acetyl-CoA carboxylase 2 antisense oligonucleotides

文档序号：816701 发布日期：2021-03-26 浏览：11次中文

阅读说明：本技术 乙酰辅酶a羧化酶2反义寡核苷酸 (acetyl-CoA carboxylase 2 antisense oligonucleotides ) 是由韩璿瑛成基浩洪铭涍姜多映许情奭张降愿于 2019-08-05 设计创作，主要内容包括：本发明提供了靶向人ACC2前体mRNA“外显子12”的5’剪接位点的肽核酸衍生物。本发明的肽核酸衍生物在细胞中强烈诱导人ACC2 mRNA的剪接变体,并且对于治疗与人ACC2蛋白相关的皮肤衰老状况或病症非常有用。(The present invention provides peptide nucleic acid derivatives that target the 5' splice site of "exon 12" of the human ACC2 precursor mRNA. The peptide nucleic acid derivatives of the present invention strongly induce splice variants of human ACC2mRNA in cells and are very useful for treating skin aging conditions or disorders associated with human ACC2 protein.)

1. A peptide nucleic acid derivative represented by formula I, or a pharmaceutically acceptable salt thereof:

[ formula I ]

Wherein the content of the first and second substances,

n is an integer between 10 and 21;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA or partially complementary to human ACC2 pre-mRNA, with one or two mismatches;

S₁、S₂、…、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIndependently represent a hydrogen group, a deuterium group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group;

x and Y independently represent hydrogen radical, deuterium radical, formyl [ H-C (═ O) -]Aminocarbonyl [ NH ]₂-C(＝O)-]Aminothiocarbonyl [ NH ]₂-C(＝S)-]Substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted alkanoyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothocarbonyl, substituted or unsubstituted alkoxythiocarbonyl, substituted or unsubstituted aryloxythiocarbonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted alkylphosphono, or substituted or unsubstituted arylphosphono;

z represents a hydrogen group, a deuterium group, a hydroxyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted amino group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group;

B₁、B₂、…、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases; and the number of the first and second groups,

B₁、B₂、…、B_n-1and B_nIs independently selected from a non-natural nucleobase having a substituted or unsubstituted amino group covalently linked to a nucleobase moiety.

2. The peptide nucleic acid derivative of claim 1, or a pharmaceutical salt thereof:

wherein the content of the first and second substances,

n is an integer between 10 and 21;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA or partially complementary to human ACC2 pre-mRNA, with one or two mismatches;

S₁、S₂、…、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIndependently represent a hydrogen group, a tritium group;

z represents a hydrogen group, a hydroxyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted amino group;

B₁、B₂、…、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases;

B₁、B₂、…、B_n-1and B_nIs independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IV:

wherein the content of the first and second substances,

R₁、R₂、R₃、R₄、R₅and R₆Independently selected from hydrogen radicals and substituted or unsubstituted alkyl radicals;

L₁、L₂and L₃Is a covalent linker represented by formula V, covalently linking a basic amino group to a nucleobase moiety:

[ formula V ]

Wherein the content of the first and second substances,

Q₁and Q_mIs a substituted or unsubstituted methylene group (-CH)₂-, and Q_mDirectly to a basic amino group;

Q₂、Q₃… and Q_m-1Independently selected from the group consisting of substituted or unsubstituted methylene, oxy (-O-), thio (-S-), and substituted or unsubstituted amino [ -N (H) -, or-N (substituent) -](ii) a And the number of the first and second groups,

m is an integer between 1 and 15.

3. The peptide nucleic acid derivative of claim 2, or a pharmaceutical salt thereof:

wherein the content of the first and second substances,

n is an integer between 11 and 16;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA;

S₁、S₂、…、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIs a hydrogen radical;

x and Y independently represent a hydrogen group, a substituted or unsubstituted alkanoyl group or a substituted or unsubstituted alkoxycarbonyl group;

z represents a substituted or unsubstituted amino group;

B₁、B₂、…、B_n-1and B_nIndependently selectFrom natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases;

B₁、B₂、…、B_n-1and B_nIs independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IV;

R₁、R₂、R₃、R₄、R₅and R₆Is a hydrogen radical;

Q₁and Q_mIs methylene, and Q_mDirectly to a basic amino group;

Q₂、Q₃… and Q_m-1Independently selected from methylene and oxy; and the number of the first and second groups,

m is an integer between 1 and 9.

4. The peptide nucleic acid derivative of claim 3, or a pharmaceutical salt thereof:

wherein the content of the first and second substances,

n is an integer between 11 and 16;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA;

S₁、S₂、…、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIs a hydrogen radical;

x is a hydrogen radical;

y represents a substituted or unsubstituted alkoxycarbonyl group;

z represents a substituted or unsubstituted amino group;

B₁、B₂、…、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases;

B₁、B₂、…、B_n-1and B_nAt least 5 of which are independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IVA group;

R₁、R₂、R₃、R₄、R₅and R₆Is a hydrogen radical;

L₁represents- (CH)₂)₂-O-(CH₂)₂-、-CH₂-O-(CH₂)₂-、-CH₂-O-(CH₂)₃-、-CH₂-O-(CH₂)₄-or-CH₂-O-(CH₂)₅-; and the number of the first and second groups,

L₂and L₃Independently selected from- (CH)₂)₂-O-(CH₂)₂-、-(CH₂)₃-O-(CH₂)₂-、-(CH₂)₂-O-(CH₂)₃-、-(CH₂)₂-、-(CH₂)₃-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-、-(CH₂)₇-and- (CH)₂)₈-。

5. The peptide nucleic acid derivative of claim 4, which is selected from the group consisting of peptide nucleic acid derivatives of the following group, or a pharmaceutically acceptable salt thereof:

(N→C)Fethoc-CTG(6)-ACG(6)-AA(5)A-TG(6)G-C(1O2)C-NH₂；

(N→C)Fethoc-TA(5)C(1O2)-TGA(5)-CGA(5)-AA(5)T-G(6)GC(1O2)-C-NH₂；

(N→C)Fethoc-TA(5)C-TG(5)A-C(1O2)GA(5)-AA(5)T-G(5)G-NH₂；

(N→C)Fethoc-AC(1O2)T-GA(5)C-GA(5)A-A(5)TG(5)-GC(1O2)-NH₂；

(N→C)Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-NH₂；

(N→C)Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-C(1O2)C-NH₂；

wherein the content of the first and second substances,

A. g, T and C are peptide nucleic acid monomers having the natural nucleobases adenine, thymine, guanine and cytosine, respectively;

c (pOq), A (p) and G (p) are peptide nucleic acid monomers having non-natural nucleobases represented by formula VI, formula VII and formula VIII, respectively;

wherein the content of the first and second substances,

p and q are integers, as defined in (N → C) Fethoc-AC (1O2) T-GA (5) C-GA (5) A-A (5) TG (5) -GC (1O2) -NH₂Wherein p is 1 or 5 and q is 2; and

"Fethoc-" is an abbreviation of "[2- (9-fluorenyl) ethyl-1-oxy ] carbonyl".

6. A method of treating a condition or disorder associated with transcription of the human ACC2 gene comprising administering to a subject the peptide nucleic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof.

7. A method for treating skin aging, comprising administering the peptide nucleic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof to a subject.

8. A pharmaceutical composition for treating a condition or disorder associated with transcription of the human ACC2 gene, comprising the peptide nucleic acid derivative of claim 1, or a pharmaceutically acceptable salt thereof.

