JP2018191617A - Methods for screening endogenous control factors using pyrrole-imidazole polyamide compounds - Google Patents

Abstract

To provide methods for efficiently identifying endogenous control factors that modulate expression of genes associated with onset of various diseases such as cancer and cell differentiation at an upstream site.SOLUTION: The invention provides a method for screening endogenous control factors comprising the steps of: 1) treating a cell with a polyamide compound of a pyrrole-imidazole polyamide compound group, where the polyamide compound is capable of recognizing and binding to a DNA sequence, and is conjugated to a substance that activates or suppresses a gene expression; 2) selecting a polyamide compound that brings about a cell response by the treatment of the step 1) from the pyrrole-imidazole polyamide compound group; and 3) identifying an endogenous control factor associated with the cell response on the basis of the DNA sequence recognized by the polyamide compound selected in the step 2).SELECTED DRAWING: None

Description

  The present invention relates to a method for screening an endogenous regulator such as a disease using a pyrrole-imidazole polyamide compound group.

  Molecular targeted drugs that control diseases such as cancer act on specific cell surface antigens, receptors, signal transduction factors, etc. on diseased cells, but the emergence of resistance due to mutations in the specific molecules is a problem. Therefore, the identification of endogenous regulators such as transcription regulators that bind and control the function expression of disease-related factor groups upstream is extremely promising in the research and development of innovative molecular targeted drugs that do not easily develop resistance. It is believed that there is. However, identification of such endogenous regulators is usually carried out using methods such as comprehensive genetic screening and state-of-the-art gene expression analysis techniques, which takes time and is difficult. There are many. For example, when a paralog exists in a certain control factor, the paralog may function in a complementary manner. In that case, it is extremely difficult to identify the control factor by genetic screening with RNAi or the like. It becomes difficult. In addition, conventional comprehensive genetic screening methods are largely subject to mutation analysis, and it is almost impossible to identify regulatory factors when no mutation exists. Even with state-of-the-art gene expression analysis techniques, it is extremely difficult to extract important regulatory factors actually responsible for disease characteristics due to their diverse gene change patterns.

  On the other hand, pyrrole-imidazole polyamide (hereinafter abbreviated as “PI polyamide”) is a synthetic oligomer, and a minor groove of DNA is formed by N-methylpyrrole (P) and N-methylimidazole (I) pairs facing each other by hairpin linking. It is known to bind to and recognize specific sequences. PI polyamides recognize P / I pairs as C · G base pairs, P / P pairs as A · T or T · A base pairs, and I / P pairs as G · C base pairs, thereby It can specifically bind to a double-stranded DNA sequence. Accordingly, the base sequence specificity of PI polyamide can be arbitrarily designed by appropriately arranging N-methylpyrrole and N-methylimidazole in the molecular structure.

  Despite its relatively large molecular weight, PI polyamide is cell membrane permeable, localizes to the cell nucleus and affects endogenous gene expression at the nanomolar level. PI polyamides bound to target genes have been studied as artificial gene switches that inhibit transcription factor binding to DNA and control specific gene expression. In recent studies, PI polyamides conjugated with suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase (HDAC) inhibitor, were synthesized (Non-patent Documents 1 and 2). The SAHA-conjugated PI polyamide can specifically upregulate the expression of the target gene by acetylation of the regulatory region of the target gene. In addition, chlorambucil, a nitrogen mustard alkylating agent, was successfully conjugated to PI polyamide (Non-patent Documents 3 and 4). The conjugate was found to have enhanced binding ability to genomic sequences, resulting in strong down-regulation of target gene expression. Furthermore, in Non-Patent Document 1, human skin fibroblasts are treated with a library of PI polyamide conjugated with SAHA, and SAHA alone has no effect, but a gene whose transcription is activated by the conjugate. Studies have been reported to screen for.

Pandian, G. N. et al., Sci Rep 4, 3843, doi: 10.1038 / srep03843 (2014) Saha, A. et al., Bioorg Med Chem 21, 4201-4209, doi: 10.1016 / j.bmc.2013.05.002 (2013) Bando, T. et al., Acc Chem Res 39, 935-944, doi: 10.1021 / ar030287f (2006) Minoshima, M. et al., Nucleic Acids Symp Ser (Oxf), 69-70, doi: 10.1093 / nass / nrp035 (2009)

  The present invention develops a method for efficiently identifying endogenous regulatory factors that control the expression of genes related to the onset of various diseases such as cancer and cell differentiation more upstream.

  As a result of diligent research to solve the above-mentioned problems, the present inventors treated cells such as diseased cells using a library of PI polyamide conjugated with a substance that activates gene expression or a substance that suppresses gene expression. Thus, it has been found that by selecting a PI polyamide that produces a cellular response by the treatment, an endogenous regulator associated with the cellular response can be identified.

That is, the present invention provides the following.
[1] 1) A pyrrole-imidazole polyamide compound group that forms a complex with a substance that activates gene expression or a substance that suppresses gene expression, and that can recognize and bind to the DNA sequence. Treating cells with a polyamide compound contained in
2) a step of selecting the polyamide compound that brings about a cellular response by the treatment in step 1) from the pyrrole-imidazole polyamide compound group, and 3) based on the DNA sequence recognized by the polyamide compound selected in step 2). A method for screening an endogenous regulator comprising the step of identifying an endogenous regulator associated with the cellular response,
[2] The pyrrole-imidazole polyamide compound group includes an activated polyamide compound that forms a complex with a substance that activates gene expression, and a suppressed polyamide compound that forms a complex with a substance that suppresses gene expression. Including
In the step 2), the activated polyamide compound and the inhibitory polyamide compound capable of producing a reciprocal cellular response and recognizing and binding to the same DNA sequence are classified into the pyrrole-imidazole polyamide compound group. Choose from,
[1] The method according to
[3] The activated polyamide compound and the repressed polyamide compound capable of causing the opposite cellular responses and recognizing and binding to the same DNA sequence have a DNA sequence recognition portion common to each other. The method according to [2],
[4] In the above step 3), from the DNA sequence recognized by the polyamide compound selected in the above step 2) and the gene expression profile of the cell subjected to the above step 1), an endogenous regulatory factor related to the cellular response. The method according to any one of [1] to [3], wherein
[5] The cellular response is a cell viability, a cell proliferation rate, an expression level of a disease-related gene or a disease-related protein, or an increase or decrease of an expression level of a differentiation marker gene or a differentiation marker, or a change in cell morphology. , The method according to any one of [1] to [4],
[6] The method according to any one of [1] to [5], wherein the cell is a disease cell or a stem cell.
[7] In the step 3), an endogenous regulator that controls cell proliferation, expression of a disease-related gene or gene group, or expression of a differentiation-related gene or gene group is identified [1] to [6] A method according to any of the above,
[8] The method according to any one of [1] to [7], for screening a factor controlling disease or cell differentiation,
[9] The method according to any one of [1] to [8], wherein the endogenous regulator is a cis element or a trans regulator.
[10] The method according to any one of [1] to [9], wherein the substance that activates gene expression is a histone deacetylase inhibitor, and the substance that suppresses gene expression is an alkylating agent. ,
[11] The method according to any one of [5] to [10], wherein the disease is cancer, neurodegenerative disease, metabolic endocrine disease, or viral infection, and [12] activate gene expression. Two or more pyrrole-imidazole polyamide compounds including a library consisting of a pyrrole-imidazole polyamide compound group conjugated with a substance and a library consisting of a pyrrole-imidazole polyamide compound group conjugated with a substance that suppresses gene expression A set of libraries, wherein the pyrrole-imidazole polyamide compound group comprises a DNA sequence recognition moiety that is common between the two or more libraries.

