermic reaction. cause. These chemical changes generate heat that distracts the initial (electrically balanced) DP signal from equilibrium, so that the DP feedback of the device repowers the cell to compensate for these changes. Thus, the DP signal display has a power unit (μcal / sec) and the peak integrated over time gives a measure of the thermal energy ΔH.

The reaction conditions for determining the profile of the hexokinase substrate were as follows: The sample cell was filled with 2 ml of the above substrate / coenzyme solution. The assay method is
The sample cell was started by injecting 6 μl of 50 units / ml hexokinase solution. The temperature during each calorimetric assay was held constant at 25 ° C for each experiment. Under the experimental conditions introduced herein, the heats of dilution and mixing (measured by the heat released in the absence of substrate) were less than 5% of the total calories measured for the enzymatic reaction (see Figure 3B). That). Throughout the enzymatic reaction, the exothermic rate was constantly monitored as shown in Figures 3A-3B. The heat flow reached its maximum shortly after adding the enzyme to the reaction cell,
Subsequent depletion of substrate levels returns to baseline (see Figure 3A). As can be seen from Figures 3A and 3B, the amount of heat generated directly reflects whether or not the substrate is present.

The actual experimental protocol is summarized in the flow chart shown in FIG. For example, the first
In one experiment, hexokinase is injected into a complete carbohydrate library. Carbohydrate libraries used include: D-glucose, arabinose, sucrose, ribose, lactose, galactose, maltose, and xylose examined in the presence of cofactor libraries, with cofactors ATP, GTP, CTP, TTP, UTP. , NADH, NAD
Included are PH, NAD, NADP, FAD, flavins, thiamine chloride monophosphate, pyridoxal 5'-phosphate, coenzyme A, and cocarboxylase.

The thermal signal is similar to that shown in Figure 3A. This signal indicates the turnover of the enzyme and thus the presence of the substrate. For the next set of experiments, the complete carbohydrate library was split into two, one containing the substrate (carbohydrate library 2A) and the other without the substrate (carbohydrate library 2B).
(Figure 4). The experiment is repeated as before, but this time only one sample produces the thermal signal for carbohydrate library 2A. Discard this harvest since no thermal signal of carbohydrate library 2B is observed (see Figure 4). Next, the carbohydrate library 2A is divided into two. The experiment is repeated as before, but again only one sample produces a thermal signal. The sample containing the substrate is divided in half again and the entire process is repeated until the elimination process reaches a single component sample containing glucose, the appropriate enzyme substrate.

All references and publications cited above are incorporated herein by reference. Other aspects are within the scope of the claims.

[Brief description of drawings]

FIG. 1 depicts a schematic of target-mediated conversion of test substance to product. Such conversion produces a heat signal. The method measures the heat output generated from the interaction between the test substance and the target (eg, target protein). As shown, target-mediated conversion of test substance to product produces a thermal signal. The thermal signal can be detected calorimetrically.

FIG. 2 depicts a schematic of a molecular detection switch for detecting binding of a test ligand. The method utilizes the generation of a thermal signal to identify the interaction of the test ligand with the target (eg, target protein). As indicated, the surrogate ligand is incubated in the presence of target so that the interaction (eg binding) occurs. As soon as the test ligand binds, the surrogate ligand is displaced. Free surrogate ligands (ie, substituted surrogate ligands) act as substrates for signal generators, such as enzymes, so that a thermal signal is generated. An important feature of the signal generator is that it generates a large amount of heat per unit time (ie, amplifies the bound signal). The thermal signal can be detected calorimetrically.

FIG. 3 depicts the measurements of the rate of heat generated during substrate screening with hexokinase (μcal / sec) with respect to time (sec). FIG. 3A shows the heat flow in the presence of substrate, reaching a maximum shortly after addition of enzyme to the reacting cells, and then decreasing to a substrate-depleted level of baseline. FIG. 3B shows a control (carbohydrate library without substrate).

FIG. 4 depicts an experimental flow chart for substrate screening with hexokinase.

