JP2006141348A - Method for selecting and acquiring domain polypeptide interacting with target substance, method for identifying structural motif sequence, and method for designing molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for selecting and acquiring a domain polypeptide interacting with a target substance, to provide a method for identifying and acquiring a structural motif, and to provide a method for designing a molecule interacting with the target substance. <P>SOLUTION: A pharmaceutical compound interacting with the target substance is designed as follows: the domain polypeptide which is necessary for interacting with the target substance is cut out of a protein and acquired, by utilizing a cell-free protein displaying procedure, in a method for analyzing a protein or a polypeptide interacting with the target protein; the structural motif comprising an amino acid sequence which is necessary and sufficient for interacting with the target substance is identified, by analyzing an amino acid sequence of the acquired domain polypeptide; and the pharmaceutical compound is designed, by estimating a combining space based on a three-dimensional structure of a complex of the structural motif with the target substance. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for selecting and obtaining a domain polypeptide that interacts with a target substance, a method for identifying a structural motif using the amino acid sequence of the domain polypeptide, and a compound that interacts with a target substance using the structural motif. The present invention relates to a molecular design method.

  Proteins, which are the most fundamental and indispensable players in the structure and function of living organisms, are generally composed of several domains consisting of tens to hundreds of amino acids. A domain is the smallest unit that controls the structure and function of a protein. In particular, relatively new proteins such as humans and rodents are known to have various domains. Proteins interact with other biomolecules through these domains, and are thus deeply involved in the regulation of gene expression, cytoskeleton formation, and intracellular signal transduction at the cellular level. In addition, this interaction is essential for the maintenance of life activity at the individual level, and on the other hand, it may cause disease.

  However, the theoretical and experimental methods of finding this domain are extremely large based on comparison with the existing information accumulated. For example, as a method for estimating a domain possessed by a protein, only an analysis method based on amino acid sequence homology using a computer is known for a protein database obtained by integrating individual experimental information. In this method, after homology analysis by a computer, it was necessary to experimentally verify whether or not the estimated domain actually has a function. For this reason, the development of an effective method for identifying an amino acid sequence region corresponding to a protein domain has been a major issue.

  On the other hand, phage display methods, ribosome display methods, mRNA display methods, and the like have been put into practical use in recent years as techniques for analyzing protein interactions. These technologies add a protein and its gene complex to the substance to be analyzed, collect the interacting complex, and then analyze the sequence of the gene part to identify the interacting protein It is a method to do. The yeast Two-Hybrid system is a method for analyzing protein interactions using the yeast intracellular transcription system, and is well known as a high-throughput analysis means. Furthermore, a method for identifying a protein that interacts by mass spectrometry after concentrating a substance to be analyzed by affinity chromatography or the like has been developed. However, the information obtained by these methods is only a fragment of a protein that interacts with the substance to be analyzed, and whether or not a domain that is the minimum unit necessary for interaction exists in the fragment. Relied on homology analysis using existing amino acid sequence databases. In addition, even if a domain is included, the polypeptide obtained by the above method has many extra amino acid sequences bound to it (FIG. 20 (A)). Requires a very complicated operation.

  Moreover, identification of the protein which interacts with a target substance by the IVV method (refer patent document 1 and nonpatent literature 1 etc.) which is the technique which analyzes the interaction of a substance and protein was also tried. According to this method, by repeating the operation of obtaining a molecule that interacts by bringing the target substance into contact with the protein, a partial polypeptide of the protein that is known to interact with the target substance is obtained ( (See Non-Patent Document 2, etc.). This polypeptide contains the above-mentioned domain, but still has an extra amino acid linked (FIG. 20 (A)), and it is similarly complicated to obtain the minimum unit necessary for interaction with the target substance. Was necessary.

On the other hand, regarding proteins that are targets of drug discovery that are known to be related to specific diseases, substances that suppress the interaction of the proteins and control their activities have been developed as therapeutic agents for the diseases. Often. It is known that a protein has a site called a hot spot at the interface of domains involved in the interaction (Non-patent Document 3, FIG. 20 (A)). Development of pharmaceutical compounds that act on spots and suppress protein interactions is desired. In general, amino acids and their side chains that exist in hot spot sites involved in protein interactions have a high degree of freedom in three-dimensional structure. It is known that it is difficult to do (Non-patent Document 3). In this sense, it has been desired to provide a simple identification or acquisition method for a domain (FIG. 20B) that is a minimum unit necessary for protein interaction.
WO98 / 16636 Publication Japanese Patent Application No. 2003-315385 Specification Protein Nucleic Acid Chemistry Vol.46, No2 (2001) 138-146 J. of Biological Chemistry, vol.276, 24, 20898-20906 (2001) Nature Reviews 301-317Vol.3 (2004)

  The present invention relates to a method for analyzing a protein or polypeptide that interacts with a target substance, and using a cell-free protein display method to obtain a domain polypeptide necessary for interaction with the target substance by cutting it out from the protein. And a method for identifying a structural motif comprising an amino acid sequence necessary and sufficient for interaction with a target substance by analyzing the amino acid sequence of the domain polypeptide obtained by the method, and the structural motif identified by the method The present invention has been made in order to provide a method for designing a pharmaceutical compound that interacts with a target substance using the above.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have (1) bound a nucleic acid construct containing puromycin to the 3 ′ end of an mRNA library, and used this as a template for cell-free protein synthesis. The protein-nucleic acid conjugate in which the protein and the mRNA encoding it are covalently bound is produced through puromycin of the nucleic acid construct, and (2) the protein-nucleic acid conjugate is brought into contact with the target substance. And selecting the above-mentioned conjugate having a protein part that specifically binds to the target substance, (3) amplifying the mRNA attached to the selected protein-nucleic acid conjugate by polymerase chain reaction (PCR), and (4) amplification The above-mentioned protein-nucleic acid conjugate is prepared using the prepared DNA as a part of the template, and the steps (2) to (4) are performed (hereinafter referred to as “concentration operation”). Is repeated several times, the average chain length of mRNA contained in the mRNA library obtained gradually decreases, the amino acid chain length of the identified protein also shortens, and several We found the phenomenon of cluster formation.

  Thus, the above enrichment operation was repeated using PPARγ2 (Peroxisome Proliferation-activated receptor gamma 2) associated with diabetes as a target substance, and as a result of analyzing the amino acid sequences of the polypeptides that formed clusters among the interacting proteins, The peptide was obtained as a partial polypeptide consisting of several tens of amino acids having a common sequence, and it was found that these substantially coincide with the PPARγ2 binding domain. Furthermore, the amino acid sequence of the polypeptide is arranged in the order of similar amino acid sequences and analyzed (multiple alignment analysis), and the amino acid sequence necessary and sufficient to interact with the target substance is obtained by determining the position and frequency at which common amino acids appear. It was found that a structural motif sequence can be determined. The present invention has been accomplished based on these findings.

That is, according to the present invention,
(1) (i) a step in which a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part is brought into contact with the target substance; ii) Obtaining the protein-nucleic acid conjugate specifically bound to the target substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding the protein part A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part, (iii) the steps (i) and (ii) above are carried out specifically for the target substance A method for selecting and obtaining a domain polypeptide that interacts with a target substance, comprising the step of repeating until the nucleic acid part of the bound protein-nucleic acid conjugate forms a cluster encoding the domain polypeptide,
(2) (i) a step of contacting a target substance with a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part; ii) Obtaining the protein-nucleic acid conjugate specifically bound to the target substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding the protein part A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part, (iii) the steps (i) and (ii) above are carried out specifically for the target substance A step of repeating until the nucleic acid part of the bound protein-nucleic acid conjugate forms a cluster that encodes the domain polypeptide, and (iv) a step of obtaining the clustered nucleic acid in step (iii) Substance and A method for selecting and obtaining a nucleic acid encoding an interacting domain polypeptide;
(3) The domain polypeptide is a polypeptide having a length of 90% or less of the full-length protein, and a polypeptide having a length of 130% or less of the interaction domain with the target substance ( 1) or the method according to (2),
(4) (i) a step of contacting a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part with a target substance ( ii) Obtaining the protein-nucleic acid conjugate specifically bound to the target substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding the protein part A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part, (iii) the steps (i) and (ii) above are carried out specifically for the target substance A step of repeating until the nucleic acid part of the bound protein-nucleic acid conjugate forms a cluster encoding the domain polypeptide, (iv) a step of analyzing the base sequence of the nucleic acid that formed the cluster in step (iii), (v) There is a base sequence Methods for identifying the structure motif sequence, which comprises the step of selecting a common sequence present frequency and / or frequency as an indicator of the amino acid sequence translated it,
(5) A structural motif having a transformed function, characterized in that a mutation is introduced into a part of the structural motif sequence described in (4), and the interaction is analyzed by bringing the mutant structural motif into contact with a target substance. How to get the
(6) (i) a step of preparing a domain polypeptide selected in (1) or (3) above, or a complex of the structural motif described in (4) or (5) and a target substance, (ii) And (iii) predicting the binding space of the target substance from the identified binding site, and analyzing the binding structure. A molecular design method for a compound that interacts with a target substance, comprising a step of designing a compound in time,
(7) The method according to any one of (1) to (6) above, wherein the target substance is related to a disease,
(8) (i) the target substance is associated with a disease, and the domain polypeptide selected in (1) or (3) above or the structural motif described in (4) or (5) above and the target substance A step of producing a complex, (ii) a step of analyzing the three-dimensional structure of the complex and specifying a binding site between the target substance and the polypeptide or structural motif, and (iii) binding of the target substance from the identified binding site Predicting space, designing a compound that fits the binding space, and (iv) synthesizing the designed compound, and a method for producing a therapeutic and / or prophylactic agent for a disease associated with a target substance,
Is provided.

  According to the present invention, a domain polypeptide necessary for interaction with a target substance is obtained by excising from a protein, and the amino acid sequence of the obtained domain polypeptide is analyzed to be necessary and sufficient for interaction with the target substance. There are provided a method for identifying a structural motif sequence comprising a simple amino acid sequence, a method for designing a compound that interacts with a target substance using the structural motif identified by the method, and the like. When the target substance is a substance related to a disease, this method is useful for obtaining a short-chain polypeptide serving as a therapeutic and / or prophylactic agent for the disease, or for molecular design / acquisition of a compound.

Hereinafter, the present invention will be described in more detail. However, the following description of the constituent elements is a representative example of the embodiment of the present invention, and the present invention is not limited only to these contents.
(1) Selection and acquisition method of domain polypeptide and nucleic acid encoding the same One of the present invention is a method of selecting and acquiring a domain polypeptide that interacts with a target substance by the method described below. The “domain polypeptide” includes a “domain” that is a necessary minimum unit that interacts with a target substance, and a polypeptide comprising an amino acid residue having a length of 30% or less of the amino acid chain length of the domain is the domain. It has the structure added to. The added polypeptide may be either the C-terminal side or the N-terminal side of the domain, or both. The “domain”, which is a necessary minimum unit that interacts with a target substance, is a partial structure of a protein that itself forms a stable three-dimensional structure, and means a unit having a function of binding to and interacting with a target substance. The domain in the present invention includes known and unknown domains.