  According to the present invention, it is possible to easily identify a DNA sequence (cis element) that controls various diseases such as cancer, and an endogenous transcriptional regulatory factor that binds to the sequence, and an innovative that hardly causes resistance. Drug discovery targets for molecular target drugs can be found. Further, the method of the present invention can be applied not only to various diseases but also to identification of new factors related to differentiation control from various stem cells or the like to specific cell lineages. According to the present invention, it is possible to identify an endogenous regulator of a disease-related gene without conducting an exhaustive genetic screen for target disease cells. In addition, according to the present invention, it is possible to identify an endogenous regulator of a disease-related gene that is not accompanied by a gene mutation.

It is a schematic diagram which shows the example of the well plate which added one type of PI polyamide compound to one well. In the figure, the description of the added PI polyamide compound is omitted for well 2-a to well 12-h, but one type of different PI polyamide compound is added to each of these wells. It is a schematic diagram showing an example of two or more polyamide compound libraries provided as two or more well plates. In the figure, the PI polyamide compound added to each well is shown on the right side. Although the description of the added PI polyamide compound is omitted for the wells 2-a to 12-h, one kind of different PI polyamide compound is added to each of these wells. It is a schematic diagram showing an example of two or more polyamide compound libraries provided as two or more well plates. In the figure, the PI polyamide compound added to each well is shown on the right side. Although the description of the added PI polyamide compound is omitted for the wells 2-a to 12-h, one kind of different PI polyamide compound is added to each of these wells. 1 is a schematic diagram of a SAHA-PI polymiad conjugate synthesized in Example 1. FIG. FIG. 2 shows a schematic diagram of the SAHA-PI polymer ad conjugate synthesized in Example 1 and the corresponding chemical formula of one SAHA-PI polymer ad conjugate. The expression levels of IL3, CSF2 and CSF2RB genes in human myeloid leukemia cells MV4-11 treated with each SAHA-PI polyamide conjugate are shown. The expression level of each gene in DMSO-treated cells is taken as 1, and the value obtained from each SAHA-PI polyamide conjugate-treated cell is shown as a relative value to DMSO-treated cells. “*” Indicates P <0.05. 2 shows a chemical formula representing the PI polyamide conjugate used in Example 1. 2 shows a chemical formula representing the PI polyamide conjugate used in Example 1. The expression levels of IL3, CSF2 and CSF2RB genes in human myeloid leukemia cells MV4-11 treated with each chlorambucil-PI polyamide conjugate are shown. The expression level of each gene in DMSO-treated cells is taken as 1, and the value obtained from each PI polyamide-chlorambucil conjugate-treated cell is shown as a relative value to DMSO-treated cells. In the figure, data are shown as mean ± SEM values, and “*” indicates P <0.05. The growth rates of various cancer cells and normal cells treated with each Chb-PI polyamide conjugate are shown.

1. PI polyamide PI polyamide is a polyamide containing an N-methylpyrrole unit (P), an N-methylimidazole unit (I), and a γ-aminobutyric acid moiety, and P, I, and γ-aminobutyric acid moiety are mutually amides. Linked by a bond (—C (═O) —NH—) (Trauger et al, Nature, 382, 559-61 (1996); White et al, Chem. Biol., 4,569-78 (1997); and Dervan , Bioorg. Med. Chem., 9, 2215-35 (2001)). In PI polyamide, the γ-aminobutyric acid moiety is a linker (γ-linker) and the whole is folded into a U-shaped conformation (hairpin type). In the U-shaped conformation, two chains including P and I are arranged in parallel across the linker. High affinity for a specific base pair in DNA when the pair of P and I facing between the two strands is a specific combination (P / I pair, I / P pair or P / P pair) Can be combined. For example, a P / I pair can bind to a C · G base pair, an I / P pair can bind to a G · C base pair, and a P / P pair can be an A · T base pair and a T · A pair. It can bind to both base pairs. In addition to P and I, PI polyamide may contain 3-hydroxypyrrole (Hp) or β-alanine. Hp / P pairs can bind to TA base pairs (White at al., Nature, 391, 468-71 (1998)). A β-alanine / β-alanine pair can bind to a T • A base pair or an A • T base pair. A β-alanine / I pair can bind to a CG base pair, and an I / β alanine pair can bind to a G · C base pair. The β-alanine / P pair and the P / β-alanine pair can bind to a T • A base pair or an A • T base pair. In addition, the γ-linker moiety and the β alanine moiety added to the N-terminus of PI polyamide can also bind to T • A base pairs or A • T base pairs. By appropriately changing the configuration, order, combination, and the like of the pair formed by P, I, Hp, and / or β-alanine, a PI polyamide that recognizes and binds to the target DNA sequence can be designed.

  In the present invention, the methyl group on the 1-position nitrogen of P or I constituting the PI polyamide may be replaced with hydrogen or an alkyl group other than a methyl group. Examples of the alkyl group other than the methyl group include linear, branched or cyclic saturated or unsaturated alkyl groups having 2 to 10 carbon atoms, preferably linear or branched chains having 2 to 5 carbon atoms. A linear, branched or cyclic saturated or unsaturated alkyl group is exemplified, and examples thereof include ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and the like. In addition, alkyl groups including methyl groups may be substituted. For example, methylene in the alkyl group may be substituted with oxygen or the like.

  Various functional groups may be added to the N-terminal and C-terminal of the PI polyamide. For example, an acetylamino group may be added to the N terminal. For example, a dimethylaminopropylamide group may be added to the C-terminus. Alternatively, a carboxyl group such as a β-alanine residue or γ-aminobutyric acid residue can be added to the end of the PI polyamide.