FIG. 5a (i) shows the heat released in binding hexokinase to glucose over time. Over time, a certain amount of hexokinase in the cells is bound, so that the addition of more glucose produces little heat. Titration was performed by injecting 5 μl each of 5 μM glucose solution into cells for calorimetry containing 2 ml of 50 nM hexokinase solution. Since there is no ATP, glucose only binds to proteins. The heat released by each injection was measured and the coupling constant was calculated from those measurements. (ii) shows the heat released when hexokinase phosphorylates glucose in the presence of ATP and ATP is converted to ADP. Add 20 μl to calorimetric cells containing 100 μM glucose and 2 ml 1 mM ATP solution.
5 μl of M hexokinase solution was injected once and titrated. A large amount of negative fever is observed when hexokinase acts on glucose. We have integrated the heat released over time to follow the time course of the reaction.

FIG. 5b compares the rates of glucose and ATP reactions catalyzed by hexokinase measured by two different techniques, UV spectrophotometry and calorimetry. The heat output of the reaction from Figure 5a (ii) is plotted against time. Responses were also measured using the second assay, both showing the same rate of reaction (within experimental error).

FIG. 6 compares the kinetics of thrombin-catalyzed cleavage of a labeled peptide substrate (SAR-PRO-ARG-paranitroanilide) using UV spectrophotometry and calorimetry. Upon cleavage, the PNA (paranitroanilide) label absorbs more UV light and releases heat. Solution containing 186 nM thrombin and 250 μM substrate 2
ml was allowed to react and the reaction was monitored calorimetrically and spectrophotometrically. again,
Almost identical velocities were measured using different assay methods.

7A-7D deconvolute a mixture of compounds.
7 illustrates the use of the invention for Hexokinase and glucose were present in each test. Various mixtures of cofactors were added to each test. A significant amount of heat was generated only when ATP was present in the mixture. Titration was performed by injecting 5 μl of 20 μM hexokinase solution into a solution containing 100 μM glucose and one or more “cofactors” and having a total concentration of 1 mM. In the first experiment with the entire library where 15 cofactors were present, enzymatic turnover was observed (see Figure 5a (ii)). Next, the inventors were able to successfully screen cofactors in subsequent experiments to determine what is actually required for hexokinase to turn glucose. A large amount of negative fever is observed on the first infusion of the protein, when the correct cofactor, ATP, is present in the intracellular mixture of cofactors. In the absence of ATP, only a slight heat of dilution is observed.

FIG. 8 shows a calorimetric method for glucose phosphorylation catalyzed by hexokinase in the presence of a complex mixture of natural products. Titration was performed by injecting 5 μl of 20 μM hexokinase solution into a solution of 100 μM glucose and 1 mM ATP and increasing concentrations of tea. Tea solution resembles a natural product extract, a complex mixture. Significant enzymatic activity (turnover) was observed in all but the highest concentrations of tea.

FIG. 9 illustrates the use of calorimetry to help determine the function of potential proteins. The E. coli protein YJEQ binds to the GTP analog GTP-γ-S. 115 μM
The K _d was measured. Titration was performed by injecting 5 μl each of 11 mM GTP-γ-S solution into 2 ml of a solution containing 385 μM Escherichia coli protein of which function was not previously known. Figure 5a (i)
As shown in, experiments have shown that the protein binds to this molecule, an analogue of GTP, allowing us to putatively confer on this protein GTP-binding properties.

[Procedure amendment]

[Submission Date] July 15, 2002 (2002.7.15)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/50 G01N 33/50 Z 33/68 33/68 (81) Designated country EP (AT, BE, CH) , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN , GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, C , CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Bruzes Frank Joseph, Boston, Massachusetts, USA 84 (72) Invention Ferman Carlos H. USA Acton Mohawk Drive 41 F-term (reference) 2G040 AB12 AB14 BA02 BA24 CA02 CB03 CB04 DA02 GA05 GA07 HA06 2G045 AA40 DA12 DA13 DA14 DA36 FA33 GC30 4B029 AA07 BB15 BB18 QR32 QQQQQBQQQQQQQQQQQQQQQQR

Claims (37)