9. A cosmetic composition for treating a condition or disorder associated with transcription of the human ACC2 gene, comprising the peptide nucleic acid derivative of claim 1, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition for treating skin aging, comprising the peptide nucleic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof.

11. A cosmetic composition for treating skin aging, comprising the peptide nucleic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof.

Technical Field

The present invention relates to peptide nucleic acid derivatives that complementarily target the human acetyl-coa carboxylase 2 precursor mRNA (pre-mRNA) to improve skin aging mediated by acetyl-coa carboxylase 2.

Background

Skin aging has received considerable attention because signs of aging are most pronounced in the skin. Skin aging begins in the mid or late 20 years of age, with a reduction in collagen and elastin in the skin, resulting in dry and low elasticity skin and even wrinkles. Obesity is an inflammatory response caused by a decrease in blood circulation due to excessive deposition of fat in the body. Internal fat on blood vessels inhibits blood circulation and secretion of various hormones, thereby promoting aging of the whole body including the skin. In this sense, the health and disease associated with obesity must be monitored to make the skin healthy and beautiful.

The biosynthesis and degradation of fatty acids is well regulated according to physiological conditions to meet the needs of the body. Acetyl-coa carboxylase (ACC), a biotin-dependent enzyme, catalyzes the carboxylation of acetyl-coa to malonyl-coa, which is the rate-limiting step in the first stage of fatty acid biosynthesis.

ACC has the function of controlling fatty acid metabolism in two ways. The most important function of ACC is to provide a malonyl-coa substrate for fatty acid biosynthesis as a new component in its active state. Another function is to block oxidation of fatty acids in mitochondria by inhibiting acyl transfer of fatty acids.

The two major isoforms of ACC, acetyl-coa carboxylase 1(ACC1, ACACA, acetyl-coa carboxylase α) and acetyl-coa carboxylase 2(ACC2, ACACB, acetyl-coa carboxylase β), are expressed in humans. The two ACCs have different functions from each other, i.e. ACC1 maintains fatty acid synthesis regulation, whereas ACC2 primarily regulates fatty acid oxidation.

ACC, which regulates fatty acid biosynthesis and oxidation, is a potential therapeutic target for many diseases, such as novel antibiotics that utilize structural differences between bacteria and human ACC, metabolic syndrome of diabetes and obesity, adipogenesis-related cancer cell growth inhibitors, and the like [ Recent Patents cardiovasc. drug discov.vol. 2,162-80 (2007); PLoS One Vol12, e0169566(2017) ].

Wherein, at ACC2^-/-A study on mutant mice has attracted much attention, among which ACC 2-deficient mice have lower fat levels, higher rates of fatty acid oxidation, reduced or maintained weight loss and reduced risk of diabetes despite increased food intake [ Science Vol 291,2613-6(2001)]. These results indicate that ACC2 inhibitors may have therapeutic effects on obesity and diabetes. In addition, inhibitor treatment of the skin can be expected to produce a degreasing effect, and ultimately prevent skin obesity and improve skin aging.

In view of the importance of obesity in the process of skin aging, it is very interesting and necessary to develop an ACC2 inhibitor or a drug or cosmetic that can improve and prevent the skin aging condition based on the ACC2 expression mechanism.

Precursor mRNA: genetic information is carried on DNA (2-deoxyribonucleic acid). The DNA is transcribed in the nucleus to produce precursor mRNA (precursor messenger ribonucleic acid). Mammalian pre-mRNA is generally composed of exons and introns, exons andthe introns are interconnected in the manner shown in the following figures. The numbering of exons and introns is shown in the following figure.

[ Structure of precursor mRNA ]

Splicing of precursor mRNA: through a complex series of reactions collectively referred to as "splicing", the precursor mRNA is processed into mRNA following intron deletion, as shown below [ Ann. Rev. biochem.72(1),291- "336 (2003); nature Rev.mol.cell biol.6(5),386-398 (2005); nature Rev.mol.cell biol.15(2),108-121(2014)]。

Splicing is initiated by the formation of a "spliceosome E complex" (i.e., an early spliceosome complex) between the precursor mRNA and the spliced aptamer factor. In the "spliceosome E complex", U1 is bound at the junction of exon N and intron N, U2AF³⁵Binding at the junction of intron N and exon (N + 1). Therefore, exon/intron or intron/exon junctions are crucial for the formation of early spliceosome complexes. After further complexation with U2, the "spliceosome E complex" evolved into the "spliceosome a complex". The "spliceosome a complex" undergoes a series of complex reactions, deletion or splicing to remove introns to adjoin adjacent exons.

Ribosomal protein synthesis: the protein is encoded by DNA (2-deoxyribonucleic acid). In response to cellular stimulation or spontaneously, DNA is transcribed in the nucleus to produce precursor mRNA (precursor messenger ribonucleic acid). Introns of the precursor mRNA are removed by enzymatic splicing to produce mRNA (messenger ribonucleic acid) which is then translocated into the cytoplasm. In the cytoplasm, a translation machinery complex called ribosome binds to mRNA and carries out protein synthesis as it scans the encoded genetic information along the mRNA [ Biochemistry vol 41,4503-4510 (2002); cancer Res.vol 48,2659-2668(1988)]。

Antisense oligonucleotides (ASO): oligonucleotides that bind in a sequence specific manner (i.e., complementary) to nucleic acids, including DNA, mRNA and pre-mRNA, are referred to as antisense oligonucleotides (ASOs).

For example, if ASO binds tightly to mRNA in the cytoplasm, ASO can inhibit ribosomal protein synthesis along the mRNA. ASO needs to be present in the cytoplasm to inhibit ribosomal protein synthesis of its target protein.

Antisense inhibition of splicing: ASO can inhibit or modulate splicing of precursor mRNA into mRNA if it binds tightly to the precursor mRNA in the nucleus. ASO needs to be present in the nucleus to inhibit or modulate splicing of precursor mRNA into mRNA. This antisense inhibition of splicing produces one or more mrnas that lack the exon targeted by the ASO. Such mrnas are referred to as "splice variants" and encode proteins that are smaller than those encoded by full-length mrnas.

In principle, splicing can be interrupted by inhibiting the formation of a "spliceosome E complex". If the ASO binds tightly to the (5' → 3') exon-intron junction, i.e. the "5 ' splice site", the ASO blocks the formation of a complex between the precursor mRNA and factor U1, and thus blocks the formation of the "spliceosome E complex". Likewise, if the ASO binds tightly (5' → 3') at an intron-exon junction, i.e., the "3 ' splice site", a "spliceosome E complex" cannot be formed.

The 3 'splice site and the 5' splice site are schematically illustrated in the following figures.

Non-natural oligonucleotides: DNA or RNA oligonucleotides are susceptible to degradation by endogenous nucleases, limiting their therapeutic use. To date, many types of non-natural (not naturally occurring) oligonucleotides [ Clin. Exp. Pharmacol. physiol. vol 33,533-540(2006) have been developed and extensively studied]. Some of them show extended metabolic stability compared to DNA and RNA. The chemical structures of some representative non-natural oligonucleotides are provided below. It is predicted that such oligonucleotides will bind complementary nucleic acids as well as DNA or RNA.