  In the present invention, the PI polyamide may be modified, for example, may be modified so as to maintain or improve the binding ability to DNA. Examples of modified PI polyamides include, but are not limited to, those in which the α-position or β-position of the γ-linker of the PI polyamide is substituted with an amino group, that is, the γ-linker is N-α-N- Those which are γ-aminobutyric acid or N-β-N-γ-aminobutyric acid, those wherein the amino group is modified with a molecule such as a fluorescent group or biotin, and the N-terminus of PI polyamide such as a fluorescent group or biotin Examples thereof include those modified with molecules, and modified products in which the C-terminus of PI polyamide is modified with molecules such as a fluorescent group, biotin, and isophthalic acid. In the present specification, examples of the fluorescent group include, but are not limited to, fluorescein, rhodamine dyes, cyanine dyes, ATTO dyes, Alexa Fluor dyes, and BODIPY. Fluorescein also includes fluorescein derivatives (eg, fluorescein isothiocyanate).

  The PI polyamide may be a cyclic PI polyamide. Examples of cyclic PI polyamides include, for example, those in which the N terminus and C terminus of PI polyamide are linked by a γ-linker, or between the chloroacetyl group added to the N terminus of PI polyamide and the cysteine group added to the C terminus. And those cyclized by sulfide bonds formed by the reaction. The PI polyamide may also be a tandem PI polyamide compound in which a hairpin PI polyamide is connected.

  The PI polyamide used in the present invention is designed to recognize any DNA sequence. The number of pairs formed by P, I, Hp, and / or β-alanine constituting the PI polyamide used in the present invention is not particularly limited as long as it is 2 or more. For example, 3 to 12, preferably Is 4 to 10, more preferably 4 to 8. When designing a PI polyamide having a DNA sequence recognition moiety having 5 or more pairs, it is preferably designed to include β-alanine.

  Methods for designing and producing PI polyamides are known (see, for example, Japanese Patent No. 3045706, Japanese Patent Application Laid-Open No. 2001-136974, WO 03/000683, Japanese Patent Application Laid-Open No. 2013-234135, and Japanese Patent Application Laid-Open No. 2014-173032). For example, it can be easily produced by an automatic synthesis method using a solid phase synthesis method (Fmoc solid phase synthesis method) using Fmoc (9-fluorenylmethoxycarbonyl).

  The PI polyamide used in the present invention forms a complex by binding a substance that activates gene expression or a substance that suppresses gene expression to its N-terminal, C-terminal, or γ-linker site. Hereinafter, in this specification, PI polyamide conjugated with the above substance (that is, PI polyamide forming a complex with the above substance) is referred to as “PI polyamide compound” or “PI polymiad conjugate”. Further, in this specification, PI polyamide that forms a complex with a substance that activates gene expression is referred to as “activated polyamide compound”, and PI polyamide that forms a complex with a substance that suppresses gene expression is referred to as “ This is referred to as “suppressed polyamide compound”. In the present specification, substances that activate gene expression and substances that suppress gene expression may be collectively referred to as “substances”.

  Examples of the substance that activates gene expression include, but are not limited to, a histone acetylase (HAT) activator and a histone deacetylase (HDAC) inhibitor. Examples of the HAT activator include N- (4-chloro-3- (trifluoromethyl) phenyl) -2-ethoxybenzamide (CTB), histone methylase regulator, histone demethylase regulator, and the like. Can be mentioned. Examples of HDAC inhibitors include suberoylanilide hydroxamic acid (SAHA).

  Examples of substances that suppress gene expression include, but are not limited to, alkylating agents, HAT inhibitors, HDAC activators, histone methylase regulators, histone demethylase regulators, and the like. Examples of the HAT inhibitor include C646. Examples of the HDAC activator include 1-benzoyl-3-phenyl-2-thiourea (TM-2-51).

  An alkylating agent is a compound having a functional group that forms a covalent bond with DNA. Examples of the alkylating agent include chlorambucil (sometimes abbreviated as “Chb” in this specification), duocarmycin, seco-CBI (1-chloromethyl-5-hydroxy-1, 2-dihydro-3H-benzo [e] indole), pyrrolobenzodiazepine, nitrogen mustard and the like. When conjugating seco-CBI to PI polyamide, an indole may be inserted between PI polyamide and seco-CBI. In a complex of PI polyamide, indole and seco-CBI (hereinafter also referred to as “conjugate”), the indole moiety is paired with two P or I units, the indole / I pair is CG base pair, indole The / P pair recognizes T · A base pairs or A · T base pairs.

  Conjugation between a substance that activates gene expression or a substance that suppresses gene expression and PI polyamide can be performed by a conventional method (see, for example, J. Am. Chem. SOC. 1995, 117, 2479-2490). ). The substance may be directly bonded to the N-terminal, C-terminal, or γ linker site of PI polyamide, or may be bonded via a linker. The linker is not particularly limited as long as it does not interfere with the action of the above substances and does not interfere with the DNA sequence recognition of PI polyamide. For example, an amide bond, phosphodisulfide bond, ester bond, coordination bond, or ether bond Or a molecule containing a functional group that forms one or more of these bonds. “Molecules containing functional groups that form one or more of these bonds” include amide bonds, phosphodisulfide bonds, ester bonds, coordination bonds, ether bonds, etc., together with the terminal portion of the PI polyamide and / or agent. A molecule containing a functional group that forms one or more bonds selected from the group consisting of: “Molecules containing functional groups that form one or more of these bonds” also include one or more bonds selected from the group consisting of amide bonds, phosphodisulfide bonds, ester bonds, coordination bonds, ether bonds, and the like. May be a molecule containing one or more. Preferred linkers include amide bonds or molecules containing functional groups that form amide bonds.

2. PI polyamide compound library and PI polyamide compound group In the present invention, “pyrrole-imidazole polyamide compound library” (hereinafter abbreviated as “PI polyamide compound library”) is a substance and / or gene that activates the above gene expression. It is a group of compounds including a PI polyamide compound conjugated with a substance that suppresses expression. Therefore, in this specification, the “PI polyamide compound library” may be referred to as a “pyrrole-imidazole polyamide compound group” (hereinafter abbreviated as “PI polyamide compound group”).

  In the present invention, the DNA sequence recognition portion of the PI polymiad compound contained in the PI polyamide compound group is arbitrarily designed. As described above, the number of pairs formed by P, I, Hp, and / or β-alanine constituting the DNA sequence recognition portion of PI polymiad is not particularly limited as long as it is 2 or more. , Preferably 4 to 10, more preferably 4 to 8. In the present invention, two or more kinds of PI polymiad compounds included in the PI polyamide compound group may have the same length of the DNA sequence recognition portion or different lengths of the DNA sequence recognition portion. Also good. Moreover, the kind of PI polymiad compound contained in a PI polyamide compound group will not be specifically limited if it is 2 or more types, What is necessary is just to determine suitably considering conditions, such as the length of a DNA sequence recognition part. For example, the PI polyamide compound group may have a DNA sequence recognition portion having the same length, and each PI polyamide may recognize a different DNA sequence. In the case of such a configuration, it is possible to obtain a number of PI polymiad compound groups covering all DNA recognition sequence patterns.

