WO2010103684A1 - Régulateur de l'expression spécifique de la séquence qui cible le gène myc en aval et méthode permettant de déterminer la cible ou les cibles du gène myc en aval - Google Patents

Régulateur de l'expression spécifique de la séquence qui cible le gène myc en aval et méthode permettant de déterminer la cible ou les cibles du gène myc en aval Download PDF

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WO2010103684A1
WO2010103684A1 PCT/JP2009/066111 JP2009066111W WO2010103684A1 WO 2010103684 A1 WO2010103684 A1 WO 2010103684A1 JP 2009066111 W JP2009066111 W JP 2009066111W WO 2010103684 A1 WO2010103684 A1 WO 2010103684A1
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myc
sequence
gene
pyrrole
region
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Japanese (ja)
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浩喜 永瀬
ミシュラ ラジーブ
真 木村
隆義 渡部
弘之 川島
省太 植草
Takeshi KUSAFUKA (草深 竹志)
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学校法人日本大学
草深ひろみ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a downstream gene of the MYC oncogene (in addition to MYC (c-myc), there are two other family members MYCN (n-myc) and MYCL (1-myc) having strong similarity).
  • the present invention relates to a sequence-specific expression regulator targeting, a therapeutic agent for a MYC gene-related disease, and an antineoplastic agent.
  • a therapeutic agent for a MYC gene-related disease and an antineoplastic agent.
  • medical agent which comprises the pyrrole imidazole polyamide (henceforth PIP) which has a specific structure.
  • the present invention provides a double helix region comprising a part or all of the E-BOX region sequence of the MYC downstream gene, a sequence comprising a base sequence upstream and / or downstream of the E-BOX region, and a complementary strand thereto.
  • the present invention relates to a method for determining a target region necessary for disease treatment and function expression by analysis of cells using a PIP compound that recognizes (hereinafter referred to as target region).
  • the MYC gene family is a proto-oncogene composed of three major genes: MYC (c-myc), MYCN (n-myc), and MYCL (1-myc), and all family genes are involved in tumor development. Is involved.
  • the protein is a transcription factor that exhibits similar properties and has a helix-loop-helix / leucine-zipper structure, and various biological processes such as cell cycle progression, transcription, protein biosynthesis and metabolism It is a transcription factor that controls cell death and untranslated transcripts (Non-patent Document 1).
  • MYC is thought to produce these effects by induction / suppression of its downstream genes, and by regulating gene expression on cells that make up a wide variety of target cells and external environments, growth of mammalian cells, tissue differentiation, stem cells It plays an important role in maintenance and aging. That is, the MYC protein forms a heterodimer with MAX, which is a partner protein of MYC, and is a DNA specific sequence, a DNA consensus sequence called E-BOX, which is a standard sequence CACGTG represented by the CACGTG motif. And the non-standard sequence CACATG, CATGTG, CATGCG, CACGCG, CTCGCG, CACGAG, or CAACGTG sequence and its complementary sequence to activate transcription (Non-patent Documents 2, 3, and 4).
  • the MAD-MAX heterodimer binds to the E-BOX sequence and suppresses transcription.
  • MYC protein binds to more than 15% of human gene promoters, and its disordered expression is involved in the development of many types of human cancers and in many biological reactions and diseases.
  • MYC protein is also a cancer protein that is deregulated in a wide variety of human tumors (Non-Patent Document 6). Therefore, downstream proteins controlled by MYC are novel anti-neoplastic agents, related diseases, and related living organisms. Can be a target for cellular reactions.
  • MYC MYC, MYCN, MYCL
  • MYC MYC, MYCN, MYCL
  • inhibition of the signal transduction affects too many genes It has been considered that it is not suitable as a target for disease treatment with simple inhibitors.
  • low molecular weight compounds, antibody drugs, and nucleic acid drugs have been studied as drug development by MYC signal suppression, but none has been successful.
  • inhibitors that specifically target each gene product the number of downstream genes of MYC is large, and it is important to identify which downstream genes are important targets for disease treatment. Because it is very difficult.
  • the treatment method targeting the MYC gene by SiRNA can be designed targeting individual MYC downstream genes, it is easily affected by nucleolytic enzymes and unstable in vitro and in vivo. In other words, it is essential to develop drug delivery system (DDS) technology that wraps a drug by SiRNA in a membrane and prevents it from being absorbed and decomposed until it reaches the target site, and there is a big problem in drug transfer to target cells. is there. In addition, there is a concern about the influence of SiRNA on other biological control mechanisms such as microRNA. Furthermore, since the treatment with SiRNA generally inhibits post-transcriptional translation with only a limited gene, it is difficult to regulate the expression of a plurality of genes downstream of MYC as a group.
  • DDS drug delivery system
  • Pyrrole imidazole polyamide is a chemically synthesized substance discovered by Dervan et al. Based on the fact that antibiotics duocarmycin-A and distamycin-A recognize DNA in a base-specific manner (Patent Document 1, Non-Patent Document 7). Non-Patent Document 8). PIP recognizes double-stranded DNA in a base sequence-specific manner and binds to a minor groove of a DNA double helix structure, so that it is possible to specifically control the expression of a target gene (Non-patent Document 8). .
  • PIP is not degraded by nucleolytic enzymes in vivo and has high ability to bind to nucleic acids. As a drug, clinical application to an antineoplastic agent is expected.
  • inactivation of gene function by reverse genetics is used to analyze the function of a specific gene, but on the other hand, viral infection, cancer, and other diseases based on abnormal gene expression There is also great potential for the treatment. That is, it is known that inactivation of gene function can be performed at the DNA level by homologous recombination, or at the RNA level by antisense oligodeoxynucleotides or ribozymes. However, homologous recombination generally has low recombination efficiency and is effective only in some cells. The antisense oligodeoxynucleotide and ribozyme methods have restrictions on the target sequence and can be transferred to tissues and cells. However, there was a problem that it was easily degraded by ribonuclease.
  • pyrrole-imidazole polyamides can specifically recognize DNA base sequences and control the expression of specific genes from outside the cell. Has been reported.
  • Pyrrole imidazole polyamide (hereinafter also referred to as Py-Im polyamide) is a group of synthetic small molecules, and is composed of N-methylpyrrole units (hereinafter also referred to as Py) and N-methylimidazole units (hereinafter also referred to as Im) which are aromatic rings.
  • Py and Im can take a U-shaped conformation in the presence of ⁇ -aminobutyric acid by sequentially coupling and folding.