Phosphorothioate oligonucleotides: phosphorothioate oligonucleotides (PTOs) are DNA analogs where one backbone phosphate oxygen atom is replaced with a sulfur atom per monomer. This small structural change renders the PTO relatively resistant to nuclease degradation [ Ann. Rev. biochem. vol.54, 367-402(1985)]。

Reflecting the structural similarity of PTO and DNA backbone, it is difficult to penetrate the cell membrane in most mammalian cell types. However, for certain types of cells that abundantly express DNA transporters, DNA and PTO exhibit good cell permeability. Systemically administered PTO is known to be readily distributed to the liver and kidney [ Nucleic Acids res. vol25,3290-3296(1997) ].

Lipofection has been commonly practiced in order to facilitate cellular penetration of PTO in vitro. Lipofection, however, physically alters the cell membrane causing cytotoxicity and is therefore not ideal for long-term in vivo therapeutic applications.

In the past 30 years, antisense PTOs and PTO variants have been evaluated clinically to treat cancer, immune system diseases, metabolic diseases, etc. [ Biochemistry vol 41,4503-4510 (2002); clin Exp. Pharmacol. Physiol. vol 33,533-540(2006) ]. Many such antisense drug candidates have not been successfully developed, in part due to the poor cellular permeability of PTO. To overcome the poor cell permeability, PTO needs to be administered at high doses for therapeutic activity. However, PTO is known to be associated with dose-limiting toxicity, including increased clotting time, complement activation, renal tubular nephropathy, Kupffer cell activation, and immune stimulation, including splenomegaly, lymphoid hyperplasia, mononuclear cell infiltration [ clin. exp. pharmacol. physiol. vol 33,533-540(2006) ].

Many antisense PTOs have been found to show expected clinical activity against diseases mainly caused by the liver or kidney. Mipomersen is a PTO analog that inhibits the synthesis of apoB-100, a protein involved in LDL cholesterol transport. Miporesen showed the expected clinical activity in atherosclerotic patients, most likely due to its preferential distribution in the liver [ Circulation vol 118(7),743-753(2008) ]. ISIS-113715 is a PTO antisense analog that inhibits the synthesis of protein tyrosine phosphate 1B (PTP1B) and was found to show therapeutic activity in type II diabetic patients [ curr. opin. mol. ther. vol.6, 331-336(2004) ].

Locked nucleic acid: in Locked Nucleic Acids (LNAs), the backbone ribose ring of RNA is structurally constrained to increase binding affinity to RNA or DNA. Therefore, LNA can be considered as a high affinity DNA or RNA analog [ Biochemistry vol45,7347-7355(2006)]。

Phosphoric acid diamide morpholino oligonucleotides: in Phosphodiamide Morpholino Oligonucleotides (PMOs), the backbone phosphate and 2-deoxyribose of DNA are replaced by phosphoramidate and morpholine, respectively [ appl. Microbiol. Biotechnol. vol 71,575-]. The DNA backbone is negatively charged, while the PMO backbone is uncharged. Thus, the binding between PMO and mRNA is not electrostatically repulsive between the backbones and tends to be stronger than the binding between DNA and mRNA. Since PMO is structurally very different from DNA, PMO is not recognized by the liver transporters that recognize DNA. PMO can show a different tissue distribution than PTO, but PMO, like PTO, cannot easily penetrate the cell membrane.

Peptide nucleic acids: peptide Nucleic Acid (PNA) is a polypeptide having a backbone of N- (2-aminoethyl) glycine unit, which was discovered by doctor Nielsen and colleagues [ Science vol 254,1497-1500(1991)]. The chemical structure and abbreviation of PNA are provided in the following figure. Like DNA and RNA, PNA also selectively binds complementary nucleic acids [ Nature (London) vol 365,566-568(1992)]. When bound to complementary nucleic acids, the N-terminus of a PNA is considered to be equivalent to the "5 'terminus" of DNA or RNA and the C-terminus of a PNA is equivalent to the "3' terminus" of DNA or RNA.

Like PMO, PNA backbone is uncharged. Thus, the binding between PNA and RNA tends to be stronger than the binding between DNA and RNA. Since PNA is significantly different in chemical structure from DNA, PNA is not recognized by liver transporters recognizing DNA, and shows different tissue distribution characteristics from DNA or PTO. PNAs, however, also hardly penetrate mammalian cell membranes [ Adv. drug Delivery Rev. vol. 55,267-280(2003) ].

Modified nucleobases improving membrane permeability of PNA: PNAs are rendered highly permeable to mammalian cell membranes by the introduction of modified nucleobases having cationic lipids or their covalently attached equivalents. The chemical structure of such modified nucleobases is provided below. Such modified nucleobases of cytosine, adenine and guanine are found to hybridize predictably and complementarily to guanine, thymine and cytosine, respectively [ PCT application nos. PCT/KR 2009/001256; EP 2268607; US8680253]。

The incorporation of such modified nucleobases into PNAs is similar to that of lipofection. By lipofection, the oligonucleotide molecule with the phosphate backbone is encapsulated by a cationic lipid molecule, such as lipofectamine, which lipofectamine/oligonucleotide complex tends to penetrate the cell membrane more easily than naked oligonucleotide molecules.

In addition to good membrane permeability, these PNA derivatives were found to have superior affinity for complementary nucleic acids. For example, introduction of 4-5 modified nucleobases on 11-13 base (13-mer) PNA derivatives readily achieves T of 20 ℃ or higher when forming duplexes with complementary DNA_mAnd (4) gain. This PNA derivative is highly sensitive to single base mismatches. Depending on the type of modified base and PNA sequence, a single base mismatch results in T_mThe loss was 11-22 ℃.

Small interfering RNA (siRNA): small interfering RNA (siRNA) refers to double-stranded RNA of 20-25 base pairs [ Microbiol. mol. biol. Rev. vo)l 67(4),657-685(2003)]. The antisense strand of the siRNA interacts with the protein in some way to form the "RNA-induced silencing complex" (RISC). RISC then binds to a specific portion of mRNA that is complementary to the siRNA antisense strand. The mRNA complexed with RISC undergoes cleavage. Thus, siRNA catalytically induces cleavage of its target mRNA, and thus inhibits protein expression of the mRNA. RISC does not always bind to the complete complement within its target mRNA, which raises concerns related to off-target effects of siRNA treatment. Like other classes of oligonucleotides having DNA or RNA backbones, sirnas have poor cellular permeability and therefore tend to exhibit poor in vitro or in vivo therapeutic activity unless properly formulated or chemically modified to have good membrane permeability.

ACC siRNA: a mixture of ACC1 siRNA and ACC2 siRNA was reported to inhibit ACC1 and ACC2mRNA and protein expression in glioblastoma cancer cell lines after transfection with 20nM of each lipid [ PLoS One Vol12, e0169566(2017)]. These results may be useful for studying ACC-related lipogenic (lipogenic) cancer metastasis.

Disclosure of Invention

Problems to be solved

Since obesity has a profound effect on skin aging, health conditions and diseases associated with obesity must be monitored to obtain healthy and beautiful skin.

Much attention has been paid to the study of obese ACC 2-/-mutant mice. In addition, although ACC sirnas have been reported to inhibit expression of ACC mRNA and proteins in cancer cell lines, sirnas are too expensive to manufacture and develop into anti-aging agents for the skin, not to mention the challenges of their delivery into the skin. Therefore, there is a need to develop a drug or cosmetic that can improve and prevent the skin aging condition based on the ACC2 expression mechanism.

