WO2002040990A1 - Procede permettant de determiner le profil d'une proteine - Google Patents
Procede permettant de determiner le profil d'une proteine Download PDFInfo
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- WO2002040990A1 WO2002040990A1 PCT/JP2001/009940 JP0109940W WO0240990A1 WO 2002040990 A1 WO2002040990 A1 WO 2002040990A1 JP 0109940 W JP0109940 W JP 0109940W WO 0240990 A1 WO0240990 A1 WO 0240990A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Definitions
- the present invention relates to a method for estimating a function of a protein, more specifically, a biological function and / or disease involving a protein, and confirming the validity as a drug discovery target.
- proteins having a certain degree of homology in amino acid sequence have similar functions, and it is possible to estimate the function of any protein from its sequence or to classify it into families. It is performed on a daily basis.
- protein kinases a protein belonging to the family of protein phosphatase or a protein phosphatase, which is a protein phosphatase, is used to determine the sequence of a protein whose function is known using a computer. By examining the homology to and the presence or absence of specific subsequences (motifs), it can be estimated to a considerable extent.
- probe means administration of ligand, antisense, monoclonal antibody, or the like, or knockout of corresponding gene or Transgenic operation
- probe means administration of ligand, antisense, monoclonal antibody, or the like, or knockout of corresponding gene or Transgenic operation
- the behavior of molecules (observed molecules) other than the target proteins is measured, analyzed, compared, and collated with known biomolecular network information.
- a probe means for a target protein is obtained by, for example, one of the following methods.
- the state of the protein function is temporarily or permanently destroyed or modulated. produce.
- a group of biomolecules other than the target protein (these are called “observed molecules”), for example, various proteins .
- the behavior of small molecules including mRNA, other hormones, etc. is measured to detect quantitative or temporal fluctuations caused by disruption or modulation of the protein function.
- the observed molecule is a molecule whose biological function is already clear and which is included in the biomolecule network information.
- the obtained experimental data is analyzed by associating the meaning of each observed molecule in the biomolecule network information and the temporal variation, thereby obtaining the target protein.
- Associated biological function or disease can be estimated. Based on the results and the sequence homology with a known protein and the biological function that can be estimated based on the presence or absence of a motif sequence, efficient and accurate experiments can be performed on the target protein without trial and error. It helps to elucidate its biological function. As a result, whether or not the target protein has any meaning in any disease and is suitable as a drug discovery target, or a drug was developed using the target protein as a drug discovery target It would be possible to predict what the expected side effects would be in such cases.
- the number of obtained reports is smaller than when the biomolecule network is used.
- the results of the modulation by the application of can be arranged in a fixed order as independent data for each observed molecule, and can be expressed and compared with the size of figures or numbers or bar codes. For example, the more the barcode notation when a specific ligand is administered to a biological material of a specific disease is closer to the normal barcode notation than the barcode notation when no specific ligand is administered, the more desirable as a drug discovery target. I can say.
- a protein whose expression level fluctuates greatly by applying the probe means or a protein derived from mRNA whose expression level fluctuates greatly by applying the probe means, and Z or a protein designated The above-described method, which is a group of observed molecules; the above-mentioned method, which is a group of molecules selected so that the observed molecule group is involved in various biological functions as much as possible; Or the above method selected from the group consisting of a molecule group related to a biological function and a molecule group expressed and / or functioning in a specific organ or tissue; a biomolecule whose biomolecule network information is known
- the above-mentioned method which is biomolecule network information indicating a connection between functions and / or interactions in biosynthesis between biomolecule networks;
- the above method is provided, which is molecular function network information including information on the relationship between biomolecules and bioevents, in addition to information indicating known functions between biomolecules and / or interactions in biosynthesis.
- the above method wherein the specific ligand for the target protein has been created using virtual screening; the virtual screening takes into account the binding mode and the total degree of freedom of the ligand conformation.
- the above-mentioned method including a step of searching for a compound data base based on the automatic docking performed; the above-described method in which virtual screening is a compound data-based search method based on the automatic docking method ADAM; By administering two or more specific ligands to one target protein, or a combination of a specific ligand and an antisense molecule at two or more doses or concentrations, the change in the expression level or abundance of the observed molecule can be used as a biological response.
- the above method is provided, comprising the steps of measuring and analyzing.
- another protein of unknown biological function whose expression fluctuates greatly by applying the probe means obtained based on the information of the target protein is searched, and a probe created for the obtained another protein Searching for one or more proteins whose expression fluctuates significantly by applying the means, and until one of the above proteins whose expression fluctuates significantly matches any of the molecules in the biomolecular network information
- a method for estimating the relationship between two or more proteins having unknown biological functions including the target protein with molecules in known biomolecular network information.