  • N-methylpyrrole units Py
  • N-methylimidazole units Im
  • ⁇ -alanine ⁇
  • ⁇ -aminobutyric acid units also referred to as ⁇ linkers
  • Such synthetic polyamides can bind with high affinity and specificity to specific base pairs in the minor groove of the double helix DNA. Specific recognition of base pairs is dependent on one-to-one pairing of Py and Im. That is, in the U-shaped conformation in the minor groove of DNA, Py / Im pairs or ⁇ / Im pairs target CG base pairs, and Im / Py pairs or Im / ⁇ pairs are GC base pairs. And Py / Py pairs, ⁇ / ⁇ pairs, Py / ⁇ pairs or ⁇ / Py pairs target both AT base pairs and TA base pairs (Non-Patent Documents 7 to 10) .
  • a part of the CG base pair is a ⁇ / Im pair
  • a part of the GC base pair is an Im / ⁇ pair
  • a part of the AT and TA base pairs is It may be targeted by a ⁇ / ⁇ pair, a Py / ⁇ pair or a ⁇ / Py pair, respectively.
  • the problem of non-selectivity of Py-Im polyamide compounds for AT and TA pairs has been to replace one pyrrole ring of Py / Py pair with 3-hydroxypyrrole (Hp). It has been found that the resulting Hp / Py pair can be overcome by preferentially binding to the T / A pair.
  • the start of transcription is considered to be an important point of gene regulation. Initiation of transcription requires several processes in which transcription factors that bind to specific recognition sequences in the gene promoter region form a complex and the complex binds to the DNA sequence.
  • the polyamide in the minor groove may interfere with gene regulation by inhibiting the binding of the transcription factor or its complex if binding to a specific sequence of the transcription factor or complex is important in gene expression. is there. This hypothesis has been proven in vitro and in vivo.
  • the 8-membered Py-Im polyamide bound inside the zinc finger recognition site (TFIIIA binding site) inhibited 5S RNA gene transcription.
  • Human cytomegalovirus (CMV) UL122-mediated early protein 2 (IE86) blocks the recruitment of promoters to RNA polymerase II and represses transcription of its associated genes. Synthetic polyamides can block the inhibition of IE86 and release the expression of its corresponding gene. Polyamides designed by Mapp et al. Act as artificial transcription factors and mediate gene transcription reactions.
  • TATA box binding protein also referred to as TBP
  • this transcription factor binding inhibition was also observed in the polyamide compound designed at a site 10 bases away from the binding sequence. It has been reported that transcription inhibition is recognized even in the polyamide designed around the factor recognition sequence (Non-patent Document 11).
  • MYC protein regulates transcription of thousands of genes as transcription factors in various signal transductions inhibition of the signal transduction is considered to affect too many genes, and disease treatment with simple inhibitors It has been considered unsuitable as a target for Although it is theoretically possible to develop inhibitors that specifically target each gene product, the number of MYC downstream genes is large, and it is very important to identify which downstream genes are important targets for disease treatment. It was difficult.
  • the methods such as antisense oligodeoxynucleotide, ribozyme, and SiRNA described above are limited in target sequences, have a problem of poor migration to target tissues and cells, and are easily degraded by nucleolytic enzymes. there were.
  • the present inventors are specific to a sequence comprising a part or all of the consensus DNA sequence of the E-BOX region of the MYC downstream gene and a gene base sequence further extending the recognition region upstream and / or downstream from the consensus DNA sequence.
  • a compound that binds to the WCGGWCW sequence Myc-1
  • a compound that binds to the WCGGWGW sequence Myc- 2
  • a compound that binds to the WCCWCGW sequence Myc-3
  • a compound that binds to the WGCWCGW sequence Myc-4
  • a compound that binds to the WCWCGWGW sequence Myc-5
  • W is A or T
  • Human cervical cancer-derived cell line HeLa cell, human B cell lymphoma cell line P493 In live cell count assay and real-time RT-PCR using 6 cells, human chronic myeloid leukemia-derived cell line K562 cell, human leukemia cell-derived cell line MOLT-4 cell, or human neuroblastoma cell line CHP134 cell, Myc-1 and Myc-2 down-regulate the MYC downstream gene E2F2, Myc-5 down
  • a target region comprising a part or all of the sequence further comprising a 1-2 base sequence upstream and / or downstream of the E-BOX region and a complementary strand thereto, ⁇ - It
  • a drug comprising the pyrrole-imidazole polyamide, wherein the Py / Py pair respectively corresponds to the AT base pair and the TA base pair.
  • the drug according to (1) further preferably comprising a ⁇ -alanine unit (hereinafter also referred to as ⁇ ).
  • a pyrrole-imidazole polyamide containing N-methylpyrrole units, N-methylimidazole units, ⁇ -alanine and ⁇ -aminobutyric acid units represented by CACGTG, CACATG, CATGTG, CATGCG, CACGCG, CTCGCG, CACGTG, or CAACGTG
  • the E-BOX region sequence of the MYC downstream gene consisting of the nucleotide sequence and the antisense sequence CACGTG, CATGTG, CACATG, CGCATG, CGCGTG, CGCGAG, CTCGTG, or CACGTTG, and part or all of the E- In a minor groove of a double helix region containing a sequence containing 1 to 2 base sequences upstream and / or downstream of the BOX region and a complementary strand thereto, the U-shaped fold is folded at the site of the ⁇ -aminobutyric acid unit.
  • Conform Py / Im or ⁇ / Im pairs for CG base pairs, Im / Py pairs or Im / ⁇ pairs for GC base pairs are defined as AT A drug comprising the above pyrrole-imidazole polyamide, each of which corresponds to a Py / Py pair, a ⁇ / ⁇ pair, a Py / ⁇ pair, or a ⁇ / Py pair for each of the base pair and the TA base pair.
  • FITC fluorescein isothiocyanate
  • the target region is a double helix region comprising a base sequence of WCGGWGW (W is A or T) and a complementary strand thereto.
  • the target region is a double helix region comprising a base sequence of ACGTGGW (W is A or T) and a complementary strand thereto.
  • the target region is a double helical region comprising a base sequence of WCCWCGW (W is A or T) and a complementary strand thereto.
  • the drug according to any one of (1) to (7), wherein the target region is a double helix region containing a base sequence of WCCACGT (W is A or T) and a complementary strand thereto.
  • the drug according to any one of (1) to (7), wherein the target region is a double helix region containing a base sequence of WGCWCGW (W is A or T) and a complementary strand thereto.