  In the present invention, the PI polyamide compound group may include two or more types of PI polyamide compounds in which the same substance is conjugated, but the DNA sequence recognition portions are different. That is, two or more kinds of PI polymer compounds included in the PI polyamide compound group may have the same structure except that they have different DNA sequence recognition portions. Preferably, the PI polyamide compound group is composed of two or more kinds of activated polyamide compounds which are substances that activate gene expression and are conjugated with the same substance but have different DNA sequence recognition portions. Alternatively, a substance that suppresses gene expression and is conjugated with the same substance, but may be composed of two or more types of suppressed polyamide compounds having different DNA sequence recognition portions. Furthermore, the PI polyamide compound group may include both an activated polyamide compound and an inhibited polyamide compound, and preferably, the activated polyamide compound and the inhibited polyamide compound have a DNA recognition portion common to each other. It may be.

3. Screening method In the present invention, 1) a cell is treated with a PI polyamide compound included in the above-mentioned PI polyamide compound group, 2) a PI polyamide compound that causes a cellular response by the treatment is selected, and 3) the selected PI polyamide compound. The endogenous regulator is screened by identifying the endogenous regulator associated with the cellular response based on the DNA sequence recognized by.

  In the above step 1), the cells to be treated with the PI polyamide compound may be diseased cells, non-disease cells, normal cells, or stem cells, and are appropriately selected according to the purpose of screening. For example, when it is desired to identify an endogenous regulator associated with a certain disease, the disease cell is preferably used, and for example, a disease model cell is used. In addition, for example, when it is desired to identify an endogenous regulatory factor related to cell differentiation, it is preferable to use stem cells. IPS cells can also be used instead of stem cells. For example, non-disease cells or normal cells can be used if one wants to identify any endogenous regulators independent of the disease. Examples of endogenous regulatory factors unrelated to the disease include, but are not limited to, factors that induce apoptosis, factors that promote iPS cell induction, and the like.

  The cell used in the present invention is preferably a mammalian cell, more preferably a human-derived cell. In the present invention, primary cultured cells can also be used, but various established cell lines are preferably used. In the present invention, non-disease cells include cells derived from individuals or tissues not affected by any disease, and individuals not affected by the target disease but affected by any other disease Or tissue-derived cells are included.

  The treatment in the above step 1) can be carried out by culturing the cells in the presence of the PI polyamide compound group. That is, cells are cultured under the same conditions in the presence of each PI polymer compound contained in the PI polyamide compound group. Culture conditions such as culture medium and culture temperature can be appropriately determined according to the type of cells used. The culture time may be any time during which any cellular response is generated by treatment with any PI polymiad compound, and can be appropriately determined by those skilled in the art.

  The step 1) can be performed using, for example, a well plate. When using a well plate, one kind of PI polyamide compound is added to one well, and cells are added to each well and cultured. An example of a well plate to which a PI polyamide compound is added is shown in FIG. For example, when a 96-well plate is used, a maximum of 96 types of PI polyamide compounds can be used.

  In the above step 2), the selection of a PI polymiad compound that brings about a cellular response is performed by selecting a PI polymiad compound that exhibits some cellular response compared to treatment with other PI polymiad compounds. For example, when the screening method of the present invention is carried out using a well plate as shown in FIG. 1, the presence or absence of a cellular response is examined for each well, and the PI polyamide compound in the well that has a cellular response is selected. Good.

  Furthermore, if necessary, cells that have not been treated with the PI polyamide compound library of the present invention, or when disease cells are used for the screening, non-disease cells or normal cells may be used as controls, and such control cells. PI polymiad compounds exhibiting some cellular response compared to may be selected. For example, when the screening method of the present invention is carried out using a well plate as shown in FIG. 1, two identical well plates are prepared, and disease cells are added to each well of one well plate. (Hereinafter referred to as “test plate”), a control cell is added to each well of the other well plate (hereinafter referred to as “control plate”), and each well of the two plates is examined for the presence of a cellular response, In a test plate, a PI polyamide compound in a well that exhibits a cellular response relative to a corresponding well in a control plate may be selected.

  In the present invention, “cell response” means that a cell brings about some event, and is not particularly limited. A cellular response includes, for example, an increase or decrease in cell viability, cell proliferation rate, or expression level of a particular gene or protein, or a change in cell morphology. Examples of the specific gene and the specific protein include a disease-related gene, a protein encoded by a disease-related gene (also referred to as “disease-related protein” in this specification), a differentiation marker gene, and a differentiation marker gene. Examples include proteins (also referred to simply as “differentiation markers” in the present specification), apoptosis-related genes, proteins encoded by apoptosis-related genes, and the like. Here, “increase or decrease” means that treatment with a certain PI polymiad compound increased or decreased the cellular response compared to treatment with another PI polymiad compound, and treatment with a certain PI polymiad compound indicates that Increased or decreased cellular response compared to cells not treated with the PI polyamide compound library, and treatment of diseased cells with a PI polymiad compound resulted in treatment of non-disease or normal cells when diseased cells were used. It includes any increase or decrease in cellular response compared to treatment with the PI polymiad compound. The change in cell morphology is that cells treated with a certain PI polymiad compound change in shape as compared with cells treated with other PI polymiad compounds, and cells treated with a certain PI polymiad compound are not affected by the PI polyamide compound live of the present invention. Changes in morphology compared to cells not treated with rally, and when diseased cells are used, diseased cells treated with a PI polymiad compound are compared to non-disease cells or normal cells treated with the PI polymiad compound. Any change in form is encompassed. Examples of changes in cell morphology include, but are not limited to, the appearance of (azur) granules, nuclear fragmentation, changes in chromatin aggregation, etc. when young myeloid cells differentiate, and Examples include increase of each cell ratio (N / C ratio) and rounding of nuclei when lymphocyte cells differentiate.

  The disease-related gene is, for example, a known related gene of the target disease. Examples of the disease-related gene include, but are not limited to, the C-Myc gene in many carcinomas, the gene HIF that controls the tumor hypoxia mechanism, the BCR-ABL fusion gene that causes CML and PhALL, pancreatic cancer, Examples thereof include Kras (mutation) gene, p53 mutation gene, C-Myb gene, C / EBPα gene and the like in colorectal cancer. Examples of the differentiation marker gene include, but are not limited to, CD11b gene, C / EBPα gene, C-Myb gene, and PAX5 gene. Examples of the apoptosis-related gene include, but are not limited to, a C-Myc gene. An increase or decrease in gene expression level can be determined, for example, by quantifying the gene expression level or quantifying the expression level of the protein encoded by the gene. The gene expression level may be quantified by a method known in the art, for example, mRNA quantification by a real-time quantitative PCR method, protein quantification by a Western blot method, or the like.