- the behavior is the expression level and Z or abundance
- the method for estimating the involvement of two or more proteins of unknown biological function including the target protein with a biological function or disease is a preferred embodiment. is there.
- molecules included in known biomolecule network information molecules related to a specific disease and Z or a biological event, molecules expressed and generated or generated in a specific organ, and present in a specific organ And a group of molecules related to as many biological events as possible.
- Method for screening proteins related to biological functions a group of proteins involved in any disease based on biomolecule network information can be used as drug target candidates, protein drug candidates, or antibody drugs.
- a method for estimating side effects in the case of a protein candidate; for the purpose of developing a therapeutic drug for any disease or condition based on biomolecule network information, 2 Drug target candidates above, proteins medicament candidates, a method for selecting a candidate of the optimum from an antibody pharmaceutical antigenic protein candidate; is provided.
- probe means based on any target protein By applying probe means based on any target protein to biological materials or animals, by measuring and analyzing the effect on the behavior of one or more observed molecules with and without known biological function or unknown.
- a method for estimating the relevant biological function and Z or disease of the target protein By applying probe means based on any target protein to biological materials or animals, by measuring and analyzing the effect on the behavior of one or more observed molecules with and without known biological function or unknown.
- a method for estimating the relevant biological function and Z or disease of the target protein By applying probe means based on any target protein to biological materials or animals, by measuring and analyzing the effect on the behavior of one or more observed molecules with and without known biological function or unknown.
- a target to which the probe means is applied is a biological material including an organelle, a cell, a tissue, an organ, or an animal.
- propping means is a noquart or transgenic manipulation of a gene corresponding to a specific ligand or an antisense molecule or a monoclonal antibody or a target protein.
- the application of the probe means includes administering a specific ligand, antisense molecule, or monoclonal antibody, and performing knockout or transgenic manipulation of the gene corresponding to Z or the target protein. 8. The method according to any one of the above 1 to 7, comprising a step of preparing an animal.
- the biomolecule whose observed molecule has a known biological function or whose relationship with a biomolecule whose biological function is known is assumed, and the observed molecule is a molecule contained in biomolecule network information. 10. The method according to any one of 1 to 10.
- the observed molecule according to any one of 1 to 11 above, wherein the observed molecule is a protein or mRNA whose expression level fluctuates greatly by applying a probe means, or Z, or a specially designated biomolecule. Method.
- biomolecule network information described in any one of the above items 1 to 14, wherein the biomolecule network information is molecular network information indicating a link between biomolecules having known biological functions and interactions in biosynthesis. the method of.
- the biomolecule network information provides information on the relationship between biomolecules and bio-events, in addition to the information indicating the functions between biomolecules with known biological functions and the interaction link in Z or biosynthesis. 16. The method according to any one of 1 to 15 above, wherein the method comprises molecular network information.
- biomolecule network information is the molecular function network information prepared by the method described in Japanese Patent Application No. 2000-2767699. The method described in.
- the specific ligand or antisense molecule or monoclonal antibody is administered to a biological material or animal at two or more doses or concentrations, and the amount or time of expression or abundance of the observed molecule is determined. 19. The method according to any one of 1 to 18 above, comprising a step of measuring and analyzing a dynamic variation.
- 25 By measuring and analyzing the fluctuation of the behavior of the observed molecule due to the application of the probe means based on the target protein, the side effects of developing a drug using the target protein as a drug target and the target protein itself can be reduced. 25. The method according to any one of 1 to 24 above, wherein a side effect when the drug is developed or a side effect when an antibody against the target protein is used as a drug is estimated.
- the target protein can be used as a candidate for drug discovery for the purpose of developing a therapeutic drug for any disease or condition, a candidate protein drug, 28.
- any protein to be subjected to the method of the present invention is referred to as “target protein”.
- the target protein needs to have amino acid sequence information.
- information on biologic functions is not necessarily required.
- a search for a protein database with a known function is performed, and a sequence related to sequence homology or function is searched. It is convenient to estimate the biochemical function and the protein family to which it belongs based on the above characteristics and the presence or absence of the motif sequence.
- New proteins have been isolated and purified without knowing their functions, and the number of cases where the structure is elucidated by crystal analysis or nmr analysis is increasing.
- Information on the biological functions estimated from the structure and information on the presence or absence of a site to which a low-molecular ligand binds are useful for estimating the relationship with the biological molecular network from the biological response data.