Solution to the problem

The present invention provides Peptide Nucleic Acid (PNA) derivatives represented by formula I:

[ formula I ]

Wherein the content of the first and second substances,

n is an integer between 10 and 21;

the compound of formula I has at least 10-base complementary overlap with the 18-base (18-mer) precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA or partially complementary to human ACC2 pre-mRNA, with one or two mismatches;

B₁、B₂、…、B_n-1and B_nIndependent of each otherIs selected from the group consisting of natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases; and the number of the first and second groups,

B₁、B₂、…、B_n-1and B_nIs independently selected from a non-natural nucleobase having a substituted or unsubstituted amino group covalently linked to a nucleobase moiety.

The compounds of formula I induce skipping of "exon 12" in human ACC2 precursor mRNA (skiping), resulting in human ACC2mRNA splice variant lacking "exon 12" and thus are useful for inhibiting the functional activity of the gene that transcribes human ACC2 precursor mRNA.

The condition "n is an integer between 10 and 21" literally means that n is an integer that may be selected from the integer groups 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

The chemical structures of natural or unnatural nucleobases in PNA derivatives of formula I are illustrated in FIGS. 1a-1 c. The invention of natural (i.e. natural) or non natural (natural absence) nucleobases including but not limited to figure 1a-1c provided in the nucleobases. Such non-natural nucleobases are provided to illustrate the permissible diversity of nucleobases and therefore should not be construed as limiting the scope of the invention.

The substituents used to describe PNA derivatives of formula I are illustrated in FIGS. 2a-2 e. Fig. 2a provides an example of a substituted or unsubstituted alkyl group. Substituted or unsubstituted alkanoyl and substituted or unsubstituted aryloyl groups are illustrated in figure 2 b. Fig. 2c shows examples of substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted aryl, substituted or unsubstituted alkylsulfonyl or arylsulfonyl, and substituted or unsubstituted alkylphosphono or arylphosphono groups. Figure 2d provides examples of substituted or unsubstituted alkoxycarbonyl or aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, or arylaminocarbonyl groups. Examples of substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothiocarbonyl, substituted or unsubstituted alkoxythiocarbonyl and substituted or unsubstituted aryloxythiocarbonyl are provided in FIG. 2 e. Such exemplary substituents are provided to illustrate the variety of permissible substituents and, therefore, should not be construed as limiting the scope of the invention. One skilled in the art will readily appreciate that the oligonucleotide sequence is a major factor in the sequence specific binding of the oligonucleotide to the target pre-mRNA sequence at the N-or C-terminal substituent.

Such as the prior art [ PCT/KR2009/001256]]The compounds of formula I bind tightly to complementary DNA. The duplexes between the PNA derivatives of formula I and their full length complementary DNA or RNA have Tm values that are too high to be reliably determined in aqueous buffers. PNA compounds of formula I having high T with shorter length complementary DNA_mThe value is obtained.

The compound of formula I binds complementarily to the 5' splice site of "exon 12" of the human ACC2 precursor mRNA. [ NCBI reference sequence: NG _046907 ]. The 16-base sequence [ (5'→ 3') GCCAUUUCGUCAGUAU ] spans the junction of "exon 12" and "intron 12" in the human ACC2 pre-mRNA and consists of 8-bases from "exon 12" and 8-bases from "intron 12". Thus, a 16-base precursor mRNA sequence can be generally represented as [ (5'→ 3') GCCAUUC ┃ gucaguuu ], wherein the exon and intron sequences are represented by "upper case" and "lower case" letters, respectively, and the exon-intron junction is represented by "┃". The conventional representation of the precursor mRNA is further illustrated by the 30-base sequence [ (5'→ 3') GGAAGAGGCCAUUUC ┃ gucaguaucuccuuc ] spanning the junction of "exon 12" and "intron 12" in the human ACC2 precursor mRNA.

The compound of formula I binds tightly to the target 5' splice site of the human ACC2 pre-mRNA transcribed from the human ACC2 gene and interferes with the formation of the "spliceosome early complex", producing ACC2mRNA splice variants lacking "exon 12" (exon 12 skipping).

The strong RNA affinity allows the compounds of formula I to induce ACC2 "exon 12" skipping even when the PNA derivative has one or two mismatches with the target 5' splice site in the ACC2 pre-mRNA. Similarly, PNA derivatives of formula I can still induce ACC2 "exon 12" skipping in ACC2 mutant precursor mRNA with one or two SNPs (single nucleotide polymorphisms) at the target splice site.

The compounds of formula I have good cell permeability and can be easily delivered into cells as "naked" oligonucleotides, as shown in the prior art [ PCT/KR2009/001256 ]. Thus, the compounds of the invention induce an "exon 12" skipping in ACC2 pre-mRNA and produce ACC2mRNA splice variants lacking ACC2 "exon 12" in cells treated with the compounds of formula I as "naked" oligonucleotides. The compounds of formula I can be delivered into a cell without any means or formulation to effectively induce target exon skipping in the cell. In cells treated with the compounds of the invention as "naked" oligonucleotides at sub-femtomolar concentrations, the compounds of formula I readily induced skipping of ACC2 "exon 12".

Due to good cell or membrane permeability, PNA derivatives of formula I can be topically applied as "naked" oligonucleotides to induce skipping of ACC2 "exon 12" in the skin. The compounds of formula I need not be formulated for a specified therapeutic or biological activity to increase transdermal delivery to the target tissue. Typically, the compounds of formula I are dissolved in water and a co-solvent, and applied topically or transdermally at a sub-picomolar concentration to elicit the desired therapeutic or biological activity in the target skin. The compounds of the present invention do not require severe or invasive formulation to elicit surface therapeutic activity. However, the PNA derivative of formula I can be formulated with cosmetic ingredients or adjuvants into topical creams or lotions. The topical cosmetic cream or lotion can be used for treating skin aging.

The compounds of the invention may be topically administered to a subject at therapeutically or biologically effective concentrations of 1aM to greater than 1nM, which may vary depending on the dosing regimen, the condition or state of the subject, and the like.

The PNA derivatives of formula I may be formulated differently, including but not limited to injections, nasal sprays, transdermal patches, and the like. In addition, the PNA derivatives of formula I may be administered to a subject in a therapeutically effective dose, which may vary according to the indication, the route of administration, the dosing regimen, the condition or state of the subject, and the like.

The compounds of formula I may be used in combination with pharmaceutically acceptable acids or bases including, but not limited to, sodium hydroxide, potassium hydroxide, hydrochloric acid, methanesulfonic acid, citric acid, trifluoroacetic acid and the like.

The PNA derivatives of formula I or pharmaceutically acceptable salts thereof may be administered to a subject in combination with pharmaceutically acceptable adjuvants including, but not limited to, citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethylene glycol, polypropylene glycol, ethanol, isopropanol, sodium bicarbonate, distilled water, preservatives and the like.

