WO2006028160A1

WO2006028160A1 - S-oligonucleotide conjugate and antisense agent

Info

Publication number: WO2006028160A1
Application number: PCT/JP2005/016483
Authority: WO
Inventors: Hideki Oba; Masayuki Fujii
Original assignee: National Institute Of Advanced Industrial Science And Technology; Kitakyushu Foundation For The Advancement Of Industry Science And Technology
Priority date: 2004-09-10
Filing date: 2005-09-08
Publication date: 2006-03-16
Also published as: JP2006075110A

Abstract

A novel conjugate which has high activation inhibitory activity especially against the telomerase contained in a human leukemic cell extract and which gives a stable duplex hybrid with a complementary DNA. It is a phosphorothioate oligonucleotide conjugate represented by the general formula [Chemical formula 1] (wherein A1 is alkylene or alkylene separated by an oxygen atom; A2 is alkylene; R is a residue of a peptide, sugar, or functional amine; and n is 0 or 1). Also provided is an antisense agent containing the conjugate as an active ingredient.

Description

Specification

S-oligonucleotide conjugates and antisense agents

Technical field

[0001] The present invention relates to a novel phosphorothioate oligonucleotide conjugate that inhibits telomerase activity in a human leukemia cell extract and an antisense agent comprising the same as an active ingredient.

Background art

[0002] A complex of an oligonucleotide and a peptide has the potential to be used as an antisense agent for gene expression, and the peptide facilitates an increase in the intercellular concentration of the active oligonucleotide. Many attempts have been made to achieve this.

[0003] For example, by introducing a thiol group at the 5 terminus of a synthetic oligonucleotide, a conjugate of DNA and a peptide siphon has been obtained (see Non-Patent Document 1). In addition, functional organic compounds using bifunctional linkers such as dialkylating reagents, dimaleimides, and dialdehydes using functional groups such as amino groups, carboxylic acid groups, hydroxyl groups, and phenol groups present in protein molecules. For example, a combination of angiotensin I, insulin, bradykinin, topramycin, and other anticancer substances is also known, and the use of aromatic dithioisocyanate as a linker is also proposed (non- See Patent Document 2

) o

[0004] However, these oligonucleotide and peptide conjugates are expensive in terms of raw materials required for their production, are complicated in the production process, have low yields, and have anti-sense characteristics. Since it was insufficient, it was not always satisfactory for practical use.

[0005] Non-Patent Document 1: “Bioconjugate Chemistry”, 1 994, Vol. 5, p. 373-378

Non-Patent Document 2: "Science", 1964, No. 144, p. 1344

Disclosure of the invention

[0006] The present invention provides a novel activity that inhibits telomerase in a human leukemia cell extract, in particular, a high activity inhibitory action, and provides a stable double-stranded hybrid with a complementary DNA. It was made for the purpose of providing such a conjugate.

[0007] The inventors of the present invention have made extensive studies to develop a novel DNA conjugate. First, a nuclear localization signal polypeptide is added to a natural oligo DNA via ω-aminoalkyl phosphoroate. By condensing, we succeeded in obtaining a DNA conjugate that has excellent antisense properties and forms stable double-stranded hybrids with complementary DNA op =

However, as a result of further research, phosphorothioate oligonucleotides (S-Oly

Human white blood by condensing a nuclear localization signal peptide to

A

It was found that an S-oligonucleotide conjugate exhibiting a high activity inhibitory action against telomerase in disease cell extract was obtained, and the present invention was made based on this finding. o =

That is, the present invention provides a general formula

H N

[Chemical 1]

A

O H

H Η Ν

s-Origo N-C 0 C = R Nucleotide

η

(Alkylene Alpha ¹ in the formula is interrupted by an alkylene group or an oxygen atom, Alpha ² alkylene group, R represents a peptide residue of the sugar or functional Amin, eta is 0 or 1)

And an antisense agent comprising the same as an active ingredient.

