WO2022019303A1 - PYRROLE-IMIDAZOLE POLYAMIDE, TGFβ GENE EXPRESSION INHIBITOR, PHARMACEUTICAL COMPOSITION, AND METHOD FOR PRODUCING PYRROLE-IMIDAZOLE POLYAMIDE - Google Patents

Abstract

Provided is a pyrrole-imidazole polyamide that binds to the promoters of two or more genes and simultaneously controls transcription of the two or more genes. Also provided is a pharmaceutical composition comprising the pyrrole-imidazole polyamide. Also provided are: a pyrrole-imidazole polyamide that simultaneously controls transcription of HGF gene and TGF-β gene; a TGF-β gene expression inhibitor comprising the pyrrole-imidazole polyamide; and a pharmaceutical composition for treating a TGF-β-associated disease or a fibrous disease.

Description

Pyrrole imidazole polyamide, TGFβ gene expression inhibitor, pharmaceutical composition, and method for producing pyrrole imidazole polyamide.

The present invention relates to a pyrrole imidazole polyamide, a TGF-β gene expression inhibitor, a pharmaceutical composition, and a method for producing a pyrrole imidazole polyamide.
This application claims priority based on Japanese Patent Application No. 2020-123988 filed in Japan on July 20, 2020, the contents of which are incorporated herein by reference.

Pyrrole imidazole polyamide (hereinafter, also referred to as "PI polyamide") is a minor groove binder that strongly hydrogen-bonds to the minor groove of double-stranded DNA in a base sequence-specific manner. By designing the PI polyamide to bind to the transcriptional control region of the promoter of the target gene, the transcription of the target gene can be suppressed by using the PI polyamide. In addition, by binding an alkylating agent to PI polyamide, it is possible to suppress the expression of the target gene in a base sequence-specific manner. PI polyamides can be distributed in vivo, taken up by cells and bound to the nucleus without the use of drug delivery systems (DDS) such as lipids or viral vectors. In this respect, PI polyamide has an advantage over DNA recognition compounds such as ordinary nucleic acid drugs. PI polyamide can be freely molecularly designed by targeting various genes. Since the transcriptional activity of the disease gene activated by the disease can be specifically suppressed, there are few side effects. In addition, PI polyamide may be used as an orally administrable gene regulator. Therefore, many drug discovery studies using PI polyamide have been conducted so far.

TGF-β1 is a responsible factor for fibrotic diseases such as progressive nephropathy, liver cirrhosis and pulmonary fibrosis. The present inventors have developed a PI polyamide that binds to the TGF-β1 gene promoter and suppresses the expression of the TGF-β1 gene (Patent Document 1).

Japanese Patent No. 4682312

The conventional PI polyamide was designed to target the promoter of one specific gene. Therefore, conventional PI polyamides could only control the transcription of one gene. However, for example, there may be cases where it is desired to simultaneously control the transcription of two or more target genes.

Therefore, an object of the present invention is to provide a novel pyrrole imidazole polyamide capable of simultaneously controlling the transcription of two or more genes, a pharmaceutical composition containing the pyrrole imidazole polyamide, and a method for producing a pyrrole imidazole polyamide. In addition, pyrrole imidazole polyamide, which increases the expression of HGF and suppresses the expression of TGF-β, which is useful as a therapeutic agent for TGF-β-related diseases or fibrotic diseases, and the TGF-β gene using the pyrrole imidazole polyamide. It is an object of the present invention to provide an expression inhibitor and a pharmaceutical composition for treating a TGF-β-related disease or a fibrous disease.

The present invention includes the following aspects.
[1] A pyrrole imidazole polyamide that binds to the promoters of two or more genes and controls the transcription of the two or more genes.
[2] In [1], the two or more kinds of genes include a first gene and a second gene, and the first gene is a gene encoding an expression regulator of the second gene. The pyrrole imidazole polyamide described.
[3] The pyrrole imidazole polyamide according to [2], which binds to the promoter of the first gene and inhibits the binding of a transcriptional regulator to the promoter of the first gene.
[4] The pyrrole imidazole polyamide according to [1], wherein the two or more genes contain an HGF gene and a TGF-β gene.
[5] The pyrrole imidazole polyamide according to [4], which promotes the transcription of the HGF gene and inhibits the transcription of the TGF-β gene.
[6] The pyrrole imidazole polyamide according to [5], which binds to a transcriptional repression region in the promoter of the HGF gene.
[7] The pyrrole imidazole polyamide according to [6], which binds to a region of the promoter of the HGF gene containing at least a part of the base sequence set forth in SEQ ID NO: 1.
[8] The pyrrole imidazole polyamide according to [7], which binds to a region containing any of the following sequences 1 to 3 in the promoter of the HGF gene.
Sequence 1: AGGTGAC
Sequence 2: ACCTTTT
Sequence 3: CTTTTCT
[9] The pyrrole imidazole polyamide according to any one of [4] to [8], which binds to the transcription promoting region in the promoter of the TGF-β gene.
[10] A pyrrole imidazole polyamide represented by the following formula (P-3), (P-5) or (P-7).

[In the formula, R ¹ and R ² each independently represent a monovalent organic group. R ¹ and R ² may be interconnected to form a divalent organic group. ]
[11] The pyrrole imidazole polyamide according to [10], which is represented by the following formula Hu-HGF-3, Hu-HGF-5 or Hu-HGF-7.

[12] A TGF-β gene expression inhibitor comprising the pyrrole imidazole polyamide according to any one of [4] to [11].
[13] A pharmaceutical composition comprising the pyrrole imidazole polyamide according to any one of [1] to [11].
[14] A pharmaceutical composition for treating a TGF-β-related disease, which comprises the pyrrole imidazole polyamide according to any one of [4] to [11].
[15] A pharmaceutical composition for treating a fibrotic disease, which comprises the pyrrole imidazole polyamide according to any one of [4] to [11].
[16] Of the steps of (a) designing a pyrrole imidazole polyamide that binds to the promoters of two or more genes, (b) synthesizing the pyrrole imidazole polyamide, and (c) the synthesized pyrrole imidazole polyamide. , A method for producing a pyrrole imidazole polyamide, comprising a step of selecting a pyrrole imidazole polyamide that controls the transcription of the two or more kinds of genes.

According to the present invention, a novel pyrrole imidazole polyamide capable of simultaneously controlling the transcription of two or more kinds of genes, a pharmaceutical composition containing the pyrrole imidazole polyamide, and a method for producing a pyrrole imidazole polyamide are provided. In addition, pyrrole imidazole polyamide, which increases the expression of HGF useful as a therapeutic agent for TGF-β-related diseases or fibrotic diseases and suppresses the expression of TGF-β, and TGF-β gene expression using the pyrrole imidazole polyamide. Inhibitors and pharmaceutical compositions for treating TGF-β-related or fibrous disorders are provided.