  “Interact” means binding to a target substance to control the function of the substance or being controlled by the substance. The “target substance” used in the method of the present invention may be any substance that interacts with a protein or polypeptide, but is associated with a specific disease, and control of the function of the substance is related to the disease. Substances known to be useful for the treatment and / or prevention of are particularly preferably used. Specific examples include proteins, polypeptides, nucleic acids, carbohydrates, lipids, compounds, known pharmaceutical compounds, and the like.

  The method for selecting and obtaining a domain polypeptide of the present invention comprises: (1) a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part; A step of contacting with a target substance, (2) obtaining the protein-nucleic acid conjugate specifically bound to the target substance, amplifying the nucleic acid part of the conjugate, and using this as a part of a template, A step of preparing a protein-nucleic acid conjugate group in which a part and a nucleic acid part encoding the same are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part, (3) in (1) and (2) above It is characterized by including the process of repeating a process until the nucleic acid part of the protein-nucleic acid coupling body couple | bonded specifically with the target substance forms one or more clusters. The method is described in detail below.

(I) Step of contacting a protein-nucleic acid conjugate and the conjugate with a target substance The “protein-nucleic acid conjugate” used in the method of the present invention is, for example, as described in WO98 / 16636 The protein part and the nucleic acid part that encodes it are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part, and can be a powerful tool for protein interaction analysis and the like.

  Here, the “protein part” includes a protein encoded by a coding sequence contained in the nucleic acid part, and the protein includes both known and unknown proteins. Specifically, the protein is expressed in an organism. Are all included, and those composed of a synthetic polypeptide and those composed of a random amino acid sequence are also included. Moreover, the fusion protein of the tag for purifying a protein-nucleic acid coupling body and the said protein is also contained. The “nucleic acid part” is a nucleic acid containing a coding sequence encoding the protein of the protein part and further containing a sequence for generating the protein in the protein synthesis system to be used, and may be RNA or DNA.

  Specifically, the nucleic acid part includes a coding region and an expression control region. Examples of expression control sequences include (1) promoter sequences, (2) sequences recognized by ribosomes during translation, and the like. The type of promoter sequence is not particularly limited as long as it is appropriately selected from those suitable for the expression system to be applied. Examples thereof include a T7 promoter sequence recognized by E. coli virus T7 RNA polymerase, an SP6 promoter sequence recognized by SP6 RNA polymerase, and the like. In addition, sequences recognized by ribosomes during translation include DNA sequences corresponding to RNA sequences (Kozak sequences) recognized by eukaryotic ribosomes during translation, and shine recognized by prokaryotic ribosomes. A sequence recognized by the ribosome of Tabacco mosaic virus, such as a Dalgarno sequence (Shine-Dalgarno), a 5 ′ cap structure (Shatkin, Cell, 9,645- (1976)), an omega sequence, and the like described in WO 03/56009 Examples thereof include a sequence, a ribosome recognition region of rabbit β-globulin, Xenopus β-globulin or bromo mosaic virus. In addition, when PCR is used to amplify the nucleic acid part of the protein-nucleic acid conjugate, those containing a PCR primer sequence are also included. In addition, when PCR is used to amplify the nucleic acid part, one containing a common sequence for PCR primer binding is also included.

The coding sequence is a sequence encoding a protein contained in the protein part of the protein-nucleic acid conjugate, and the type is not particularly limited and can be appropriately selected depending on the purpose. Further, when preparing a protein-nucleic acid conjugate group, a base sequence or the like possessed by each element of a cDNA library is also preferred.
In addition, the “nucleic acid construct” bound to the 3 ′ end of the nucleic acid part has a “nucleic acid derivative” bound to the end, and the protein in a cell-free protein translation system or living cell using the nucleic acid part as a template. When translation is performed, it means that the nucleic acid derivative (for example, puromycin) has a function of binding to a protein by entering the A site of the ribosome after translation proceeds to the vicinity of the end of mRNA. Specifically, those including a spacer, a labeling part for labeling the protein-nucleic acid conjugate, a structure for binding the conjugate to a solid phase, a primer part for reverse transcription of the nucleic acid part, and the like are preferable.

  The “nucleic acid derivative” is not limited as long as it is a compound having the ability to bind to the C-terminus of the synthesized protein when the protein is translated in a cell-free protein translation system or living cells, but the 3 ′ Those having a chemical structure skeleton similar to aminoacyl-tRNA at the end can be selected. Representative compounds include puromycin and 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), that is, PANS-Gly having an amino acid part of glycine and an amino acid part of valine. PANS-Val, PANS-Ala whose amino acid part is alanine, and other PANS-amino acid compounds whose amino acid part corresponds to all amino acids.

In addition, 3′-N-aminoacyl adenosine aminonucleoside (AANS-amino acid) in which the amino group of 3′-aminoadenosine and the carboxyl group of amino acid are linked by dehydration condensation, that is, the amino acid part is glycine. AANS-Gly, AANS-Val whose amino acid part is alanine, AANS-Ala whose amino acid part is alanine, and other AANS-amino acid compounds corresponding to each amino acid whose amino acid part is all amino acids can be used. In addition, nucleic acid or nucleic acid and amino acid ester-bonded ones can be used. Furthermore, the nucleic acid derivatives used in the present invention are all compounds in which a nucleic acid or a substance having a chemical structure skeleton similar to a nucleic acid and a base is chemically bonded to a substance having a chemical structure skeleton similar to an amino acid. . The nucleic acid derivative is more preferably a compound in which puromycin, PANS-amino acid or AANS-amino acid is bound to a nucleoside via a phosphate group. Of these compounds, puromycin derivatives such as puromycin, ribocytidylpuromycin, deoxycytidylpuromycin, deoxycytidyldeoxycytidylpuromycin, and deoxyuridylpuromycin are particularly preferred.
Such a nucleic acid derivative can be produced by a chemical bonding method known per se. Specifically, when the synthesis unit is bound by a phosphodiester bond, the synthesis unit can be synthesized by solid phase synthesis by a phosphoramidide method or the like generally used in DNA synthesizers. When a peptide bond is introduced, a synthesis unit is bound by an active ester method or the like, but when a complex with DNA is synthesized, a protecting group capable of supporting both synthesis methods is required.

  As the “spacer”, a polymer substance such as polyethylene or polyethylene glycol or a derivative thereof, a biopolymer substance such as an oligonucleotide, a peptide or a derivative thereof, or the like is used, and preferably polyethylene glycol is used. The length of the spacer is not particularly limited, but preferably the molecular weight is 150 to 6000, or the number of atoms in the main chain is 10 to 400 atoms, more preferably the molecular weight is 600 to 3000, The number of atoms in the chain is 40 to 200 atoms.

  Examples of the “labeling part” include affinity substances, covalent substances, fluorescent substances, and degradable substances. These are used as “a structure for binding the linked substance to a solid phase” and also used as a “structure for purifying the linked substance”. Examples of the affinity substance include poly A sequences, poly T sequences, various antigens or antibodies such as biotin and FLAG, His tags, ligands such as NTA, receptor ligands, and the like. Examples of the covalently binding substance include nucleic acid terminal portions such as deoxyribonucleotide and ribonucleotide, functional groups such as hydrazide, ketone, and thioester, and crosslinkable substances such as psoralen. Examples of the fluorescent substance include fluorescein, Oregon green, rhodamine, tetramethylrhodamine, Texas red, Cy3, Cy5, Alexa488 and the like. Examples of the degradable substance include derivatives having a 1- (2-nitrophenyl) -ethyl group that decomposes by a photoreaction, amino acid sequences recognized by proteases and peptidases, and the like. These labeling substances are known and used in general and can be easily obtained, and can be labeled by binding to a nucleic acid or the like by a conventional method.

  Preferred structures of a preferred nucleic acid part and a nucleic acid construct or nucleic acid derivative (hereinafter sometimes referred to as a “protein-nucleic acid conjugate template”) bound to the 3 ′ end thereof include RNA as a nucleic acid part and nucleic acid It has a structure in which a single-stranded nucleic acid (hereinafter sometimes referred to as a “spacer chain”) to which a spacer having a derivative at its end is bound in a branched state is annealed. Examples include those in which a single-stranded nucleic acid and a nucleic acid part are contained. Specifically, the method described in the specification of PCT / JP2004 / 013399 and the method described in the specification is preferably used as the production method. By having such a structure, the binding efficiency of the nucleic acid part and the nucleic acid construct by RNA ligase or the like is high, the protein-nucleic acid conjugate can be purified by the labeling part, the RNA of the nucleic acid part can be reverse-transcribed, The nucleic acid part can be double-stranded DNA.

A protein-nucleic acid conjugate can be produced by introducing the thus constructed protein-nucleic acid conjugate template into a protein translation system. Translation systems for artificially generating the protein that it encodes from nucleic acids are known to those skilled in the art. Specifically, a cell-free protein synthesis system in which a component having protein synthesis ability is extracted from an appropriate cell, and the target protein is synthesized using the extract. Such a cell-free protein synthesis system includes elements necessary for translation such as ribosomes, initiation factors, elongation factors, and tRNAs. Examples of such cell-free protein synthesis systems include eukaryotic cell-free protein synthesis systems, and more specifically, rabbit reticulocyte extract and wheat germ extract. It is not limited. As the cell-free protein synthesis system, a commercially available kit can be used. For example, the kit rabbit reticulocyte lysate, Rabbit Reticulocyte Lysate Systems, Nuclease Treated (Promega Corporation) and the like are used, and as the wheat germ ^{extract, PROTEIOS TM Wheat germ cell-free} protein synthesis core kit (TOYOBO Etc.). As the protein translation system, live cells may be used. Specifically, prokaryotic or eukaryotic organisms such as cells of E. coli can be used. The cell-free protein translation system or living cell is not particularly limited as long as protein synthesis is performed by adding or introducing a nucleic acid encoding a protein therein. Immediately before introducing the nucleic acid construct of the present invention into a cell-free protein synthesis system, a step of heating at 60 to 90 ° C. and then rapidly cooling is preferable because the synthesis efficiency of the protein-nucleic acid conjugate is increased.

When purifying a protein-nucleic acid conjugate from the above translation reaction solution, if an affinity substance or a covalent binding substance is bound to the labeled chain, the purification is performed via the affinity substance or the covalent binding substance. Can do. The purification method can be appropriately selected according to the affinity substance and covalent bond substance to be used, and a known method per se can be used.
The protein-nucleic acid conjugate produced above is brought into contact with the target substance and subjected to a step of selectively obtaining a conjugate that interacts with the substance. This selection method means that a protein that interacts with a target substance is selected as the conjugate using the function (biological activity) of the protein in the protein-nucleic acid conjugate. As a method for analyzing such interaction, for example, the method described in WO98 / 16636 can be used. The protein-nucleic acid conjugate to be contacted with the target substance is usually a plurality of conjugate groups produced from a cDNA library or the like.