Of interest are PNA derivatives of formula I or pharmaceutically acceptable salts thereof:

wherein the content of the first and second substances,

n is an integer between 10 and 21;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA or partially complementary to human ACC2 pre-mRNA, with one or two mismatches;

S₁、S₂、…、S_n-1、S_n、T₁、T₂、...、T_n-1and T_nIndependently represent a hydrogen group, a tritium group;

x and Y independently represent a hydrogen group, a deuterium group, a formyl group [ HC (═ O) -]Aminocarbonyl [ NH ]₂-C(＝O)-]Aminothiocarbonyl [ NH ]₂-C(＝S)-]Substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted alkanoyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylaminothiocarbonyl, substituted or unsubstituted arylaminothocarbonyl, substituted or unsubstituted alkoxythiocarbonyl, substituted or unsubstituted aryloxythiocarbonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted alkylphosphono, or substituted or unsubstituted arylphosphono;

z represents a hydrogen group, a hydroxyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, or a substituted or unsubstituted amino group;

B₁、B₂、...、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil and non-natural nucleobases;

B₁、B₂、...、B_n-1and B_nAt least four of (a) are independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IV;

wherein the content of the first and second substances,

R₁、R₂、R₃、R₄、R₅and R₆Independently selected from hydrogen radicals and substituted or unsubstituted alkyl radicals;

L₁、L₂and L₃Is a covalent linker represented by formula V, covalently linking a basic amino group to a nucleobase moiety:

[ formula V ]

Wherein the content of the first and second substances,

Q₁and Q_mIs a substituted or unsubstituted methylene group (-CH)₂-) a group, and Q_mDirectly to a basic amino group;

Q₂、Q₃._m-1Independently selected from the group consisting of substituted or unsubstituted methylene, oxy (-O-), thio (-S-), and substituted or unsubstituted amino [ -N (H) -, or-N (substituent) -](ii) a And the number of the first and second groups,

m is an integer between 1 and 15.

Of high interest are PNA oligomers of formula I, or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

n is an integer between 11 and 16;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA;

S₁、S₂、...、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIs a hydrogen radical;

x and Y independently represent a hydrogen group, a substituted or unsubstituted alkanoyl group or a substituted or unsubstituted alkoxycarbonyl group;

z represents a substituted or unsubstituted amino group;

B₁、B₂、…、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

B₁、B₂、…、B_n-1and B_nIs independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IV;

R₁、R₂、R₃、R₄、R₅and R₆Is a hydrogen radical;

Q₁and Q_mIs methylene, and Q_mDirectly to a basic amino group;

Q₂、Q₃…, and Q_m-1Independently selected from methylene and oxy; and the number of the first and second groups,

m is an integer between 1 and 9.

Of further interest are PNA derivatives of formula I, or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

n is an integer between 11 and 16;

the compound of formula I has at least 10-base complementary overlap with the 18-base precursor mRNA sequence [ (5'→ 3') GGCCAUUUCGUCAGUAUC ] in human ACC2 precursor mRNA;

the compound of formula I is fully complementary to human ACC2 pre-mRNA;

S₁、S₂、...、S_n-1、S_n、T₁、T₂、…、T_n-1and T_nIs a hydrogen radical;

x is a hydrogen radical;

y represents a substituted or unsubstituted alkoxycarbonyl group;

z represents a substituted or unsubstituted amino group;

B₁、B₂、…、B_n-1and B_nIndependently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and non-natural nucleobases;

B₁、B₂、…、B_n-1and B_nIs independently selected from the group consisting of non-natural nucleobases represented by formula II, formula III or formula IV;

R₁、R₂、R₃、R₄、R₅and R₆Is a hydrogen radical;

L₁represents- (CH)₂)₂-O-(CH₂)₂-、-CH₂-O-(CH₂)₂-、-CH₂-O-(CH₂)₃-、-CH₂-O-(CH₂)₄-or-CH₂-O-(CH₂)₅-; and the number of the first and second groups,

Of particular interest are PNA derivatives of formula I selected from the group of compounds provided below (hereinafter referred to as ASO1, 2, 3,4, 5 and 6, respectively) or pharmaceutically acceptable salts thereof:

(N→C)Fethoc-CTG(6)-ACG(6)-AA(5)A-TG(6)G-C(1O2)C-NH₂；

(N→C)Fethoc-TA(5)C(1O2)-TGA(5)-CGA(5)-AA(5)T-G(6)GC(1O2)-C-NH₂

(N→C)Fethoc-TA(5)C-TG(5)A-C(1O2)GA(5)-AA(5)T-G(5)G-NH₂；

(N→C)Fethoc-AC(1O2)T-GA(5)C-GA(5)A-A(5)TG(5)-GC(1O2)-NH₂；

(N→C)Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-NH₂；

(N→C)Fethoc-CTG(6)-AC(1O2)G-A(5)AA(5)-TG(6)G-C(1O2)C-NH₂

wherein the content of the first and second substances,

A. g, T and C are PNA monomers with the natural nucleobases adenine, thymine, guanine and cytosine, respectively;

c (pOq), A (p) and G (p) are PNA monomers having non-natural nucleobases represented by formula VI, formula VII and formula VIII, respectively;

wherein the content of the first and second substances,

p and q are integers, for example p is 1 or 5 and q is 2 in the case of ASO 4; and

"Fethoc-" is "[2- (9-fluorenyl) ethyl-1-oxyl]Abbreviation of carbonyl, "-NH₂"is an unsubstituted" amino "group.

FIG. 3 provides in general the explicit chemical structures of PNA monomers abbreviated A, G, T, C, C (pOq), A (p) and G (p). Such as the prior art [ PCT/KR2009/001256]]As discussed above, a "modified cytosine" PNA monomer is considered to be due to the hybridization of C (pOq) to "guanine". A (p) is considered a "modified adenine" PNA monomer due to hybridization to "thymine" and G (p) is considered a "modified guanine" PNA monomer due to hybridization to "cytosine". In addition, to illustrate abbreviations for such PNA derivatives, ASO 1"(N → C) CTG (6) -ACG (6) -AA (5) A-TG (6) G-C (1O2) C-NH is provided in FIG. 4₂Chemical structure of。

ASO1 is equivalent to the DNA sequence "(5 '→ 3') CTG-ACG-AAA-TGG-CC", and binds complementarily to the pre-mRNA. 14-base PNA with 30-base RNA sequence spanning the junction of "exon 12" and "intron 12" in human ACC2 precursor mRNAThe 14-base sequences denoted by "bold" and "underlined" in (A) have 14-base complementary overlap.

In some embodiments, the present invention provides a method of treating a condition or disorder associated with transcription of the human ACC2 gene in a subject, comprising administering to the subject a peptide nucleic acid derivative of the present invention, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a method of treating skin aging in a subject, comprising administering to the subject a peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceutical composition for treating a condition or disorder associated with transcription of the human ACC2 gene, comprising a peptide nucleic acid derivative of the present invention, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a cosmetic composition for treating a condition or disorder associated with transcription of the human ACC2 gene, comprising a peptide nucleic acid derivative of the present invention, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceutical composition for treating skin aging, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a cosmetic composition for treating skin aging, comprising the peptide nucleic acid derivative of the present invention or a pharmaceutically acceptable salt thereof.

Effects of the invention

Conditions or disorders associated with the transcription of the human ACC2 gene may be treated by administering PNA derivatives of formula I or a pharmaceutically acceptable salt thereof.

Skin aging may be treated by administering a PNA derivative of formula I or a pharmaceutically acceptable salt thereof.

Brief Description of Drawings

FIGS. 1a-1 c: examples of natural or non-natural (modified) nucleobases which may be selected for peptide nucleic acid derivatives of formula I.

FIGS. 2a-2 e: examples of alternative substituents for peptide nucleic acid derivatives of formula I.

FIG. 3: chemical structure of PNA monomers with natural or modified nucleobases.

FIG. 4: chemical structure of "ASO 1".

FIG. 5: chemical structure of Fmoc-PNA monomers used for the synthesis of PNA derivatives of the present invention.

FIGS. 6a-6 b: c of "ASO 1" before and after HPLC purification, respectively₁₈Reversed phase HPLC chromatogram.

FIG. 7: through C₁₈ESI-TOF mass spectrum of RP preparative HPLC purified "ASO 1".