  • the target region is a double helix region comprising a WCWCGWGW base sequence (W is A or T) and a complementary strand thereto.
  • the target region is a double helix region comprising a base sequence of WCACGTGGW (W is A or T) and a complementary strand thereto.
  • a pharmaceutical composition comprising the drug according to any one of (1) to (18), (20), (22), (24) or (26) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the drug according to (18) and / or (20) and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to (34) for regulating eIF4 gene expression (6) A pharmaceutical composition comprising the drug according to (20) and a pharmaceutically acceptable carrier. (37) The pharmaceutical composition according to (36) for regulating MDM2 gene expression. (38) A drug comprising the pyrrole-imidazole polyamide according to any one of (19), (21), (23), (25) or (27), and a pharmaceutically acceptable carrier. (39) A drug comprising the pyrrole-imidazole polyamide according to (19) and / or (21) and a pharmaceutically acceptable carrier.
  • the gene expression can be specifically regulated, so that there is no side effect like a conventional chemotherapeutic agent, and since it is a stable compound, it does not have the disadvantage of being degraded by ribonuclease.
  • a drug for regulating expression, a drug for treating a MYC gene-related disease, and an antineoplastic agent can be obtained.
  • the target or target group of the MYC downstream gene can be determined.
  • a functional test reagent used for molecular biological research and drug development research can be obtained by the selective regulatory effect of the compound according to the present invention.
  • the N-methylpyrrole unit, the N-methylimidazole unit, and the ⁇ -aminobutyric acid unit (hereinafter also referred to as ⁇ linker) are connected to each other by an amide bond (—C ( ⁇ O) —NH—).
  • ⁇ linker ⁇ -aminobutyric acid unit
  • the general structure and manufacturing method thereof are known (see, for example, Patent Documents 1 to 3).
  • pyrrole-imidazole polyamide can be produced by an automatic synthesis method using a solid phase method (solid phase Fmoc method) using Fmoc (9-fluorenylmethoxycarbonyl) (Patent Document 3).
  • solid phase Fmoc method solid phase Fmoc method
  • Fmoc (9-fluorenylmethoxycarbonyl) Patent Document 3
  • the terminal of pyrrole-imidazole polyamide can be cut out from the solid support as a carboxylic acid residue, so that various functional groups can be introduced into the molecular terminal to produce derivatives of pyrrole-imidazole polyamide.
  • a compound having an alkylating ability for DNA such as duocarmycin, pyrrolobenzodiazepine, bleomycin, enediyne compound, nitrogen mustard, and derivatives thereof can be introduced as necessary.
  • the solid phase Fmoc method is an automatic synthesis method using a commercially available protein (peptide) synthesizer, it is also possible to synthesize conjugates (conjugates) of naturally occurring proteins or non-natural proteins and pyrrole imidazole polyamides.
  • the Fmoc method has milder reaction conditions than the t-BOC method, it is possible to introduce organic compounds other than proteins (including compounds having a functional group unstable under acidic conditions). For example, it is also possible to automatically synthesize a conjugate of pyrrole-imidazole polyamide and DNA or RNA (or their derivatives).
  • a pyrrole-imidazole polyamide having a carboxyl group at the terminal can be synthesized.
  • Specific examples thereof include pyrrole imidazole polyamide having a ⁇ -alanine residue ( ⁇ -aminopropionic acid residue) or a ⁇ -aminobutyric acid residue at the terminal.
  • a pyrrole-imidazole polyamide having a ⁇ -alanine residue or a ⁇ -aminobutyric acid residue at the end is, for example, an aminopyrrole carboxylic acid, aminoimidazole carboxylic acid, ⁇ -alanine or ⁇ -aminobutyric acid, each having an amino group protected with Fmoc.
  • aminopyrrole carboxylic acid examples include, for example, 4-amino-2-pyrrole carboxylic acid, 4-amino-1-methyl-2-pyrrole carboxylic acid, 4-amino-1-ethyl-2-pyrrole carboxylic acid, Examples include 4-amino-1-propyl-2-pyrrole carboxylic acid and 4-amino-1-butyl-2-pyrrole carboxylic acid.
  • aminoimidazole carboxylic acid examples include, for example, 4-amino-2-imidazole carboxylic acid, 4-amino-1-methyl-2-imidazole carboxylic acid, 4-amino-1-ethyl-2-imidazole carboxylic acid, Examples include 4-amino-1-propyl-2-imidazolecarboxylic acid, 4-amino-1-butyl-2-imidazolecarboxylic acid, and the like.
  • a conjugate of pyrrole imidazole polyamide and FITC fluorescein isothiocyanate
  • FITC fluorescein isothiocyanate
  • the resulting conjugate can be used to prove that the pyrrole-imidazole polyamide recognizes a specific DNA sequence.
  • the MYC downstream gene expression regulator of the present invention is a pyrrole-imidazole polyamide containing N-methylpyrrole unit (Py), N-methylimidazole unit (Im) and ⁇ -aminobutyric acid unit, and is CACGTG, CACATG, CATGTG, CATGCG , CACGCG, CTCGCG, CACGAG, or CAACGTG, the consensus DNA sequence of the E-BOX region of the MYC downstream gene, and its antisense sequence CACGTG, CATGTG, CACATTG, CGCATTG, CGCGTG, CGCGGG, Among the sequences including a part or all of CACGTTG and a gene base sequence having a recognition region extending upstream and / or downstream from the consensus DNA sequence, a WCGGWGCW sequence (Myc 1 recognition sequence), WCGGWGW sequence (Myc-2 recognition sequence), WCCWCGW sequence (Myc-3 recognition sequence), WGCWCGW sequence (Myc-4 recognition sequence), or WCWCGWGW sequence (
  • the pyrrole-imidazole polyamide is included, each corresponding to a Py / Py pair.
  • the present invention is a pyrrole imidazole polyamide comprising N-methylpyrrole units, N-methylimidazole units, ⁇ -alanine and ⁇ -aminobutyric acid units, CACGTG, CACATG, CATGTG, CATGCG, CACGCG, CTCGCG, CACGAG Or a consensus DNA sequence of the E-BOX region of the MYC downstream gene consisting of the base sequence represented by CAACCGTG, and one of its antisense sequences CACGTG, CATGTG, CACATG, CGCATTG, CGCGTG, CGCGAG, CTCGTG, or CACGTG
  • sequences comprising a part or all of the gene base sequence further extending the recognition region upstream and / or downstream from the consensus DNA sequence
  • WCGGWGCW sequence Myc-1 recognition sequence
  • WCGGWGW sequence Myc-2 recognition sequence
  • WCCWCGW sequence Myc-3 recognition sequence
  • WGCWCGW sequence Myc-4 recognition sequence
  • WCWCGWGW sequence (Myc-1 recognition
  • Myc-1 recognizes the ACGTG sequence of the E-BOX region sequence of the MYC downstream gene, and further recognizes the CA or CT 2-base sequence outside the E-BOX region
  • Myc-3 is designed to recognize the CACGT sequence of the E-BOX region sequence, and to recognize the AC or TC 2 base sequence outside the E-BOX region.