It is a figure explaining the relationship between HGF and TGF-β. The base sequence of the human HGF promoter is shown. The underlined part indicates the COUP-TF1 binding site (-108 to -96). The design of two kinds of PI polyamides (Hu-HGF-1, Hu-HGF-2) targeting the vicinity of the COUP-TF1 binding site is shown. The underlined part indicates the COUP-TF1 binding site. Py: N-methylpyrrole unit; Im: N-methylimidazole unit; β: β-alanine unit; Ac: acetyl group; Dp: dimethylaminopropyl group. The design of four PI polyamides (Hu-HGF-3, Hu-HGF-4, Hu-HGF-5, Hu-HGF-6) targeting the vicinity of the COUP-TF1 binding site is shown. The underlined part indicates the COUP-TF1 binding site. The design of two kinds of PI polyamides (Hu-HGF-7, Hu-HGF-8) targeting around the COUP-TF1 binding site is shown. The underlined part indicates the COUP-TF1 binding site. The structure of Hu-HGF-3 is shown. The structure of Hu-HGF-5 is shown. The structure of Hu-HGF-7 is shown. The base sequence of the human TGF-β1 promoter is shown. "+1" indicates the transcription start point. The underlined part shows the transcription factor binding site. The dotted line indicates the expected transcription factor binding site. The sequence surrounded by a solid line indicates the binding site of Hu-HGF-3. The sequence enclosed by the broken line indicates the Hu-HGF-7 binding site. The human TGF-β1 promoter around the Hu-HGF-3 binding site and the Hu-HGF-7 binding site is shown. The sequence surrounded by a solid line indicates the binding site of Hu-HGF-3. The sequence enclosed by the broken line indicates the Hu-HGF-7 binding site. The broken line indicates the transcription factor binding prediction site. The results of the gel shift assay for the HGF promoter of PI polyamide are shown. Hu-HGF-1 and Hu-HGF-2 were used as PI polyamides. The results of the gel shift assay for the HGF promoter of PI polyamide are shown. Hu-HGF-3 and Hu-HGF-4 were used as PI polyamides. The results of the gel shift assay for the HGF promoter of PI polyamide are shown. Hu-HGF-5 and Hu-HGF-6 were used as PI polyamides. The outline of the test protocol of HGF expression level analysis and TGF-β1 expression level analysis is shown. The operation indicated as "in the case of TGF-β1 expression analysis" was performed only in the case of TGF-β1 expression analysis. The effect of Hu-HGF-3 on the mRNA expression level of HGF is shown. HDF cells were used. The effect of Hu-HGF-5 on the mRNA expression level of HGF is shown. HDF cells were used. The effect of Hu-HGF-7 on the mRNA expression level of HGF is shown. HDF cells were used. The effect of Hu-HGF-3 on the mRNA expression level of TGF-β1 is shown. HDF cells were used. The effect of Hu-HGF-5 on the mRNA expression level of TGF-β1 is shown. HDF cells were used. The effect of Hu-HGF-7 on the mRNA expression level of TGF-β1 is shown. HGF cells were used. The effect of Hu-HGF-5 on the mRNA expression level of TGF-β1 is shown. MC cells were used. The effect of Hu-HGF-3 on the expression level of HGF protein is shown. HDF cells were used. The effect of Hu-HGF-5 on the expression level of HGF protein is shown. HDF cells were used. The effect of Hu-HGF-7 on the expression level of HGF protein is shown. HDF cells were used. The effect of Hu-HGF-3 on the expression level of TGF-β1 protein is shown. HDF cells were used. The effect of Hu-HGF-5 on the expression level of TGF-β1 protein is shown. HDF cells were used. The effect of Hu-HGF-7 on the expression level of TGF-β1 protein is shown. HDF cells were used. The effect of Hu-HGF-3 on the TGF-β1 protein expression level in the presence of HGF siRNA is shown. HDF cells were used. # Indicates the result of the significance test for "1 DMSO" (# p <0.05). * Indicates the result of the significance test for "2 DMSO + PMA + HGF siRNA" (*: p <0.05). The effect of Hu-HGF-6 on the TGF-β1 protein expression level in the presence of HGF siRNA is shown. HDF cells were used. ** and *** show the results of the significance test for "2 DMSO + PMA + HGF siRNA" (**: p <0.01, ***: p <0.0001). The result of the mRNA expression test of TGF-β1 using the mismatched PI polyamide (HGF mismatch) is shown. HDF cells were used.

[Pyrrole imidazole polyamide (PI polyamide)]
In one aspect, the invention provides a pyrrole imidazole polyamide that binds to the promoter regions of two or more genes and controls transcription of the two or more genes.

PI polyamide is a polyamide having an N-methylpyrrole unit (Py) and an N-methylimidazole unit (Im), and Py and Im are linked by an amide bond (-C (= O) -NH-). PI polyamides generally have a linker moiety (eg, a γ-aminobutyric acid linker) that is entirely folded by the linker moiety to form a U-shape. In the U-shaped conformation, two chains containing Py and Im are arranged in parallel with the linker portion in between. The combination of specific pairs in this double strand binds to a specific base pair of DNA with high affinity. As a result, the PI polyamide enters the minor groove of the DNA double strand and binds to the DNA double strand in a sequence-specific manner.

In PI polyamide, Py / Im pair binds to C—G base pair, Im / Py pair binds to G—C base pair, and Py / Py pair binds to AT base pair or TA base pair. do. PI polyamide may use 3-hydroxypyrrole unit (Hp) and β-alanine unit (β). Hp / Py pairs recognize TA base pairs, Py / Hp pairs recognize AT base pairs, β / β pairs recognize TA base pairs or AT base pairs, and β / Im pair recognizes C—G base pair, Im / β pair recognizes G—C base pair, and β / Py pair and Py / β pair recognize TA base pair or AT base pair. .. By appropriately changing the composition and order of the pair formed by the combination of Py, Im, β, and Hp, it is possible to design a PI polyamide that binds to a specific base sequence of DNA.

The number of pairs consisting of a combination of Py, Im, Hp and β constituting the PI polyamide is not particularly limited as long as it is 2 or more, but 3 to 12 is preferable, 4 to 10 is more preferable, and 4 to 10 is more preferable. Eight are more preferred. When the PI polyamide has 5 or more pairs, it is preferable to have 1 or more pairs containing β.

The methyl group bonded to nitrogen at the 1-position of Py and Im may be replaced with a hydrogen atom or an aliphatic hydrocarbon group having 2 to 10 carbon atoms. The aliphatic hydrocarbon group may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. The aliphatic hydrocarbon group is preferably an alkyl group, and examples thereof include an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group and a tert-butyl group.

When the linker moiety of PI polyamide is a γ-aminobutyric acid linker, it may have a substituent at the α-position or the β-position. For example, it may be an N-α-N-γ-diaminobutyric acid linker in which the α-position is substituted with an amino group, or an N-β-N-γ-diaminobutyric acid linker in which the β-position is substituted with an amino group. May be. The amino group may be modified with a molecule such as a fluorescent group or biotin.

Various molecules may be introduced into the terminal portion of PI polyamide. The molecule introduced into the terminal portion of PI polyamide is not particularly limited. Various molecules can be introduced into the end of the PI polyamide, for example, via an amide bond. Examples of such molecules include, but are not limited to, fluorescent dyes, biotins, alkylating agents and the like. For example, an acetyl group may be introduced into the N-terminal portion of PI polyamide, and a dimethylaminopropylamino group may be introduced into the C-terminal portion.

PI polyamide can be synthesized by a known method. The PI polyamide can be produced, for example, by an automatic synthesis method by a solid phase method (solid phase Fmoc method) using Fmoc (9-fluorenylmethoxycarbonyl) (International Publication No. 03/000683). According to the solid-phase Fmoc method, the terminal of PI polyamide can be cut out from the solid support as a carboxylic acid residue. Therefore, various functional groups can be introduced into the molecular ends to produce PI polyamide derivatives. For example, compounds having an alkylating ability for DNA such as duocarmycin, pyrolobenzodiazepine, bleomycin, enediyne compound, nitrogen mustard, and derivatives thereof can be introduced into the terminal of PI polyamide. Since the solid-phase Fmoc method is an automatic synthesis method using a commercially available protein (peptide) synthesizer, it is also possible to synthesize a conjugate of a naturally occurring protein or an unnatural protein and a pyrrole imidazole polyamide. In addition, since the reaction conditions of the solid-phase Fmoc method are more relaxed than those of the t-BOC method, it is possible to introduce organic compounds other than proteins (including compounds having unstable functional groups under acidic conditions). .. For example, it is also possible to automatically synthesize conjugates of pyrrole imidazole polyamide with DNA or RNA (or derivatives thereof).