  Moreover, the protein-nucleic acid conjugate of the present invention and the target substance can be used by being bound to a solid phase (support). The protein-nucleic acid conjugate can be bound to the solid phase when an affinity substance or a covalent substance is bound to the labeling portion. Specifically, the protein-nucleic acid conjugate is brought into contact with a solid phase on which an affinity substance or a covalently bound substance has an affinity or binds a substance in advance, thereby bringing the protein-nucleic acid conjugate into contact. It can be easily immobilized on a solid phase. Binding of the target substance to the solid phase is described, for example, in Scott, J. et al. K. & Smith, G.M. P. (1990) Science, 249, 386-390; Devlin, P. et al. E. et al. (1990) Science, 249, 404-406; Mattheakis, L .; C. et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 9022-9026, etc.

  The solid phase (support) is not particularly limited as long as it is a support that can be used for immobilizing normal nucleic acids, proteins, carbohydrates, lipids, compounds, and the like. The shape of the support is not particularly limited as long as it does not adversely affect the formation of a bond between affinity substances or covalent substances, or the binding of a target substance. For example, a plate, a microwell, a bead, etc. It can take any form. Examples of the support material include glass, cement, ceramics such as ceramics, polyethylene terephthalate, cellulose acetate, bisphenol A polycarbonate, polystyrene, polymethyl methacrylate, and other polymers, silicon, activated carbon, porous glass, and porous ceramics. And porous materials such as porous silicon, porous activated carbon, woven and knitted fabric, non-woven fabric, filter paper, short fiber, and membrane filter.

  The protein-nucleic acid conjugate to be subjected to the above selection method may be one in which the nucleic acid part is RNA, or may be a protein-reverse transcription nucleic acid conjugate in which the mRNA chain is reverse-transcribed into DNA. Reagents and reaction conditions necessary for the reverse transcription reaction are well known to those skilled in the art, and can be appropriately selected as necessary. Furthermore, the double-stranded DNA-protein conjugate can be prepared and used by degrading the RNA of the obtained protein-reverse transcription nucleic acid conjugate using an RNase or the like and subjecting the DNA to a polymerase reaction. . Use of such a protein-reverse transcription nucleic acid conjugate or a double-stranded DNA-protein conjugate is preferred because the stability of the nucleic acid moiety is good and there is no non-specific adsorption of single-stranded RNA.

(Ii) Acquisition of a protein-nucleic acid conjugate specifically bound to a target substance, amplification of the nucleic acid portion, and a protein-nucleic acid conjugate preparation step using the nucleic acid portion as a part of a template Protein selected by the above selection method -The nucleic acid conjugate is subjected to a step of selecting it again based on the interaction with the target substance. In order to further amplify the nucleic acid part of the protein-nucleic acid conjugate selected once to make a protein-nucleic acid conjugate further, (a) a single-stranded RNA portion of the protein-nucleic acid conjugate obtained by selection is necessary. After being decomposed accordingly, this is amplified by PCR or the like (amplification step), (b) an mRNA chain is produced based on the amplified DNA chain, and the above-mentioned protein-nucleic acid conjugate template is further produced. This can be done by translating this to prepare a protein-nucleic acid conjugate.

  (A) It is also preferable to perform an amplification process as follows, for example using PCR. In the nucleic acid of the protein-nucleic acid conjugate, the region to be amplified contains at least the coding sequence. The DNA sequence encoding the protein selected by the above selection method can be identified by analyzing the base sequence of the DNA thus amplified by a known method per se, so that DNA or RNA can be identified based on the sequence. Can also get. The PCR primer used for amplifying the region is not particularly limited, but as a sequence commonly used for all protein-nucleic acid conjugates, the 5 ′ primer is located 5 ′ upstream of the coding sequence. A linked sequence is preferably used. Specifically, in the case of the above-described protein-nucleic acid conjugate, the 5 ′ primer is preferably a DNA sequence recognized by a ribosome during translation, and the 3 ′ primer is a tag sequence or common A sequence is preferably used.

  Since the DNA thus obtained contains only the coding sequence, (1) the promoter sequence described above and (2) the DNA sequence recognized by the ribosome during translation (hereinafter referred to as “5 ′”). Sometimes referred to as "additional sequence"). A tag sequence, a common sequence, etc. (hereinafter, these may be referred to as “3 ′ additional sequences”) are combined. The binding of these sequences can be performed using DNA ligase, the overlap extension method described below, or the PCR method. PCR primers include a 5'-end additional sequence having a sequence common to the 5 'end of the amplified DNA at the 3' end, and a sequence common to the 3 'end of the amplified DNA as the 5' end. What consists of 3 'addition arrangement | sequence which it has in is used.

  In the binding method by the overlap extension method, first, approximately 5 moles of 5 ′ additional sequence having a sequence common to the 5 ′ end of the amplified DNA at the 3 ′ end is prepared, annealed, and then DNA polymerase, etc. After preparing an equimolar number of 3 ′ additional sequence having a sequence common to the 3 ′ end of the amplified DNA at the 5 ′ end and annealing the DNA, In this method, double-stranded DNA is synthesized using a polymerase or the like. The 5'-side additional sequence and 3'-added sequence may be combined one by one or both at the same time. The double-stranded DNA thus synthesized may be further amplified by PCR using a primer having a nucleotide sequence at both ends.

  The protein-nucleic acid conjugate template thus prepared is translated by the method described in (i) above to prepare a protein-nucleic acid conjugate.

(Iii) Concentration operation In the method of the present invention, the steps described in (i) and (ii) above are carried out using a cluster in which the nucleic acid part of the protein-nucleic acid conjugate specifically bound to the target substance encodes a domain polypeptide. (This may be referred to herein as a “concentration operation”). A “cluster encoding a domain polypeptide” (hereinafter, sometimes referred to as “cluster”) means a cluster of nucleic acids having substantially the same length, including the nucleic acid encoding the domain polypeptide as a common sequence. A cluster of almost the same length including a common sequence means that 50% or more of the protein-nucleic acid conjugate group obtained above contains the common sequence, and the nucleic acid having the shortest chain length and the longest nucleic acid are 1 to 10 times. It means to enter the double range. The formation of such a cluster is not particularly limited as long as the formation can be confirmed, but for example, the nucleic acid part of a protein-nucleic acid conjugate obtained by interacting with a target substance is selected, It can be confirmed by observing a band when the RNA is separated as a DNA or agarose gel or SDS-polyacrylamide gel is separated by electrophoresis or the like.
When a cluster is formed, nucleic acids encoding all polypeptides contained in the reaction solution are obtained, translated using the above method, etc., contacted with the target substance again, and bound to the target substance. It can also be confirmed that the concentration operation is sufficient due to the high ratio of the polypeptide.

  Usually, this enrichment operation is performed 2 to 15 times to obtain a conjugate of the domain polypeptide of the present invention and the nucleic acid encoding it. By amplifying the nucleic acid part of the obtained linked body by the above method and analyzing the base sequence by a conventional method, the base sequence encoding the domain polypeptide can be identified. Further, if a nucleic acid having the base sequence is synthesized by a conventional method, a nucleic acid encoding a domain polypeptide can be obtained, and this nucleic acid can be obtained by translational synthesis using a known method such as the protein synthesis method described above. A domain polypeptide can be obtained.

  Examples of the domain polypeptide thus obtained include those having the amino acid sequences described in SEQ ID NOs: 1 to 14 in the Sequence Listing as domain peptides that interact with PPARγ2. Furthermore, those including those registered in the UNIGENE database as the genes described in FIGS. 1 and 2 were obtained. Domain peptides that interact with Smad3 include those having the amino acid sequences described in SEQ ID NOs: 15 to 19, and those registered in the UNIGENE database as the genes described in FIGS. 3 and 4 Things were acquired. In addition, as domain polypeptides that interact with FK506, those having the amino acid sequences described in SEQ ID NOs: 20 to 23 are obtained, and those registered in the UNIGENE database as genes in FIGS. What was included was acquired.

(2) Identification method of structural motif sequence From the base sequence encoding the domain polypeptide group selected and obtained in (1) above, or from the translated amino acid sequence, a common sequence is determined using the presence frequency and / or appearance frequency as an index. The structural motif sequence can be identified by the selection process or the like. An outline of this structural motif sequence identification method is shown in FIG.

The “structural motif” means a polypeptide that is necessary and sufficient for interaction with a target substance consisting of a shorter chain amino acid than a domain that interacts with the target substance. The “structural motif sequence” means an amino acid sequence constituting the structural motif or a base sequence encoding the amino acid sequence.
The structural motif sequence is analyzed and identified from the base sequence encoding the domain polypeptide group selected and obtained above, or the amino acid sequence obtained by translating it. Hereinafter, although the method of identifying from an amino acid sequence is demonstrated, it can identify similarly from a base sequence. As an identification method, a method by extracting an amino acid sequence that is substantially common to the obtained domain polypeptide group is used. The “almost common amino acid sequence” is identified using (i) presence frequency and (ii) appearance frequency as indices. (I) In the selection method based on the presence frequency, the amino acid sequence of the obtained domain polypeptide group is first converted into commercially available software (Clustal W: Thompson JD, et al., Nucleic Acids Res., 22 (22), 4673-80 (1994)) to perform multiple alignment analysis (FIG. 7). Here, centering on the amino acid sequence that many domain peptides have in common (FIG. 7), for each of the adjacent amino acids one after the other, analyze whether each domain polypeptide exists, A range that is present in 50%, preferably 80%, more preferably 90%, and most preferably 100% of the total domain polypeptide is selected (hereinafter referred to as “polypeptide selected by frequency”). Sometimes). In FIG. 7, the range from the N end (left end) of the amino acid sequence B1 to the C ends (right end) of the amino acid sequences A-1 and A-2 is in the range of 100% existence frequency.

  (Ii) Furthermore, the amino acid type is analyzed for each of the amino acid sequences shared by the domain peptides subjected to the multiple alignment analysis and the adjacent amino acids adjacent to each other, and 50% of all domain peptides, preferably Is referred to as selecting a range in which 80%, more preferably 90%, and most preferably 100% have the same amino acids (hereinafter referred to as “polypeptides selected by frequency of appearance”). In FIG. 7, it can be said that amino acids indicated by black ellipses are amino acids having a high appearance frequency.