[0009] The S-oligonucleotide in the present invention is obtained by changing the phosphodiester structure in a natural oligonucleotide to a thiophosphate diester structure.

As the oligonucleotide, nucleotide residues having 10 to 15 base unit power, for example, SEQ ID NO: 1-3 'and SEQ ID NO: 2-3' are preferred.

[0010] With regard to the origin of the S-oligonucleotide in the above general formula (I), there is no particular limitation. Oligonucleotides derived from various animal cells, oligonucleotides derived from various bacteria, or they are shredded with an enzyme. Use the nucleotide segment obtained from It can be arbitrarily selected depending on the purpose of use, but in the present invention, it is necessary to use a 5-terminal hydroxyl group reacted with an aminating agent to introduce an amino group.

Next, as the alkylene group having an 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, Hexylene group and the like 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.

In addition, 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.

[0012] The peptides introduced into the S-oligonucleotide via a linker include peptides obtained by degradation of various proteins, such as a nuclear export signal peptide, an antigen-derived nuclear localization signal peptide, Examples of the synthesized amphipathic α-helix and β-sheet peptide include sucrose and galactosamine as saccharides, and spermine and lipofectamine as functional amines.

Of the S-oligonucleotide conjugates of the present invention, those having η = 1 in the general formula (I) are in accordance with the solid phase fragment condensation method, for example, a solid phase carrier such as porous glass, controlled porous glass (CPG). ), S-oligonucleotide having a hydroxyl group at the 5 ′ end is condensed with polyethylene glycol-polystyrene, etc., and then produced as follows.

[0014] That is, in the presence of a catalyst, the general formula

[Chemical 2]

OR ¹

I

HO— P-0— A ¹ -NHR ² (II)

(Wherein R ¹ and R ² are protecting groups, A ¹ has the same meaning as above)

Is reacted with a phosphite represented by the following formula: OR ¹

I

S-Oligo — 5 '— O— P— O— A ¹ — NHR ² (ill) Nucleotide

(Wherein R ¹ R ² and A ¹ have the same meaning as above)

To form a DNA derivative represented by Next, the DNA derivative is oxidized to form a general formula.

[Chemical 4]

OR ¹

S-Origo _ 5 '_0— P— O— A ¹ — NHR ² (IV)

Nuku Leo de Do II

o

(Wherein R ¹ R ² and A ¹ have the same meaning as above)

S-oligonucleotide derivative represented by the 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

[Chemical 5]

OR ¹

I H H H

S-Oligo ― — υ _ A '_ IN _ 1 Ν— Α Ν _ C— Η (V

ooo

A ¹ and ΑΊ have the same meaning as above)

Is produced. It can be produced by reacting this with a peptide, sugar or functional amine, and finally treating with an alkali such as aqueous ammonia to separate the solid phase carrier, and removing the protecting group (hereinafter referred to as synthesis method 1). And u).

On the other hand, in the general formula (I), those having η = 0 are aliphatic dioxins represented by the general formula (V). Instead of isocyanate, the formula

[Chemical 6]

A eight

N N-C-N N (V I I)

0

Using carbodiimidazole represented by the general formula

[Chemical 7]

OR ¹ O

I H II

S-oligo — P— O— A ¹ — N_C— NN (VIII) nucleotide II \ = /

O

(R ¹ and A ¹ in the formula have the same meaning as 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 carried out using an appropriate solvent such as acetonitrile or dimethylformamide.

[0016] Next, ammonia water 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 a 2-cyanoethyl group or a trimethyl derivative residue such as a 4-methoxyphenyldimethyl group is used.

[0017] Among the S-oligonucleotide conjugates of the general formula (I) thus obtained,

ΟΗ

I

S—Oligo _P_0_CH ₂ CH ₂ OCH ₂ CH ₂ — ΝΗ— C— R Nucleotide II II

oo Examples of those corresponding to include S-oligonucleotide and C to C in which R has the structure shown in Table 1 below.