- a specific antibody can be prepared and used as a pro- plication tool. If the target protein does not have the crystal structure of the relevant analog protein (eg, structure estimated by nmr), which is the type II model for modeling, probe the specific ligand obtained by experimental random screening. It can also be used as a means.
- Specific ligand or “ligand” refers to a small molecule or small molecule compound that binds to a target biopolymer (mainly a protein) more than to some extent.
- Biomolecule network information refers to a network that shows functional, biosynthetic, or metabolic connections between biomolecules. In principle, there should be a connection between directly binding or interacting biomolecules. For example, the case where molecule A becomes a substrate for molecule B or the signal is transmitted by binding of molecule A to molecule C is called a functional connection, and molecule C is generated from molecule A by enzyme molecule B. In such cases, the molecules A and C are called biosynthetic links.
- molecular network-based databases that focus on molecular connections include metabolic pathways such as KEGG (Kaneku et al., Kyoto University), Biochemical Pathways (Boehringer Mannheim), WIT (Indian Academy) of Sciences), Biofrontier ( ⁇ ubadani), Protein Pathway (AxCell), bioSCOUT (LION), EcoCyc (DoubleTwist), UM-BBD (Minnesota Univ.).
- metabolic pathways such as KEGG (Kaneku et al., Kyoto University), Biochemical Pathways (Boehringer Mannheim), WIT (Russian Academy) of Sciences), Biofrontier ( ⁇ ubadani), Protein Pathway (AxCell), bioSCOUT (LION), EcoCyc (DoubleTwist), UM-BBD (Minnesota Univ.).
- KEGG's PATHWAY database contains metabolic pathways of general low-molecular-weight compounds involved in substance metabolism and energy metabolism, as well as proteins involved in signal transduction.
- CSNDB National Institute of Health Sciences, Japan
- SPAD Korean et al., Kyushu University
- Gene Net Institute of Cytology & Genetics Novosibirsk, Russia
- GeNet GeNet (Maria G. Samsonova).
- DIP UCLA
- PathCalling CuraGen
- ProNet Myriad
- the present inventors have filed a patent application for a method for generating a molecular function network that includes information on events caused by specific biomolecules in living organisms, organs, tissues, and cells, in addition to information on connections between biomolecules (" Method for Generating Molecular Function Network ”, Japanese Patent Application No. 2000-276699, Japanese Patent Application.
- the molecular function network described in the above specification is also included in the biomolecular network of the present specification.
- “Virtual screening” refers to a method in which a large compound group is used to search for a large number of compounds in a compound database, instead of experimental screening (HTS) by binding to an arbitrary protein or inhibiting the reaction using a large compound library. This means selecting compounds that are theoretically likely to become ligands in the evening. Structural Features of Compounds In addition to methods based on the presence or absence of a partial structure, there are methods such as estimating the possibility of phyto-binding to the ligand binding site of the target biopolymer.
- Biomolecule refers to a molecule that exists in a living body, including an organism that is parasitic on the living body, such as an organelle, a cell, a tissue, an organ, a living individual, or an aggregate.
- an organelle such as an organelle, a cell, a tissue, an organ, a living individual, or an aggregate.
- Biosynthesis is used as a concept that includes all reactions or processes in which biomolecules are produced in a living body, and includes not only the metabolism and chemical conversion of small molecules but also the entire process of protein synthesis.
- Bio event is a concept that includes all phenomena, responses, reactions, and symptoms that occur endogenously or exogenously in a living body. Specific examples include transcription, cell migration, cell adhesion, cell division, neural circuit excitation, vasoconstriction, increased blood pressure, hypoglycemia, fever, convulsions, infection by foreign organisms and parasites such as viruses, and the like. it can.
- Bio response should be interpreted in the broadest sense, including all biological phenomena caused by organelles, cells, tissues, organs, animals, etc. by applying probe means to any biopolymer. Examples include improving disease and pathology indicators, fluctuating the abundance of observed molecules, and affecting protein and gene expression. Can be.
- Biochemical functions of proteins refer to the functions of proteins at the molecular level, such as the mode of transmission of enzymatic reactions and signal transduction.
- the enzyme that phosphorylates proteins is a protein kinase
- the enzyme that hydrolyzes proteins is a protease
- Receptors are classified by enzymatic reaction mode or signal transduction mode, such as seven transmembrane receptors and nuclear receptors that regulate transcription by binding sex hormones in the cell nucleus.
- Biological functions of proteins are functions that are observed at the cellular level or higher.