  The structural motif sequence of the present invention can be identified by selecting the common part of the polypeptide selected by the occurrence frequency and the polypeptide selected by the appearance frequency. In FIG. 7, the part shown at the bottom can be selected as the structural motif. In addition, when performing multiple alignment, grouping domain polypeptides that completely match the partial amino acid sequences of the acquired domain polypeptides as a family can also analyze the above structural motifs within these families. it can. For example, in FIG. 7, family A is a family such that the amino acid sequence of the black portion is completely matched, Family B is the amino acid sequence of the shaded portion is completely matched, Family C is the amino acid sequence of the vertical portion is completely matched. can do.

  As the structural motif sequence thus identified, for example, the amino acid sequence described in SEQ ID NO: 24 or 25 in the sequence listing as a structural motif that interacts with PPARγ2, and the structural motif that interacts with the intracellular signal transduction factor smad3 of TGFβ A structure in which the amino acid sequence described in SEQ ID NO: 26 or 27 in the sequence listing further interacts with FK506 (tacrolimus, J. Antibiot. (Tokyo), 40 (9), 1249-55 (1987)), which is an immunosuppressant Examples of the motif include the amino acid sequence set forth in SEQ ID NO: 28 in the Sequence Listing. Such a structural motif sequence can be prepared and obtained by preparing a DNA encoding it by a well-known method and translating it by a well-known method as described above, or as a synthetic polypeptide by chemical synthesis. It can also be acquired.

  In addition, a structural motif having a modified function can be obtained by adding a mutation to the structural motif sequence. Such structural motifs are also within the scope of the present invention. Examples of the “transformed function” include those having a strong binding force to the target substance and those having a strong interaction. As the amino acid to be introduced with a mutation, the amino acid having the highest appearance frequency of 60% or less in the analysis of the appearance frequency can be selected. The structural motif into which the mutation has been introduced can be brought into contact with the target substance, and its interaction can be analyzed by the above method. By comparing with the wild type, the transformed function of the structural motif can be identified. it can. The method for introducing a mutation into a structural motif can be carried out by a commonly used method known per se. For example, it can be prepared by introducing a mutation into a DNA encoding a structural motif using a commercially available point mutation introduction kit, etc., and translating this using a well-known method, or by chemical synthesis as a polypeptide. be able to.

(3) Three-dimensional structure analysis of domain polypeptide or structural motif and target substance complex One of the present inventions is (i) a step of producing a complex of the domain polypeptide or structural motif obtained above and the target substance. (Ii) performing a three-dimensional structure analysis of the complex and identifying a binding site between the target substance and the polypeptide or structural motif; (iii) predicting a binding space of the target substance from the identified binding site; A molecular design method for a compound that interacts with a target substance, comprising a step of designing a compound that fits the binding space.

  “Complex of domain polypeptide or structural motif and target substance” means a complex formed with a three-dimensional structure and arrangement in which both molecules interact with each other, and a plurality of different domain polypeptides or structures The motif may be bound to the target substance. Since the present invention is finally a method for designing the structure of a compound that interacts with a target substance based on the three-dimensional structure when the domain polypeptide or structural motif binds to the target substance, As long as the structural motif has a function of binding to and interacting with the target substance, it is preferably a shorter chain (stereostructure has a low volume).

The structural motifs obtained by the present invention, particularly short-chain motifs, may themselves have pharmaceutical activity. By further optimizing the structure of this polypeptide, it can be made a more excellent medicine. These are sometimes referred to herein as “short polypeptides”.
The bound `` target substance and the binding site between the target substance and the polypeptide or structural motif '' means a binding position (hereinafter referred to as `` hot position '') for maintaining a three-dimensional structure and arrangement in which both molecules interact. It may be referred to as “spot”).
“Target substance binding space” means a protein, compound, or polypeptide binding space that interacts with the target substance when the target substance is a protein, and when the target substance is a compound or polypeptide. , And means the binding space of proteins that interact with this. Specifically, when the target substance is a protein and the substance that interacts with it is a polypeptide, the amino acid side chain present at the hot spot site of the protein that binds to the target substance is the amino acid side of the polypeptide bound to the protein. Forms a conformationally stable complex with the chain. This makes it possible to analyze the exact spatial positions of amino acids and their side chains present at the hot spot site of the target protein, which is extremely important information for the molecular design of compounds that act on the site.

The above steps can be performed by appropriately combining methods known per se. For example, (i) a step of producing a complex of the domain polypeptide or structural motif obtained above and a target substance, (ii) a three-dimensional structure analysis of the complex, and the target substance and the polypeptide or structural motif As for the step of identifying the binding site, X-ray analysis, NMR method, etc. can be used to analyze the three-dimensional structure. For example, Duncan E. McRee et al., Practical Protein Crystallography (second edition (1999), Academic Pr) (Iii) The process of predicting the binding space of a target substance from the identified binding site and designing a compound that fits the binding space is a drug discovery chemistry (first edition), Tetsuo Nagano. , Hideaki Natsume, Hiroshi Hara, Tokyo Chemical Doujin (2004), or the latest drug discovery chemistry (top) (bottom), CG Wermth,
The method described in Technomic (1998) can be used.

  Whether the short polypeptide or compound thus designed is synthesized by a well-known method and brought into contact with the target substance, whether or not it shows the same interaction as the domain polypeptide or structural motif from which it was designed. Check. Here, when the target substance is related to a disease, the drug candidate compound designed by the above-described method is used for the drug candidate compound in a test tube or a pharmacological or physiological test in vivo by a method known per se. It is possible to obtain a therapeutic agent for the disease by screening for activity and safety. In this case, a method for producing a pharmaceutical composition containing the compound obtained in the above step as an active ingredient is also included in the present invention.

  The pharmaceutical compound thus obtained can itself be used alone, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The ratio of the active ingredient to the carrier at this time can vary between 1 and 90% by weight. The pharmaceutical compound obtained in the present invention is in the form of tablets, capsules, soft capsules, powders, granules, fine granules, syrups, emulsions, suspensions, liquids, etc. May be administered orally or parenterally as an injectable solution such as a sterile solution or suspension form with a pharmaceutically acceptable liquid, eg intravenous, intramuscular, topical Administration or subcutaneous administration may be used. Administration as a suppository is also possible.

  When an oral, enteral or parenteral composition is prepared, it can be prepared by mixing in an organic or inorganic solid or liquid carrier or diluent in a unit volume form usually used. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained. Examples of excipients used in producing the solid preparation include lactose, sucrose, starch, talc, cellulose, dextrin, kaolin, calcium carbonate and the like. Liquid preparations for oral administration, i.e. syrups, suspensions, solutions, etc., contain generally used inert diluents such as water, vegetable oils and the like. In addition to the inert diluent, the preparation can also contain adjuvants such as wetting agents, suspending aids, sweetening agents, flavoring agents, coloring agents, preservatives, stabilizers and the like.

Examples of the solvent or suspending agent used for the preparation of parenteral preparations, that is, injections, suppositories, and the like include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin and the like. Examples of bases used for suppositories include cocoa butter, emulsified cocoa butter, laurin butter, witepsol and the like.
In this specification, the nucleic acid isolation, preparation, nucleic acid ligation, nucleic acid synthesis, PCR, plasmid construction, and cell-free genetic manipulation techniques described above are the same when a commercially available kit is used. According to the instruction manual, unless otherwise specified, Sambrook et al. (1998) Molecular Clonimg, 2nd Edition, Cold Spring Harbor Laboratory Press, or a method analogous thereto.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples.
Example 1 Acquisition of a correspondence library containing proteins that interact with a target protein and DNA encoding the protein (1) Preparation of a correspondence molecule library (1-i) For creation of a correspondence molecule using random primers Preparation of cDNA library Reverse transcription reaction was performed by random priming method using 0.5 μg of human brain-derived mRNA (CLONTECH) purified by oligo dT column as a template. As a random primer used in the random priming method, a primer (SEQ ID NO: 29) containing a random sequence consisting of 9 bases and a sequence that becomes the 3 ′ common sequence in PCR described below (0.2 or 0.4 pmol, or 2 or 4 pmol), SuperScript Double Strand A complementary single-stranded cDNA library was synthesized from mRNA using the reverse enzyme attached to cDNA Synthesis Kit (manufactured by Invitrogen). In accordance with the manual attached to the kit, the RNA double-stranded with cDNA was partially decomposed using RNaseH, and the second strand DNA was synthesized with E. coli DNA polymerase I. Next, after ligating the second strand synthesized with E. coli DNA ligase, the cDNA ends obtained from T4 DNA polymerase were smoothed, and the double-stranded cDNA was purified by ethanol precipitation.

  On the other hand, a translation enhancer sequence for cell-free protein synthesis, a single-stranded DNA (SEQ ID NO: 30) containing a gene encoding FLAG and a DNA (SEQ ID NO: 31) consisting of a sequence complementary to its 3 ′ end were annealed. An adapter was prepared. Mix this adapter (100 μM) with cDNA (4 μl), add 5 μl of DNA ligase (ligationhigh; manufactured by TOYOBO), react at 16 ° C overnight, bind both, and purify using QIAGEN DNA purifucation Kit And dissolved in 50 μl of nucleasefree water.

  Next, the SP6 promoter, a forward primer consisting of a translation enhancer sequence and a start codon (SEQ ID NO: 32), and a reverse primer (SEQ ID NO: 33) having a sequence for binding to the puromycin linker described later were used with TOYOBO KODplus. After the reaction at 94 ° C. for 2 minutes by the conventional PCR method, the reaction at 98 ° C. for 10 seconds, 60 ° C. for 30 seconds and 68 ° C. for 5 minutes was performed 16 to 18 times, and further reacted at 68 ° C. for 3 minutes. The DNA amplified by this PCR was used as a cDNA library for creating a corresponding molecule.

(1-ii) Creation of a mapping molecule library from a cDNA library for creating mapping molecules Using the Ribomax Large Scale RNA Production System manufactured by Promega, according to the method of this system, the cDNA library for creating mapping molecules prepared above is used. MRNA was synthesized from the rally. Then, a cap analog (7.2 mM, RNA Capping Analog; manufactured by Gibco BRL) was added to the mRNA, and the 5 ′ side of the mRNA was modified by a conventional method. Next, T4RNA ligase buffer (50 mM Tris-HCl) was prepared so that the mixing molar ratio of puromycin linker (T-splint5.9FA) described in Japanese Patent Application No. 2003-315385 and mRNA was 1.2: 1 to 3.0: 1. , PH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP), and dimethyl sulfoxide was added to a final concentration of 5%. This solution was heated to 94 ° C. and then annealed by cooling to 25 ° C. over 10 minutes. After that, an optimal amount of T4RNA ligase (Takara) was added and allowed to react at 25 ° C. for about 1 hour, and then purified using RNeasy Mini Kit (QIAGEN) to obtain an mRNA library for creating a corresponding molecule. .

