CN115960901A - Phosphamide morpholino antisense oligonucleotide for resisting virus and application thereof - Google Patents

Abstract

The invention provides a phosphoramide morpholino antisense oligonucleotide for resisting virus, which comprises a Base sequence shown in SEQ ID NO.1, wherein the antisense oligonucleotide is a phosphoramide morpholino antisense oligonucleotide, the structural formula of a phosphoramide morpholino nucleotide monomer in the antisense oligonucleotide is shown in the following formula, and Base is a Base; r is N, N-dimethylamino or piperazinyl. The antisense oligonucleotide can be combined with a specific region in the 5 th segment virus RNA of the influenza virus, the transcription of the virus RNA is blocked through steric hindrance, the antiviral effect is exerted, a PMO structure with good nuclease stability, high antisense efficiency and good water solubility is used, meanwhile, the introduction of a piperazine group with positive charge under physiological conditions is beneficial to the transmembrane entry of a PMO compound into cells, and the compound can be effectively enhancedAnd (4) biological activity.

Description

Phosphamide morpholino antisense oligonucleotide for resisting virus and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a phosphoramide morpholino antisense oligonucleotide for resisting virus and application thereof.

Background

Influenza viruses cause a global epidemic every year, and seasonal influenza causes up to 65 million deaths each year. At present, the medicines for treating influenza are few, and neuraminidase inhibitors such as oseltamivir, peramivir and the like are mainly used; ion channel M2 blockers such as amantadine; an RNA-dependent RNA polymerase inhibitor such as fapivvir. In addition, the problem of drug resistance of the small molecular chemical drugs is increasingly serious, and the development of novel anti-influenza virus drugs has important application value.

Oligonucleotide therapy is considered as a new generation of effective drug discovery technology platform following small molecule drugs and protein drugs. PMO refers to phosphoramide morpholino antisense oligonucleotide, wherein morpholine ring is used to replace five-membered sugar ring in nucleic acid structure, and neutral dimethyl phosphoramide is used to replace negatively charged phosphate group in PMO structure. Has the advantages of good nuclease stability, strong water solubility, low toxicity and the like. Particularly, piperazine groups with positive charges under physiological conditions are used for replacing part of dimethylamino groups in the structure of the PMOs main chain, and the obtained piperazine groups with positive charges modify PMO (PMOplus), so that the target sequences can be effectively promoted to enter cells. Based on the technology, the Sarepta Therapeutics in the United states has 3 approved medicines for treating Duchenne muscular dystrophy and shows good effectiveness and safety. The specific PMO antisense sequence can be specifically combined with corresponding nucleic acid fragments on RNA through the base complementary pairing principle, and the protein synthesis is blocked through steric hindrance, so that the corresponding biological effect is generated. This action characteristic makes the PMO technique especially suitable for the antiviral treatment field, and there are many antiviral drugs developed based on the PMO technique to carry out preclinical or clinical studies, such as AVI-7537 for treating Ebola virus infection, AVI-7288 for treating Marburg virus infection, and AVI-7100 for treating influenza, etc. The above drugs play an antiviral role mainly by blocking the AUG initiation codon region contained in the virus specific protein mRNA and blocking the protein translation process. There is a research report on a method for designing an antisense sequence directly based on the influenza virus segment 5 virus genome sequence. By studying the secondary structure of the influenza virus segment 5 genome, the corresponding antisense sequence was designed against the RNA secondary structure of the region not binding to the nucleoprotein. Wherein the 2 '-methoxy modified antisense oligonucleotide and the 2' -methoxy modified antisense locked nucleoside aiming at the 878-888 region show the best antiviral activity, which indicates that the region is a potential antisense oligonucleotide drug design target. However, the antisense oligonucleotide designed in the paper has a short sequence comprising only 11 bases, and the 2 '-methoxy modified antisense oligonucleotide and the 2' -methoxy modified antisense locked nucleoside are used to promote cellular uptake by means of transfection reagents. The above problems limit further research as anti-influenza virus drugs.