According to the solid-phase Fmoc method or the like, PI polyamide having a carboxy group at the terminal can be synthesized. Specific examples thereof include PI polyamide having a β-alanine residue (β-aminopropionic acid residue) or a γ-aminobutyric acid residue at the terminal. PI polyamides having β-alanine residues or γ-aminobutyric acid residues at the ends include, for example, aminopyrol carboxylic acid, aminoimidazole carboxylic acid, β-alanine or γ-aminobutyric acid, each of which has an amino group protected by Fmoc. The supported solid phase carrier can be used for synthesis by the solid phase Fmoc method using a peptide synthesizer.

Specific examples of the aminopyrrole carboxylic acid include 4-amino-2-pyrrole carboxylic acid, 4-amino-1-methyl-2-pyrrole carboxylic acid, 4-amino-1-ethyl-2-pyrrole carboxylic acid, and the like. Examples thereof include 4-amino-1-propyl-2-pyrrolecarboxylic acid and 4-amino-1-butyl-2-pyrrolecarboxylic acid. Specific examples of the aminoimidazole carboxylic acid include 4-amino-2-imidazole carboxylic acid, 4-amino-1-methyl-2-imidazole carboxylic acid, 4-amino-1-ethyl-2-imidazole carboxylic acid, and the like. Examples thereof include 4-amino-1-propyl-2-imidazole carboxylic acid and 4-amino-1-butyl-2-imidazole carboxylic acid.

According to the solid-phase Fmoc method, for example, a conjugate of PI polyamide and FITC (fluorescein isothiocyanate) can be synthesized. FITC has been conventionally known as a fluorescent labeling reagent for antibodies. The conjugate of PI polyamide and FITC can be used to prove that the PI polyamide recognizes a particular DNA sequence.

The PI polyamide of this embodiment binds to the promoters of two or more kinds of genes and controls the transcription of the two or more kinds of genes. Traditionally, PI polyamides have been designed to target the promoter of the particular gene for the purpose of controlling the expression of that particular gene. On the other hand, the PI polyamide of this embodiment is designed based on a completely new concept of controlling the expression of two or more genes at the same time. The PI polyamide of this embodiment is designed to bind to the promoters of two or more genes. By binding to the promoters of two or more genes, it is possible to control the transcription of two or more genes with one type of PI polyamide. The PI polyamide of this embodiment is preferably designed to bind to a transcription promoting region or a transcription repressing region in the promoters of two types of genes.

As used herein, the term "promoter" means a region located upstream of a gene and regulating transcription of the gene.
As used herein, the term "transcription promoting region" means a region located upstream of a gene and in which transcription of the gene is promoted by binding to a transcription factor, a complex protein, or the like. Transcription-promoting regions can usually be present in the promoter.
As used herein, the term "transcription-suppressing region" means a region located upstream of a gene and in which transcription of the gene is suppressed by binding to a transcription factor, a complex protein, or the like. The transcriptional repression region can usually be present in the promoter.
In the present specification, the transcription promoting region and the transcription suppression region may be collectively referred to as a "transcriptional control region".
As used herein, the term "transcriptional regulatory factor" means a protein (including a transcription factor, a complex protein, etc.) that binds to a transcription promoting region or a transcriptional repressing region of a specific gene and promotes or suppresses the transcription of the gene. do.
As used herein, the term "expression control factor" means a protein (including a transcription factor, a complex protein, etc.) that promotes or suppresses the expression of a specific gene. The expression regulator may be any protein whose expression of the specific gene is upregulated or downregulated by the presence of the protein, and includes a transcriptional regulator.

When a transcriptional regulator binds to the transcriptional promoting region of the promoter, gene transcription is initiated or promoted. When a transcriptional regulator binds to the transcriptional repressive region of a promoter, gene transcription is repressed. Therefore, it is possible to inhibit the transcription of a gene by inhibiting the binding of a transcriptional regulatory factor to the transcription promoting region in the promoter. In addition, gene transcription can be promoted by inhibiting the binding of transcriptional regulators to the transcriptional repressive region of the promoter. Therefore, when PI polyamide binds to the transcription promoting region in the promoter, the binding of the transcriptional regulator to the transcription promoting region is inhibited, and the expression of the gene is inhibited. When the PI polyamide binds to the transcriptional repressor region of the promoter, the binding of the transcriptional regulator to the transcriptional repressor region is inhibited, and gene expression is initiated or promoted.

In the present specification, "controlling the transcription of a gene" means changing the transcriptional activity of the gene. Transcription control may be upward control or downward control. When PI polyamide binds to the transcription-promoting region of the promoter, the binding of transcriptional regulators to the transcription-promoting region is inhibited and gene expression is down-regulated. When the PI polyamide binds to the transcriptional repressive region in the promoter, the binding of the transcriptional regulator to the transcriptional repressor region is inhibited and the gene expression is upwardly regulated.

The design of PI polyamide that binds to the promoters of two or more genes can be performed, for example, as follows.

First, select two or more genes targeted by PI polyamide. Two or more kinds of genes can be arbitrarily selected according to the purpose. Examples of the two or more genes include a combination of two or more genes whose expression is upregulated or downregulated in a specific disease; a combination of a gene whose expression is upregulated in a specific disease and a gene whose expression is downregulated in the specific disease, and the like. Will be.

For example, in cancer, the existence of oncogenes whose expression is enhanced in a cancer-specific manner is known. Examples of genes whose expression is enhanced in a cancer-specific manner include CA125, CEA, CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD70, CD8, CLL-1, c-Met, PSA, PSMA, ROR1, HER2, MAGE, p53 and the like can be mentioned. When PI polyamide is used for cancer treatment, two or more of these oncogenes may be selected as target genes. Alternatively, an immune checkpoint molecule gene (PD1, PD-L1, CTLA-4, etc.) and an oncogene may be selected as two or more target genes.

Examples of the combination of the gene whose expression is enhanced in a specific disease and the gene whose expression is decreased in the specific disease include a combination of a TGF-β gene and an HGF gene.

Next, the promoter sequences of two or more target genes are analyzed to identify the transcription-promoting region and the transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired.

Next, for one of the two or more target genes, a PI polyamide that binds to a specific sequence in the acquired transcriptional control region is designed. Next, it is confirmed whether the designed PI polyamide can bind to the transcriptional control region of the remaining target genes. If the PI polyamide is capable of binding to the transcriptional control regions of all target genes, then that PI polyamide is selected.
If there is a target gene that is not bindable to the transcriptional control region, select other sequences in the transcriptional control region and redesign the PI polyamide. It is confirmed whether the redesigned PI polyamide can bind to the transcriptional control region of the remaining target genes. Hereinafter, the above steps are repeated until a PI polyamide capable of binding to the transcriptional control region of all target genes can be obtained.

As described above, PI polyamide that binds to the transcriptional control regions of two or more genes can be obtained.

It was confirmed by culturing cells having a target gene in the presence of the acquired PI polyamide that the acquired PI polyamide binds to the transcriptional control region of two or more kinds of genes and exerts a desired expression control function. can do.

The number of target genes of PI polyamide is not particularly limited, but is, for example, 2 to 5, 2 to 4, 2 to 3, or 2.

In one embodiment, PI polyamide can control the transcription of both the first gene and the second gene. In one embodiment, the first gene is the gene encoding the transcriptional regulator of the second gene.

When the first gene is an expression regulator that suppresses the expression of the second gene, the second gene is promoted by PI polyamide by promoting the transcription of the first gene and inhibiting the transcription of the second gene. Transcription of the gene can be suppressed more strongly. In this case, the PI polyamide is preferably bound to the transcriptional repression region in the promoter of the first gene. In addition, PI polyamide preferably binds to the transcription promoting region in the promoter of the second gene.
When the first gene is an expression regulator that suppresses the expression of the second gene, PI polyamide suppresses the transcription of the first gene and promotes the transcription of the second gene. It can more strongly promote the transcription of the second gene. In this case, the PI polyamide preferably binds to the transactivation region in the promoter of the first gene. In addition, PI polyamide preferably binds to the transcriptional repression region in the promoter of the second gene.