13

[table 1]

[0019] 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 the nuclear export signal peptide can be conjugated. When gated, it is easily introduced into the cell 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 an S-oligonucleotide can be easily introduced into cells without a cell introduction agent, and the force can be reduced with the cytoplasm. Localizes both in the nucleus.

BEST MODE FOR CARRYING OUT THE INVENTION

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

[0021] Example 1

First, using an automated DNA synthesizer [product name “PS250” manufactured by Cruachem, Inc., product name “500 A support” (product name “500 A support”, hereinafter abbreviated as CPG)] S-oligonucleotide having a hydroxyl group protected with a dimethoxytrityl group (supported by 5 SEQ ID NO: 3-3) and then treated with a 3% by weight acetonitrile solution of trichlorodiacetic acid to remove the protecting group, N-methoxytrityl-2- (2-aminoethyloxy) ethyl phosphoramidite, a commercially available amination reagent, was aminated by reacting the free hydroxyl group with a dimethylformamide solution containing 1H tetrazole at room temperature for 30 minutes. .

[0022] Next, the unreacted 5 hydroxyl group was capped with acetic anhydride, and then dissolved in iodoacetic acid. Phosphoric acid ester was formed by acidifying the phosphorus atom portion with the liquid. The intermediate compound thus obtained is reacted with a 3% by weight acetonitrile solution of trichlorodiacetic acid to remove the methoxytrityl group, which is a protecting group for the terminal amino group, and then hexamethylene diisocyanate. Nitrate (H Ml) or carbodiimidazole (CDI) and dimethylformamide containing diisopropylethylamine were reacted at room temperature for 2-12 hours, and then SV40T antigen nuclear localization signal (SEQ ID NO: SEQ ID NO: 7) was reacted in dimethylformamide containing diisopropylethylamine at room temperature for 24 hours.

[0023] Next, the reaction product thus obtained is treated in concentrated aqueous ammonia at 55 ° C for 4 hours, whereby excision from CPG and removal of the protecting group bound to the oligonucleotide and peptide are performed. The desired S-oligonucleotide conjugate was obtained as a crude product.

The crude product was purified using a reverse phase liquid chromatograph, and the resulting compound (No. 1) was analyzed by liquid chromatography and laser-excited time-of-flight mass spectrometry (MALDI—T OF MS). Yield ί or 30.45 ^_0/0 during this, TOF MSi or 5642.63 der ivy o

[0024] Comparative Example

An oligonucleotide conjugate No. 2 was produced in the same manner as in Example 1 except that a natural oligonucleotide (SEQ ID NO: 2-3) was used in place of the S-oligonucleotide in Example 1. The yield was 18.21% and TOF-MS was 5384.26.

[0025] Example 2

The S-oligonucleotide conjugate sample No. 1 obtained in Example 1 and the oligonucleotide conjugate No. 2 obtained in the comparative example were tested for stability with complementary strand DNA as follows.

Using a UV detector (product name “V-560UVZVis Spectrometer” manufactured by JASCO Corporation), a buffer solution consisting of lOOmM NaCl, 50mM Tris-HC1, 20mM MgCl (ρΗ7

2

0), each sample is dissolved at a concentration of 1 μΜ, and the complementary strand DNA is added to the sample and heated at 92 ° C for 5 minutes. Slowly cool to recombine the two strands Let

[0026] Next, these were heated from 5 ° C to 80 ° C at an increase rate of 0.5 ° CZ, and the change in absorbance at 260nm was measured with a UV detector to melt the temperature at the 50% absorbance increase point. Calculated as temperature. The results are shown in Table 2.

In the case + Mg ²⁺ in the table of MgCl is present in the measurement solution, -Mg ²⁺ is MgCl

2 Indicates the case where 2 does not exist.

[Table 2]

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

[0027] Example 3

The resistance test of the S-oligonucleotide conjugate of the present invention against DNase 1 was performed.