  Next, using PROYOIOS (wheat germ cell-free protein synthesis system) manufactured by TOYOBO, protein synthesis was carried out from the mRNA library (64 μg) for creating a corresponding molecule bound with the puromycin linker according to the method attached to the kit. . As a result of this reaction, an association molecule in which mRNA with a puromycin linker is bound to a protein and a protein are covalently bound (in this specification, this may be referred to as a “protein-nucleic acid conjugate” or “association molecule”) is produced. It was done. In addition, a library of mapping molecules may be referred to as a “matching molecule library”.

(1-iii) Reverse transcription reaction and purification of mapping molecule library The solution containing the mapping molecule library obtained in (2) above is 1 M NaCl, 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.25% Triton-X100 was prepared, and MAGNOTEX-SA (Takara) 360 μl bound with 9.6 nmol Biotinylated Oligo (dT) Probe (Promega) was bound at 4 ° C. for about 1 hour. After this, the supernatant was removed, washed 3 times with washing buffer A (1M NaCl, 100 mM Tris-HCl (pH 8.0), 0.25% Triton-X100), and then buffer B (500 mM NaCl, 100 mM Tris-HCl (pH 8.0)). Wash once with 0.25% Triton-X100), then once with buffer C (250 mM NaCl, 100 mM Tris-HCl (pH 8.0), 0.25% Triton-X100), then elute three times with 90 μl MilliQ water. The corresponding molecule was purified using poly-A.

  Next, reverse transcription reaction was performed using SuperScript III Reverse Transcriptase (manufactured by Invitrogen) using the reverse transcription sequence contained in the linker of the purified mapping molecule. That is, for 270 μl of the elution fraction in the above purification step, 108 μl of 5 × RT Buffer, 54 μl of 10 mM dNTP, 27 μl of 0.1 M DTT, 21.6 μl of 40 U / μl RNase Inhibitor (WAKO), 27 μl of 200 U / μl RTase Then, 32.4 μl of nuclease free water was added, reacted at 50 ° C. for 2 minutes, and then the reverse transcription reaction was performed by decreasing the temperature by 0.4 ° C. per second until reaching 26 ° C.

  Next, 540 μl of the reaction solution subjected to the reverse transcription reaction described above was added 20 mM HEPES-KOH (pH 7.8), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Np40, 10% glycerol, 50 μg / ml BSA, 0.5 μg / ml tRNA. It was adjusted to become. This was combined with 40 μl of anti-FLAG M2 antibody agarose beads (Sigma) at 4 ° C. overnight, and 100 μl of Binding Buffer (20 mM HEPES-KOH (pH 7.8), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1 After washing 3 times with% Np40, 10% glycerol, 50 μg / ml BSA, 0.5 μg / ml tRNA), 3 times with 40 μl of 3 × FLAG peptide (manufactured by Sigma) Buffer (binding buffer containing a maximum concentration of 100 μM 3 × FLAG peptide) An association molecule library in which the eluted and purified nucleic acid portion is a hybrid of DNA and RNA (in the present specification, this is referred to as “protein-reverse transcription linkage group” or “RNA-DNA association library”. Sometimes obtained).

(2) Acquisition of a mapping molecule having a protein that interacts with the target protein (PPARγ2) and a gene encoding the protein (2-i) Contact between the target protein and the mapping molecule library and acquisition of a fusion target protein for screening A nuclear receptor (PPARγ2) was used as a screen, and a protein (polypeptide) interacting with this was screened from the correspondence molecule library prepared in Example 1. PPARγ2 is a nuclear receptor consisting of 506 amino acids and is known to be involved in diabetes and hyperlipidemia.
First, according to a conventional method, glutathione S-transferase (GST) is encoded as a fusion protein at the N-terminus of human PPARγ2 (GenBank accession No. D83233.1, AF012874.1, L40904.2 ,: SEQ ID NO: 34). The DNA to be inserted was added to prepare a DNA inserted into an expression vector for E. coli, which was expressed in E. coli (BL21 (DE3) pLysS). At this time, a site capable of being cleaved by TEV protease was introduced between GST and PPARγ2. The obtained fusion protein was purified using Glutathione Sepharose 4B beads (manufactured by Amersham Pharmacia) to remove glutathione used for elution on a desalting column. For the detailed purification method, the protein (25 μg) obtained in the above synthesis reaction was bound with Binding Buffer (100 mM KCl, 20 mM HEPES-KOH (pH 7.8), 0.1% NP40, 0.1 mM EDTA, 1 mM DTT, 0.5 μg / It was dissolved in ml tRNA, 50 μg / ml BSA, 10% glycerol and bound to Glutathione Sepharose 4B beads (Amersham Pharmacia), followed by washing with 100 μl of Binding Buffer three times. For the negative control, only GST protein was synthesized and purified by the same method.

  The GST-PPARγ protein and GST protein obtained above were brought into contact with the corresponding molecule library prepared in Example 1. First, the correspondence molecule library obtained in Example 1 was mixed with Glutathione Sepharose 4B beads (beads not bound with protein) at room temperature for 30 minutes, and after removing the beads, the Glutathione Sepharose 4B beads bound with the above PPARγ2 were used. And allowed to react at room temperature for 1 to 2 hours, and then washed 3 to 10 times with Binding Buffer.

  Next, in order to obtain the GST-PPARγ protein obtained by the above contact or the fusion of the GST protein and the corresponding molecule, 100 μl of BindingBuffer added with 2 μl of TEV protease (manufactured by Invitrogen) was added for 1 hour at room temperature. The operation of reacting and collecting the supernatant was repeated twice.

(2-ii) Retrieval of a mapping molecule having a protein that interacts with a target protein and DNA encoding the protein From the fusion of GST-PPARγ2 or GST and the mapping molecule obtained in (1) above, mapping Only the nucleic acid part of the molecule was obtained as DNA. Specifically, 40 μl of 100 μg / ml RNase A (Qiagen) or 10 μl of 10 U / μl RNase H (TOYOBO) was added to 200 μl of the eluate obtained above, and the mixture was stirred for 5 minutes at room temperature. Alternatively, the reaction was allowed to proceed at 37 ° C. for 30 minutes to remove the RNA strand portion where the corresponding molecule was double-stranded (RNA / DNA), purified by ethanol precipitation, and suspended in 20 μl of NucleaseFree water. Here, the DNA contained in the obtained molecule encodes a protein that interacts with the target protein. The series of operations so far may be hereinafter referred to as “DNA acquisition operation”.

  Using this DNA, the method described in Example 1 was performed again, and the corresponding molecules having proteins that interacted with the target protein were concentrated.

Example 2 Concentration of a mapping molecule having a protein that interacts with a target protein and a gene encoding the protein 1 μl of a DNA solution encoding a protein that interacts with PPARγ or GST obtained in Example 1 (2) above As a template, using a primer consisting of the sequences of SEQ ID NOs: 35 and 36, after reaction at 94 ° C. for 2 minutes by PCR using TOYOBO KODplus, further 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, The reaction was carried out 18 times at 68 ° C. for 5 minutes, and further reacted at 68 ° C. for 3 minutes. Next, using this PCR product as a template, PCR was performed by the method described in Example 1 using primers (SEQ ID NO: 32 and SEQ ID NO: 33) containing an annealing sequence with the SP6 promoter and spacer, and a corresponding molecule was prepared. A cDNA library was prepared.

With respect to the obtained cDNA library for creating an associating molecule, a series of operations described in Example 1 (1-ii), (1-iii) and (2) were performed. The above operation may be referred to as “concentration operation of the mapping molecule having a protein that interacts with the target protein and a gene encoding the protein” or “concentration operation”.
This concentration operation was repeated three more times. By this operation, the correspondence molecule having a protein that interacts with the target protein PPARγ2 or GST and the concentration of the gene encoding the interacting protein are totaled four times in total (the acquisition of the first correspondence molecule or gene (execution) (Described in Example 1) is not included in the concentration operation).

  The DNA obtained by these operations (first DNA acquisition operation, 1st to 4th concentration operations) was made into an mRNA library for producing a corresponding molecule by the method described in Example 2, and each 100 ng was treated with 4% urea. Electrophoresis was performed using a denaturing polyacrylamide gel. After this, the fluorescence (Fluoroscein) introduced into the puromycin linker bound to the mRNA library for creating the mapping molecule is detected using MolecularImager (Bio Rad), and after each DNA acquisition and concentration step The migration pattern of the mRNA library was analyzed.

  The result is shown in FIG. Lane 0 shows the results obtained from the DNA obtained by the DNA acquisition operation, and lanes 1 to 4 show the results after the concentration operation 1 to 4 times. As is apparent from the figure, the DNA (RNA) after 3 and 4 enrichment operations is more likely to have several singles compared to the heterogeneous mRNA library (smear electropherogram described in lane 0). It was found that a good band was formed (lanes 3 and 4). That is, it was found that DNA (RNA) obtained by the above concentration operation 3 to 4 times was concentrated into several fragments.

Example 3 Analysis of protein interacting with PPARγ2 by base sequence analysis (1) Base sequence analysis A cDNA library for creating a correspondence molecule obtained by performing the enrichment operation 2 to 4 times obtained by the operations up to Example 2 was prepared. Then, after purification according to a conventional method, a cloning operation for insertion into a pGEM-Teasy vector (Promega) was performed. Then, clones containing the inserted sequence were selected, and base sequence analysis of about 100 clones was performed each time. As a base sequence obtained by the concentration operation 3 to 4 times, there was one encoding a gene registered in the UNIGENE database shown in FIG. 1 and FIG.

(2) Concentration analysis of a specific amino acid sequence The base sequence obtained above is converted into an amino acid sequence, and multiple alignment analysis is performed using software (Clustal W) based on the obtained amino acid sequence information. did. Representative of these are shown in FIG. 9 and SEQ ID NOs: 1-14. As a result of this analysis, it was found that the proteins obtained by the enrichment operation using GST-PPARγ2 as the target protein were aggregated into relatively short peptides of about 14 to 55 amino acids containing the same amino acid sequence. It was. In addition, a characteristic amino acid sequence known to interact with nuclear receptors (LXXL ^* L ^* -like sequence, X represents any amino acid. L ^* L ^* is both L or either It was found that a novel protein containing one of L was selectively obtained.

FIG. 10 shows the results showing the frequency of appearance of the LXXL ^* L ^* -like sequence out of about 100 clones whose base sequences were analyzed above. As is clear from the figure, it was found that the ratio of the protein having the LXXL ^* L ^* -like sequence increased each time the concentration operation was repeated (FIG. 10, white bar: PPARγ2). On the other hand, in the results of screening in the same manner using GST instead of the target protein (GST-PPARγ2) in Examples 1 and 2, the protein having the LXXL ^* L ^* -like sequence was not concentrated or obtained at all ( FIG. 10, black bar: GST). From this, it was found that the protein obtained by the concentration operation was obtained by being specifically selected by the target protein GST-PPARγ2.