When the first gene is an expression regulator that promotes the expression of the second gene, the second gene is promoted by promoting the transcription of the first gene by PI polyamide and promoting the transcription of the second gene. Transcription of the gene can be promoted more strongly. In this case, the PI polyamide is preferably bound to the transcriptional repression region in the promoter of the first gene. In addition, PI polyamide preferably binds to the transcriptional repression region in the promoter of the second gene.
When the first gene is an expression regulator that promotes the expression of the second gene, PI polyamide suppresses the transcription of the first gene and suppresses the transcription of the second gene. Transcription of the second gene can be suppressed more strongly. In this case, the PI polyamide preferably binds to the transactivation region in the promoter of the first gene. In addition, PI polyamide preferably binds to the transcription promoting region in the promoter of the second gene.

The first gene and the second gene are not particularly limited, and any gene can be used. As the combination of the first gene and the second gene, for example, any combination may be selected depending on the purpose from those listed as the combinations of the above two or more target genes. Examples of the first gene include the HGF gene. Examples of the second gene include the TGF-β gene.

When the first gene is the HGF gene and the second gene is the TGF-β gene, PI polyamide may have a function of promoting the transcription of the HGF gene and inhibiting the transcription of the TGF-β gene. preferable. Hereinafter, a PI polyamide in which the first gene is an HGF gene and the second gene is TGF-β may be referred to as “PI polyamide (P)”.

FIG. 1 is a diagram illustrating the relationship between HGF and TGF-β. TGF-β has actions such as fibroblast activation, mesangial cell activation, and epithelial-mesenchymal transition, and promotes tissue fibrosis. HGF exhibits angiogenic action, anti-apoptotic action, anti-fibrotic action, and has an action of suppressing the expression of TGF-β. Therefore, by increasing the expression level of HGF, the expression level of TGF-β is suppressed, and the progress of fibrosis is suppressed.

PI polyamide (P) promotes the transcription of the HGF gene and inhibits the transcription of the TGF-β gene. When the expression level of HGF is increased by promoting the transcription of the HGF gene, the inhibitory effect of HGF on the expression of TGF-β becomes stronger. Furthermore, since PI polyamide (P) directly inhibits the transcription of the TGF-β gene, it strongly suppresses the expression of the TGF-β protein by the additive effect with the TGF-β expression inhibitory action of HGF. Therefore, according to PI polyamide (P), the progress of fibrosis due to TGF-β can be efficiently suppressed.

PI polyamide (P) is an HGF-mediated expression suppression mechanism of TGF-β (hereinafter, also referred to as “expression suppression mechanism 1”) by promoting transcription of the HGF gene, and TGF-β by inhibiting the transcription of the TGF-β gene. The expression of TGF-β can be suppressed by two expression suppression mechanisms of the expression suppression mechanism (hereinafter, also referred to as “expression suppression mechanism 2”). Therefore, the PI polyamide (P) can suppress the expression of TGF-β at a lower concentration as compared with the conventional PI polyamide that suppresses the expression of TGF-β only by the expression suppression mechanism 2.

It is preferable that PI polyamide (P) inhibits the binding of the transcriptional regulatory factor to the transcriptional repressive region in the HGF promoter by binding to the transcriptional repressive region in the promoter of the HGF gene (hereinafter, also referred to as “HGF promoter”). .. By inhibiting the binding of the transcriptional regulator to the transcriptional repressor region in the HGF promoter, the transcriptional repression of the HGF gene by the binding of the transcriptional regulator to the transcriptional repressor region does not occur. Therefore, transcription of the HGF gene is promoted.

The PI polyamide (P) is preferably bound to a region of the HGF promoter containing at least a part of the base sequence (AGGTGACCTTTTC) shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1 is a sequence predicted as a binding site of COUP-TF1 by analysis of the human HGF promoter. It has been reported that the binding site of COUP-TF1 is often a transcriptional repression region. It has been reported that COUP-TF1 often acts as a transcriptional repressor by binding to the binding site.

Specific examples of the sequence to which PI polyamide (P) binds in the HGF promoter include a region containing any of the following sequences 1 to 3. As shown in Examples below, PI polyamides (Hu-HGF-3, Hu-HGF-5, Hu-HGF-7) designed to bind to the following sequences 1-3 are the expression of TGF-β1. Can be effectively suppressed.
Sequence 1: AGGTGAC
Sequence 2: ACCTTTT
Sequence 3: CTTTTCT

It is preferable that PI polyamide (P) inhibits the transcription of the TGF-β gene by binding to the transcriptional repression region in the promoter of the TGF-β gene (hereinafter referred to as "TGF-β promoter"). Preferably, PI polyamide (P) inhibits transcription of the TGF-β gene by inhibiting the binding of transcriptional regulators to the transcriptional repressor region of the TGF-β promoter. TGF-β may be any of TGF-β1, TGF-β2, and TGF-β3, but TGF-β1 is preferable. PI polyamide (P) changes the three-dimensional structure of the TGF-β promoter by binding to the peripheral region of the TGF-β promoter, and inhibits the binding of transcriptional regulators to the transcriptional repressor region of the TGF-β promoter. May be.

A suitable example of PI polyamide (P) is given below.

[In the formula, R ¹ and R ² each independently represent a monovalent organic group. R ¹ and R ² may be interconnected to form a divalent organic group. ]

In the formulas (P-3), (P-5) and (P-7), R ¹ and R ² each independently represent a monovalent organic group. R ¹ and R ² are not particularly limited and may be any organic group. Any molecule may be introduced into R ¹ and R ² , for example, via an amide bond. Examples of the introduced molecule include those listed as the introduced molecule to the terminal of the PI polyamide.

R ¹ may be, for example, ^{a group represented by -NHCO-R 11} (R ¹¹ is a monovalent organic group). R ¹¹ is not particularly limited, and examples thereof include an aliphatic hydrocarbon group. The aliphatic hydrocarbon group may be linear, may be branched, or may be cyclic. The aliphatic hydrocarbon group may be saturated or unsaturated. Examples of the number of carbon atoms of the aliphatic hydrocarbon group include 1 to 10 carbon atoms, 1 to 5 carbon atoms, 1 to 3 carbon atoms, 1 or 2 carbon atoms, and the like. The R ¹¹ is preferably an alkyl group, and examples thereof include a methyl group and an ethyl group.

R ² may be, for example, ^{a group represented by —CONH-R 21} (R ²¹ is a monovalent organic group). The R ²¹ is not particularly limited, and examples thereof include an aliphatic hydrocarbon group which may have a substituent. The aliphatic hydrocarbon group may be linear, may be branched, or may be cyclic. The aliphatic hydrocarbon group may be saturated or unsaturated. Examples of the number of carbon atoms of the aliphatic hydrocarbon group include 1 to 15 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, and the like. Examples of the substituent that the aliphatic hydrocarbon group may have include, but are not limited to, an amino group, a hydroxy group, a carboxy group and the like. The aliphatic hydrocarbon group may have a part of carbon atoms constituting the carbon chain substituted with —O—, —CO—, —COO—, —CONH—, or the like. R ²¹ may be a group represented by −β-R ²² (β is a β-alanine unit; R ²² is a monovalent organic group). The R ²² is not particularly limited, and examples thereof include an aliphatic hydrocarbon group which may have a substituent. The aliphatic hydrocarbon group may have the substituent include the same ones as exemplified in R ^21. Examples of R ²² include a dimethylaminoalkyl group and an aminoalkyl group. Specific examples of R ²² include, for example, a dimethylaminopropyl group and an aminopropyl group.