  • Myc-1 is ACGTG It is thought that Myc-3 regulates (inhibits) the control of 1/8 (12.5%) of different MYC downstream genes in the E-BOX-regulated MYC gene having the CACGT sequence. .
  • a DNA helical skeleton forms two types of grooves, and a wide and deep groove is called a main groove (major groove), and a narrow and shallow groove is called a minor groove.
  • the pyrrole-imidazole polyamide can be bonded in a non-conjugated manner with high affinity and specificity to a minor groove formed by a specific base pair.
  • the pyrrole imidazole polyamide Py / Im pair or ⁇ / Im pair is applied to the minor groove CG base pair, and the Im / Py pair or Im / ⁇ pair is applied to the GC base pair.
  • Each of the pair corresponds to an AT base pair and a TA base pair by a Py / Py pair, a ⁇ / ⁇ pair, a Py / ⁇ pair, or a ⁇ / Py pair, respectively. Then, the molecule is folded at the site of the ⁇ -aminobutyric acid unit in the pyrrole-imidazole polyamide molecule to take a U-shaped conformation.
  • a part of the CG base pair is a ⁇ / Im pair
  • a part of the GC base pair is an Im / ⁇ pair
  • a part of the AT and TA base pairs is It may be targeted by a ⁇ / ⁇ pair, a Py / ⁇ pair or a ⁇ / Py pair, respectively.
  • a pyrrole imidazole polyamide in which the minor groove base pair and Py-Im pair do not correspond as described above is referred to as a mismatch or mismatch polyamide in the present application.
  • the base sequence of the E-BOX region used by the MYC gene family to regulate the expression of its downstream gene, and the schematic diagram of the transcriptional regulation of the MYC downstream gene group by the MYC-MAX dimer and the MAG-MAX dimer are shown in FIG. 1A. , B as shown.
  • Py targeting a sequence comprising a part or all of the consensus DNA sequence of the E-BOX region of the MYC downstream gene of the present invention and a gene base sequence further extending the recognition region upstream and / or downstream from the consensus DNA sequence -Im polyamides, Myc-1, Myc-2, Myc-3, Myc-4 and Myc-5 are as shown below.
  • Myc-1 has the molecular formula C 64 H 92 N 26 O 13 , molecular weight 1423.6 and its target sequence is the WCGGWGCW sequence.
  • Py represents an N-methylpyrrole unit
  • Im represents an N-methylimidazole unit (Im)
  • ⁇ represents ⁇ -alanine
  • Dp represents dimethylaminopropylamide
  • Ac represents acetyl.
  • Myc-2 has the molecular formula C 64 H 92 N 26 O 13 and a molecular weight of 1423.6, and its target sequence is the WCGGWGW sequence.
  • Py represents an N-methylpyrrole unit
  • Im represents an N-methylimidazole unit (Im)
  • ⁇ represents ⁇ -alanine
  • Dp represents dimethylaminopropylamide
  • Ac represents acetyl.
  • Myc-3 has the molecular formula C 64 H 82 N 26 O 13 , molecular weight 1422.8, and its target sequence is the WCCWCGW sequence.
  • Py represents an N-methylpyrrole unit
  • Im represents an N-methylimidazole unit (Im)
  • ⁇ represents ⁇ -alanine
  • Dp represents dimethylaminopropylamide
  • Ac represents acetyl.
  • Myc-4 has the molecular formula C 64 H 83 N 26 O 13 , molecular weight 1424.5, and its target sequence is the WGCWCGW sequence.
  • ⁇ is N-methylpyrrole unit
  • ⁇ is N-methylimidazole unit
  • ⁇ is ⁇ -alanine
  • ) represents ⁇ -aminobutyric acid
  • Ac represents acetyl.
  • Myc-5 has the molecular formula C 73 H 94 N 29 O 15 , molecular weight 1617.7, and its target sequence is the WCWCGWGW sequence.
  • ⁇ is N-methylpyrrole unit
  • ⁇ is N-methylimidazole unit
  • ⁇ is ⁇ -alanine
  • ) represents ⁇ -aminobutyric acid
  • Ac represents acetyl.
  • W represents A or T.
  • mismatched polyamide Py-Im No. which does not bind to the E-BOX region. 92 (mismatch A), mismatch polyamide Py-Im No. 120 (mismatch B) is as shown below.
  • Mismatch Py-Im No. 92 (mismatch A) has the molecular formula C 67 H 85 N 23 O 13 , molecular weight 1421.0, and its target sequence is the WWWWWW sequence.
  • Py represents an N-methylpyrrole unit
  • Im represents an N-methylimidazole unit (Im)
  • ⁇ represents ⁇ -alanine
  • Dp represents dimethylaminopropylamide
  • Ac represents acetyl
  • W represents A or T is represented.
  • Py-Im No. 120 (mismatch B) has the molecular formula C 65 H 84 N 25 O 13 , molecular weight 1668.0, and its target sequence is the WCWGGCWGW sequence.
  • Py represents an N-methylpyrrole unit
  • Im represents an N-methylimidazole unit (Im)
  • ⁇ represents ⁇ -alanine
  • Dp represents dimethylaminopropylamide
  • Ac represents acetyl
  • W represents A or T is represented.
  • the present inventors target a sequence including a part or all of the consensus DNA sequence of the E-BOX region of the MYC downstream gene and a gene base sequence further extending the recognition region upstream and / or downstream from the consensus DNA sequence. Py-Im polyamides were synthesized.
  • the Py-Im polyamide compounds Myc-1 and Myc-2 of the present invention alone Or the combined use of Myc-1 + Myc-2 down-regulates the E2F2 gene, which is one of the important c-MYC downstream genes and proved to play an important role in cell proliferation, and It was shown that the expression of the Myc downstream gene eIF4 is down-regulated by the inventive Py-Im polyamide compound Myc-5.
