JP2006075110A - New s-oligonucleotide conjugate and anti-sense agent containing the same as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new conjugate which especially exhibits a high activity-inhibiting action against a telomerase in human leukemic cell extracts and gives a double strand hybrid between complementary DNAs in a stable state. <P>SOLUTION: This phosphorothioate oligonucleotide conjugate is represented by the general formula [A<SP>1</SP>is an alkylene or an alkylene interrupted by an oxygen atom; A<SP>2</SP>is an alkylene; R is the residue of a peptide, sugar or functional amine; (n) is 0 or 1]. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel phosphorothioate oligonucleotide conjugate that inhibits telomerase activity in human leukemia cell extract and an antisense agent comprising the same as an active ingredient.

  Oligonucleotide and peptide complexes have the potential to be used as antisense agents for gene expression, and the peptide facilitates an increase in the intercellular concentration of active oligonucleotides, so many Attempts have been made.

  For example, a conjugate of DNA and peptide siphon has been obtained by introducing a thiol group at the 5′-end of a synthetic oligonucleotide (see Non-Patent Document 1). In addition, functional organic compounds such as amino groups, carboxylic acid groups, hydroxyl groups, phenol groups, etc. present in protein molecules are utilized via bifunctional linkers such as dialkylating reagents, dimaleimides, dialdehydes, etc. A combination of angiotensin I, insulin, prazikinin, topramycin, and other anticancer substances is also known, and the use of aromatic dithioisocyanate as a linker has also been proposed (see Non-Patent Document 2).

  However, these oligonucleotide and peptide conjugates are expensive because of the high cost of raw materials required for their production, complicated production processes, low yields, and insufficient antisense properties. However, it was not always satisfactory.

“Bioconjugate Chemistry”, 1994, Vol. 5, p. 373-378 “Science”, 1964, vol. 144, p. 1344

  The present invention provides a novel conjugate that exhibits a high activity inhibitory action, particularly on telomerase in human leukemia cell extract, and that stably provides a double-stranded hybrid with complementary DNA. It was made for the purpose.

  The present inventors have conducted extensive research to develop a novel DNA conjugate, and first condensed a nuclear localization signal polypeptide to natural oligo DNA via ω-aminoalkyl phosphoroate. Have succeeded in obtaining DNA conjugates that have excellent antisense properties and form stable double-stranded hybrids with complementary DNA. As a result of further research, phosphorothioate oligonucleotides (hereinafter referred to as “phosphorothioate oligonucleotides”) It was found that by condensing a nuclear localization signal peptide to (abbreviated as S-oligonucleotide), an S-oligonucleotide conjugate exhibiting a high activity inhibitory action against telomerase in human leukemia cell extract was obtained, The present invention has been made based on this finding.

（式中のＡ¹はアルキレン基又は酸素原子で中断されたアルキレン基、Ａ²はアルキレン基、Ｒはペブチド、糖又は機能性アミンの残基、ｎは０又は１である）
で表わされるＳ‐オリゴヌクレオチドコンジュゲート、及びそれを有効成分としてなるアンチセンス剤を提供するものである。 That is, the present invention has the general formula

(In the formula, A ¹ is an alkylene group or an alkylene group interrupted by an oxygen atom, A ² is an alkylene group, R is a residue of a peptide, sugar or functional amine, and n is 0 or 1)
And an antisense agent comprising the same as an active ingredient.

The S-oligonucleotide in the present invention is obtained by changing a phosphodiester structure in a natural oligonucleotide to a phosphophosphate diester structure.
The oligonucleotide is preferably a nucleotide residue consisting of 10 to 15 base units, such as SEQ ID NO: 1-3 ′ and SEQ ID NO: 2-3 ′.

  The origin of the S-oligonucleotide in the general formula (I) is not particularly limited, and oligonucleotide segments derived from various animal cells, oligonucleotides derived from various bacteria, or nucleotide segments obtained by chopping them with enzymes. In the present invention, it is necessary to use an amino group introduced by reacting a 5'-terminal hydroxyl group with an aminating agent. It is.