R ¹ and R ² may be interconnected to form a divalent organic group. In this case, the PI polyamide represented by the formula (P-3), (P-5) or (P-7) forms a cyclic structure. Cyclic peptides are known to be suitable for oral administration (Keiichi Masuya, Journal of Pharmacology, 148.322-328 (2016)). It has also been reported that the binding properties of PI polyamide do not change significantly even when PI polyamide is used as a cyclic structure (David M. Chenoweth, J Am Chem Soc. 2009 May 27; 131 (20): 7182-7188.). .. Therefore, the cyclic structure PI polyamide is promising as an orally administered drug.

Specific examples of the PI polyamide (P) represented by the above formula (P-3), (P-5), or (P-7) are given below.

[TGF-β gene expression inhibitor]
In one aspect, the invention provides a TGF-β gene expression inhibitor comprising PI polyamide (P).

PI polyamide (P) can effectively suppress TGF-β gene expression as described above. Therefore, PI polyamide (P) can be used as a TGF-β gene expression inhibitor.

As used herein, "gene expression" means that the protein encoded by the gene is produced by transcription and translation of the gene. The "gene expression inhibitor" means an agent having an action of suppressing the production of a protein encoded by the gene.

The TGF-β gene expression inhibitor of this embodiment may be used in vitro or in vivo. When used in vitro, it is preferably used for human cells or common marmoset cells. When used in vivo, it is preferably administered to humans or common marmosets. When used in vivo, it may be formulated as a pharmaceutical composition described later.

[Pharmaceutical composition]
In one aspect, the invention provides a pharmaceutical composition comprising said PI polyamide.

Since the PI polyamide of the above embodiment can control the transcription of two or more target genes, it can be used as a pharmaceutical composition for treating a disease associated with abnormal gene expression. Two or more target genes can be appropriately selected depending on the disease to be treated.

In one aspect, the invention provides a pharmaceutical composition for treating a TGF-β related disease, comprising PI polyamide (P).
In one aspect, the invention provides a pharmaceutical composition for treating a fibrotic disease, comprising PI polyamide (P).

As used herein, the term "TGF-β-related disease" means a disease caused by an increase in TGF-β. Examples of TGF-β-related diseases include, but are not limited to, fibrotic diseases, various renal diseases, and male-type frontal hair loss. "Fibrotic disease" means a disease that occurs with fibrosis of a tissue or organ. The fibrotic disease is not particularly limited, and is, for example, liver cirrhosis, pulmonary fibrosis, renal fibrosis, pancreatic fibrosis, myocardial fibrosis, myeloid fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, mammary fibrosis, cyst. Examples include sexual fibrosis, gastrointestinal fibrosis, adipose tissue fibrosis, systemic fibrosis, localized fibrosis, keroids, hypertrophic scars, post-skin wound or post-skin ulcer scars, skin fibrosis, etc. , Not limited to these. Preferred examples of the fibrotic disease include various liver diseases, various renal diseases, pancreatic fibrosis, myocardial fibrosis, and various skin fibrosis diseases described later.

In the process of liver fibrosis, hepatic stellate cells play an important role in extracellular matrix production (Bataller R et al, Gastroenterology 118,1149,2000). Activation of stellate cells is carried out by TGF-β1, and further activated stellate cells cause the secretion of TGF-β1 from inflammatory cells in the injured liver. At the same time, TGF-β1 receptor expression in activated stellate cells is enhanced, and extracellular matrix protein is increased by the autocrine mechanism by TGF-β1 (Hisatake Watanabe et al., Hyundai Medical, Vol. 35 (No. 2), 2003). From these facts, the above-mentioned various liver diseases can be exemplified as TGF-β-related diseases.

In addition, model animals for renal diseases such as IgA nephropathy, focal glomerulonephritis, crescentic nephritis, focal sclerosing lupus nephritis, diffuse proliferative lupus nephritis, diabetic nephropathy, and hypertensive nephropathy. Or, the expression of TGF-β is increased in parallel with the extracellular substrate in the renal biopsy tissue of patients with glomerulonephritis and diabetic nephropathy (Yamamoto T et al: Kidney Int 49: 461, 1996, Border WA). et al: Kidney Int 51: 1388, 1997). At the same time, Border et al. Reported that administration of anti-TGF-β to Thy-1 nephritis rats suppressed the accumulation of extracellular matrix in renal glomeruli (Border WA et al: Kidney Int 51: 1388, 1997). .. From these facts, the above-mentioned various renal diseases can be exemplified as TGF-β-related diseases.

In addition, in the animal myocardial infarction model, the expression of TGF-β is continuously enhanced in the infarct lesion during the scar formation stage and is involved in the promotion of myocardial fibrosis (Ono et al: Circulation 98: 149, 1998). From these facts, myocardial fibrosis after myocardial infarction can be exemplified as a TGF-β-related disease.

In addition, pulmonary fibrosis is improved by administering an anti-TGF-β antibody or TGF-β soluble receptor to a pulmonary fibrosis model animal (Giri SN al: Thorax 1993). From these facts, pulmonary fibrosis can be exemplified as a TGF-β related disease.

In addition, although many reports have been made on the high expression of TGF-β1 in human chronic pancreatitis, when recombinant TGF-β is administered to a model animal of recurrent acute pancreatitis, fibrosis of the inflamed part of the pancreatic part and high fibronectin mRNA are reported. It has been clarified that administration of a neutralizing antibody of TGF-β1 at the time of creating a model of pancreatitis, which causes expression, suppresses extracellular matrix production and mRNA expression of type I / III collagen and fibronectin (). Naohiko Makino et al .: Contemporary Medicine Vol.35, No.2, 2003). From these facts, fibrosis in chronic pancreatitis can be exemplified as a TGF-β related disease.

In addition, TGF-β was proposed as a cause of scleroderma, and Mori et al. Reported that TGF-β induces skin fibrosis in skin fibrosis model mice (Mori et al: J Cell Physiol 181: 153). , 1999). From these facts, various skin fibrosis diseases can be exemplified as TGF-β related diseases.

In addition, the expression of TGF-β mRNA is enhanced in megakaryocytes of patients with myeloid fibrosis (Reilly Jet al: Clin Haematol: 11751-767, 1998), and the TGF-β concentration in platelets is high (Martyre MC). It has been reported that the plasma TGF-β concentration in patients is significantly high (Rameshwar Pet al: Am J Haematol 59: 133-142, 1998) in et al: Br J Haematol 77: 80-86, 991). According to Rameshwar et al., Monocytes in patients with bone marrow fibrosis activate NF-k by adhesion to induce IL-1 production, and IL-1 enhances TGF-β production to induce bone marrow fibrosis. (Rameshwar et al: J Immunol 165: 2271-2277,2000). From these facts, myeloid fibrosis can be exemplified as a TGF-β related disease.

In addition, it has been reported that male hormone induces TGF-β1 from cutaneous papillary cells and this TGF-β1 suppresses epidermal cell proliferation in the cultured cell line of male-type frontal hair loss patients (Shigeki et). al: FASEB J 16: 1967-1969, 2002). From these facts, male-type frontal hair loss can be exemplified as a TGF-β-related disease.

The administration target of the pharmaceutical composition of this embodiment is preferably human. The pharmaceutical composition of this embodiment is preferably administered to a human or common marmoset. When the pharmaceutical composition of this embodiment is administered to humans, the effect of the pharmaceutical composition of this embodiment can be confirmed by animal experiments using primates. Common marmosets can be preferably used as primates.