[Claims] 1. A method of analyzing a target comprising the steps of: (1) assigning a putative function to the target; (2) providing a library of candidate interactors; (3) including the target. Providing a reaction mixture; (4) contacting the target with a member of the library; (5) assessing the change in heat output of the reaction mixture; (6) selectively altering the resulting heat change. Analyzing the target by comparing the value with a default value. 2. The method of claim 1, further comprising repeating steps (3)-(6) for the second library member. 3. The method of claim 1, wherein the analyzing step comprises identifying a library member that is a target substrate or ligand. 4. The method of claim 1, wherein the target is a naturally occurring protein or fragment thereof. 5. The method of claim 1, wherein the target is a nucleic acid. 6. The library of candidate interactors comprises a plurality of members,
The method of claim 1, wherein each is known to interact with a target having an assigned putative function. 7. A library of candidate interactors is at least 10, 10 ² , 1
The method of claim 1, comprising 0 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ compounds. 8. The method of claim 1, wherein the library of candidate interactors comprises a plurality of members having common structural or functional characteristics. 9. A library of candidate interactors is a substrate library, cofactor library, carbohydrate library, purine and pyrimidine library, amino acid library, lipid library, vitamin and hormone library, nucleic acid library. , And a natural product library. 10. A member of a library of candidate interacting substances is an enzyme substrate, metabolite, cofactor, natural product, carbohydrate, polysaccharide, nucleic acid, amino acid, vitamin, hormone, lipid, small organic molecule, metal, peptide. 2. The method of claim 1, wherein the method is selected from the group consisting of :, proteins, lipids, glycoproteins, glycolipids, transition state analogs, and combinations thereof. 11. The method of claim 1, further comprising testing the target against at least one member of the second library. 12. The method of claim 11, wherein the testing of the first and second libraries occurs simultaneously. 13. The method of claim 11, wherein the target modifies a member of the library. 14. The change in heat output is measured by a microcalorimeter.
The method described. 15. A library of candidate interactors comprising at least one member that interacts with a target to produce a heat output. 16. A group consisting of a substrate library, a cofactor library, a carbohydrate library, a purine and pyrimidine library, an amino acid library, a lipid library, a vitamin and hormone library, a nucleic acid library, and a natural product library. The library of claim 15, which is selected from: 17. The library of claim 15, comprising multiple members having a common feature or function. 18. The library of claim 17, wherein all members of the plurality are enzyme cofactors or substrates for biosynthetic or degradative enzymes. 19. At least 10, 10²,Ten³,Ten^Four,Ten^Five,Ten⁶,Ten⁷, Or 10 ⁸ 16. The library of claim 15, which comprises a number of compounds. 20. The member is an enzyme substrate, metabolite, cofactor, natural product, carbohydrate, polysaccharide, nucleic acid, amino acid, vitamin, hormone, lipid, small organic molecule, metal,
16. The library of claim 15, selected from the group consisting of peptides, proteins, lipids, glycoproteins, glycolipids, and transition state analogs. 21. An apparatus for analyzing a biomolecular target, including: a calorimeter for assessing the change in heat of a reaction mixture containing at least one biomolecular target in contact with at least one candidate interactant. 22. The apparatus of claim 21, wherein the calorimeter comprises a microcalorimeter. 23. The apparatus of claim 22, wherein the microcalorimeter comprises a differential scanning microcalorimeter. 24. The apparatus of claim 21, wherein the calorimeter comprises an isothermal titration calorimeter. 25. The apparatus of claim 21, wherein the calorimeter comprises a reaction vessel for holding polymer beads having at least one candidate interactor. 26. The apparatus of claim 21, wherein the calorimeter comprises a molecular detection switch. 27. The apparatus of claim 1, wherein the calorimeter comprises a flow cell. 28. The calorimeter includes a sample holder having a plurality of compartments.
21. The device as described in 21. 29. The device of claim 28, wherein each compartment comprises a calorimetric cell and a thermopill. 30. The device of claim 29, wherein the calorimeter further comprises channels for transporting liquid to the plurality of compartments. 31. The apparatus of claim 30, wherein the calorimeter further comprises channels for transporting liquid to the plurality of compartments. 32. The device of claim 31, wherein the compartment is a channel for liquid transport and is formed in a microchip. 33. The apparatus of claim 28, wherein the sample holder comprises a multiwell plate. 34. The apparatus of claim 33, wherein the multiwell plate comprises a microtiter plate. 35. The apparatus of claim 34, wherein the microtiter plate comprises a 96 well plate. 36. The device of claim 21, wherein the calorimeter comprises an artificial neural network. 37. The device of claim 21, wherein the calorimeter comprises a two cell differential device.