Next, the ratio of DNA encoding a specific amino acid sequence was analyzed with respect to the entire DNA obtained by the above DNA acquisition and concentration operation. The specific amino acid sequences were analyzed by using examples classified into the PPAR1 family and PPAR2 family shown in FIG. Here, the PPAR1 family and the PPAR2 family are those in which multiple alignment analysis is aligned using software (ClustalW) and then the LXXL ^* L ^* -like sequences are the same amino acid sequence as a family. It is.

  First, according to a conventional method, the abundance of DNA encoding the PPAR1 family and PPAR2 family in each enrichment round was quantified by a quantitative PCR method using Cyber Green (SYBER GREEN Master Mix: manufactured by ABI). As a negative control, βActin was used as an index. For gene quantification, primers having the sequences of SEQ ID NOs: 37 and 38 are used for the PPAR1 family, primers having the sequences of SEQ ID NOs: 39 and 40 are used for the PPAR2 family, and primers having the sequences of SEQ ID NOs: 41 and 42 are used for βActin. Carried out.

  The result is shown in FIG. FIG. 11 (1) shows the number of molecules of DNA encoding the PPAR1 family in 10 ng of DNA obtained by each enrichment operation, and FIG. 11 (2) is the DNA encoding the PPAR2 family in 10 ng of DNA obtained by each enrichment operation. Indicates the number of molecules. FIG. 11 (3) shows the number of molecules of DNA encoding βActin in 10 ng of DNA obtained by each concentration operation. As is clear from the figure, the number of molecules of DNAs encoding the PPAR1 family and PPAR2 family increased with the progress of enrichment, indicating that these DNAs were concentrated. On the other hand, the number of molecules of βActin-encoding DNA was found to decrease with the progress of concentration.

(3) Binding analysis with PPARγ2 (3-i) Protein synthesis The peptides obtained in (1) above (PPAR1-6 (SEQ ID NO: 1), 1-7 (SEQ ID NO: 2) described in FIG. 9), 1- 13 (SEQ ID NO: 3), 2-1 (SEQ ID NO: 4), 2-5 (SEQ ID NO: 5), 2-7 (SEQ ID NO: 6), 2-8 (SEQ ID NO: 7), 3-3 (SEQ ID NO: 8) ), 4-1 (SEQ ID NO: 9), 4-2 (SEQ ID NO: 10), 5-3 (SEQ ID NO: 11), 5-5 (SEQ ID NO: 12), 7-5 (SEQ ID NO: 13), 10-1 (SEQ ID NO: 14) was synthesized by the following method. First, a base sequence encoding the amino acid sequence of GST and FLAG epitope is bound by a standard method to the 5 ′ side of the base sequence encoding the amino acid sequence, and Proc. Natl. Acad. Sci. USA, 99 : A PCR reaction was performed according to the method described in 14652-14657 (2002) to prepare a DNA fragment for transcription. Using this DNA as a template, a transcription reaction was performed using SP6RNA Polymerase (Promega) to synthesize mRNA, and the mRNA obtained by ethanol precipitation was purified. Protein synthesis using this mRNA was performed using a cell-free protein synthesis system by a batch method according to Japanese Patent Application Laid-Open No. 2002-204689 and Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002).

  As a positive control, RxRα known to form a heterodimer with PPARγ and a negative control having only GST and FLAG epitope were prepared in the same manner as described above. As a result of quantifying the GST fusion protein contained in the synthesis solution obtained by the cell-free protein synthesis system using the GST96-well Detection Module (Amersham), it was found that almost constant amount of protein was synthesized. It was.

(3-ii) Binding analysis with target protein After purifying the PPARγ2 protein expressed in E. coli in Example 1, it was diluted with PBS (−) to a concentration of 5 to 10 μg / ml, and a 96-well plate (manufactured by CORNING, (Clear, high binding type) was added in an amount of 100 μl (0.5 to 1 μg) per well. This plate was allowed to stand for 12 hours or more at 4 ° C., and PPARγ2 protein was immobilized on the plate. Next, the plate was washed twice with PBS, 200 μl of Blocking Buffer (1% BSA / PBS) was added per well, and a blocking reaction was performed in an incubator at 37 ° C., 200 rpm for 2 to 3 hours. After this, the plate was washed twice with PBS, BindingBuffer (L +) containing 10 μM thiazolidine compound (rosiglitazone, dissolved in ethanol) as a ligand (20 mM Hepes-KOH (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 95% per well of 0.1% NP40, 10% (v / v) glycerol, 50 μg / ml BSA, 10 μM rosiglitazone), or Binding Buffer (L-) without ligand (20 mM Hepes-KOH (pH 7.9), 100 mM KCl, 95 mM of 0.1 mM EDTA, 0.1% NP40, 10% (v / v) glycerol, 50 μg / ml BSA, 0.4% (v / v) ethanol) was added per well.

  Subsequently, 5 μl of the synthesis reaction solution containing the GST fusion protein prepared in the above (3-i) was added per well and reacted at room temperature for 1 hour. Next, after washing this plate 3 times with WashBuffer (0.05% Tween20 in PBS), add 100 μl of Anti-FLAG M2 Antibody-Peroxidase Conjugate (SIGMA) diluted 10,000 times with Blocking Buffer for 1 hour at room temperature. Reacted. Next, after washing the plate 3 times with WashBuffer, add 100 μl per well of color developing solution (24 mM citric acid, 51 mM Na2HPO4, 0.4 mg / ml orthophenylenediamine (OPD, SIGMA), 0.014% H2O2) for about 5 minutes. After that, the reaction was stopped by adding 100 μl of 1N sulfuric acid solution per well. For the absorbance at a wavelength of 490 nm of the reaction solution, the reference wavelength was measured at 650 nm. The result is shown in FIG.

  As is clear from the figure, PPAR1-6, 1-7, 1-13, 2-1, 2-5, 2-7, 2-8, 3-3, 4-1, 4-2, 5-3, 5-5, 7-5, and 10-1 all showed higher binding activity to PPARγ2 than GST-FLAG (FIG. 12: Negative Control) which is a negative control (FIG. 12: white bar). It was also found that the binding activity of these binding activities increased with addition of rosiglitazone (FIG. 12: black bar).

  Furthermore, among these, regarding the polypeptides PPAR1-7, PPAR 1-13, and PPAR 2-7, a synthetic peptide in which biotin is bonded to the C-terminal side of the amino acid sequence via K is prepared by chemical synthesis, and BIACORE3000 ( SPR measurement and analysis using Biacore) were performed. According to BIAapplications handbook, chapter 4.4, the biotinylated peptides were individually immobilized on the sensor chip surface. The sensor chip was an SA type (manufactured by Biacore). The flow cell attached to one sensor chip is divided into four. However, nothing was immobilized on the flow cell 1 and used as a control section, and one type of biotinylated peptide was immobilized on each of the flow cells 2, 3 and 4.

  Next, let the running buffer flow at a constant flow rate (5 μL / min), stabilize the SPR measurement value, and after the baseline value of each flow cell (SPR-baseline), flow various concentrations of PPARγ2 at the same flow rate. Specific binding was formed between each polypeptide on the sensor chip, the SPR response value (SPR-bound) of each flow cell was measured, and the dissociation constant (Kd) was analyzed. As a result, it was found that the Kd value for PPARγ2 of PPAR1-7 was 132 nM, the Kd value for PPARγ2 of PPAR 1-13 was 241 nM, and the Kd value for PPARγ2 of PPAR 2-7 was 92.6 nM.

(3-iii) PPARγ2 Ligand Binding Inhibitory Activity Measurement SRC-1 binding inhibitory activity, which is a PPARγ2 coactivator of each polypeptide, was measured. SRC-1 has an LXXL ^* L ^* -like sequence in its molecule, and is known to have enhanced binding power to PPARγ2 in the presence of rosiglitazone. Therefore, the effect of the above-mentioned polypeptides (PPAR1-7, PPAR 1-13, PPAR 2-7) on the interaction between PPARγ and SRC-1 was analyzed by a pull-down assay.

10 μl (bed volume 7.5 μl) of Glutathione Sepharose 4B beads were mixed with 50 μl (10-20 μg of GST-PPARγ2 (a fusion protein in which a GST tag was bound to the N-terminal side of PPARγ) synthesized and purified using E. coli in Example 1. ) Added to bind. As a negative control, 50 μl (10 to 20 μg) of GST was added and bound. Human SRC-1 (GenBank accession No. 1101269074203_0.1
, 1101269074203_1.1.), A DNA in which the gene SEQ ID NO: 43 encoding the 565 to 770 residue part is inserted into the pET15b vector, and the mRNA is prepared according to the method described in Japanese Patent Application No. 2003-173634. A DNA template serving as a template for the transcription reaction was prepared. Thereafter, mRNA is prepared by a transcription reaction using RiboMAXLarge Scale RNA ProductionSystem-SP6 (Promega), the obtained mRNA is purified using RneasyKit (QIAGEN), and further, Japanese Patent Application No. 2003-173634 A reaction solution containing SRC-1 labeled at the C-terminus with Cy3-Puro (hereinafter sometimes referred to as “C-terminal labeled SRC-1”) was obtained.

  Binding buffer containing 25 μM rosiglitazone on beads with GST-PPARγ2 or GST immobilized (20 mM Hepes-KOH (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 0.1% NP40, 10% (v / v) glycerol, 50 μg / Binding buffer without rosiglitazone (20 mM Hepes-KOH (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 0.1% NP40, 10% (v / v) glycerol, 50 μg / ml BSA, 0.25 mlBSA, 10 μM rosiglitazone) After adding 100 μl of ˜0.4% (v / v) ethanol), the beads were washed with the same buffer as the added binding buffer.

As a positive control, beads bound with GST-PPARγ2 plus 50 μl of binding buffer containing rosiglitazone and 50 μl of reaction solution containing C-terminal labeled SRC-1, and 50 μl of binding buffer containing rosiglitazone and C-terminal label A reaction solution containing 50 μl of a reaction solution containing SRC-1 was prepared.
Meanwhile, 50 μl of binding buffer containing rosiglitazone and 50 μl of reaction solution containing C-terminal labeled SRC-1 and 4 μM of the above polypeptide (PPAR1-7, PPAR 1- 13, prepared by adding PPAR 2-7) individually.

  Protect the sample from light, place it on a rotary shaker, react at room temperature and 200 rpm for 2 hours, wash the beads with the same binding buffer, add 15 μl of 2 × SDSloading Buffer, and heat-treat at 95 ° C for 5 minutes. After that, C-terminal labeled SRC-1 bound to the beads was recovered. The collected solution was separated by SDS polyacrylamide electrophoresis, and the fluorescent band of C-terminal labeled SRC-1 was analyzed with MolecularImager (Bio Rad). The result is shown in FIG.