- both the acetylcholine receptor and the oxytocin receptor are seven transmembrane proteins, but the acetylcholine receptor is involved in neurotransmission, and the oxytocin receptor is involved in the biological function of uterine contractility.
- Angiotensin-converting enzyme is a protein (or peptide) carohydrolytic enzyme that converts angiotensin-I to angiotensin-I I and is involved in the biological function of regulating blood pressure.
- low molecular weight or “low molecular weight compound” refers to an organic conjugate molecule having a molecular weight of preferably 200 or more and 100 or less.
- Antisense molecule refers to an oligonucleotide or oligoribonucleotide having a sequence complementary to an arbitrary gene or mRNA sequence and having a residue of 10 to 30 residues. It is used for the purpose of specifically binding to and suppressing transcription and protein expression.
- Probe means includes a ligand that specifically binds to a target protein, an antisense molecule against mRNA encoding the protein, a vector for knocking out the target protein gene, and a gene sequence encoding the target protein.
- “Application of probe means” refers to inhibiting or modulating the function of a target molecule by causing the probe means to act on a living body.
- singular Riga Applying antisense molecules or monoclonal antibodies to cells, creating mice with knockout of target molecules, creating mice with overexpressed target molecules, or applying specific antibodies against target molecules to cells Injection, etc.
- Response of biological response there are expression of biomolecules, changes in abundance, and physical parameters. Examples of the former include protein expression, modification, mRNA expression, and changes in the abundance of one diagnostic marker molecule, and examples of the latter include blood pressure, body temperature, urine volume, and the like.
- observation molecule refers to a group of molecules selected or specifically designated for observing a biological response, or containing one or more molecules, preferably two or more molecules.
- the observation molecule is preferably, but not necessarily, a biomolecule included in known biomolecule network information. In this case, it is desirable that the link to the molecule contained in the known biomolecular network information has been clarified by some method such as the yeast two hybrid method.
- Different monitoring molecules can be set for each target protein, such as a protein group whose expression is greatly reduced by application of the probe means or an mRNA group having such properties, but a large number of target proteins can be set in advance with a certain intention.
- the biological response can also be measured using a specified group of molecules as an observation molecule.
- Observed molecules are not necessarily limited to proteins and mMA, but can be selected from all other types of small molecules, such as glucose, creatinine, and other disease-related diagnostic macromolecules.
- Bio event is a concept that includes all phenomena, responses, reactions, and symptoms that occur endogenously or exogenously in a living body. Specific examples include transcription, cell migration, cell adhesion, cell division, neural circuit excitation, inflammation, vasoconstriction, increased blood pressure, hypoglycemia, and others.
- behavior of the observed molecule or “behavior of the biomolecule” refers to changes in the abundance, expression level, modification state, localization, cofactor binding state, etc. of the observed molecule or biomolecule.
- Bio material refers to the organelles, cells, tissues, organs, etc. Means any level of sample.
- Step (1) Selection of candidate proteins for drug discovery
- a drug discovery target candidate protein can be selected by the following method. It is a feature of the method of the present invention that the biological function of each protein does not necessarily need to be known as a protein group of drug discovery target candidates. There is no need for the target proteins to be related to each other, and they may be unrelated proteins collected by the drug discovery researcher in the literature, or may be used between patients with specific diseases (symptoms) and normal individuals. Departure It is also possible to identify and select a group of proteins having a remarkable increase or decrease from the two-dimensional electrophoresis map of the expressed protein.
- biomolecule network information which indicates a direct relationship between biomolecules in terms of function or biosynthesis, enables the identification of proteins that are directly or indirectly associated with them, rather than proteins whose expression significantly increases or decreases due to a specific disease. It can be easily estimated and selected as drug discovery target candidate group. Furthermore, by using biomolecule network information including information on bioevents represented by specific biomolecules such as vasoconstriction, proteins on the biomolecule network that lead to bioevents related to the pathology and symptoms of the target disease A group can be selected as a candidate for drug discovery. However, it goes without saying that the present invention can be applied to drug discovery evening get candidates that are not included in known biomolecule network information selected by any other method. An example of selecting a protein group as a candidate for drug discovery using a molecular network is shown below.
- Proteins present on disease-related molecular nests are considered drug discovery target candidates.
- Typical probing methods include a method using a specific ligand, a method using an antisense molecule, and a method using a monoclonal antibody.
- a specific ligand binds specifically to a ligand binding pocket or other site of a target protein and controls its function.