The pharmaceutical composition of this embodiment may contain an arbitrary component in addition to PI polyamide. Examples of the optional component include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the administration subject thereof. "Not substantially toxic" means that the ingredient is not toxic to the subject at the dose normally used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the binding of the PI polyamide to the promoter and does not show substantial toxicity to the administration subject thereof. .. When the PI polyamide is PI polyamide (P), the pharmaceutically acceptable carrier does not inhibit the HGF gene transcription promoting ability and the TGF-β gene transcription inhibitory ability of PI polyamide (P), and the carrier thereof is to be administered. On the other hand, it is a carrier that does not show substantial toxicity. The pharmaceutically acceptable carrier includes any known pharmaceutically acceptable ingredient, which is typically considered an inactive ingredient. The pharmaceutically acceptable carrier is not particularly limited, and is, for example, a solvent, a diluent, a vehicle, an excipient, a flow accelerator, a binder, a granulator, a dispersant, a suspending agent, a wetting agent, and the like. Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (eg antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned. As the pharmaceutically acceptable carrier, one type may be used alone, or two or more types may be used in combination.

The pharmaceutical composition of this embodiment may contain an arbitrary component other than a pharmaceutically acceptable carrier. The arbitrary ingredient is not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Further, the pharmaceutical composition of this embodiment may contain an active ingredient other than PI polyamide. Examples of the active ingredient include vitamins and their derivatives, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulants, analgesics, cell activators, plant / animal / microbial extracts, and antipruritics. Agents, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensive agents, antihypertensive diuretics, antibiotics, anesthetics, antibacterial agents, antiepileptic agents, coronary vasodilators, crude drugs, cessation Examples include, but are not limited to, antipruritics, keratin softening and stripping agents. As for the other components, one kind may be used alone, or two or more kinds may be used in combination.

The dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation. Examples of the oral preparation include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drop loves, emulsions and the like. Examples of the parenteral preparation include injections, suppositories, ointments, sprays, external solutions, ear drops, eye drops, nasal drops, inhalants and the like. Pharmaceutical compositions of these dosage forms can be formulated according to a conventional method (for example, the method described in the Japanese Pharmacopoeia).

The administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be administered by an oral or parenteral route. Parenteral routes include all non-oral routes of administration, such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, eye drops, intracerebral, rectal, intravaginal and intraperitoneal administration. The administration may be local administration or systemic administration. Preferred routes of administration of the pharmaceutical composition of this embodiment include, for example, intravenous injection or intramuscular injection. When orally administered, the PI polyamide (P) contained in the pharmaceutical composition of this embodiment preferably has a cyclic structure.

The pharmaceutical composition of this embodiment can be administered with a therapeutically effective amount of PI polyamide. "Therapeutically effective amount" means the amount of a drug effective for the treatment or prevention of a target disease. For example, a therapeutically effective amount of PI polyamide (P) can be an amount that can delay the onset and / or progression of TGF-β-related or fibrotic disease. The therapeutically effective amount may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. For example, the pharmaceutical composition of this embodiment can have a single dose of PI polyamide of 0.001 to 1000 mg per 1 kg of body weight of the subject to be administered. The dose may be 0.01 to 800 mg / kg, 0.1 to 500 mg / kg, 1 to 100 mg / kg, or 1 to 50 mg / kg. May be good.

The pharmaceutical composition of this embodiment may contain a therapeutically effective amount of PI polyamide per unit dosage form. For example, the content of PI polyamide in the pharmaceutical composition of this embodiment may be 0.01 to 90% by mass, 0.1 to 80% by mass, or 1 to 50% by mass. May be good.

The administration interval of the pharmaceutical composition of this embodiment may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like. The administration interval may be, for example, every few hours, once a day, once every two to three days, once a week, or the like.

The pharmaceutical composition of this embodiment may be used in combination with other pharmaceuticals. For example, it can be used in combination with other therapeutic agents for fibrotic diseases.

[Manufacturing method of PI polyamide]
In one aspect, the present invention provides a method for producing a pyrrole imidazole polyamide. The methods of this embodiment include (a) a step of designing a pyrrole imidazole polyamide that binds to promoters of two or more genes, (b) a step of synthesizing the pyrrole imidazole polyamide, and (c) the step of synthesizing the synthesized pyrrole imidazole imidazole. Among the polyamides, a step of selecting a pyrrole imidazole polyamide that controls the transcription of the two or more kinds of genes is included.

<Step (a)>
In step (a), a PI polyamide that binds to the promoters of two or more genes is designed.

The step (a) may include, for example, the following (i) to (iv).
(I) Select two or more genes targeted by PI polyamide.
(Ii) A transcription promoting region or a transcription repressing region is specified for the two or more kinds of genes.
(Iii) For the first gene among the two or more kinds of genes, a PI polyamide that binds to the transcription promoting region or the transcription suppression region is designed.
(Iv) From the designed PI polyamide, a PI polyamide that binds to a transcription promoting region or a transcription repressing region of a gene other than the first gene among the two or more kinds of genes is selected.

About (i):
Select any two or more genes targeted by PI polyamide. Examples of the combination of two or more genes include the combinations mentioned in the above section [Pyrrole imidazole polyamide (PI polyamide)].

About (ii):
For each of the two or more genes, the promoter sequence is analyzed to identify the transcription-promoting region or transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired.

Next, the promoter sequences of the two or more target genes are analyzed to identify the transcription-promoting region and the transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired. For example, the promoter sequences of the first gene and the second gene are analyzed to identify the transcription-promoting or transcription-repressing region of the first gene and the transcription-promoting or transrepression region of the second gene.

About (iii):
For the first gene among the two or more target genes, a PI polyamide that binds to a transcription promoting region or a transcription repressing region is designed. When the purpose is to suppress the expression of the first gene, a PI polyamide that binds to the transcription promoting region is designed. For the purpose of promoting the expression of the first gene, a PI polyamide that binds to the transcriptional repression region is designed. At this time, it is preferable to design a plurality of PI polyamides. As a method of designing a plurality of PI polyamides, a method of designing a PI polyamide for each of a plurality of transcription-promoting regions or transcription-suppressing regions; the target sequence is shifted with respect to one transcription-promoting region or transcription-suppressing region. Methods for designing multiple PI polyamides; and combinations thereof.

About (iv):
From the PI polyamide designed in (iii), a PI polyamide that binds to a transcription promoting region or a transcription repressing region of a target gene other than the first gene among the two or more target genes is selected. For example, a sequence to which the PI polyamide designed in (iii) can be bound (hereinafter, also referred to as “candidate PI polyamide binding sequence”) is acquired. Next, using the candidate PI polyamide binding sequence as a query sequence, a sequence of a transcription promoting region or a transcription repressing region of a target gene other than the first gene is searched. As a result, if a candidate PI polyamide-binding sequence is present in the transcription-promoting region or transcription-suppressing region of all target genes other than the first gene, the PI polyamide targeting the candidate PI polyamide-binding sequence is selected. .. For example, from the PI polyamide designed in (iii), a PI polyamide capable of binding to the transcription promoting region or the transcription suppression region of the second gene is selected.

Alternatively, step (a) may include, for example, the following (iii') and (iv') in place of the above (iii) and (iv).
(Iii') Search for a target sequence to which the same PI polyamide can bind in the transcription-promoting region or transcription-suppressing region of the two or more genes.
(Iv') Design a PI polyamide capable of binding to the target sequence detected by (iii').

About (iii'):
In PI polyamide, Py / Im pair binds to C—G base pair, Im / Py pair binds to G—C base pair, and Py / Py pair binds to AT base pair or TA base pair. do. Hp / Py pair binds to TA base pair, Py / Hp pair binds to AT base pair, β / β pair binds to TA base pair or AT base pair, β / Im pair binds to C—G base pair, Im / β pair binds to G—C base pair, and β / Py pair and Py / β pair bind to TA base pair or AT base pair. .. Based on the binding law, a target sequence to which the same PI polyamide can bind is searched for in the transcription promoting region or the transcription suppression region of all target genes. For example, a target sequence to which the same PI polyamide can bind is searched for in the transcription-promoting region or the transcription-suppressing region of the first gene and the transcription-promoting region or the transcription-suppressing region of the second gene.