  • the expression of the MDM2 gene which is a gene that controls the p53 gene of the tumor suppressor gene, is down-regulated by the Py-Im polyamide compound Myc-2 of the present invention. Therefore, it is shown that the Py-Im polyamide compound according to the present invention has an effect of regulating the expression of a MYC downstream gene having an E-BOX sequence to which the MYC transcription factor complex (MYC-MAX) binds. It was.
  • the cell growth is changed by changing the expression of the downstream gene by inhibiting the function regulation while dividing into each group. Therefore, this indicates the possibility that the function of the MYC downstream gene group can be predicted by detailed analysis of cells after addition of each Py-Im polyamide.
  • the PIP compounds synthesized here are thought to regulate the expression of genes called 3000 to 4000 downstream of the MYC (genes and microRNAs related to cell cycle, protein synthesis, cell adhesion / cytoskeleton, intracellular metabolism, etc.). Therefore, it was considered that the compound itself can be used as a therapeutic drug for MYC-related diseases, a histogenesis differentiation control drug, or the like.
  • MYC downstream gene target pyrrole imidazole polyamide Myc-1, Myc-2, Myc-3, Myc-1 and Myc-2, Myc-2 and Myc-3, Myc-1 and Myc-3, negative control: mismatch Py- Im No. 92 and untreated control
  • cultivation of the HeLa cell by Myc-1, Myc-2, Myc-3 treatment is shown.
  • the cell growth inhibitory effect of Myc-5 was shown because the increase in the number of living cells accompanying growth was suppressed in a concentration-dependent manner.
  • the result of quantitative expression analysis is shown.
  • Myc-5 is shown to down-regulate the expression levels of eIF4G1 and CCND1 genes in HeLa cells in a concentration-dependent manner.
  • the result of quantitative expression analysis is shown.
  • Myc-5 shows that the expression level of the eIF4G1 gene is down-regulated in a concentration-dependent manner in P493-6 cells.
  • Lane 1 single stranded DNA
  • lane 2 double stranded DNA
  • lane 3 eIF4G gene promoter double stranded DNA and Myc-5
  • lane 4 double stranded DNA with mutant sequence inserted and Myc-5
  • lane 5 EIF4G gene promoter double-stranded DNA and mismatch B (Py-ImNo. 120) (recognizes WCWGGCWGW)
  • the result of HPLC of Myc-1 is shown.
  • the result of HPLC of Myc-2 is shown.
  • the result of HPLC of Myc-3 is shown.
  • the result of HPLC of Myc-4 is shown.
  • the HPLC results of Myc-5 are shown.
  • the target region WCACGTGW of Myc-5 exists in the E-BOX region of the promoter of the human CCND1 gene, the E-BOX region of the promoter of the human eIF4G1 gene, and the E-BOX2 region of the promoter of the human CDK4 gene.
  • Western blotty analysis protein measurement
  • Nuclear localization of FITC-bound Myc-5 (5 ⁇ M) in MCF-7 cells
  • Chromatin immunoprecipitation assay in K562 cells The growth inhibitory effect of CHP134 tumor cells by Myc-2 and Myc-3 targeting E-BOX.
  • mismatched polyamide Py-Im No. 92, and no. 120 was also designed and synthesized.
  • Machine Assisted (Synthesis Assisted) Automatic Synthesis of Py-Im Polyamide Using Fmoc Method Machine assisted automated synthesis of pyrrole-imidazole polyamide was performed using a continuous flow peptide synthesizer Pioneer (trademark) (Applied Biosystems). Performed on a 1 mmol scale (200 mg Fmoc- ⁇ -alanine-CLEAR acid resin, 0.50 meq / g, Peptide Institute, Inc.).
  • FITC coupling A 4-fold excess of isothiocyanated fluorescein (0.40 mmol) and DIEA dissolved in DMF was flushed through the column for 60 minutes.
  • General procedure After removing the Fmoc group of the Fmoc- ⁇ -alanine-Wang resin, the resin was washed successively with DMF. The coupling step was performed with Fmoc amino acid followed by washing with DMF. These steps were repeated many times until the entire sequence was introduced. After completing the coupling step, the N-terminal amino group was protected as necessary or coupled with FITC, washed with DMF, and the reaction vessel was removed.
  • Decomposition as carboxylic acid The synthetic polyamide was isolated by cold ethyl ether precipitation after the decomposition step (5 ml of a mixture of 91% TFA-3% / TIS-3% DMS-3% water / 0.1 mmol of resin).
  • Synthetic polyamide was isolated by cold ethyl ether precipitation after the decomposition step (N, N-dimethylaminopropylamine 5 ml / resin 0.1 mmol, 50 ° C., overnight).
  • HeLa cells human cervical cancer-derived cells
  • ATCC American Type Culture Collection
  • HeLa cells were seeded in 96-well plates at 3,000 cells per well, and 24 hours in 100 ⁇ l Dulbecco's modified Eagle medium (DMEM) containing 1% PSG under 10% calf serum (Invitrogen). After culturing, the medium is changed, and Myc-1, Myc-2, Myc-3 at a concentration of 10 ⁇ M alone, or Myc-1 and Myc-2, Myc-2 and Myc-3, Myc-1 and Myc-3 at a concentration of 10 ⁇ M.
  • DMEM Dulbecco's modified Eagle medium
  • HeLa cells were cultured for 72 hours at 37 ° C., 5% CO 2, 95% to 100% relative humidity in a medium supplemented with 10% calf serum containing Py-Im polyamide or similar medium without PIP. .
  • untreated HeLa cells were used as controls, and mismatched polyamide Py-Im No. Cells treated with 92 served as a negative control. The number of viable cells was measured 24 hours, 48 hours and 72 hours after the culture, and the effect of Py-Im polyamide was evaluated.
  • Myc-2 alone and the combination of Myc-1 and Myc-2 showed a minimum relative survival rate (%) compared to the cell survival rate on day 0 (at the start of culture). This indicates that the growth of HeLa cells is most inhibited by Myc-2 alone or in combination with Myc-1 and Myc-2.
  • the results are shown in FIG.
  • MOLT-4 cells human leukemia cell-derived cells
  • RIKEN Cell Bank RIKEN Cell Bank.
  • MOLT-4 cells were cultured in Myc-5 at a concentration of 100, 500 nM, 1, 5, 10 ⁇ M, and Myc-2 at a concentration of 10,500 nM. Untreated MOLT-4 cells were used as a control. In the case of Myc-5 treatment, the number of viable cells was measured on the third day after culture, and in the case of Myc-2 treatment, the number of viable cells was measured on the second and fifth days after the culture. II.