- any method may be used to create a specific ligand, there is a method of experimentally finding the compound by random screening and a method of using virtual screening. To find ligands experimentally, it is not necessary to know the three-dimensional structure of the target protein, but it is necessary to prepare a protein sample and a vast library of compounds and perform large-scale random screening.
- any method may be used as long as it can find as many ligands with various structures as possible. From the viewpoint of a small molecule that stably binds to the ligand binding pocket of the target protein, it is desirable to use a virtual screening method based on automatic docking that can search for a new ligand having a wide structure without any prejudice. More preferably, a compound database utilizing the automatic docking method (Japanese Patent No. 2621884) of the invention titled “Searching method for stable complex of biopolymer-ligand molecule". The method of the invention title "Method of searching for novel ligand compounds from a three-dimensional structure database" (Japanese Patent Application No.
- 8_5144542 can be used. According to this method, a stable complex with the target protein can be estimated for each conjugate, taking into account the binding mode and all degrees of freedom derived from the ligand conformation, and the stability and characteristics of the structure can be estimated. Since it is possible to search using as a search condition, it has already been proven for many protein systems that the selected compound exhibits the desired activity at a high rate.
- Any compound database may be used as a target for virtual screening. If a compound database with two-dimensional structure information is available, it is automatically converted to a three-dimensional database with three-dimensional structure and other necessary information using structural three-dimensionalization software, and the virtual It can be used for screening. ⁇ If the target is the night of a sales product, the promising compound (hit) selected in the search can be obtained without synthesis and used for subsequent experiments, which is convenient. Even if the compound is not a commercially available compound or a real compound, the search may be performed on a data base based on a group of compounds having a chemical structure suitable for exploring a biological response as a ligand used as a probe means. In that case, the hit compound must be synthesized.
- the compound selected by virtual screening When the compound selected by virtual screening is obtained, it may be used for subsequent experiments as it is, but it may be possible depending on the reliability of the virtual screening method used, the shape of the ligand binding pocket of the target protein, and other conditions. If the target It is desirable to confirm the binding activity to the protein experimentally before proceeding to the next step.
- the binding activity of a compound selected and obtained by virtual screening to a target protein can be confirmed by, for example, a method based on surface plasmon resonance or other methods.
- the crystal structure or nmr structure (three-dimensional coordinates of each atom) of the target protein itself is available from the Protein De Bank, etc., it can be used as it is for virtual screening. Even if the crystal structure of the protein has not been analyzed, or even if it has been analyzed but its three-dimensional coordinates cannot be used, if information such as the crystal structure of a related protein is available, the crystal A structure modeled on a combo by a method such as substituting a side chain with the structure as ⁇ can be used in the same manner as the crystal structure. The higher the sequence homology, the more likely that the three-dimensional structures are similar and the easier the modeling.
- an antisense molecule can be used instead of a specific ligand.
- antisense molecules are oligonucleotides with 10 to 30 residues, which selectively bind to mRNA having a complementary nucleotide sequence, inhibit the translation of genetic information, and suppress protein expression. Used in. Whereas a specific ligand directly inhibits or activates the function of a target protein without affecting the expression or abundance of the target protein, an antisense molecule has the effect of directly suppressing the protein synthesis of the protein. .
- an antisense molecule having a nucleotide sequence complementary to the mRNA corresponding to the amino acid sequence of the target protein is synthesized and administered, and as a result of suppressing the protein synthesis of the protein, other biomolecules may be affected. From this, the role of the protein in the living body can be explored. Therefore, when a specific ligand, an antisense molecule, and a monoclonal antibody are administered to a living body, the biological response is different, and performing two or three kinds is inferring the function or disease related to the protein. Useful. For target proteins for which it is not possible to create small specific ligands, antisense molecules can be used as a probe.
- antisense molecules have the advantage that they can be easily synthesized as long as they have an mRNA sequence that encodes the target protein, and that they do not require any information on iffi construction.
- the target protein is a protein that has already been synthesized, a protein that has a slow overnight set-up, or a protein that has an important physiological action by converting the precursor protein synthesized by hydrolysis or the like.
- a clue to estimate the type of the target protein can be obtained. Probability is high. Therefore, a preferred embodiment of the method of the present invention includes a method in which two or more specific ligands are prepared as probe means, and an antisense molecule or a monoclonal antibody is created. Step (3): Setting of observed molecules
- Any data may be used as the data of the biological response when the probe means is applied to cells, tissues, organs, animals, etc. as long as the difference due to the presence or absence of the probe means can be measured and compared.