Next, as the alkylene group of the A ¹ bond in the general formula (I), a polymethylene group having 2 to 10 carbon atoms, preferably 4 to 8 carbon atoms such as an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, etc. Can be mentioned. The alkylene group may be one having a carbon chain interrupted by an oxygen atom, such as a 2-oxapropylene group, a 3-oxapentylene group, or a 4-oxaheptylene group.
Further, the alkylene group having an A ² bond in the general formula (I) is preferably a polymethylene group having 4 to 12 carbon atoms or a branched alkylene group.

  The peptides introduced into the S-oligonucleotide via a linker include peptides obtained by degradation of various proteins, such as nuclear export signal peptides, antigen-derived nuclear localization signal peptides, synthesized parents Examples of the medium α-helix and β-sheet peptide include saccharides such as sucrose and galactosamine, and functional amines include spermine and lipofectamine.

（式中のＲ¹及びＲ²は保護基、Ａ¹は前記と同じ意味をもつ）
で表わされる亜リン酸エステルを反応させ、５´末端を化学修飾し、一般式

（式中のＲ¹、Ｒ²及びＡ¹は前記と同じ意味をもつ）
で表わされるＤＮＡ誘導体を形成させたのち、酸化して、一般式

（式中のＲ¹、Ｒ²及びＡ¹は前記と同じ意味をもつ）
で表わされるＳ‐オリゴヌクレオチド誘導体とし、次いで一般式
ＯＣＮ−Ａ²−ＮＣＯ （Ｖ）
（Ａ²は前記と同じ意味をもつ）
で表わされる脂肪族ジイソシアネートを反応させることにより、一般式

（式中のＲ¹、Ａ¹及びＡ²は前記と同じ意味をもつ）
で表わされる化合物を製造したのち、これにペプチド、糖又は機能性アミンを反応させ、最後にアルカリ例えばアンモニア水で処理して固相担体からの切りはなし、及び保護基の脱離を行うことにより製造することができる（以下合成法１という）。 Among the S-oligonucleotide conjugates of the present invention, those with n = 1 in the general formula (I) are in accordance with a solid phase fragment condensation method, such as solid phase carriers such as porous glass, controlled porous glass (CPG), polyethylene. After condensing an S-oligonucleotide having a hydroxyl group at the 5 ′ end to glycol-polystyrene, etc., in the presence of a catalyst, the general formula

(Wherein R ¹ and R ² are protecting groups, and A ¹ has the same meaning as above)
A phosphite ester represented by the following formula:

(Wherein R ¹ , R ² and A ¹ have the same meaning as above)
After the formation of a DNA derivative represented by

(Wherein R ¹ , R ² and A ¹ have the same meaning as above)
S-oligonucleotide derivatives represented by the general formula OCN-A ² -NCO (V)
(A ² has the same meaning as above)
Is reacted with an aliphatic diisocyanate represented by the general formula

(Wherein R ¹ , A ¹ and A ² have the same meaning as described above)
By reacting this with a peptide, sugar or functional amine, and finally treating with an alkali such as ammonia water to cut off the solid phase carrier and removing the protecting group. Can be produced (hereinafter referred to as Synthesis Method 1).

で表わされるカルボニルジイミダゾールを用いて、一般式

（式中のＲ¹、Ａ¹は前記と同じ意味をもつ）
で表わされる化合物を製造したのち、これにペプチド、糖又は機能性アミンを反応させ、さらにアルカリ処理することにより製造することができる（以下合成法２という）。
これらの反応は、適当な溶媒、例えばアセトニトリル、ジメチルホルムアミドなどを用いて行われる。 On the other hand, in the general formula (I), those in which n = 0 are substituted with the aliphatic diisocyanate represented by the general formula (V).

Using the carbonyldiimidazole represented by the general formula

(Wherein R ¹ and A ¹ have the same meaning as described above)
Can be produced by reacting it with a peptide, sugar or functional amine and further subjecting it to an alkali treatment (hereinafter referred to as synthesis method 2).
These reactions are performed using a suitable solvent such as acetonitrile, dimethylformamide and the like.

Next, aqueous ammonia is added to the reaction product thus obtained to excise the DNA conjugate from the solid support, and optionally remove the protecting group of the peptide present.
As the protecting group represented by R ¹ and R ² , for example, a ω-cyanoalkyl group such as 2-cyanoethyl group or a triphenylmethyl derivative residue such as 4-methoxyphenyldiphenylmethyl group is used.