  In the screening method of the present invention, a PI polyamide compound may be selected based on a plurality of cellular responses.

  In the step 3), an endogenous regulatory factor related to the cellular response is identified based on the DNA sequence recognized by the selected PI polymiad compound. “Endogenous regulatory factor” means a factor that controls the expression of a target gene endogenous to a cell, and is a factor that regulates the expression of a target gene mainly through binding of a protein and DNA. In the present invention, endogenous regulatory factors include cis elements and trans regulatory factors (transcription factors). The cis element is the DNA sequence itself that is recognized and bound by the PI polymer ad conjugate. A transregulatory factor is a factor that regulates transcription by binding to a DNA sequence (cis element) to which a PI polymiad conjugate binds.

  The DNA sequence recognized by the selected PI polymiad compound is identified as an endogenous regulator (cis element) associated with the cellular response. Furthermore, in step 3), based on the DNA sequence recognized by the selected PI polymer compound, an endogenous regulatory factor related to the cellular response is identified from the gene expression profile of the cell subjected to the treatment. Also good. A known gene expression profile can be used. Gene expression profiles of various cells have been published, and for example, NCBI (National Center for Biotechnology Information) database, TRANSFAC, TFSEARCH, CorePromoter, etc. can be used. For example, in the gene expression profile, an endogenous control factor containing the same sequence as the DNA sequence recognized by the selected PI polymiad compound is searched, and the endogenous control factor (cis element associated with the cellular response is searched for. ) Or an endogenous regulator that binds to a sequence containing the same sequence as the DNA sequence recognized by the selected PI polymiad compound, and that is associated with the cellular response. Identified as (transcription factor). Furthermore, in the cells treated with the selected PI polymer compound, the expression level of the plurality of target genes of the identified transcription factor is quantified by RT-PCR, so that the PI polyamide compound is actually endogenous. You can see if it works instead of a control factor. In addition, by creating Conditional shRNAi vector or Conditional Over-expression vector of the above identified transcription factor, and verifying the validity of the cell response by the PI polymiad compound, the target endogenous regulator is determined. Is possible.

  In the screening method of the present invention, two or more PI polyamide compound libraries may be used in the above step 1). Preferably, the pattern of the DNA sequence recognition part of the PI polymiad compound constituting two or more PI polyamide compound libraries is common between the two or more libraries. That is, preferably, one PI polyamide compound library is composed of two or more kinds of PI polymer compounds having different DNA sequence recognition parts, and the DNA sequence recognition part of the PI polymer compound contained in one library Corresponding PI polyamide compounds with the same DNA sequence recognition moiety are also present in other libraries. Preferably, the PI polyamide compounds constituting two or more PI polyamide compound libraries are conjugated with the same substance in each library and different between the libraries. More preferably, the two or more PI polyamide compound libraries cover a series of DNA recognition sequence patterns common between the libraries, and the PI polyamide compounds constituting the two or more PI polyamide compound libraries Are conjugated to the same substance in each library and different materials between the libraries. For example, a library of two or more PI polyamide compounds can be provided as two or more well plates in which one library is contained in one well plate. Examples of the two or more well plates are shown in FIGS. 2-1 and 2-2 (hereinafter collectively referred to as FIG. 2). For example, as shown in FIG. 2, it is preferable to add a PI polyamide compound having the same PI polyamide portion (DNA recognition portion) to the corresponding well of each well plate (ie, in FIG. The PI polyamide compound added to the well 1-a, the PI polyamide compound added to the well 1-a of the library II, and the PI polyamide compound added to the well 1-a of the library III are the same PI polyamide. The same applies to the well 1-b to the well 12-h). Further, as shown in FIG. 2, the substance conjugated to the PI polyamide compound added to the well plate is different for each well plate, but the PI in which the same substance is conjugated to the wells in the same well plate. It is preferred that a polyamide compound is added (ie, in FIG. 2, all PI polyamide compounds added to library I are conjugated with substance X, and all PI polyamide compounds added to library II are substances. Y is conjugated and all PI polyamide compounds added to library III are conjugated with substance Z).

  For example, but not limited to, one library of two or more PI polyamide compound libraries may be different types of DNA sequence recognition moieties indicated by A to φ as represented by the schematic diagram of FIG. 3-1. And is composed of a group of PI polyamide compounds conjugated with SAHA, and another library contains different types of DNA sequence recognition moieties, also denoted by A to φ, but substances other than SAHA, For example, but not limited to, it may be composed of a group of PI polyamide compounds conjugated with Chb or seco-CBI.

  The two or more PI polyamide compound libraries are combinations of two or more libraries composed of two or more kinds of activated polyamide compounds, each of which activates gene expression conjugated to each PI polyamide compound. It may be a combination of libraries that are the same within a library and that differ between the two or more libraries. Explaining with reference to FIG. 2, the substances X, Y and Z are all substances that activate gene expression. Further, the two or more PI polyamide compound libraries are combinations of two or more libraries composed of two or more types of suppressed polyamide compounds, and a substance that suppresses gene expression conjugated to each PI polyamide compound. A combination with a library that is the same in each library and that is different between the two or more libraries may be used. Explaining with reference to FIG. 2, the substances X, Y, and Z are all substances that suppress gene expression. The two or more PI polyamide compound libraries may be a combination including both a library composed of two or more types of activated polyamide compounds and a library composed of two or more types of suppressed polyamide compounds. Referring to FIG. 2, for example, the cases where the substances X and Y are substances that activate gene expression, and the substance Z is a substance that suppresses gene expression.

  Preferably, in the above step 1), a PI polyamide compound group including an activated polyamide compound and a suppressed polyamide compound may be used. For example, in the above step 1), two or more PI polyamide compound libraries, wherein at least one of the two or more PI polyamide compound libraries is a library comprising an activated polyamide compound group, and , At least one uses a library of two or more PI polyamide compounds, which is a library consisting of a group of suppressed polyamide compounds.

  The above-mentioned two or more PI polyamide compound libraries are not limited, but, for example, two PIs consisting of one library consisting of an activated polyamide compound group and one library consisting of a suppressed polyamide compound group. It may be a polyamide compound library or three PI polyamide compound libraries consisting of two libraries consisting of activated polyamide compounds and one library consisting of suppressed polyamide compounds.

  When two or more PI polyamide compound libraries are used in the screening method of the present invention, in step 2), PI polymiad compounds that cause cellular responses in at least two libraries and recognize the same DNA sequence are selected. Preferably, a PI polyamide compound is selected that produces a cellular response in at least two libraries and has a DNA sequence recognition moiety common to each other. For example, when the screening method of the present invention is carried out using a well plate as shown in FIG. 2, the presence or absence of a cellular response is examined for each well of each well plate, and the cells in the corresponding wells of two or more well plates If there is a response, select the PI polyamide compound in that well (eg, if any cellular response is detected in well 1-b of library I and well 1-b of library II, the wells of each plate The PI polyamide compound added to 1-b is selected).