- Such data may include 1) effects on blood pressure, body temperature, pulse, cell shape, growth rate, etc., and 2) effects on expression and abundance of biomolecules.
- 1) is difficult to interpret in a short time, so the effect on the expression and abundance of biomolecules, which is easy to measure and can obtain many data at a time, should be considered as 2) .
- a molecule for measuring the biological response to the application of the probe means based on 2) is called an “observed molecule”.
- the easiest way to select the observed molecule is to select a protein or mRNA whose expression or modification is significantly altered by applying the probe means. It is a routine technique to determine the amino acid sequence of a group of proteins whose expression level remarkably increases or decreases depending on patients, healthy subjects, the presence or absence of drug administration, and the like, using a mass spectrum. The controversy is that even if there are a large number of proteins whose expression is greatly changed, they cannot be an effective means for estimating a biological function because their relationship is not known. The only way to know the function of a protein whose biological function is unknown is to relate the biological function or disease to a known protein.
- the observed molecule is a protein whose expression or modification is greatly changed by applying the probe means or a protein corresponding to mRNA, and shows a functional or biosynthetic link between biomolecules. It is desirable to select molecules included in the biomolecular network. Biomolecule network information can directly and indirectly associate a large number of seemingly unrelated proteins whose expression significantly changes by applying probe means, and place target proteins among biomolecules with known biological functions. Can be installed. In this method, the combination of proteins to be observed molecules changes for each target protein, and the set of observed molecules is determined after observing the change in protein expression due to the application of the probe means.
- the proteins on the protein expression map are Correspond to the molecule on the biomolecule network.
- correspondence with molecules on the known biomolecule network is prepared, and for all target proteins, analysis of the relationship between the target protein and biomolecules with known functions Becomes easier.
- the observed molecule can be converted into mRNA having a large variation in the mRNA expression map by applying the probe means without using the protein expression map.
- the method of selecting an observed molecule based on protein expression or mRNA expression can only target proteins.
- the observed molecules can be any group of biomolecules including arbitrarily selected low molecules, without being based on the protein expression map ⁇ ⁇ mRNA expression map. These molecules are desirably included in the biomolecule network information, and are desirably associated with molecules on a known biomolecule network.
- the number of observed molecules is specified as 1 or more, and preferably tens or more, for one target protein, but any combination of molecules may be used.
- molecules that are indicators of a specific disease or molecules that are known to increase or decrease in patients with a specific disease proteins that significantly increase or decrease expression in patients with a specific disease and normal individuals, specific organs or specific tissues
- One or more molecule groups can be appropriately selected from a group of molecules characteristically present at a specific site, a group of biomolecules binding to a receptor, and a group of molecules included in a certain molecular pathway in a biomolecular network.
- the biological response of a cultured cell, cultured tissue, cultured organ, or animal individual to the application of the probe means is the behavior of the observed molecule in the administered cell, tissue, organ, animal, etc., e.g., a change in expression and / or abundance.
- the biological response to the application of the probe means administered to an animal individual can be measured by the behavior of the observed molecule in a specific biological material (eg, organ, tissue, cell, etc.) (eg, expression level and Z or presence of Changes in the amount).
- the detection and / or quantification of the observed molecule may be performed by any method, but a method with high sensitivity and accuracy is preferable for detecting and / or quantifying the molecule having a small amount. If an antibody is prepared using each observed molecule as an antigen, the amount of the observed molecule can be easily quantified with high sensitivity by a technique such as ELISA or Western blottin. Simultaneous measurement of many observed molecules is also possible. Therefore, as a preferred embodiment of the method of the present invention, it is desirable to prepare a set of antibodies for a set of observed molecules (observed molecule group) selected with a specific intention, and to use them appropriately according to the purpose.
- the relationship between the target protein and the abundance of the target protein. can be estimated.
- a set of antibodies of a group of observed molecules consisting of molecules related to a specific disease is used, whether or not the target protein is a protein related to the specific disease can be determined by the effect on the molecules in the set.
- a new drug discovery gate can be found.
- the behavior of the observed molecule may be measured by any means.For example, when measuring the fluctuation of mRNA level, a DNA chip on which the corresponding DNA probe is immobilized can be used.
- a method can be used in which the extracted protein is developed by means such as two-dimensional electrophoresis or liquid chromatography, and its abundance is measured.
- a method of comparing and identifying the spectrum of each observed molecule measured in advance with the spectrum of the sample using the electronic spectrum and the like is also considered.