That is, sample No. 1 obtained in Example 1 was adjusted to a concentration of M with 0.1 M NaCl, and 5 ml of DNase 1 (manufactured by Sigma) in 160 Kunits was stored therein at 37 ° C. When the decomposition rate was measured over time, the decomposition rate in 180 minutes was 13%. For comparison, when the degradation rate was similarly measured using the natural oligonucleotide conjugate obtained in the comparative example, the degradation rate was 100%.

IKunit is a unit that increases the absorbance of UV spectrum at 260 nm by 0.001 per minute for nucleotides in lml at 25 ° C and ρΗ5.0.

[0028] Example 4

The sample No. 1 obtained in Example 1 and the sample No. 2 obtained in the comparative example were subjected to a degradation test for ribonuclease H.

In other words, the 5 'end of RNA complementary to each sample was modified with a fluorescent label, adjusted to a concentration of 10 μΜ to form a hybrid duplex, and then ribonuclease Η (Sigma' (Aldrich) 2.5 units were added, and when the degradation rate in the reaction buffer was measured over time by 20% polyacrylamide gel electrophoresis, they showed exactly the same activity.

[0029] Example 5

Sample No. 1 obtained in Example 1 and Sample No. 2 obtained in Comparative Example were examined for stability in serum.

Dissolve each sample in 120 1 ultrapure water at InM concentration, add 10% non-immobilized urine fetal serum 20 μ1, and use RP-HPLC (Hewlett Packard, Series 1100) for oligonucleotides The degradation state of was monitored over time. At this time, the sample is added to a reaction solution (pH 8.4) consisting of 0.1 M Tris-HCl, 0.09 M boric acid, and 7 M urea at predetermined time intervals, and the reaction is performed by freezing with liquid nitrogen. Stopped.

The monitoring was performed by measuring the change at 260 nm using the above RP-HPLC and ODS column (5 × 125 × 4 mm). As a result, sample No. 2 decomposed almost 100% after 24 hours, while sample No. 1 only decomposed 15%.

[0030] Example 6

The sample No. 1 obtained in Example 1 and the sample No. 2 obtained in the comparative example were measured for human telomerase inhibitory activity in the cell extract. The human telomerase used here is an enzyme that is specifically expressed in cancer cells and causes immortalization of the cells.

Jurkat cell force derived from leukemia cells was used and expressed as the concentration required for 50% inhibition (IC).

50

As a result, sample No. 2 was 1030 nM, while sample No. 1 showed sufficient activity at a very low concentration of 0.5 nM.

From this, the S-oligonucleotide conjugate of the present invention exhibits significantly higher human telomerase inhibitory activity than the oligonucleotide conjugate.

Industrial applicability

[0031] According to the present invention, by a simple reaction operation, a particularly high inhibitory action is exerted on telomerase activity, an excellent antisense property, and a stable double-stranded hybrid is formed with complementary DNA. S-oligonucleotide conjugates suitable as antisense agents can be produced. The s-oligonucleotide conjugates of the present invention have excellent antisense properties and form stable hybrid duplexes with complementary RNA, and thus are useful as antisense agents.

In addition, it inhibits the activity of telomerase that contributes to the immortalization of cancer cells, so it is effective for inhibiting the proliferation of cancer cells when administered to cancer patients.

Claims

Claim [1] General formula

[Chemical 1]

Phosphorothioate--R oli goon leotide

(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:

[2] Expression

[Chemical 2]

O H

I

Phosphorothioate P_0—CH ₂ CH. OCH ₂ CH ₂ — NH— C— R Oligonucleotide II II

O O

(Wherein R is a residue of a peptide, sugar or functional amine)

The phosphorothioate oligonucleotide conjugate according to claim 1 represented by claim 1.

[3] An antisense agent comprising the phosphorothioate oligonucleotide conjugate according to claim 1 or 2 as an active ingredient.