About (iv'):
When a target sequence to which the same PI polyamide can be bound is found by (iii'), a PI polyamide capable of binding to the target sequence is designed.

<Step (b)>
In the step (b), the PI polyamide designed in the step (a) is synthesized.
The PI polyamide can be synthesized by a known method such as the solid phase Fmoc method.

<Step (c)>
In the step (c), among the PI polyamides synthesized in the step (b), the PI polyamide that controls the transcription of the two or more kinds of genes is selected.

In the presence of PI polyamide synthesized in step (b), cells expressing all target genes are cultured. When the expression of the target gene is promoted or suppressed as compared with the cells cultured in the absence of PI polyamide, it can be determined that PI polyamide regulates the transcription of the target gene. For example, assume that the PI polyamide is designed to bind to the transcription-promoting region of the first gene and to the transcription-repressing region of the second gene. In this case, when cultured in the presence of PI polyamide, transcription of the first gene is suppressed and transcription of the second gene is promoted. Gene expression may be confirmed by mRNA or protein. The transcription amount of mRNA can be measured by, for example, Northern blotting, RT-qPCR, or the like. The expression level of the protein can be measured by, for example, Western blotting, ELISA or the like.

By the method according to this embodiment, it is possible to produce a PI polyamide that binds to the promoters of two or more kinds of genes and controls the transcription of the two or more genes. By the method according to this aspect, the PI polyamide exemplified above can be produced.

[Other aspects]
In one aspect, the invention provides the use of PI polyamide (P) in the manufacture of pharmaceutical compositions for treating or preventing TGF-β related or fibrotic diseases.
In one aspect, the invention provides PI polyamide (P) for use in the treatment or prevention of TGF-β related or fibrotic diseases.
In one aspect, the invention comprises administering PI polyamide (P) to a subject (eg, a patient suffering from a TGF-β related disease or fibrous disease, etc.), comprising administering the PI polyamide (P) to a TGF-β related disease or fibrous. Provide a method of treating a disease.
In one aspect, the invention provides PI polyamide (P) for treating or preventing TGF-β related or fibrotic diseases.

Hereinafter, the present invention will be described with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Design of PI polyamide]
(Analysis of human HGF gene promoter)
The promoter of the human HGF gene was analyzed using the gene analysis software PROMO to predict the binding site of the transcriptional regulator. As a result, a binding site (-108 to -96; AGGTGACCTTTTC: SEQ ID NO: 1) of COUP-TF1 functioning as a transcriptional regulator was detected (see FIG. 2). Eight types of PI polyamides were designed targeting the peripheral region of this COUP-FT1 binding site (Hu-HGF-1, Hu-HGF-2: FIG. 3; Hu-HGF-3, Hu-HGF-4, Hu). -HGF-5, Hu-HGF-6: FIG. 4; Hu-HGF-7, Hu-HGF-8: FIG. 5). Assuming the use of common marmosets in in vivo tests, regions homologous to humans and common marmosets were selected as target regions for PI polyamides.

The structures and compositions of Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 are shown in FIGS. 6 to 8, respectively. The sequences to which Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 can bind are shown in Table 1. In Table 1, the underlined sequences are the sequences present in the TGF-β1 promoter.

(Analysis of human TGF-β1 promoter)
The sequence of the human TGF-β1 promoter is shown in FIG. In FIG. 9, the sequence surrounded by a solid line shows the binding sequence of Hu-HGF-3, and the sequence surrounded by a broken line shows the binding sequence of Hu-HGF-7.

FIG. 10 shows a transcriptional regulatory factor binding prediction site around the binding region of PI polyamide. There are multiple transcriptional regulator binding prediction sites around the binding regions of Hu-HGF-3 and Hu-HGF-5.

(Synthesis of PI polyamide)
Eight kinds of PI polyamides designed above (Hu-HGF-1, Hu-HGF-2, Hu-HGF-3, Hu-HGF-4, Hu-HGF-5, Hu-HGF-6, Hu-HGF- 7. Hu-HGF-8) was synthesized in Fmoc solid phase using a peptide synthesizer PSSM8 (Shimazu). Each synthesized PI polyamide was purified by a C18 HPLC column.

[Gel shift assay]
Oligonucleotides of the human HGF promoter were synthesized, and gel shift assay was performed using the eight PI polyamides synthesized above. Double-stranded DNA was ^{labeled with T4 polynucleotide kinase using [γ-32} P] -ATP and bound buffer (40 mM Tris, pH 7.9, 250 mM NaCl, 25 mM EDTA, 25 mM DTT, for 15 minutes at 37 ° C., Incubated with PI polyamide in 100 mM KCl). The resulting complex was electrophoresed on a 20% polyacrylamide gel and visualized by autoradiography.

As a result of the gel shift assay, gel shift was confirmed in all PI polyamides by incubation with the double-stranded DNA of the HGF promoter. The results of the gel shift assay of Hu-HGF-1 and Hu-HGF-2 are shown in FIG. The results of the gel shift assay of Hu-HGF-3 and Hu-HGF-4 are shown in FIG. The results of the gel shift assay of Hu-HGF-5 and Hu-HGF-6 are shown in FIG.
As a result of gel shift assay, it was confirmed that all eight PI polyamides bind to the human HGF promoter.

[Evaluation of HGF mRNA expression level]
The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of HGF mRNA was evaluated.
FIG. 14 shows an outline of the test protocol. Human skin-derived fibroblasts (HDF cells) were maintained in 10% FBS-DMEM medium. The medium was replaced with 0.5% FBS-DMEM medium and cultured for 24 hours. ^10-11 to ^10-7 M PI polyamide was added. In the negative control, DMSO was added instead of PI polyamide. After the addition of PI polyamide or DMSO, the cells were cultured for 15 hours and the cells were collected.

Total RNA was extracted from the collected cells using ISOGEN (Nippon Gene), and reverse transcription reaction was performed using PrimeScript (registered trademark) Reverse Transscriptase (Takara Bio) to prepare cDNA. Using the cDNA prepared above as template DNA, RT-qPCR was performed to quantify HGF mRNA. For RT-qPCR, Power SYBR (registered trademark) Green PCR Master Mix (manufactured by Applied Biosystems) was used. The expression level of HGF mRNA was leveled by the expression level of 18S rRNA.

The primers used for RT-qPCR are shown below.
Human HGF
Forward: TGACCCTATTGAAAAAAAAGACTACA (SEQ ID NO: 4)
Reverse: GCTGACATTTGATGCCACTCTTAG (SEQ ID NO: 5)
Human 18S rRNA
Forward: TCAAGAACGAAAGTCGGACG (SEQ ID NO: 6)
Reverse: GGACATCTAAGGGCATCACA (SEQ ID NO: 7)

The results of using Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 as PI polyamides are shown in FIGS. 15 to 17, respectively. In all three species, an increase in HGF mRNA expression level was confirmed as compared with negative control (DMSO). In all PI polyamides, the expression level of HGF mRNA tended to increase at ^{low doses (10-11} to ^{10-9 M).}

[Evaluation of TGF-β1 mRNA expression level]
The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of TGF-β1 mRNA was evaluated.
FIG. 14 shows an outline of the test protocol. The cells were cultured and recovered in the same manner as above, except that 0.1 μM PMA (phorbol 12-myristate 13-acetylate) was added 3 hours after the addition of PI polyamide or DMSO.
In the same manner as above, total RNA was extracted from the collected cells and RT-qPCR was performed. The expression level of TGF-β1 mRNA was leveled by the expression level of 18S rRNA.

