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Sequence-selective gene expression regulators

Foreign code F200010115
File No. 6151
Posted date May 18, 2020
Country United States of America
Application number 201916410046
Gazette No. 20190343873
Date of filing May 13, 2019
Gazette Date Nov 14, 2019
Priority data
  • P2018-093273 (May 14, 2018) JP
Title Sequence-selective gene expression regulators
Abstract A conjugate comprising a pyrrole-imidazole polyamide that recognizes a specific DNA sequence and a bromodomain inhibitor, a composition for regulating biochemical activity, specifically for regulating histone modification, e.g., for inducing histone acetylation, which comprises the conjugate, a composition for recruiting a bromodomain-containing protein which comprises the conjugate, a method for nucleosome acetylation which comprises using the conjugate, and a method for bromodomain-containing protein recruitment which comprises using the conjugate are provided.
Outline of related art and contending technology BACKGROUND OF THE INVENTION
Eukaryotic genomes have their DNAs packed in form of chromatin into the nucleus. The chromatin has nucleosomes as the fundamental repeating units. Each of the units consists of a segment of DNA and histones. It is believed that the structure of chromatin and gene expression are regulated by epigenetic modifications of histones and DNA.
Posttranslational modifications (PTMs) on histone proteins play significant roles in epigenetic regulation of eukaryotic chromatin. Acetylation of lysine residues is one of the major PTMs on histones, and is strongly correlated with transcriptional activation. The code governing histone acetylation is regulated by histone acetyltransferases (HATs) as writers and by histone deacetylases (HDACs) as erasers. Acetylated histones are generally found in euchromatin and activated gene regions, and known to operate via two mechanisms. One is to decrease the positive charge of histone proteins, resulting in weakened interaction between histones and DNA, which in turn open up the chromatin structure. The other is mediated by bromodomain (BD)-containing proteins, which selectively bind to acetylated lysine residues. Although locus-specific (i.e., DNA sequence-specific) regulation of histone acetylation has been shown to be essential in biological processes, techniques for controlling histone acetylation at any region of interest remain limited.
Pyrrole-imidazole polyamides (hereinafter, referred to as “PI polyamides” or “PIP”) which consist of N-methyl pyrrole and N-methyl imidazole are synthetic oligomers that recognize specific DNA sequences located within the minor groove by virtue of their pyrrole (P) and imidazole (I) pairs interlocked by a hairpin linkage. In the anti-parallel structure, a pair of pyrrole and imidazole (P/I) recognizes C⋅G base pair, a pair of pyrrole and pyrrole (P/P) recognizes A⋅T or T⋅A base pair, and a pair of imidazole and pyrrole (I/P) recognizes G⋅C base pair. PI polyamides can specifically bind to any double-stranded DNA sequence by virtue of the above recognitions. Thus, designing the order of PI pairs enables in vivo delivery of PI polyamides to the targeted site in genome. PI polyamides targeting nuclear DNAs have been variously applied.
In recent study, PI polyamides were conjugated with a potent histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid-conjugated (SAHA) (see Pandian, G. N. et al., Sci Rep 4, 3843, doi:10.1038/srep03843 (2014); and Saha, A. et al., Bioorg Med Chem 21, 4201-4209, doi:10.1016/j. bmc. 2013. 05. 002 (2013)). PI polyamides were also conjugated with a HAT-activating compound, N-(4-chloro-3-(trifluoromethyl)phenyl)-2-ethoxybenzamide-conjugated (CTB) (see WO2016/129680). The SAHA-conjugated PI polyamides and the CTB-conjugated PI polyamides were shown to specifically upregulate the expression of a targeted gene. However, the level of gene expression activation is not stable.
Scope of claims [claim1]
1. A conjugate comprising a pyrrole-imidazole polyamide that recognizes a specific DNA sequence and a bromodomain inhibitor.

[claim2]
2. The conjugate according to claim 1, wherein the bromodomain inhibitor is a compound that interacts with a bromodomain contained in a protein selected from the group consisting of a histone acetyltransferase, a histone methyltransferase, an ATP-dependent chromatin-remodeling complex protein, and a BET protein.

[claim3]
3. The conjugate according to claim 2, wherein the bromodomain inhibitor is a compound that interacts with a histone acetyltransferase.

[claim4]
4. The conjugate according to claim 1, wherein the bromodomain inhibitor is a 5-isoxazolyl-benzimidazole compound.

[claim5]
5. The conjugate according to claim 4, wherein the 5-isoxazolyl-benzimidazole compound is a compound represented by the following formula:
wherein,
R is NH2 or NHCOCH2CH2CH2COOH,
R′ is H, CH3, F, Cl, or NO2,
R″ is a group represented by the following formula:
R′″ is H or CH3,
R″″ is H or CH3,
X is CH or N,
Y is CH or O, and
Z is CH or O.

[claim6]
6. The conjugate according to claim 3, which is represented by Formula I:
or Formula II:

[claim7]
7. A composition for regulating biochemical activity, comprising the conjugate according to claim 1.

[claim8]
8. A composition for regulating modification of histone, comprising the conjugate according to claim 1.

[claim9]
9. A method of DNA sequence-selectively acetylating a nucleosome, the method comprising bringing the conjugate according to claim 3 into contact with a sample containing a nucleosome.

[claim10]
10. A composition for recruiting a bromodomain-containing protein, comprising the conjugate according to claim 1.

[claim11]
11. A method of DNA sequence-selectively recruiting a bromodomain-containing protein, the method comprising bringing the conjugate according to claim 1 into contact with a sample.
  • Inventor, and Inventor/Applicant
  • SUGIYAMA Hiroshi
  • SERIE Kazuo
  • KYOTO UNIVERSITY
IPC(International Patent Classification)
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