Formula S- oligonucleotides -PO thus obtained _{3 CH 2 CH 2 OCH 2 CH} 2 NH-R
As examples of the S-oligonucleotide conjugates of the present invention represented by the formula (1), C _{1 to} C ₃ having the structures shown in Table 1 below can be mentioned.

Natural oligonucleotides are not introduced into cells without a cell introduction agent.
However, when a nuclear localization signal peptide is conjugated to this, it can be easily introduced into a cell without a cell introduction agent and localized in the nucleus, and when a nuclear export signal peptide is conjugated to a cell Easily introduced into and localized in the cytoplasm (extranuclear).
In contrast, the S-oligonucleotide conjugates of the present invention in which a nuclear localization signal peptide is conjugated to S-oligonucleotides can be easily introduced into cells without a cell introduction agent, and can be introduced into the cytoplasm and nucleus. Localizes to both.

  According to the present invention, it is possible to form a stable double-stranded hybrid with a complementary DNA by a simple reaction operation, particularly exhibiting a high inhibitory effect on telomerase activity, having excellent antisense characteristics, S-oligonucleotide conjugates suitable as antisense agents can be produced.

  Next, the present invention will be described in more detail with reference to examples.

  First, using an automatic DNA synthesizer (product name “PS250” manufactured by Kurachem), a control porous glass (product name “500 mm support”, hereinafter abbreviated as CPG) is added to the 5 ′ end. After supporting the S-oligonucleotide (5'-Sequence Listing SEQ ID NO: 3-3 ') whose hydroxyl group was protected with a dimethoxytrityl group, the protective group was removed by treatment with a 3% by mass acetonitrile solution of trichloroacetic acid. The aminated hydroxyl group was aminated by reacting a commercially available amination reagent N-methoxytrityl-2- (2-aminoethyloxy) ethyl phosphoramidite with a 1H tetrazole-containing dimethylformamide solution at room temperature for 30 minutes. .

  Next, after capping the unreacted 5'-hydroxyl group with acetic anhydride, the phosphorus atom portion was oxidized with an ioacetic acid solution to form a phosphate ester. The intermediate compound thus obtained was reacted with a 3% by mass acetonitrile solution of trichloroacetic acid to remove the methoxytrityl group, which is a protecting group for the terminal amino group, and then hexamethylene diisocyanate (HMI) or carbonyldiimidazole ( CDI) and dimethylformamide containing diisopropylethylamine for 2 to 12 hours at room temperature, and then reacting SV40T antigen nuclear localization signal (SEQ ID NO: 7) in dimethylformamide containing diisopropylethylamine for 24 hours at room temperature. I let you.

Next, the reaction product thus obtained is treated in concentrated aqueous ammonia at 55 ° C. for 4 hours to excise from CPG and remove the protecting group bonded to the oligonucleotide and peptide. The S-oligonucleotide conjugate was obtained as a crude product.
This crude product was purified using a reverse phase liquid chromatograph, and the resulting compound was analyzed by liquid chromatography and laser-excited time-of-flight mass spectrometry (MALDI-TOF MS). The yield at this time was 30.45%, and the TOF-MS was 564.63.

Comparative Example An oligonucleotide conjugate No. 1 was treated in the same manner as in Example 1 except that a natural oligonucleotide (SEQ ID NO: 2-3 ′) was used instead of the S-oligonucleotide in Example 1. 2 was produced. The yield at this time was 18.21 wt%, and TOF-MS was 5384.26.

The S-oligonucleotide conjugate sample No. 1 obtained in Example 1 was used. 1 and the oligonucleotide conjugate No. obtained in Comparative Example. 2 was tested for stability with complementary DNA as follows.
Using a UV detector (manufactured by JASCO Corporation, product name “V-560 UV / Vis spectrometer”), each sample was 1 μM in a buffer solution (pH 7.0) composed of 100 mM NaCl, 50 mM Tris-HCl, 20 mM MgCl _2. After dissolving at a concentration and adding complementary strand DNA thereto and heating at 92 ° C. for 5 minutes, the double strand was once melted and then slowly cooled to 4 ° C. to recombine the double strand.
Next, this was heated from 5 ° C. to 80 ° C. at a rate of 0.5 ° C./min, the change in absorbance at 260 nm was measured with a UV detector, and the temperature at the 50% absorbance rise point was calculated as the melting temperature. The results are shown in Table 2.
Note that + Mg in the table indicates that MgCl ₂ is present in the measurement solution, and −Mg indicates that MgCl ₂ is not present.