  The binding of PPARγ and SRC-1 was enhanced by the addition of rosiglitazone (10 μM) compared to the case where rosiglitazone was not added (FIG. 13 Ligand (−)) (FIG. 13: Ligand (+)). As a result of analyzing the influence of synthetic peptides (PPAR1-7, PPAR 1-13, PPAR 2-7) in the presence of rosiglitazone, these peptides have the activity of inhibiting the binding of PPARγ and SRC-1 I found out.

  That is, it was found that these polypeptides obtained by the enrichment operation of the present invention were obtained by cutting out a portion necessary and sufficient to bind to and interact with the target protein PPARγ2.

Example 4 Identification of Structural Motif Sequence of Protein Interacting with PPARγ2 by Multiple Alignment Analysis As described above, according to the enrichment operation of the present invention, a partial polypeptide necessary and sufficient to bind to and interact with a target protein As a result, it was found that it is possible to obtain a shorter polypeptide with stronger binding force and interaction activity by introducing mutation into the obtained polypeptide. did.

(1) Selection of structural motif sequence candidates For the amino acid sequence derived from a clone having an LXXL ^* L ^* -like sequence obtained 3 or 4 times after the enrichment operation using PPARγ2 as a target protein in Example 3, software Multiple alignment analysis was performed using Wear (ClustalW). In these amino acid sequences, by determining the position and frequency of occurrence of common amino acids, an attempt was made to determine a preferred amino acid sequence and a necessary and sufficient polypeptide chain length for binding and interacting with PPARγ2.

(1-i) Analysis of the amino acid sequence of the polypeptide obtained by the enrichment operation using PPARγ2 as the target protein In FIGS. 14 (A) and (B), various amino acid sequences obtained by the enrichment operation 3 and 4 times. For all of the amino acid sequences containing LXXL ^* L ^* -like sequences, multiple alignment analysis was performed, and for these amino acid sequences, the first leucine (L) position of the LXXL ^* L ^* -like sequences was amino acid number 0, and the C-terminal side Are arranged as plus (+) and the N-terminal side is minus (−).

  First, in the aligned amino acid sequences described above, the type of amino acid at a specific position (number) is analyzed, and the proportion of amino acids with the largest number of amino acids at each position in each clone (hereinafter referred to as this) (Sometimes referred to as “appearance frequency of most amino acids”) is indicated by a black bar. The results are shown in FIGS. 14 (A) and (B). (A) shows the result of the polypeptide obtained at the third enrichment, and (B) shows the result of the polypeptide obtained at the fourth enrichment.

In this figure, the amino acid numbers are on the horizontal axis of the graph, and the appearance frequency of the most frequently occurring amino acid is on the vertical axis. In addition, FIG. 14 (D) shows the most amino acids in the third round (upper) and the fourth round (upper). For example, in the clone containing the LXXL ^* L ^* -like sequence obtained by the third enrichment, the most common amino acid at amino acid number-8 was serine (S), and its appearance frequency was about 75%. I understand that. FIG. 14 (C) shows the difference between the appearance frequency of the above-mentioned amino acid after 4 times of the concentration operation and the result of 3 times of the concentration operation, and shows a specific position between the polypeptide after 3 and 4 times of concentration. The presence or absence of changes in the appearance frequency of amino acids was shown.

  From FIG. 14 (A) and FIG. 14 (B), it was found that both the purine (P) of amino acid number-2 and the leucine (L) of amino acid number 0 showed an appearance frequency of 100%. In addition, the appearance frequency of amino acids with the highest amino acid numbers of amino acid numbers -2 to 0, 3, 4, and 10 to 13 is 90% or more, and the amino acid numbers of -13 to -7 and 9 to 13 with the highest number of amino acids. It was found that the frequency of appearance of specific amino acids was high, with an appearance frequency of 65% or more. In other words, the polypeptide obtained by the enrichment operation using PPARγ2 as the target protein has a highly common amino acid sequence in each clone, and conversely, several different amino acids are used. I found out that there are some places.

On the other hand, the obtained clones containing LXXL ^* L ^* -like sequences contained amino acid sequences of various lengths. For this reason, in the amino acid sequences subjected to the above alignment, there were clones in which an amino acid was present and a clone in which an amino acid was not present. Therefore, a graph in which the ratio of the number of clones that actually had amino acids in each amino acid number to the total number of clones (hereinafter, this may be referred to as “presence frequency”) is plotted with white circles in FIGS. It showed in. This existence frequency serves as an index for knowing the range of the region length that is commonly present in the amino acid sequences between the analyzed clones. As is apparent from the figure, it was found that the amino acid numbers -4 to 4 have a presence frequency of 100%. Further, it was found that the existence frequency was 80% or more in the range of -7 to 9 and 50% or more in the range of -11 to 13 and the existence frequency was relatively high. These ranges are shown surrounded by a frame line in FIG.

  In addition, as amino acids having high appearance frequency and existence frequency, those with 65% or more are surrounded by a square, those with 50 to 65% are circled, and those with 40 to 50% are indicated by a broken line. It was. These amino acid sequences exist as a common sequence in the polypeptide obtained by the enrichment operation using PPARγ2 as a target protein, and this amino acid sequence was considered to be selected as a structural motif sequence candidate.

(1-ii) Selection of Structural Motif Sequence Candidate Further, FIG. 15 (A) shows the result of the above analysis performed on the polypeptide obtained after performing the concentration operation with PPARγ2 as the target protein 2 to 5 times. It was. The uppermost number is the amino acid number in which the position of the first leucine (L) of the LXXL ^* L ^* -like sequence is amino acid number 0, the C-terminal side is plus (+), and the N-terminal side is minus (-) In the amino acid sequence, amino acids having the highest frequency of appearance are arranged in numerical order in the polypeptide obtained after all the concentration operations. Below that, amino acids appearing in other clones are arranged in descending order of appearance frequency. In addition, amino acid sequences (−4 to 4) with an occurrence frequency of 100% are shown surrounded by a frame, and the occurrence frequencies are 80% or more (−7 to 9) and 50% or more (−11 to 13) with broken lines Enclosed in In addition, the amino acids having the highest occurrence frequency with an occurrence frequency of 50% or more are enclosed by a square, and the amino acids having both the appearance frequency and the occurrence frequency are indicated by asterisks. In addition, amino acids having an occurrence frequency of 50% or more and the appearance frequency of 50% or more in any of the concentration operations are circled.

From this result, structural motif sequence candidates were selected as follows. A polypeptide of amino acid Nos. -2 to 4 having both occurrence frequency and appearance frequency of 50% or more, and Proline (P) of amino acid No.-2 having appearance frequency of 100% and lysine (L) of amino acid No. 0 are fixed Then, the amino acid number 4 shown in FIG. 15B, which is a hydrophobic amino acid (X ₂ ) and the other amino acids (X ₁ ), was selected as a basic type. Furthermore, a polypeptide having amino acid number 1 as tryptophan (T) and amino acid number 2 as phenylalanine (F) or serine (S) in this sequence was also selected as a more preferred structural motif candidate (FIG. 15B (2)). In addition, amino acid number-2 is purine (P), -1 is lysine (L), 0 is lysine (L), 3 is lysine (L), 4 is lysine (L), and any other amino acid (X ₁ A polypeptide having the sequence of) was also selected (FIGS. 15B and 3). Furthermore, in this sequence, a polypeptide having amino acid number 1 of threonine (T) and 2 of phenylalanine (F) or serine (S) (FIGS. 13B and 4) was also selected.

Furthermore, as a structural motif candidate in which the sequence is further added to the structural motif candidate having the amino acid sequence described in FIG. 13B, amino acid number -7 is purine (P), and amino acid numbers -6 to -3 are arbitrarily selected. The basic type of structural motif (FIG. 13C (1)) was selected as amino acid (X ₁ ). In the basic sequence of this added structural motif, further candidate structural motifs in which amino acid number-4 is arginine (R) or lysine (L) and amino acid number-3 is tyrosine (Y) or histidine (H) (FIG. 13). (C) (2)) was also selected. Furthermore, the sequences described in FIG. 13 (C) (3) were also selected.

Example 5 Analysis of the amino acid sequence of a protein that interacts with a transcription factor (Smad3) and selection and confirmation screening of candidate structural motifs of the transcription factor Smad3 is used as a target protein to screen for proteins that interact with this. This was carried out from the mapping molecule library prepared in 1. Smad3 is a protein consisting of 425 amino acids that plays an important role in the TGFβ signaling pathway and is highly associated with diseases. Human Smad3 (GenBank Accession No. 1101269074203_2.1, 1101269074203_3.1, 1101269074203_4.1, 1101269074203_5.1, 1101269074203_6.1, 1101269074203_7.1, 1101269074203_8.1, 1101269074203_9.1, 1101269074203_10.1, 1101269074203_12.1, 1101269074203_12.1 , 1101269074203_13.1, 1101269074203_14.1) create a normal active DNA (SEQ ID NO: 44) in which the acid sequence (SSVS) of the C-terminal network is replaced with DDVD, and GST (gulutathione S-transferase at the N-terminus thereof. ) Was added to Escherichia coli in the same manner as in Example 1 as a fusion protein to which) was added. At this time, a site that can be cleaved by thrombin was introduced between GST and Smad3. And according to Example 2, DNA acquisition operation and concentration operation which used Smad3 as target protein were implemented.

  100 ng each of the mRNA library for creating a corresponding molecule obtained in the process of DNA acquisition operation (FIG. 16: 0) and each concentration operation (FIG. 16: 1 to 5) was electrophoresed on 4% urea-denatured PAGE. Thereafter, fluorescence (Fluoroscein) introduced into the spacer of the mRNA library was detected using MolecularImager (manufactured by Bio Rad), and the migration pattern of the mRNA library in the DNA acquisition operation and each concentration operation was analyzed.

The results are shown in FIG. As can be seen from the figure, 4 and 5 enrichment operations formed several single bands (lanes 4 and 5) compared to the heterogeneous mRNA library (smear electropherogram described in lane 0). 5). As a result of this analysis, it was found that the mRNA library for creating a corresponding molecule was concentrated by 4 to 5 concentration operations.
After cloning the cDNA library for creating a corresponding molecule obtained by performing the concentration operation 4 times and 5 times according to the method of Example 3, the nucleotide sequence was analyzed. Further, the base sequence was converted into an amino acid sequence, and multiple alignment analysis was performed according to the method described in Example 4 (1). As a result, amino acid sequences having a high appearance frequency are shown in FIGS. 17 (1) to (5) and Shown in SEQ ID NOs: 15-19. As a result of analyzing the amino acid sequence shown in FIG. 17, it was found that these proteins are part of SARA (SmadAnchor for Receptor Activation) consisting of 1425 amino acids. This protein is known to interact with the Smad3 MH2 domain and suppress Smad3 nuclear translocation.

That is, the enrichment operation of the present invention was confirmed by analysis using two different types of target proteins that are obtained by cutting out and obtaining a polypeptide necessary and sufficient to bind to and interact with the target protein.
Further, among the amino acid sequences analyzed above, the sequence surrounded by a square with the amino acid sequences of FIGS. 17 (1) to (5) is used as an indicator that the presence frequency and appearance frequency are high according to the method of Example 4. Selected as a structural motif sequence candidate.