  In the screening method of the present invention, when a PI polyamide compound group including an activated polyamide compound and a suppressed polyamide compound is used, in step 2), an opposite cellular response is caused and the same DNA sequence is recognized and the DNA is recognized. An activated polyamide compound and an inhibited polyamide compound that can bind to the sequence are selected. Preferably, activated polyamide compounds and inhibited polyamide compounds are selected that produce conflicting cellular responses and have DNA sequence recognition moieties in common with each other. For example, in the screening method of the present invention, when two or more PI polyamide compound libraries including both an activated polyamide compound library and an inhibited polyamide compound library are used, in step 2), in the two or more libraries, An activated polyamide compound and a repressed polyamide compound are selected that produce conflicting cellular responses and are able to recognize and bind to the same DNA sequence. Preferably, activated polyamide compounds and inhibited polyamide compounds are selected that produce conflicting cellular responses and have DNA sequence recognition moieties in common with each other. Referring to FIG. 2, for example, when substances X and Y are substances that activate gene expression and substance Z is a substance that suppresses gene expression, cells in either well of library I or II If a response is detected and a cellular response is detected in the corresponding well of library III, as opposed to that detected in library I or II, a PI polyamide compound in the well is selected (eg, If conflicting cellular responses are detected in well 1-b of library I or II and well 1-b of library III, the PI polyamide compound added to well 1-b of each plate is selected). Preferably, a cellular response is detected in the corresponding well of either library I and II and a cell response is detected in the corresponding well of library III that is contrary to that detected in libraries I and II If so, select a PI polyamide compound in the well (eg, if opposing cell responses are detected in wells 1-b of library I and II and well 1-b of library III, Select PI polyamide compound added to well 1-b).

  The above-mentioned “reciprocal cellular responses” include, for example, but not limited to, cellular events in which there is a treatment with a certain PI polymiad compound contained in a PI polyamide compound library conjugated with a substance that activates gene expression. And treatment with a corresponding PI polymiad compound contained in a PI polyamide compound library conjugated with a substance that suppresses gene expression results in the same reduction in cellular events. Thus, by screening using a group of PI polyamide compounds containing two or more kinds of PI polymiad compounds conjugated with substances having conflicting actions, the target endogenous regulatory factor can be identified more efficiently. Can do.

  According to the screening method of the present invention, it is possible to screen for factors that control disease or cell differentiation, or other useful endogenous control factors. For example, the screening method of the present invention can identify an endogenous regulator that controls cell proliferation, expression of a disease-related gene or gene group, or expression of a differentiation-related gene or gene group.

  According to the screening method of the present invention, an endogenous regulator of any disease can be screened, and the target disease is not particularly limited. For example, various diseases such as various cancers, neurodegenerative diseases (Alzheimer, Parkinson's disease, etc.), metabolic endocrine diseases (diabetes, hyperlipidemia, hypertension, etc.), viral infections (HIV, etc.) can be mentioned. For example, in the case of lipid-related metabolic diseases, screening can be performed using the above-mentioned PI polyamide compound group in the 3T3-L1 cell line (which can induce fatty acids in the cytoplasm) to identify metabolic disease control factors that increase or decrease fatty acids. is there. For example, when screening for an endogenous regulator of Alzheimer, the diseased cells are treated with the PI polyamide compound group, and the expression level of the disease-related gene or protein such as tau protein or beta amyloid is reduced or increased. A PI polymiad compound is selected.

  For example, when screening for an endogenous regulator of cancer using the screening method of the present invention, cancer cells are treated with the above-mentioned PI polyamide compound group, and cell viability, cell proliferation rate, or expression level of cancer-related gene or protein PI polymiad compounds that increase or decrease or kill cells in culture are selected. For example, when the PI polyamide compound group includes both an activated polyamide compound and an inhibited polyamide compound, the activated polyamide compound and the inhibited polyamide compound capable of recognizing and binding to the same DNA sequence One of the activated polyamide compound and the inhibited polyamide compound increases cell viability, cell proliferation rate, or expression level of a cancer-related gene or protein, and the other increases cell viability, cell proliferation rate, or cancer PI polymiad compounds are selected that reduce the expression level of the relevant gene or protein or kill the cells in culture.

  For example, when screening a factor that controls cell differentiation using the screening method of the present invention, cells such as stem cells are treated with the above-mentioned PI polyamide compound group, and the expression level of the differentiation marker gene or differentiation marker, cell viability or cell PI polymiad compounds that increase or decrease proliferation rate or kill cells during culture are selected. For example, when the PI polyamide compound group includes both an activated polyamide compound and an inhibited polyamide compound, the activated polyamide compound and the inhibited polyamide compound capable of recognizing and binding to the same DNA sequence One of the activated polyamide compound and the suppressed polyamide compound increases the expression level of the differentiation marker gene or differentiation marker, cell survival rate or cell proliferation rate, and the other increases the expression level of the differentiation marker gene or differentiation marker, PI polymiad compounds are selected that reduce cell viability or cell proliferation or kill cells during culture. For example, when screening with the above-mentioned PI polyamide compound group using KG1 cells (very weak myeloblastic leukemia cell line), a PI polyamide compound that increases the expression level of myeloid maturation markers such as CD11b, or cells A PI polymer compound is selected in which granules representing morphological differentiation are observed macroscopically. For example, it is possible to treat primary hematopoietic stem cells such as CD34 positive stem cells of umbilical cord blood with the above-mentioned PI polyamide compound group, and to select a PI polymiad compound that induces differentiation into a specific cell line (lineage) by surface marker analysis It is.

  After identifying the endogenous regulator by the screening of the present invention, identification of the binding site on the genome of the endogenous regulator (transcription factor), or identification of the position of the endogenous regulator (cis element) on the genome, The DNA sequence to which the PI polymiad compound is bound may be determined by a method known in the art, for example, the ChIP-Seq method, the Chem-Seq method, the ChIP-qPCR method, the Bind n Seq method, etc. it can.

  Endogenous regulators such as transcription factors and transcription factor binding sequences identified by the screening of the present invention include drug therapeutic targets such as disease therapeutic agents, for example, anticancer agents, or cell differentiation regulators (differentiation inducers, differentiation inhibitors). can do. In addition, a conjugate of a PI polymiad that binds to an endogenous regulator (cis element) identified by the screening method of the present invention and a drug can also be used as a disease therapeutic agent such as an anticancer agent or a differentiation inducer.