The primers used for RT-qPCR are shown below.
Human TGF-β1
Forward: CTCGGCTGGAAGTGGATCCA (SEQ ID NO: 8)
Reverse: TGTACAGGGCCAGGACCTTG (SEQ ID NO: 9)

The results using Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 as PI polyamides are shown in FIGS. 18 to 20, respectively. Addition of 0.1 μM PMA induced the expression of TGF-β1 mRNA. When any of the PI polyamides was used, the expression of TGF-β1 mRNA stimulated by PMA was suppressed at an extremely low concentration of ^10-12 to ^{10-10 M.}

FIG. 21 shows the results when human renal mesandium (MC) cells were used instead of HDF cells. Hu-HGF-5 was used as the PI polyamide. It was also confirmed that the expression of TGF-β1 mRNA stimulated by PMA was suppressed by the addition of PI polyamide in MC cells.

[Evaluation of HGF protein expression level]
The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of HGF protein was evaluated.
The cells were cultured and recovered in the same manner as in [Evaluation of HGF mRNA expression level]. The cells were disrupted and Western blotting was performed using an anti-HGF antibody (Catalog (MAB294), Monoclonal Mouse, R & D System). The expression level of HGF protein was leveled by the expression level of β-actin.

The results using Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 as PI polyamides are shown in FIGS. 22 to 24, respectively. In all three types, an increase in HGF protein expression level was confirmed as compared with negative control (DMSO). In all PI polyamides, the HGF protein expression level tended to increase at ^{low doses (10-11} to ^{10-10 M).}

[Evaluation of TGF-β1 protein expression level]
The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of TGF-β1 protein was evaluated.
The cells were cultured and recovered in the same manner as in [Evaluation of TGF-β1 mRNA expression level]. The cells were disrupted and subjected to Western blotting using an anti-TGF-β antibody (Catalog (Y241), Polyclonal, Rabbit, Peptide Research Institute, Inc.). The expression level of TGF-β protein was leveled by the expression level of β-actin.

The results using Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 as PI polyamides are shown in FIGS. 25 to 27, respectively. The addition of 0.1 μM PMA induced the expression of TGF-β1 protein. It was confirmed that when any of the PI polyamides was used, the expression of TGF-β1 protein stimulated by PMA was significantly suppressed at an extremely low concentration of ^{10-11 M.}

[Evaluation of TGF-β1 expression level in the presence of HGF siRNA]
A test using HGF siRNA was performed to confirm whether PI polyamide directly reduced the expression level of TGF-β1. In the presence of HGF siRNA, HGF expression is suppressed. Therefore, the effect of increasing the HGF expression level can be eliminated. Therefore, it can be confirmed whether PI polyamide directly suppresses the expression of TGF-β1 by allowing PI polyamide to act on cells in the presence of HGF siRNA.

As HGF siRNA, HSS179212 (Thermo Fisher SCIENTIFIC) was used. It was confirmed that HGF siRNA specifically suppresses the expression of HGF mRNA (data not shown).

The cells were cultured and recovered in the same manner as in [Evaluation of HGF mRNA expression level] except that 10 nM HGF siRNA was added. The cells were disrupted and Western blotting was performed using an anti-HGF antibody (Catalog (MAB294), Monoclonal Mouse, R & D System). The expression level of HGF protein was leveled by the expression level of β-actin.

The results of using Hu-HGF-3 and Hu-HGF-5 as PI polyamides are shown in FIGS. 28 to 29, respectively. In FIGS. 28 to 29, the numbers on the horizontal axis indicate the following. The concentration of PMA was 0.1 μM, and the concentration of HGF siRNA was 10 nM.
1: DMSO
2: DMSO + PMA + HGF siRNA
3: DMSO + PMA + HGF siNRA + 10 -11 M PI polyamide 4: DMSO + PMA + HGF siNRA + 10 -10 M PI polyamides 5: DMSO + PMA + HGF siNRA + 10 -9 M PI polyamides

It was confirmed that both Hu-HGF-3 and Hu-HGF-5 significantly suppressed TGF-β protein expression in the presence of HGF siRNA. These results suggest that Hu-HGF-3 and Hu-HGF-5 bind to the promoter region of TGF-β and directly inhibit the expression of TGF-β.

[Evaluation of TGF-β1 mRNA expression level using mismatched PI polyamide]
A mismatched PI polyamide (HGF mismatch) that was not set to bind to the transcriptional repression region of the HGF gene and the transcription promotion region of the TGF-β1 gene was synthesized. The cells were cultured in the same manner as in [Evaluation of TGF-β1 mRNA expression level] except that the mismatched PI polyamide was used, and the TGF-β1 mRNA expression level was evaluated. The structure of HGF mismatch is shown below.

The results are shown in FIG. HGF mismatch did not affect the expression level of TGFβ mRNA.

According to the present invention, there is provided a formula pyrrole imidazole polyamide capable of simultaneously controlling transcription of two or more kinds of genes, and a pharmaceutical composition containing the pyrrole imidazole polyamide. In addition, pyrrol imidazole polyamide useful as a therapeutic agent for TGF-β-related diseases or fibrous diseases, TGF-β gene expression inhibitor using the pyrrol imidazole polyamide, and TGF-β-related diseases or fibrous diseases are treated. A pharmaceutical composition for this is provided.

Claims (16)

1. Pyrrole imidazole polyamide that binds to the promoters of two or more genes and controls the transcription of the two or more genes.
2. The two or more genes include a first gene and a second gene.
   The first gene is a gene encoding an expression regulator of the second gene.
   The pyrrole imidazole polyamide according to claim 1.
3. Binds to the promoter of the first gene and inhibits the binding of transcriptional regulators to the promoter of the first gene.
   The pyrrole imidazole polyamide according to claim 2.
4. The two or more genes include an HGF gene and a TGF-β gene.
   The pyrrole imidazole polyamide according to claim 1.
5. Promotes transcription of the HGF gene and inhibits transcription of the TGF-β gene.
   The pyrrole imidazole polyamide according to claim 4.
6. Binds to the transcriptional repression region in the promoter of the HGF gene,
   The pyrrole imidazole polyamide according to claim 5.
7. The pyrrole imidazole polyamide according to claim 6, which binds to a region of the promoter of the HGF gene containing at least a part of the base sequence set forth in SEQ ID NO: 1.
8. The pyrrole imidazole polyamide according to claim 7, which binds to a region containing any of the following sequences 1 to 3 in the promoter of the HGF gene.
   Sequence 1: AGGTGAC
   Sequence 2: ACCTTTT
   Sequence 3: CTTTTCT
9. The pyrrole imidazole polyamide according to any one of claims 4 to 8, which binds to the transcription promoting region in the promoter of the TGF-β gene.
10. A pyrrole imidazole polyamide represented by the following formula (P-3), (P-5) or (P-7).
    

    

    

    

    [In the formula, R ¹ and R ² each independently represent a monovalent organic group. R ¹ and R ² may be interconnected to form a divalent organic group. ]
11. The pyrrole imidazole polyamide according to claim 10, which is represented by the following formula Hu-HGF-3, Hu-HGF-5 or Hu-HGF-7.
    

    

    

    
12. A TGF-β gene expression inhibitor containing the pyrrole imidazole polyamide according to any one of claims 4 to 11.
13. A pharmaceutical composition comprising the pyrrole imidazole polyamide according to any one of claims 1 to 11.
14. A pharmaceutical composition for treating a TGF-β-related disease, which comprises the pyrrole imidazole polyamide according to any one of claims 4 to 11.
15. A pharmaceutical composition for treating a fibrotic disease, which comprises the pyrrole imidazole polyamide according to any one of claims 4 to 11.
16. (A) A step of designing a pyrrole imidazole polyamide that binds to promoters of two or more genes, and
    (B) The step of synthesizing the pyrrole imidazole polyamide and
    (C) Among the synthesized pyrrole imidazole polyamides, a step of selecting a pyrrole imidazole polyamide that controls transcription of the two or more genes, and a step of selecting the pyrrole imidazole polyamide.
    A method for producing a pyrrole imidazole polyamide, which comprises.

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