The structural motif candidate selected above is the SARA interaction part shown by X-ray crystal structure analysis of the complex of Smad3 and SARA shown in the literature (Genes Dev. 16 pp. 1950 (2002)). And almost completely matched. That is, it was confirmed that the structural motif sequence candidate selected above was an actual structural motif sequence.
Furthermore, the site and binding indicated by the double line part of FIG. 17 (5) sequence have been reported (Nat Genet. 2002 Aug; 31 (4): 419-23.) PPP1CC was obtained by this enrichment operation and analysis. I found out that This indicates that proteins that interact indirectly with the target protein for screening can be obtained in the same manner.

  Moreover, what was registered in the UNIGENE database by the domain polypeptide which interacts with the said Smad3 acquired 4 to 5 times after concentration operation was described in FIG.

Example 6 Analysis of the amino acid sequence of a protein that interacts with the pharmaceutical compound FK506 and selection and confirmation of its structural motif candidate FK506, an immunosuppressive agent, was used as a target compound for screening a protein that interacts with this. This was carried out from the correspondence molecule library prepared in (1). FK506 forms a complex with the protein FKBP, a peptidylprolyl isomerase, binds to calcineurin, and inhibits T cell activation by dephosphorylation of NFAT (nuclear factor of activated T cells) and its nuclear translocation. Isolated immunosuppressant. Among FKBPs, 12 kDa FKBP12 is the main target of FK506.

  The target compound FK506 was first reacted in 5 equivalents of succinic anhydride and dimethylformamide (DMF) at room temperature for 2 days in the presence of 1 equivalent of dimethylaminopyridine, and purified by reverse phase HPLC as a carboxylic acid derivative. did. After dissolving this in DMF and adding 1 equivalent each of Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP), 1-Hydroxybenzotriazole hydrate (HOBt), triethylamine and reacting at room temperature for 5 minutes, 3 equivalents of Biotin-POE3-amine (Molecular Bioscience) was added and reacted for 1 hour. The resulting biotinylated FK506 was purified by reverse phase HPLC and confirmed by MALDI-TOF-MS.

  Add 16 μl of the biotinylated FK506 prepared above to 1 mM in 50% ethanol aqueous solution to TBKT (150 mM KCl, 50 mM Tris-HCl (pH 7.5), 0.2% Tween20) 384 μl, stir, and MAGNOTEX- SA beads (Takara Co., Ltd.) equivalent to 100 μl were added and mixed at room temperature for 30 minutes. The beads were collected, washed 5 times with 150 μl of TBKT, and washed 3 times with 300 μl of FH506 binding buffer (150 mM KCl, 50 mM Tris-HCl (pH 7.5), 0.1% Tween20, 1 mM EDTA, 1 mM DTT) to obtain FK506 beads. . In the above method, Biotin-POE3-amine (manufactured by Molecular Bioscience) was used instead of biotinylated FK506, and this was used as a biotin bead as a negative control.

  To this FK506 bead and biotin bead, the mapping molecule library prepared in Example 1 was added, and the protein interacting with FK506 and the DNA encoding the same according to the method described in Example 2 were added. Acquisition and concentration operations were performed. As a result, bands that could be confirmed by SDS-PAGE were formed after 7 and 8 enrichment operations. The obtained cDNA library for creating attachment molecules was obtained and cloned, and then the nucleotide sequence was analyzed. did. On the other hand, biotin beads as a negative control did not aggregate into specific DNA.

  As a result of converting the base sequence obtained above into an amino acid sequence and conducting multiple alignment analysis according to the method described in Example 4 (1), the amino acid sequence having a high appearance frequency is shown in FIG. 23. Here, when the obtained sequence was analyzed, a sequence corresponding to the full length of FKBP12 (FIGS. 18 and 19: FKBP12) was obtained. In addition, a partial amino acid sequence of FKBP5 (FIGS. 18 and 19: FKBP5) was obtained. This partial amino acid sequence completely contained the N-terminal FK506 binding domain (FK1), which is considered to have relatively high FK506 binding activity (FIG. 19: FKBP5). It was confirmed that the concentration operation using can be carried out even if the target substance is a compound, and that the obtained peptide has a sufficient polypeptide cut out to bind to and interact with the compound.

In the above enrichment operation, proteins (Fig. 18: Homolog1 (SEQ ID NO: 22), Homolog2 (SEQ ID NO: 23)) presumed to be homologues of FKBP12 were obtained. From these amino acid sequences, the structural motif candidate sequences of proteins that bind to FK506 are shown in the lower part of FIG. 18 and SEQ ID NO: 28. X represents any amino acid.
5 and 6 show the gene numbers and gene names registered in the UNIGENE database among nucleic acids encoding polypeptides interacting with FK506 obtained 7 to 8 times after the concentration operation.

  In the method of the present invention, a domain polypeptide necessary for interaction with a target substance is obtained by cutting out from the protein (FIG. 20B), and the amino acid sequence of the obtained domain polypeptide is analyzed, and the target substance is analyzed. For identifying a structural motif sequence consisting of amino acid sequences necessary and sufficient for interaction with a target substance (FIG. 21A), and designing a compound that interacts with a target substance using the structural motif identified by the method A method (FIG. 21B) and the like are provided. When the target substance is a substance related to a disease, this method is useful for obtaining / designing a short polypeptide or compound that is a therapeutic agent for the disease.

It is the figure which showed the gene which codes the domain polypeptide which interacts with PPARγ2. FIG. 2 shows a gene encoding a domain polypeptide that interacts with PPARγ2, and is a continuation of FIG. It is the figure which showed the gene which codes the domain polypeptide which interacts with Smad3. FIG. 4 shows a gene encoding a domain polypeptide that interacts with Smad3, which is a continuation of FIG. It is the figure which showed the gene which codes the domain polypeptide which interacts with FK506. FIG. 6 shows a gene encoding a domain polypeptide that interacts with FK506, and is a continuation of FIG. It is the schematic diagram which showed the identification method of the structural motif of this invention. It is an electrophoresis photograph which shows the result of the concentration operation which used PPARγ2 as a target substance. It is the figure which showed the example of the amino acid sequence of the domain polypeptide obtained by using PPARγ2 as a target substance. It is a graph which shows the relationship between concentration operation and the ratio of the nucleic acid which has a common sequence. It is a graph which shows the relationship between concentration operation and the amount of nucleic acids which have a common sequence. It is the graph which showed the binding degree with PPARγ2 of the domain polypeptide obtained by the concentration operation using PPARγ2 as a target substance. It is the graph which showed the grade which the domain polypeptide obtained by the concentration operation which used PPARγ2 as the target substance inhibits the binding of the natural ligand of PPARγ2. It is a graph which shows the presence frequency and appearance frequency of each amino acid of the domain polypeptide obtained by the concentration operation which used PPARγ2 as a target substance. It is a figure which shows the structure motif sequence obtained from the domain polypeptide obtained by the concentration operation which used PPARγ2 as the target substance. It is an electrophoretic photograph showing the result of concentration operation using Smad3 as a target substance. It is the figure which showed the example of the amino acid sequence of the domain polypeptide obtained by using Smad3 as a target substance. It is the figure which showed the example of the amino acid sequence of the domain polypeptide obtained by using FK506 as a target substance. It is a figure which shows the position of the domain polypeptide obtained by the concentration operation of this invention in the natural ligand sequence of FK506. It is a schematic diagram which shows the domain polypeptide of this invention. It is a schematic diagram which shows the structural motif of this invention.

Claims (8)

(1) A step of bringing a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part into contact with a target substance, (2) target Obtaining the protein-nucleic acid conjugate specifically bound to the substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding it are A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of (3), a protein specifically bound to a target substance in the steps (1) and (2) above A method for selecting and obtaining a domain polypeptide that interacts with a target substance, comprising a step of repeating until the nucleic acid part of the nucleic acid conjugate forms a cluster encoding the domain polypeptide. (1) A step of bringing a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part into contact with a target substance, (2) target Obtaining the protein-nucleic acid conjugate specifically bound to the substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding it are A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of (3), a protein specifically bound to a target substance in the steps (1) and (2) above A target substance comprising a step of repeating until the nucleic acid part of the nucleic acid conjugate forms a cluster encoding a domain polypeptide, and (4) a step of obtaining the nucleic acid having a cluster formed in step (3) Selection method of obtaining nucleic acid encoding the domain interacting polypeptides. The domain polypeptide is composed of a polypeptide having a length of 90% or less of the full-length protein, and a polypeptide having a length of 130% or less of the interaction domain with the target substance. The method described in 1. (1) A step of bringing a protein-nucleic acid conjugate group in which a protein part and a nucleic acid part encoding the protein part are directly bound via a nucleic acid construct bound to the 3 ′ end of the nucleic acid part into contact with a target substance, (2) target Obtaining the protein-nucleic acid conjugate specifically bound to the substance, amplifying the nucleic acid part of the conjugate, and using this as part of the template, the protein part and the nucleic acid part encoding it are A step of preparing a group of protein-nucleic acid conjugates directly bound via a nucleic acid construct bound to the 3 ′ end of (3), a protein specifically bound to a target substance in the steps (1) and (2) above -A step of repeating until the nucleic acid part of the nucleic acid conjugate forms a cluster encoding the domain polypeptide, (4) a step of analyzing the base sequence of the nucleic acid that formed the cluster in step (3), (5) the base sequence Method for identifying structural motifs sequences have the which comprises the step of selecting a common sequence present frequency and / or frequency as an indicator of the amino acid sequence translated it. A method for obtaining a structural motif having a transformed function, wherein a mutation is introduced into a part of the structural motif sequence according to claim 4 and the interaction is analyzed by contacting the mutant structural motif with a target substance. (1) a step of producing a domain polypeptide selected in claim 1 or 3 or a complex of the structural motif according to claim 4 or 5 and a target substance; (2) three-dimensional structure analysis of the complex; Performing a step of specifying a binding site between the target substance and the polypeptide or structural motif, and (3) predicting a binding space of the target substance from the identified binding site and designing a low molecular compound that fits the binding space. A molecular design method for a compound that interacts with a target substance, comprising: The method according to claim 1, wherein the target substance is associated with a disease. (1) The target substance is associated with a disease, and a domain polypeptide selected in claim 1 or 3 or a complex of the structural motif of claim 4 or 5 and the target substance, (2 (3) a step of analyzing the three-dimensional structure of the complex to identify the binding site between the target substance and the polypeptide or structural motif; (3) predicting the binding space of the target substance from the identified binding site, and A method for producing a therapeutic and / or prophylactic agent for a disease associated with a target substance, comprising a step of designing a compound in time and (4) synthesizing the designed compound.