4). Set of PI polyamide compound library The present invention further provides a set of PI polyamide compound library comprising a combination of two or more of the above PI polyamide compound libraries. The set of PI polyamide compound libraries preferably comprises a library consisting of activated polyamide compounds and a library consisting of inhibited polyamide compounds. Preferably, the pattern of the DNA sequence recognition part of the PI polymiad compound constituting each library included in the set of PI polyamide compound libraries may be common between the libraries.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 Identification of Runt-related Transcription Factors (RUNX) (1) Preparation of PI Polyamide Compound Library Formed from P, I and β-alanine by placing imidazole at various positions 4 32 types of PI polyamides containing two pairs were designed and PI polymiads conjugated with suberoylanilide hydroxamic acid (SAHA) via β-alanine linkers at their N-termini (hereinafter referred to as “SAHA-PI polymiad conjugates”). Was also synthesized. The synthesis of the SAHA-PI polymiad conjugate was performed according to the method described in Tetrahedron Letters 50 (2009) 7288-7292. The 32 kinds of PI polymer ad conjugates (A to Z and α to φ) synthesized are shown in FIGS. 3-1 and 3-2.

  Briefly, PI polyamides with terminal amino groups were prepared by a stepwise reaction by Fmoc solid phase synthesis using a CLEAR-acid resin linked with β-alanine. N, N-dimethyl-1,3-propanediamine was added and treated overnight at 55 ° C. to cut out the PI polyamide from the solid phase. 4- (8-Methoxy-8-oxooctanamido) benzoic acid was prepared by coupling aminobenzoic acid and methyl 8-chloro-8-oxooctanoate to produce pentafluorophenyldiphenylphosphinate (FDPP) and N, Conversion to the activated ester using N-diisopropylethylamine (DIEA), followed by coupling with PI polyamide, produced a PI polyamide conjugated with 4- (8-methoxy-8-oxooctanamido) benzoyl. . A SAHA-PI polymiad conjugate was then prepared from the PI polyamide conjugate by amino-dealkoxylation using a hydroxylamine solution in water / dimethylformamide (DMF) (1/1). The crude product was purified by reverse phase high performance liquid chromatography and then confirmed by ESI-TOF MS (Electrospray ionization time-of-flight mass spectrometry). Reagents and solvents were obtained from standard suppliers and used without further purification. ESI-TOF MS (Electrospray ionization time-of-flight mass spectrometry) was performed using a Bio-TOF II (Bruker Daltonics) mass spectrometer using a positive ionization mode. Mechanical polyamide synthesis was performed on a computer controlled PSSM-8 (Shimadzu) system.

(2) Screening of endogenous regulators in myeloid leukemia cells Human myeloid leukemia cell MV4 with 20 μM of each SAHA-PI polymer conjugate library prepared in (1) above. -11 cells [obtained from ATCC (American Type Culture Collection, USA)] were treated for 12 hours. Total RNA was extracted from the treated cells, and the expression levels of RUNX1 target genes IL3, CSF2, and CSF2RB were quantified by real-time quantitative PCR (qRT-PCR). As a control, cells treated with DMSO were used, and the value obtained with each treated cell was normalized to the value of DMSO-treated cells (n = 3). RUNX1 is also known as acute myeloid leukemia 1 protein (AML1), and is a transcription factor that regulates the expression of blood-related genes and hematopoietic stem cell-related genes.

  Real-time quantitative PCR (qRT-PCR) was performed as follows. Total RNA was isolated with the RNeasy mini kit (Qiagen) and reverse transcribed with the Reverse script kit (TOYOBO) to generate cDNA. Real-time quantitative polymerase chain reaction (PCR) was performed using a 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Results were normalized to GAPDH levels. The relative expression level was calculated using the 2-ΔΔCt method. Table 1 shows primers used for qRT-PCR.

  The results are shown in FIG. As is clear from FIG. 4, in particular, the SAHA-PI polymer ad conjugate M increased the expression levels of IL3, CSF2, and CSF2RB. The recognition sequence of SAHA-PI polymiad conjugate M (hereinafter referred to as “conjugate M”) is 5′-WGGWGW-3 ′ (W is either A or T). As a result of searching for the sequence 5'-WGGWGW-3 'in the NCBI database of MV4-11 cells, it was found that the sequence was a consensus binding sequence of RUNX1. Thus, it was confirmed that the PI polyamide compound library of the present invention can be used for screening of endogenous regulators.

  Furthermore, the DNA binding sequence of conjugate M was determined by the Bind-n-Seq method. Briefly, biotin-labeled conjugate M and double-stranded random oligonucleotide were synthesized and biotin-labeled conjugate M was bound to double-stranded random oligonucleotide. Conjugate M-bound DNA was concentrated by biotin-streptavidin affinity purification. The concentrated DNA was amplified by PCR and sequenced. As a result, it was found that the sequence to which conjugate M was bound was “5′-TGTGGT-3 ′”.

(3) Confirmation of screening result Next, PI that specifically recognizes the RUNX consensus binding sequence of 5′-TGTGGT-3 ′ and 5′-TGCGGT-3 ′ by sequential binding of four pyrrole-imidazole pairs. Polyamides were designed to synthesize PI polymiad conjugates (SAHA-M ′, SAHA-50, Chb-M ′, Chb-50) with SAHA or chlorambucil (Chb) at the N-terminus (FIGS. 5-1 and Fig. 5-2). Here, SAHA-M ′ and Chb-M ′ target 5′-TGTGGT-3 ′, and SAHA-50 and Chb-50 target 5′-TGCGGT-3 ′. The method for synthesizing the SAHA-PI polymiad conjugate was performed in the same manner as described above.

  The synthesis of chlorambucil-PI polymiad conjugate was performed as follows. PI polyamides supported by oxime resin were prepared by stepwise reaction by Fmoc solid phase synthesis method. N, N-dimethyl-1,3-propanediamine (1.0 mL) was added to the product having an oxime resin, and the mixture was treated at 45 ° C. for 3 hours, and cut out. The resin was removed by filtration and the residue was dissolved in a minimum amount of dichloromethane and washed with diethyl ether to give 59.6 mg. To this crude compound (59.6 mg, 48.1 μmol) was added chlorambucil (32.6 mg, 107 μmol), PyBOP (benzotriazol-1-yl-oxy-tris-) in N, N-dimethylformamide (DMF) (300 μL). Pyrrolidino-phosphonium hexafluorophosphate) (101 mg, 195 μmol) and N, N-diisopropylethylamine (100 μL, 581 μmol) were added. The reaction mixture was incubated at room temperature for 1.5 hours, washed 3 times with diethyl ether and DMF and dried in vacuo. The crude product was purified by reverse phase flash column chromatography (0.1% aqueous trifluoroacetic acid / MeCN). The product was obtained after lyophilization (30.2 mg, 19.8 μmol).

  MV4-11 cells were treated with SAHA-M 'and SAHA-50 in the same manner as in (2) above, and the expression levels of myeloid leukemia related genes IL3, CSF2 and CSF2RB were quantified. The results are shown in FIG. As is clear from FIG. 4, SAHA-M ′ and SAHA-50 increased the expression levels of IL3, CSF2 and CSF2RB in the same manner as conjugate M.