FIG. 8: exon skipping of ACC2mRNA by "ASO 1" in C2C 12.

FIG. 9: inhibition of ACC2mRNA levels by "ASO 1" in C2C 12.

FIG. 10: inhibition of ACC2mRNA levels by "ASO 6" in C2C 12.

FIG. 11: inhibition of ACC2mRNA levels by "ASO 5" in C2C 12.

Best Mode for Carrying Out The Invention

General procedure for preparation of PNA oligomers

According to the prior art [ US6,133,444; WO96/40685]The disclosed method was slightly modified to synthesize PNA oligomers by Solid Phase Peptide Synthesis (SPPS) based on Fmoc-chemistry. Fmoc is { (9-fluorenyl) methoxy } carbonyl. The solid support used in this study was H-Rink Amide-ChemMatrix available from PCAS BioMatrix Inc. (Quebec, Canada). Such as the prior art [ PCT/KR2009/001256]The Fmoc-PNA monomer with modified nucleobase was synthesized as described in (1) or with slight modifications. Such Fmoc-PNA monomers with modified nucleobases and Fmoc-PNA monomers with naturally occurring nucleobases are used for the synthesis of PNA derivatives of the present invention. Through C₁₈Reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) purification of PNA oligomersAnd identified by mass spectrometry including ESI/TOF/MS.

Scheme 1 illustrates a typical monomer extension cycle employed in the SPPS of this study, the details of which are provided below. However, it is apparent to those skilled in the art that there are many small variations to efficiently perform such SPPS reactions on an automated peptide synthesizer or a manual peptide synthesizer. Each reaction step in scheme 1 is briefly described below.

[ scheme 1]

[ activation of H-Rink-ChemMatrix resin ] when the amine on the resin is not protected with Fmoc, 0.01mmol (close to 20mg resin) of ChemMatrix resin in 1.5mL of 20% piperidine/DMF is vortexed in a Libra tube for 20 min, and the DeFmoc solution is filtered off. The resin was washed successively with 1.5mL of dichloromethane (MC), 1.5mL of Dimethylformamide (DMF), 1.5mL of MC, 1.5mL of DMF, and 1.5mL of MC for 30 seconds each. The resulting free amine on the solid support was coupled with Fmoc-PNA monomer.

[ DeFmoc ] when the amine on the resin was Fmoc protected, a suspension of 0.01mmol (approx. 20mg) of the resin in 1.5mL of 20% piperidine/DMF was vortexed for 7 minutes, and then the DeFmoc solution was filtered off. The resin was washed successively with 1.5mL of MC, 1.5mL of DMF, 1.5mL of MC, 1.5mL of DMF and 1.5mL of MC for 30 seconds each. The resulting free amine on the solid support was immediately coupled to the Fmoc-PNA monomer.

[ coupling with Fmoc-PNA monomer ] free amine on solid support was coupled with Fmoc-PNA monomer as described below. 0.04mmol of PNA monomer, 0.05mmol HBTU and 0.1mmol DIEA were incubated in 1mL anhydrous DMF for 2 min and added to the resin along with the free amine. The resin solution was vortexed for 1 hour and the reaction medium was filtered off. The resin was then washed successively with 1.5mL of MC, 1.5mL of DMF, and 1.5mL of MC each for 30 seconds. FIG. 5 provides the chemical structure of Fmoc-PNA monomer with modified nucleobases for use in the present invention. FIG. 5 provides Fmoc-PNA monomers with modified nucleobases as an example and therefore should not be considered as limiting the scope of the invention. Those skilled in the art can readily understand numerous variations of Fmoc-PNA monomers to synthesize PNA derivatives of formula I.

[ capping ] after the coupling reaction, the unreacted free amine was capped by shaking for 5 minutes in 1.5mL of a capping solution (5% acetic anhydride and 6% 2,6-leutidine in DMF). The capping solution is then filtered off and washed successively with 1.5mL of MC, 1.5mL of DMF and 1.5mL of MC each for 30 seconds.

[ introduction of a Fethoc group into the N-terminus]The "Fethoc-" group was introduced into the N-terminus by reacting the free amine on the resin with "Fethoc-OSu" as follows. A suspension of the resin in 0.1mmol of Fethoc-OSu and 0.1mmol of DIEA in 1mL of anhydrous MDF was vortexed for 1 hour and the solution was filtered off. The resin was washed successively with 1.5mL of MC, 1.5mL of DMF and 1.5mL of MC for 30 seconds each. "Fethoc-OSu" [ CAS No.179337-69-0, C ] used in the present invention₂₀H₁₇NO₅，MW 351.36]The chemical structure of (a) is as follows.

Cleavage from resin PNA oligomers bound to resin were cleaved from resin by shaking in 1.5mL of cleavage solution (2.5% triisopropylsilane and 2.5% water in trifluoroacetic acid) for 3 hours. The resin was filtered off and the filtrate was concentrated under reduced pressure. The resulting residue was triturated with ether and the resulting precipitate was collected by filtration for purification by reverse phase HPLC.

[ HPLC analysis and purification]After cleavage from the resin, by C₁₈The crude PNA derivative product was purified by reverse phase HPLC eluting with water/acetonitrile or water/methanol containing 0.1% TFA (gradient method). Fig. 6a and 6b are exemplary HPLC chromatograms of "ASO 1" before and after HPLC purification, respectively.

Synthetic examples of PNA derivatives of formula I

In order to complement the 5' splice site targeting "exon 12" in the human ACC2 precursor mRNA, PNA derivatives of the invention were prepared according to the synthetic methods provided above or with minor modifications. Such PNA derivatives targeting the pre-mRNA of human ACC2 are provided to exemplify PNA derivatives of formula I and should not be construed as limiting the scope of the invention.

Table 1 PNA derivatives complementary targeting the 5' splice site of "exon 12" in human ACC2 precursor mRNA and structure identification data by mass spectrometry.

^a)The theoretical accurate mass is obtained by the method,^b)accurate quality of observation

Table 1 provides complementary targeting of the gene from human ACC2 [ NCBI reference sequence: NG — 046907], of the 5' splice site of "exon 12" in human ACC2 pre-mRNA, and structure identification data by mass spectrometry. The peptide nucleic acid derivatives of the invention in table 1 are provided to illustrate PNA derivatives of formula I and should not be construed as limiting the scope of the invention.

"ASO 1" has a 30-base RNA sequence that spans the junction of "exon 12" and "intron 12" in the human ACC2 precursor mRNAThe 14-base sequences denoted in "bold" and "underlined" are complementary overlapping 14-base sequences. Thus, "ASO 1" has 9-base overlap with "exon 12" and 5-base overlap with "intron 12" within the human ACC2 pre-mRNA.

Binding affinity of "ASO" to complementary DNA

PNA derivatives of formula I were evaluated for binding affinity to complementary N-terminal or C-terminal targeted 10-base DNA. T by duplex between PNA and 10-base complementary DNA_mValues assess binding affinity. Duplexes between PNA derivatives and fully complementary DNA show T_mToo high a value cannot be reliably determined in aqueous buffer solutions, since the buffer solution is at T_mTending to boil during the measurement. T which can be based on a duplex between PNA and 10-base complementary DNA_mValues to predict and compare T for full-length PNA_mThe value is obtained.