この表から、本発明のＳ‐オリゴヌクレオチドコンジュゲート分子は、相補的なＲＮＡとの間でハイブリッド２本鎖を形成しうることが分る。

From this table it can be seen that the S-oligonucleotide conjugate molecules of the present invention can form hybrid duplexes with complementary RNA.

The resistance test of the S-oligonucleotide conjugates of the present invention against DNase 1 was performed.
That is, the sample No. obtained in Example 1 was obtained. 1 was adjusted to a concentration of 1 μM with 0.1 M NaCl, and 5 ml of 160 Kunit units of DNase 1 (manufactured by Sigma) was added thereto and maintained at 37 ° C., and the degradation rate was measured over time. The degradation rate in minutes was 13%.
For comparison, when the degradation rate was measured in the same manner using the natural oligonucleotide conjugate obtained in the comparative example, the degradation rate was 100%.
1 Kunit is a unit that increases the absorbance of the UV spectrum at 260 nm by 0.001 per minute for nucleotides in 1 ml at 25 ° C. and pH 5.0.

Sample No. obtained in Example 1 1 and the sample No. obtained in the comparative example. 2 was subjected to a degradation test against ribonuclease H.
That is, the 5′-end of RNA complementary to each sample was modified with a fluorescent label, adjusted to a concentration of 10 μM to form a hybrid duplex, and then ribonuclease H (manufactured by Sigma-Aldrich) 2.5 When units were added and the degradation rate in the reaction buffer was measured over time by 20% polyacrylamide gel electrophoresis, they showed exactly the same activity.

Sample No. obtained in Example 1 1 and the sample No. obtained in the comparative example. 2 was examined for stability in serum.
Each sample was dissolved in 120 μl of ultrapure water at a concentration of 1 nM, 20 μl of 10% non-immobilized fetal bovine serum was added, and the degradation state of the oligonucleotide was monitored over time using RP-HPLC. At this time, the sample was added to a reaction solution (pH 8.4) composed of 0.1 M Tris-HCl, 0.09 M boric acid and 7 M urea every predetermined time, and the reaction was stopped by freezing with liquid nitrogen. It was.
The monitoring was performed by measuring changes at 260 nm using RP-HPLC (manufactured by Hewlett-Packard, series 1100) and ODS column (5 × 125 × 4 mm). As a result, sample no. 2 decomposed almost 100% after 24 hours, whereas sample no. 1 stopped at 15% decomposition.

Sample No. obtained in Example 1 1 and the sample No. obtained in the comparative example. Regarding 2, the human telomerase inhibitory activity in the cell extract was measured. The human telomerase used here is an enzyme that is specifically expressed in cancer cells and causes immortalization of the cells.
Using a solution extracted from leukemia cell-derived Jurkat cells, it was expressed as the concentration required for 50% inhibition (IC ₅₀ ).
As a result, sample no. 2 was 1030 nM, whereas sample no. 1 was found to show sufficient activity at very low concentrations of 0.5 nM.
From this, the S-oligonucleotide conjugate of the present invention exhibits a significantly higher human telomerase inhibitory activity than the oligonucleotide conjugate.

The S-oligonucleotide conjugates of the present invention are useful as antisense agents because they have excellent antisense properties and form stable hybrid duplexes with complementary RNA.
Moreover, since it inhibits the activity of telomerase that contributes to immortalization of cancer cells, it is effective to inhibit the proliferation of cancer cells when administered to cancer patients.

Claims (2)

General formula

(In the formula, A ¹ is an alkylene group or an alkylene group interrupted by an oxygen atom, A ² is an alkylene group, R is a residue of a peptide, sugar or functional amine, and n is 0 or 1)
A phosphorothioate oligonucleotide conjugate represented by An antisense agent comprising the phosphorothioate oligonucleotide conjugate according to claim 1 as an active ingredient.