- the biological response can be obtained by changing the time after the application of the probe means and performing measurements over time, for example, 5 minutes, 20 minutes, 1 hour, and 4 hours. Furthermore, it is desirable to measure the biological response data by applying the probe means at two or more doses or concentrations and use it for analysis.
- biological response data is measured by applying both an antisense molecule and a specific ligand as probe means,
- two or more specific ligands having different structural characteristics can be used for analysis by measuring the biological response data.
- the biological response data described in two or more observed molecules on the known biomolecular network is the time when the strength and fluctuation of the biological response appear.
- the stronger the biological response and the shorter the time the change appears the closer the association of the target protein with the molecule on the known biomolecular network can be considered.
- the biochemical function of the target protein estimated by motif search, three-dimensional structure, etc. is considered.
- the relationship between the target protein and a molecule on a known biomolecular network can be estimated.
- the biological function associated with the target protein can be inferred. If biomolecule network information containing bio-event information can be used, it is possible to estimate the involvement in disease based on bio-event information that connects the closest molecular pathways and molecules. Evaluation scores can be calculated based on the number of biomolecules passing through and the reliability of information on direct interactions between biomolecules.
- the network-based database of existing biomolecules includes KEGG (Kyoto University, Kanehisa et al.), GeneNet (Institute of Cytology & Genetics, FA Kolpakov) targeting molecules on metabolic pathways and known molecules involved in signal transduction. ).
- these existing databases contain information on the action of each molecule at the molecular level (eg, phosphorylation or binding to the receptor), but do not show biological event information at the cell level or higher (eg, However, it does not include information on muscle contraction or hypoglycemia, and does not suggest any association with disease, symptoms, or side effects.
- the biomolecule network information used in the method of the present invention includes not only information on connections between biomolecules and action information on a biochemical level, but also bio-events such as a bio-response at a cell level or higher brought about by a biomolecule.
- Information that includes information on whether the event is increased or decreased by a specific molecule and that can be easily understood from a biological response in which the target protein is directly or indirectly related to a disease is convenient.
- a biomolecule network type database there is KeyMolnet (Title of Invention "Method for Generating Molecular Function Network", Japanese Patent Application No. 2000-276699).
- a probe method is obtained for the other protein as well as the target protein, and the biological response data is measured and compared. By doing so, the positioning of the target protein on the biological molecular network and the presumption of its association with functions and diseases can be confirmed, and the association of two or more proteins with unknown biological functions with functions and diseases can be confirmed. Can be estimated.
- This step can be additionally performed as necessary. Judgment of the validity of the target protein as a drug discovery target is sufficient if it can be easily determined by improving the disease state index in animals or the abundance of observed molecules.
- the type of biomolecule network information to be used is not particularly limited, but in particular, as in the biomolecule network information in “Method for Generating Molecular Function Network” (Japanese Patent Application No.
- the biomolecule network information includes a biological response at a cell level or higher as biological event information.
- the molecular connection from the drug discovery target protein to the bio-event or bio-molecule that directly causes the target disease or condition is clarified, and the connection to other bio-events can be estimated.
- Low risk of side effects is also a requirement for drug targets.
- Side effects include the side effects that are inevitably caused by setting the drug target, and those that occur depending on the structure of the compound (ligand). In the latter case, which causes side effects by binding to completely unrelated biopolymers on the biomolecular network, it can be avoided by subsequent structural modification or search for ligands with completely different structures.
- the former can be predicted to some extent from biomolecule network information, but it is useful to actually confirm the balance with the main effect using specific ligands and / or antisense molecules.
- biomolecule network information By using the biomolecule network information with the bioevent, it is possible to understand the existence of another biomolecule network related to the bioevent as a side effect.
- biomolecule network information By associating biomolecule network information with a known biological function that indicates a functional or biosynthetic link between biomolecules, information other than sequence cannot be used.
- Biological functions related to the target protein and / or Disease can be estimated.
- a specific ligand of the protein or an antisense molecule is used as a probe means, and preferably, the effect of the abundance of a group of observed molecules, which is a molecule on a known biomolecular network, on the biological response data. Measure and analyze as evening You.
- an arbitrarily selected group of observed molecules can be used as the observed molecule. This makes it possible to detect and / or quantify proteins with low expression levels.
- proteins related to a specific disease or a specific biological function can be screened from a large number of proteins with unknown functions.
- biomolecule network information including bioevent information
- bioevent information it is possible to estimate a relationship with a disease from the association between a target protein and a molecular pathway in the network.
- the target molecule is appropriate as a drug discovery target, and what are the side effects of developing a drug using the target molecule as a drug discovery target.
- One embodiment of the method of the present invention is as follows.