  • K562 Cell Viability Assay
  • K562 cells human chronic myeloid leukemia cell-derived cell line
  • ATCC American Type Culture Collection
  • K562 cells human chronic myeloid leukemia cell-derived cell line
  • Untreated K562 cells were used as a control.
  • Viable cell counts were measured 24, 48, and 72 hours after culture using the trypan blue staining exclusion assay.
  • Results Myc-5 inhibited the growth of K562 cells in a concentration-dependent manner compared to the control (FIG. 16).
  • CHP134 cells Cell Viability Assay
  • CHP134 cells Cell Viability Assay
  • HeLa cells were seeded in 1 well of a 6-well plate to 120,000 cells and 24 hours in Dulbecco's modified Eagle medium (DMEM) containing 1% PSG under 10% calf serum (Invitrogen). After culturing, the medium is changed, and Myc-1, Myc-2, Myc-3 at a concentration of 10 ⁇ M alone, or Myc-1 and Myc-2, Myc-2 and Myc-3, Myc-1 and Myc-3 at a concentration of 10 ⁇ M. HeLa cells treated with a 10% calf serum-supplemented medium containing Py-Im polyamide or a similar medium not containing PIP were cultured for 72 hours.
  • DMEM Dulbecco's modified Eagle medium
  • RT-PCR and quantitative real-time RT-PCR (measurement of mRNA expression) I. Materials and Methods HeLa cells were seeded in 6-well plates at 120,000 cells per well and 24 hours in Dulbecco's modified Eagle medium (DMEM) containing 1% PSG under 10% calf serum (Invitrogen). After culturing, the medium is changed, and Myc-1, Myc-2, Myc-3 at a concentration of 10 ⁇ M alone, or Myc-1 and Myc-2, Myc-2 and Myc-3, Myc-1 and Myc-3 at a concentration of 10 ⁇ M.
  • DMEM Dulbecco's modified Eagle medium
  • GADPH human endogenous glyceraldehyde-3-phosphate dihydrogenase
  • TAKARA DICE real time system (trade name) (TaKaRa Bio Inc.)
  • PrimeScript (trade name) RT Enzyme Mix I (TaKaRa Bio Inc.) is used to perform reverse transcription reaction E x Ta (Product name) (TaKaRa Bio Inc.)
  • E2F2 and GAPDH primers purchased from TAKARA Perfect Real Time Primer (initial denaturation) 95 ° C, 20 seconds (PCR reaction: 28 cycles) 94 ° C 20 seconds , 55 ° C. for 20 seconds and 72 ° C. for 20 seconds.
  • E2F2 sense primer (5′-AGCGGGCGCATCTATGACATC-3 ′) (SEQ ID NO: 1)
  • E2F2 antisense primer (5′-TCCAAGGCCTGCTCCGTGTT-3 ′)
  • GAPDH sense primer 5'-GCACCGTCAAGGCTGGAGAAC-3)
  • GAPDH antisense primer 5'-TGGTGAAGACGCCATGTG-3-3)
  • E2F2 mRNA expression of E2F2 gene in HeLa cells was most down-regulated by Myc-1, Myc-2Py-Im polyamide alone, and Py-Im polyamide combined with Myc-1 and Myc-2 (FIG. 4).
  • the expression level of E2F2 gene mRNA was 96.2 for Myc-1 compared to the expression level of E2F2 gene mRNA in HeLa cells without Py-Im polyamide control.
  • Myc-2 is 94.6%
  • Myc-1 and Myc-2 combination is 64.8%
  • Myc-2 and Myc-3 combination is 21.7%
  • Myc-1 and Myc-3 combination is Each decreased by 18.0%.
  • Myc-3 increased the expression level of mRNA by 74.1% compared to the control, and negative control (mismatch Py-Im No. 92) increased by 39.0% compared to the control (FIG. 5).
  • Myc-3 and mismatch Py-Im No. It was found that the expression level of mRNA of the E2F2 gene was decreased by all Py-Im polyamide compounds other than 92.
  • Myc-1, Myc-2 alone or a combination of Myc-1 and Myc-2 is one of the important c-MYC downstream genes and plays an important role in cell proliferation. It has been shown to down-regulate the E2F2 gene that has been proven to be responsible.
  • TAKARA DICE real time system (trade name) (TaKaRa Bio Inc.)
  • PrimeScript (trade name) RT Enzyme Mix I (TaKaRa Bio Inc.) is used to perform reverse transcription reaction E x Ta (Trade name) (TaKaRa Bio Inc.)
  • E2F2 and GAPDH primer purchased from TAKARA Perfect Real Time Primer (initial denaturation) 95 ° C, 20 seconds (PCR reaction: 28 cycles) 94 ° C, 20 Second, 55 ° C., 20 seconds, 72 ° C., 20 seconds.
  • Specific primer base sequences are eIF4G1 sense primer (5′-CTCATGACGGCTGTCTGCTA-3 ′) (SEQ ID NO: 5), eIF4G1 antisense primer (5′-TGGGAGGCTGTTCTAGAGTC-3 ′) (SEQ ID NO: 6), MYC sense primer ( 5′-TACCCCTCTCAACGACAGCAG-3 ′) (SEQ ID NO: 7), MYC antisense primer (5′-TCTTGACATTCTCCTCGGGTG-3 ′) (SEQ ID NO: 8), CDK4 sense primer (5′-CATCGTTCACCGAGATCTGA-3 ′) (SEQ ID NO: 9) CDK4 antisense primer (5′-CCAACACTCCACATGTCCAC-3 ′) (SEQ ID NO: 10), CCND1 sense primer (5′-CACTTGCATG TCGTGGCCT-3 ′) (SEQ ID NO: 11), CCND1 antisense primer (5′-GGAGAGGAAGTGTTCAATGA-3 ′
  • Adherent cells were detached from the dish using cultured cell trypsin, and floating cells were collected from the culture medium, and the centrifuged cell pellet was washed with PBS and washed with PBS.
  • Qiagen and RNeasy total RNA kit RNA extraction was performed. The concentration of the extracted RNA was measured for ultraviolet absorbance using NanoDrop. 1 ⁇ g of purified RNA was converted to cDNA using PrimeScript TM RT regent kit from TAKARA, and quantitative PCR was performed using SYBER premix EX Taq kit. The reaction was carried out using Thermal Cycler Dice TM Real Time System TP800 for 40 cycles of 95 ° C. for 2 minutes, 94 ° C.