Determination of T on a UV/Vis spectrometer as described below_mThe value is obtained. A mixed solution of 4. mu.M PNA oligomer and 4. mu.M complementary 10-base DNA in 4mL aqueous buffer (pH 7.16, 10mM sodium phosphate, 100mM NaCl) in a 15mL polypropylene Falcon tube was incubated at 90 ℃ for several minutes and then slowly cooled to ambient temperature. The solution was then transferred to a 3mL quartz UV cuvette equipped with a gas-tight cap and the cuvette was mounted on an Agilent 8453 UV/visible spectrophotometer. The change in absorbance at 260nm was recorded as the cuvette temperature increased by 0.5 ℃ or 1 ℃ per minute. From the absorbance versus temperature curve, the temperature showing the greatest increase in absorbance was read as T between PNA and 10-base DNA_m. For T_mThe DNA measured was purchased from Bioneer (www.bioneer.comDajeon, Republic of Korea) without further purification.

As shown in Table 2, PNA derivatives of formula I were observed for T binding complementary to 10-base DNA_mThe value is very high.

[ Table 2 ]]PNA T with 10-base complementary DNA targeting N-terminus or C-terminus of PNA_mValue of

For example, "ASO 1" is shown and targeted at T of a 10-base complementary DNA duplex of N-terminal 10-base in PNA denoted by "bold" and "underline_mThe value was 72.80 ℃. At the same time, "ASO 1" shows and targets In which the "bold" and "Underlined "T of duplexes of 10-base complementary DNA of C-terminal 10-base in PNA_mThe temperature was 79.60 ℃.

Examples of the biological Activity of PNA derivatives of formula I

The in vitro ACC2 antisense activity of the PNA derivatives of the present invention in C2C12 skeletal muscle cells was evaluated by using real-time quantitative polymerase chain reaction (RT-qPCR) and the like. The biological examples are provided merely to illustrate the biological profile of the PNA derivatives of formula I and therefore should not be construed as limiting the scope of the invention.

Example 1: "ASO 1" induced exon skipping in C2C12

The ability of "ASO 1" to induce ACC2 "exon 12" skipping in C2C12 cells was evaluated as described below.

[ cell culture and ASO treatment]5% CO in 60mm dishes containing DMEM medium (Dulbecco's modified Eagle Medium: DMEM) (catalog No. 12-604F, Lonza) supplemented with 10% FBS (fetal bovine serum) (catalog No. 10099-41, GIBCO) and 1% streptomycin/penicillin (catalog No. 15140-122, GIBCO)₂Atmospheric growth of C2C12 cells (2X 10) at 37 deg.C⁵) (catalog number CRL-1772, ATCC). Cells were either not treated at all (negative control) or treated with aliquots of "ASO 1" aqueous storage stock at 100zM to 1fM for 5 hours.

[ RNA extraction and nested PCR]Total RNA was extracted from "ASO 1" treated cells using the RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions and by using SuperScript^TMIII One-step RT-PCR System (Cat. No. 12574-018, Invitrogen) cDNA was prepared from 200ng RNA. 200ng RNA, 25. mu.l 2 × Reaction Mix buffer, 2. mu.l SuperScript III in PCR tubes^TMTo a mixture of RT/Platinum Taq Mix, 1 microliter 10. mu.M (micromolar concentration) exon 9 forward primer (5'-TTTTCCGACAAGTGCAGAG-3') and 1 microliter 10. mu.M exon 15 reverse primer (5'-AACGTCCACAATGTTCAG-3') was added autoclaved distilled water to a total volume of 50 microliters. After reaction at 60 ℃ for 30 minutes and at 94 ℃ for 2 minutes, 30 PCR cycles were entered as follows: at 94 ℃ for 15 seconds, at 50 ℃ for 30 seconds and at 68 ℃ for 1 minute to obtain a first crudeAnd (5) preparing a product. Using PyroHostStart Taq polymerase kit (Cat. No. K-2611-FCG) according to the manufacturer's instructions, a mixture of 1. mu.l of the crude product, 1. mu.l of 10. mu.M exon 10 forward primer (5'-GAG TAC TTA TAC AGC CAG G-3') and 1. mu.l of 10. mu.M exon 14 reverse primer (5'-TTC TGA ACA TCG CGT CTG-3') was reacted at 95 ℃ for 2 minutes and then reacted in the following PCR program: 30 seconds at 95 ℃,1 minute at 47 and 20 seconds at 72 ℃.

[ electrophoretic identification of exon skipping products ] the PCR products (10. mu.l) were electrophoretically separated on a 2% agarose gel. Target bands from "ASO 1" treatment were collected and analyzed by Sanger sequencing to assess exon skipping sequences.

[ exon skipping induced by "ASO 1] As shown in FIG. 8, the splice variant ACC2mRNA lacking exon 11 was produced concentration-dependently by treating cells with" ASO1 "at 0.1aM to 1 fM.

Example 2: inhibition of ACC2mRNA formation by "ASO 1" in C2C12

The ability of "ASO 1" to down-regulate ACC2mRNA formation in C2C12 was assessed by real-time qPCR as described below.

[ cell culture&ASO treatment]C2C12 cells (catalog number CRL-1772, ATCC) were stored in Dulbecco's modified Eagle's medium (DMEM, catalog number 12-604F, Lonza) supplemented with 10% fetal bovine serum (catalog number 10099-41, GIBCO) and 1% streptomycin/penicillin (catalog number 15140-122, GIBCO) at 37 ℃ and 5% CO 1514₂Growing under the condition. C2C12 cells (2X 10) stabilized in a 60mm dish for 24 hours⁵) Incubate with 0 (negative control) and 100zM to 1fM of "ASO 1" for 24 hours.

[ RNA extraction&cDNA Synthesis]Total RNA was extracted from "ASO 1" treated cells using the RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions and PrimeScript was used^TM 1^stStrand cDNA Synthesis kit (Takara, Cat. No. 6110A) prepared cDNA from 400ng RNA. To a mixture of 400ng RNA, 1. mu.l random hexamer and 1. mu.l dNTP (10mM) in a PCR tube, DEPC treated water was added to a total volume of 10. mu.l, and the reaction was carried out at 65 ℃ for 5 minutes. By adding to the reaction mixtureTo this was added 10. mu.l of PrimeScript RTase and the reaction was continued at 30 ℃ for 10 minutes and at 42 ℃ for 60 minutes to synthesize cDNA.

[ quantitative real-time PCR ] to assess the expression level of human ACC2mRNA, real-time qPCR was performed on the synthesized cDNA using Taqman probe. A mixture of cDNA, Taqman probe (Thermo, Mm01204651), IQ supermix (BioRad, Cat. No. 170- & 8862) and nuclease-free water in PCR tubes was reacted using CFX96 Touch real-time system (BioRad) according to cycling conditions as specified below: 3 minutes at 95 ℃ (primary denaturation) followed by 50 cycles as follows: 10 seconds at 95 ℃ (denaturation) and 30 seconds at 60 ℃ (annealing and polymerization). Fluorescence intensity was measured at the end of each cycle and the results of PCR were evaluated by melting curves. After normalization of the threshold cycle (Ct) for each gene with GAPDH, changes in Ct were compared and analyzed.

[ "ASO 1" reduces ACC2mRNA ] as can be seen in figure 9, the amount of ACC2mRNA decreased concentration-dependently upon treatment with 100zM to 1fM of "ASO 1" compared to the control experiment, with a statistically significant 30% decrease observed upon treatment with 1fM of "ASO 1". (Student T-test was performed to check the statistical significance of the findings).