  MV4-11 cells were treated with 5 μM Chb-M ′ or Chb-50 for 6 hours, total RNA was extracted from the treated cells, and IL3, CSF2 and CSF2RB were extracted by qRT-PCR as in (2) above. The expression level was quantified. As a control, cells treated with DMSO were used, and the value obtained with each treated cell was normalized to the value of DMSO-treated cells (n = 3). The results are shown in FIG. As is apparent from FIG. 6, contrary to the SAHA-PI polymiad conjugate, Chb-M ′ and Chb-50 suppressed the expression of IL3, CSF2RB, and CSF2.

  Furthermore, in order to specifically suppress the transcription factor RUNX1 estimated as a candidate transcription factor from the recognition sequence “5′-WGGWGW-3 ′” of the conjugate M, a Conditional shRNAi vector targeting the RUNX1 gene is prepared by a conventional method. It was confirmed that the cell growth was suppressed by a cell growth experiment in which the vector was prepared and introduced into MV4-11 cells. Therefore, RUNX1 could be determined as a transcription factor more suitable than “5′-WGGWGW-3 ′”.

Example 2: Identification of endogenous regulators that control the growth of specific disease cells . A library of chlorambucil-PI polymiad conjugates was synthesized in a manner similar to that described in Example 1. The PI polyamide portion of the 16 synthesized PI polymer conjugates (Chb-A to P) is the same as the PI polyamide portion of the SAHA-PI polymer conjugate A to P synthesized in Example 1. is there.

  Normal breast cell line (MCF10A), triple negative (TNBC) breast cancer cell line (MDA-MB-231), prostate cancer cell line (PC3), renal cancer cell line (786O), colon cancer cell line (HCT116, LOVO), Skills gastric cancer cell line (44As3), gastric cancer cell line (MKN7), esophageal cancer cell lines (TE11, TE5), pancreatic cancer cell lines (panc1, AsPC1, MiaPaca2), tyrosine kinase resistant chronic myeloid leukemia (TK resistant CML) cells Strain (MyL-R), MLL-related acute myeloid leukemia (MLL-AML) cell line (MV4-11), MLL-AML M cell line (MV4-11R), MDS-AML cell line (MOLM13), lung cancer cell line (RERF-LC-MS), neuroblastoma cell line (NB), and malignant childhood brain tumor cell lines (MRT-NS, M RT-yM) was cultured at 37 ° C. for 2 days in a medium containing 5 μM of various Chb-PI polymiad conjugates. The cell growth rate in the presence of various Chb-PI polymiad conjugates was determined with the cell growth rate when cultured under the same conditions in a medium containing no Chb-PI polymiad conjugates as 100. The results are shown in FIG.

  Based on the results of FIG. 7, Chb-PI polyamide conjugates with particularly low cell growth rates were selected. Subsequently, the binding sequence of the selected PI polymiad conjugate is determined by the Bind-n-Seq method, or the DNA recognition sequence of the conjugate is searched in the gene expression profile of the cell, thereby allowing the endogenous cancer to exist. Sex control factors are identified. In addition, endogenous regulators involved in a wide variety of cancers can be identified by selecting Chb-PI polyamide conjugates that reduce the cell growth rate in many cancer cells.

  According to the present invention, DNA sequences (cis elements) that control various diseases such as cancer by using a PI polymer compound library conjugated with a substance that activates gene expression or a substance that suppresses gene expression, and Candidates for endogenous transcription factors that bind to the sequence can be easily identified. Such an endogenous regulator can be used as a drug discovery target of an innovative molecular target drug in which resistance hardly appears. Further, the method of the present invention can be applied not only to various diseases but also to identification of new factors related to differentiation control from various stem cells or the like to specific cell lineages. In addition, PI polymiad conjugates that target sequences to which endogenous transcriptional regulatory factors identified by the screening method of the present invention can be used as anticancer agents, drugs for treating various diseases, cell differentiation regulators, and the like.

Claims (12)

1) pyrrole-imidazole polyamide compound group that forms a complex with a substance that activates gene expression or a substance that suppresses gene expression and that can recognize and bind to the DNA sequence Treating cells with a polyamide compound;
2) a step of selecting the polyamide compound that brings about a cellular response by the treatment in step 1) from the pyrrole-imidazole polyamide compound group, and 3) based on the DNA sequence recognized by the polyamide compound selected in step 2). A method for screening an endogenous regulator, comprising the step of identifying an endogenous regulator associated with the cellular response. The pyrrole-imidazole polyamide compound group includes an activated polyamide compound that forms a complex with a substance that activates gene expression, and a suppressed polyamide compound that forms a complex with a substance that suppresses gene expression,
In the step 2), the activated polyamide compound and the inhibitory polyamide compound capable of producing a reciprocal cellular response and recognizing and binding to the same DNA sequence are classified into the pyrrole-imidazole polyamide compound group. Choose from,
The method of claim 1.   The activated polyamide compound and the repressed polyamide compound capable of causing the conflicting cellular responses and recognizing and binding to the same DNA sequence have a DNA sequence recognition portion common to each other. 2. The method according to 2.   In step 3), an endogenous regulatory factor related to the cellular response is identified from the DNA sequence recognized by the polyamide compound selected in step 2) and the gene expression profile of the cell subjected to step 1). The method of any one of Claims 1-3.   The cell response is an increase or decrease in cell viability, cell proliferation rate, expression level of a disease-related gene or disease-related protein, or expression level of a differentiation marker gene or differentiation marker, or a change in cell morphology. The method of any one of 1-4.   The method according to any one of claims 1 to 5, wherein the cell is a disease cell or a stem cell.   7. In the step 3), an endogenous regulator that controls cell proliferation, expression of a disease-related gene or gene group, or expression of a differentiation-related gene or gene group is identified. The method described.   The method of any one of Claims 1-7 for screening the factor which controls a disease or cell differentiation.   The method according to any one of claims 1 to 8, wherein the endogenous regulator is a cis element or a trans regulator.   The method according to claim 1, wherein the substance that activates gene expression is a histone deacetylase inhibitor, and the substance that suppresses gene expression is an alkylating agent.   The method according to any one of claims 5 to 10, wherein the disease is cancer, neurodegenerative disease, metabolic endocrine disease, or viral infection.   2 or more including a library composed of a pyrrole-imidazole polyamide compound group conjugated with a substance that activates gene expression and a library composed of a pyrrole-imidazole polyamide compound group conjugated with a substance that suppresses gene expression A set of pyrrole-imidazole polyamide compound libraries, wherein the pyrrole-imidazole polyamide compound group comprises a pattern of DNA sequence recognition moieties that are common between the two or more libraries.