  • Specific primer base sequences are eIF4G1 sense primer (5′-CTCATGACGGCTGTCTGCTA-3 ′) (SEQ ID NO: 5), eIF4G1 antisense primer (5′-TGGGAGGCTGTTCTAGAGTC-3 ′) (SEQ ID NO: 6), MYC sense primer ( 5′-TACCCCTCTCAACGACAGCAG-3 ′) (SEQ ID NO: 7), MYC antisense primer (5′-TCTTGACATTCTCCTCGGGTG-3 ′) (SEQ ID NO: 8), CDK4 sense primer (5′-CATCGTTCACCGAGATCTGA-3 ′) (SEQ ID NO: 9) CDK4 antisense primer (5′-CCAACACTCCACATGTCCAC-3 ′) (SEQ ID NO: 10), CCND1 sense primer (5′-CACTTGCATG TCGTGGCCT-3 ′) (SEQ ID NO: 11), CCND1 antisense primer (5′-GGAGAGGAAGTGTTCAATGA-3 ′
  • Myc-5 decreased the expression of the cyclin D1 (CCND1) gene in HeLa cells in a concentration-dependent manner (Myc-5, 5 ⁇ M decreased 95.1% of the control, Myc-5, 10 ⁇ M compared to the control). 95.6% decrease), whereas P493-6 cells showed the opposite reaction (Myc-5, 1 ⁇ M decreased 56.7% vs. control, Myc-5, 5 ⁇ M increased 64.8% vs. control) Myc-5, 10 ⁇ M, 46.7% increase over control). Also in K562 cells, expression of eIF4G1 gene and CCND1 gene was suppressed by Myc-5.
  • CDK4 gene expression was not affected by Myc-4 and / or Myc-5 in any of the cells (FIGS. 7A, 7B, and 7C).
  • the eIF4G1 gene and the CCND1 gene have one E-BOX sequence in the upstream promoter region and are recognized by Myc-5.
  • the CDK4 gene has four E-BOXs in its promoter region, but only one of the four E-BOX sequences of the CDK4 gene (E-Box 2 in FIG. 19) is recognized by Myc-5 ( FIG. 19). That is, it is considered that inhibition of MYC protein binding with only E-Box2 did not affect the expression, and other E-BOX regions (E Box1, E Box3, and E Box4 in FIG.
  • Myc-5 down-regulates the expression of Myc downstream gene eIF4G1 in P493-6 cells, HeLa cells and K562 cells, and down-regulates the expression of CCND1 gene in HeLa cells and K562 cells. It was shown that.
  • Myc-2 was shown to down-regulate MDM2 gene expression and up-regulate p53 gene expression in K562 cells (FIG. 7-D).
  • MDM2 is a gene that controls the p53 gene, which is a tumor suppressor gene, and the suppression of MDM2 gene expression by Myc-2 is considered to be due to upregulation of the p53 gene.
  • K562 cells human chronic myeloid leukemia-derived cells
  • RPMI 1640 Nacalai Tesque
  • 3 ⁇ 10 5 K562 cells were cultured for 72 hours in a medium containing 1, 5, 10 uM Myc-5 polyamide or mismatch B polyamide and a culture solution (control) to which those polyamides were not added.
  • the cultured cells were centrifuged at 2500 ⁇ g for 10 minutes, the cell pellet was washed with PBS solution, and 100 ⁇ l of 4 ° C.
  • the cell lysate was mixed with 33X Laemmle sample buffer (WaKo Junyaku) at a ratio of 1: 2, treated at 95 ° C for 5 minutes, and then the reaction solution containing 40 ⁇ g of the equivalent protein was added to 12.5% SDS-polyacrylamide gel. Electrophoresis was performed. The protein in the gel after electrophoresis was transferred to a PVDF membrane using an electric transfer device i-blot (Invitrogen), soaked in a 5% skim milk solution for 1 hour, and washed with a PBS solution for 10 minutes.
  • 33X Laemmle sample buffer WiKo Junyaku
  • Anti-cyclin D1 (CCND1) antibody (sc 246 Santa Cruz Lot # D2108), anti-eIF4G antibody (L-22, sc100730 Santa Cruz Lot # 1609), anti-CDK4 antibody (sc-53636 Santa Cruz Lot # D0507) and anti- ⁇ -actin
  • CCND1 (CCND1) antibody sc 246 Santa Cruz Lot # D2108)
  • anti-eIF4G antibody L-22, sc100730 Santa Cruz Lot # 1609
  • anti-CDK4 antibody sc-53636 Santa Cruz Lot # D0507
  • Anti- ⁇ -actin Each diluted solution of antibody (Sigma A5441) was immersed in a transfer PVDF membrane for 1 hour, washed with PBS solution for 10 minutes three times, and then the transferred PVDF membrane was immersed in a diluted solution of peroxidase-polymerized anti-mouse IgG antibody for 1 hour.
  • MCF-7 cells human breast cancer-derived cells
  • FBS Invitrogen
  • HeLa cells human cervical cancer-derived cells were seeded at 4 ⁇ 3 ⁇ 10 4 in each well of a 6-well plate. The cells were cultured in RPMI 1640 medium containing 2 ml of 10% FBS (Invitrogen) at 37 ° C., 5% CO 2 .
  • FITC-conjugated Myc-5 was added to HeLa cells at a final concentration of 5 ⁇ M in the growth medium and cultured for 2 hours. Cells were washed and FBS free medium was added. At a predetermined time, viable cells were observed at x20 magnification and fixed with 4% paraformaldehyde for 10 minutes. Nuclei were stained with Hoechst 3342 (Invitrogen Life Technologies, Corp., Carlsbad, Calif.) And observed again. II. Results FITC-conjugated Myc-5 was observed to be localized in the nucleus in all cells 30 minutes after being added to the growth medium in which MCF-7 cells were cultured. Even 2 hours after the addition, it was confirmed that FITC-bound Myc-5 was stably present in the nuclei of MCF-7 cells and HeLa cells (FIGS. 21 and 22).
  • An eIF4G oligo DNA having a two-base mutation (mismatch-1) (5′-FITC-TGGCTG TCAGCTGA CGGGGCGTTTTCGCCCCGTCAGCTGACAGCCCA-3 ′) (SEQ ID NO: 16) was synthesized in the same manner, A mutant eIF4G double-stranded oligonucleotide having a two-base mutation was used.