- the biological function of the target protein estimated by the method of the present invention is not necessarily limited to one, and by performing the method of the present invention, the target protein may be associated with any function or any disease. Can be estimated. Those that can be deduced from the sequence information about the demonstrative function can also be used in the method of the present invention.
- the biological function of a biomolecule is determined by its relationship with other biomolecules, and it is not known how much the molecule itself (only that molecule) is studied. Using a specific ligand or an antisense molecule against the gene of the protein as a probe means to measure and analyze the effect of the living body itself on the expression or abundance of biomolecules other than the protein as a biological response Therefore, it has a feature that the function of the target protein can be easily estimated.
- biomolecule network information that indicates the functional or biosynthetic relationship between biomolecules whose biological functions are known, it is possible to analyze which molecule on the network the target protein is closely related to. By doing so, it becomes possible to perform function estimation more efficiently.
- the amount and function of a biomolecule are complicatedly controlled by a mechanism such as feedback control, and the effect on one molecule may eventually be widened.
- a mechanism such as feedback control
- the effect on one molecule may eventually be widened.
- the present invention is also useful as a validation method for drug discovery.
- drug target evaluation information with drug lead information is obtained, which is more and more useful.
- the function of a protein more specifically, the biological function and Z or disease involved in the protein can be estimated.
- whether the protein is appropriate as a drug discovery target Can be easily confirmed.
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01982776A EP1343006A4 (en) | 2000-11-15 | 2001-11-14 | METHOD FOR DETERMINING THE PROFILE OF A PROTEIN |
JP2002542867A JPWO2002040990A1 (ja) | 2000-11-15 | 2001-11-14 | 蛋白質のプロファイリング方法 |
CA002431655A CA2431655A1 (en) | 2000-11-15 | 2001-11-14 | Method of profiling protein |
AU2002214286A AU2002214286A1 (en) | 2000-11-15 | 2001-11-14 | Method of profiling protein |
US10/416,157 US20040054144A1 (en) | 2000-11-15 | 2001-11-15 | Method of profiling protein |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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JP2000-348054 | 2000-11-15 | ||
JP2000348054 | 2000-11-15 | ||
JP2000-396950 | 2000-12-27 | ||
JP2000396950 | 2000-12-27 |
Publications (1)
Publication Number | Publication Date |
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WO2002040990A1 true WO2002040990A1 (fr) | 2002-05-23 |
Family
ID=26604002
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2001/009940 WO2002040990A1 (fr) | 2000-11-15 | 2001-11-14 | Procede permettant de determiner le profil d'une proteine |
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Country | Link |
---|---|
US (1) | US20040054144A1 (ja) |
EP (1) | EP1343006A4 (ja) |
JP (1) | JPWO2002040990A1 (ja) |
AU (1) | AU2002214286A1 (ja) |
CA (1) | CA2431655A1 (ja) |
WO (1) | WO2002040990A1 (ja) |
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JP2007521799A (ja) * | 2003-11-07 | 2007-08-09 | ノバルティス アクチエンゲゼルシャフト | 線維芽細胞増殖因子フラグメントの使用 |
WO2018003995A1 (ja) * | 2016-07-01 | 2018-01-04 | 国立大学法人東北大学 | 免疫チェックポイント阻害薬使用における免疫関連副作用の予測方法 |
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US7930262B2 (en) * | 2007-10-18 | 2011-04-19 | International Business Machines Corporation | System and method for the longitudinal analysis of education outcomes using cohort life cycles, cluster analytics-based cohort analysis, and probabilistic data schemas |
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CN109712678B (zh) * | 2018-12-12 | 2020-03-06 | 中国人民解放军军事科学院军事医学研究院 | 关系预测方法、装置及电子设备 |
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- 2001-11-14 EP EP01982776A patent/EP1343006A4/en not_active Withdrawn
- 2001-11-14 WO PCT/JP2001/009940 patent/WO2002040990A1/ja active Application Filing
- 2001-11-14 AU AU2002214286A patent/AU2002214286A1/en not_active Abandoned
- 2001-11-14 JP JP2002542867A patent/JPWO2002040990A1/ja active Pending
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Also Published As
Publication number | Publication date |
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EP1343006A1 (en) | 2003-09-10 |
US20040054144A1 (en) | 2004-03-18 |
CA2431655A1 (en) | 2002-05-23 |
JPWO2002040990A1 (ja) | 2004-03-25 |
AU2002214286A1 (en) | 2002-05-27 |
EP1343006A4 (en) | 2007-06-06 |
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