Example 3: inhibition of ACC2mRNA formation by "ASO 6" in C2C12

The ability of "ASO 6" to down-regulate ACC2mRNA formation in C2C12 was assessed by real-time qPCR as described below.

[ RNA extraction&cDNA Synthesis]Total RNA was extracted from "ASO 6" treated cells using the RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions and PrimeScript was used^TM 1^stStrand cDNA Synthesis kit (Takara, Cat. No. 6110A) prepared cDNA from 400ng RNA. To a mixture of 400ng RNA, 1. mu.l random hexamer and 1. mu.l dNTP (10mM) in a PCR tube, DEPC treated water was added to a total volume of 10. mu.l, and the reaction was carried out at 65 ℃ for 5 minutes. cDNA was synthesized by adding 10. mu.l of PrimeScript RTase to the reaction mixture and reacting successively at 30 ℃ for 10 minutes and at 42 ℃ for 60 minutes.

[ "ASO 6" reduction of ACC2mRNA ] as can be seen in figure 10, the amount of ACC2mRNA decreased concentration-dependently upon treatment with 100zM to 1fM of "ASO 6". A 30% and 50% reduction in statistical significance, respectively, was observed when treated with 1aM and 1fM of "ASO 6" compared to the control experiment. (Student T-test was performed to check the statistical significance of the findings).

Example 4: inhibition of ACC2mRNA formation by "ASO 5" in C2C12

"ASO 5" was evaluated by the same method as described below.

[ cell culture&ASO treatment]C2C12 cells (catalog number CRL-1772, ATCC) were stored in Dulbecco's modified Eagle's medium (DMEM, catalog number 12-604F, Lonza) supplemented with 10% fetal bovine serum (catalog number 10099-41, GIBCO) and 1% streptomycin/penicillin (catalog number 15140-122, GIBCO) at 37 ℃ and 5% CO 1514₂Growing under the condition. C2C12 cells (2X 10) stabilized in a 60mm dish for 24 hours⁵) With 0 (negative control) and 100zM to 1f"ASO 5" incubation of M for 24 hours.

[ RNA extraction&cDNA Synthesis]Total RNA was extracted from "ASO 5" treated cells using the RNeasy Mini kit (Qiagen, Cat. No. 714106) according to the manufacturer's instructions and PrimeScript was used^TM 1^stStrand cDNA Synthesis kit (Takara, Cat. No. 6110A) prepared cDNA from 400ng RNA. To a mixture of 400ng RNA, 1. mu.l random hexamer and 1. mu.l dNTP (10mM) in a PCR tube, DEPC treated water was added to a total volume of 10. mu.l, and the reaction was carried out at 65 ℃ for 5 minutes. cDNA was synthesized by adding 10. mu.l of PrimeScript RTase to the reaction mixture and reacting successively at 30 ℃ for 10 minutes and at 42 ℃ for 60 minutes.

[ "ASO 5" reduction of ACC2mRNA ] as can be seen in fig. 11, the amount of ACC2mRNA was concentration-dependently reduced upon "ASO 5" treatment at 100zM to 1 fM. A 30% and 42% reduction in statistical significance, respectively, was observed when treated with 1aM and 1fM of "ASO 5" compared to the control experiment. (Student T-test was performed to check the statistical significance of the findings).

Example 5: preparation of a skin lotion containing the Compound of formula I (w/w%)

Compounds of formula I, such as "ASO 1," are formulated as a lotion for topical application to a subject. The skin lotion was prepared as follows. In view of the many variations possible for a lotion, this preparation should be taken as an example and should not be construed as limiting the scope of the invention.

In separate beakers, the mixed substances of component A and component B were dissolved at 80 ℃ respectively. Component A and component B were mixed and emulsified at 80 ℃ for 5 minutes using a 3,600rpm homogenizer. The emulsified component C was filtered through a 50 mesh screen and the filtrate was added to the mixture of component a and component B. The resulting mixture was emulsified at 80 ℃ for 5 minutes by using a 3,600rpm homogenizer. After component D was added to the mixture of components A, B and C at 35 deg.C, the resulting mixture was emulsified with a 2,500rpm homogenizer at 25 deg.C for 3 minutes. Finally ensuring uniform dispersion and complete defoaming.

TABLE 3 example of the composition of skin lotion containing the compound of formula I (w/w%)

Example 6: preparation of a cream containing a Compound of formula I (w/w%)

The compounds of formula I, e.g., "ASO 1", are formulated as a cream for topical administration to a subject. The cream was prepared as follows. Given that many variations of the topical creams are possible, this preparation should be taken as an example and should not be construed as limiting the scope of the invention.

TABLE 4 example of composition of cream containing Compound of formula I (w/w%)

The mixed substances of part A and part B were dissolved in a separate beaker at 80 ℃ respectively. Part A and part B were mixed and emulsified at 80 ℃ for 5 minutes using a 3,600rpm homogenizer. After adding part C to the mixture of parts a and B, the resulting mixture was emulsified for 5 minutes at 80 ℃ by using a 3,600rpm homogenizer. After addition of part D to the mixture of parts A, B and C at 35 deg.C, the resulting mixture was emulsified with a 3,600rpm homogenizer for 5 minutes at 35 deg.C. Finally, uniform dispersion and complete defoaming at 25 ℃ are ensured.

Sequence listing

<110> Orlitong Co

Han 29887yuying

Chengyao tea

Hongmo 28045

Zingiber officinale Makino

Xu qing 22893

Desire to open and descend

<120> acetyl-CoA carboxylase 2 antisense oligonucleotide

<130> 2018dp102

<150> KR10-2018-0095124

<151> 2018-08-14

<160> 6

<170> PatentIn version 3.2

<210> 1

<211> 14

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(14)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (3)..(3)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (6)..(6)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (8)..(8)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (11)..(11)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (13)..(13)

<223> n is a, g, c or t

<400> 1

ctnacnanat ngnc 14

<210> 2

<211> 16

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(16)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> modified _ base

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (2)..(3)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (6)..(6)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (9)..(9)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (11)..(11)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (13)..(13)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (15)..(15)

<223> n is a, g, c or t

<400> 2

tnntgncgna ntngnc 16

<210> 3

<211> 14

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(14)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (2)..(2)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (5)..(5)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (7)..(7)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (9)..(9)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (11)..(11)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (13)..(13)

<223> n is a, g, c or t

<400> 3

tnctnangna ntng 14

<210> 4

<211> 14

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(14)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (2)..(2)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (5)..(5)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (8)..(8)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (10)..(10)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (12)..(12)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (14)..(14)

<223> n is a, g, c or t

<400> 4

antgncgnan tngn 14

<210> 5

<211> 12

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(12)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (3)..(3)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (5)..(5)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (7)..(7)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (9)..(9)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (11)..(11)

<223> n is a, g, c or t

<400> 5

ctnangnant ng 12

<210> 6

<211> 14

<212> DNA

<213> Artificial

<220>

<223> PNA-modified oligonucleotide

<220>

<221> misc_feature

<222> (1)..(14)

<223> PNA-modified oligonucleotide, human acetyl-CoA carboxylase 2 targeting artificial sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> carbamation of N-terminal

<220>

<221> modified _ base

<222> (3)..(3)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (5)..(5)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (7)..(7)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (9)..(9)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (11)..(11)

<223> n is a, g, c or t

<220>

<221> modified _ base

<222> (13)..(13)

<223> n is a, g, c or t

<400> 6

ctnangnant ngnc 14

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acetyl-CoA carboxylase 2 antisense oligonucleotides

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