  • As the single-stranded DNA (5′-FITC-ATGGGAAATCAGGGTGGGCGGGGT-3 ′) (SEQ ID NO: 17) was used. No. which recognizes 5′-WCWGGCWGW-3 ′ (W is A or T) as a mismatched polyamide. 120 polyamide (mismatch B) was used.
  • Chromatin Immunoprecipitation Assays (Chromatin Immunoprecipitation Assays) I. Materials and Methods 3 ⁇ 10 6 human chronic myeloid leukemia-derived cells K562 cells were seeded and cultured with Myc-5 at a concentration of 10 ⁇ M for 72 hours. Chromatin immunoprecipitation assay was performed using Upstate Biotechnology's Chromatin Immunoprecipitation (ChIP) Assay Kit. Formalin was added to K562 cells cultured for 72 hours to a final concentration of 1%, and the cells were fixed by allowing to stand at 37 ° C. for 10 minutes. Thereby, protein and DNA are cross-linked in the cell.
  • ChIP Chromatin Immunoprecipitation Assays
  • glycine was added to a final concentration of 0.25 M, the supernatant was removed, and the cell layer was washed.
  • 220 ⁇ l of lysis buffer was added to the cell layer, and the cell lysate was recovered using a scraper. While submerging the obtained cell lysate in water at 4 ° C, the cells in the cell lysate were crushed using BIORUPTOR (SANYO) (strength is H, 30 seconds of sonication and 1 minute of suspension for one cycle) And this was repeated 10 times). Next, centrifuge at 15000 xg for 10 minutes at 4 ° C.
  • BIORUPTOR BIORUPTOR
  • sepharose was suspended in 250 ⁇ l of 0.1 M NaHCO 3 -1% SDS solution, and 15 minutes later, centrifugation was performed at 1000 ⁇ g for 1 minute to precipitate the sepharose again.
  • the precipitated sepharose was suspended again in 250 ⁇ l of 0.1 M NaHCO 3 -1% SDS solution, and 15 minutes later, centrifugation was performed at 1000 ⁇ g for 1 minute to precipitate sepharose.
  • a total of 500 ⁇ l of eluate was collected, NaCl was added to a final concentration of 200 mM, and a treatment was performed at 65 ° C. for 4 hours. Next, treatment was carried out at 45 ° C.
  • a GAPDH gene primer set forward: 5′-TACTAGCGGTTTTACGGGCG-3 ′ (SEQ ID NO: 25), reverse: 5′-TCGAACAGGAGGAGCAGAGAGCGA-3 ′ (SEQ ID NO: 26) was used. The presence of immunoprecipitation was confirmed.
  • the primers around the EIFBOX binding region of the eIF4G1 promoter are the forward: 5′-GAATCGGTCTGGGAGTTTCA-3 ′ (SEQ ID NO: 27) and reverse: 5′-CACAGACGTAGTCCACAACCA- which amplify the region from ⁇ 1092 to -8645 from the first ATG codon of eIF4G1.
  • Caspase 3 activity assay Materials and Methods The caspase 3 activity effects of 10 nM, 500 nM Myc-2, and 1 ⁇ M, 10 ⁇ M Myc-5 in human chronic myeloid leukemia-derived cells K562 cells were compared to control and positive controls.
  • a colorimetric caspase-3 assay kit (Sigma) kit was used to measure caspase 3 activity. K562 cells were seeded to 3 ⁇ 10 6 cells, and Myc-2 and Myc-5 were added to the medium so as to obtain the specified final concentration, and 10% fetal calf serum-containing RPMI 1640 (Nacalai Tesque) medium at 37 ° C., Culturing was carried out for 72 hours in a 5% CO2 incubator.
  • Cells were collected, adjusted to 10 7 cells in 100 microliter lysate (50 mM HEPES, pH 7.4, 5 mM CHAPS, 5 mM DTT), maintained on ice for 20 minutes, 20,000 Xg, 4 ° Centrifuge at C for 20 minutes, collect supernatant into 96 plates, add 100 microliter of reaction mixture and freshly prepared 10 microliter of DTT, and add 5 microliters of caspase-3 target acetyl- Asp- A solution containing Glu-Val-Asp p-nitroaniline was added and reacted overnight at 37 degrees. Absorbance at 405 nanometer was measured with a plate reader to measure caspase 3 activity. II.
  • the expression of a gene having the sequence as a regulatory region can be specifically regulated, and the mechanism is different from that of conventional chemotherapeutic agents. Therefore, it is considered that the side effects (depletion of white blood cells, nausea, vomiting, etc.) of conventional chemotherapeutic agents can be theoretically avoided.
  • the present invention is a compound, it does not have the disadvantage of being degraded by ribonuclease, such as a drug for regulating MYC downstream gene expression, a drug for treating a MYC gene-related disease and an antineoplastic agent, induction of stem cells, etc. It can be used as a cell differentiation inducer.
  • the target or target group of the MYC downstream gene can be determined.
  • a functional test reagent used for molecular biological research and drug development research can be obtained by the selective regulatory effect of the compound according to the present invention.

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Abstract

La présente invention concerne un agent comprenant un pyrrole-imidazole polyamide contenant une unité N-méthylpyrrole (nommée « Py », ci-après dans ce document), une unité N-méthylimidazole (nommée « Im », ci-après dans ce document) et une unité d'acide γ-aminobutyrique. Le pyrrole-imidazole polyamide est plié dans la région de l'unité de l'acide γ-aminobutyrique pour adopter une conformation en U dans un sillon mineur d'une région en double hélice (nommée « région cible », ci-après dans ce document), la région en double hélice étant composée d'une séquence contenant une séquence de région E-Box d'un gène MYC en aval comprenant une séquence nucléotidique spécifique, tout ou partie d'une séquence spécifique qui est une séquence antisens de la séquence de la région E-Box et une séquence nucléotidique constituée de 1 à 2 nucléotides en amont et/ou en aval de la région E-Box; et d'une séquence complémentaire de la séquence susmentionnée. Dans le pyrrole-imidazole polyamide, une paire Py/Im correspond à une paire de bases C-G, une paire Im/Py correspond à une paire de bases G-C, et une paire Py/Py correspond à la fois à une paire de bases A-T et à une paire de bases T-A.
PCT/JP2009/066111 2009-03-13 2009-09-15 Régulateur de l'expression spécifique de la séquence qui cible le gène myc en aval et méthode permettant de déterminer la cible ou les cibles du gène myc en aval WO2010103684A1 (fr)

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