Disclosure of Invention

The invention solves the problem of serious drug resistance of small molecule chemical drugs, and the problem that the antisense oligonucleotide sequence is difficult to be absorbed by cells in the existing oligonucleotide therapy, thereby providing the phosphamide morpholino antisense oligonucleotide for resisting virus and the application thereof.

In order to solve the above problems, the present invention provides a phosphoramide morpholino antisense oligonucleotide for antiviral use, comprising a base sequence shown in SEQ ID No.1, the antisense oligonucleotide being a phosphoramide morpholino antisense oligonucleotide, wherein a structural formula of a phosphoramide morpholino monomer in the antisense oligonucleotide is shown in the following formula 1:

wherein, base is Base; r is N, N-dimethylamino or piperazinyl.

Preferably, the base sequence of the antisense oligonucleotide is shown as SEQ ID NO. 1.

Preferably, the 5 '-end of the antisense oligonucleotide is-OH, and the-OH of the 5' -end is linked to any one of the following formulas 2 to 6:

preferably, in the antisense oligonucleotide, all the R of the phosphoramide morpholino nucleotide monomer with C base are N, N-dimethylamino; all R of the phosphoramide morpholino nucleotide monomer with the basic group of G is N, N-dimethylamino; r of the phosphoramide morpholino nucleotide monomer with the basic group of T is the same or different and is selected from N, N-dimethylamino and piperazinyl; the R of the phosphoramide morpholino nucleotide monomer with the basic group of A is the same or different and is selected from N, N-dimethylamino and piperazinyl.

Preferably, the number of piperazinyl groups in the antisense oligonucleotide is 1 to 9.

Preferably, the structural formula of the antisense oligonucleotide is shown as the following formula 7:

in a second aspect, the present invention provides a pharmaceutical composition for anti-viral use, comprising the above antisense oligonucleotide and a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides the use of the antisense oligonucleotide described above in the preparation of a medicament for combating viruses.

Preferably, the virus is an influenza virus that is at least one of H1N1, H2N2, H3N2, H5N1, H7N7, H7N9, and H9N 2.

Compared with the prior art, the invention has the following beneficial effects:

the phosphoramide morpholino antisense oligonucleotide for resisting virus can be combined with a specific region in the RNA of the 5 th segment virus of influenza virus, and blocks the transcription of the RNA of the virus through steric hindrance to play an antiviral role; and the PMO structure with good nuclease stability, high antisense efficiency and good water solubility is used, and meanwhile, the piperazine group with positive charge under physiological conditions is introduced, so that the PMO compound can enter cells through a membrane, and the biological activity of the compound can be effectively enhanced.

Drawings

FIG. 1 is a mass spectrum of PMO-1 in an example of the present invention;

FIG. 2 is a mass spectrum of PMO-2 in an example of the present invention;

FIG. 3 is a graph showing the results of the cytotoxicity test of PMO-1 and PMO-2 in example 1 of the present invention;

FIG. 4 is a graph showing the results of an experiment on the anti-influenza virus activity of PMO-1 and PMO-2 in example 2 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first aspect of the embodiments of the present invention provides a phosphoramide morpholino antisense oligonucleotide for antiviral use, the phosphoramide morpholino antisense oligonucleotide comprising a base sequence as shown in SEQ ID No.1, the antisense oligonucleotide being a phosphoramide morpholino antisense oligonucleotide, wherein a structural formula of a phosphoramide morpholino monomer in the antisense oligonucleotide is shown in the following formula 1:

wherein Base is a basic group; r is N, N-dimethylamino or piperazinyl.

The sequence structure of SEQ ID NO.1 is as follows:

5’-TGGAAGAAGAACAAGGAGTTGC-3’。

the phosphoramide morpholino antisense oligonucleotide for resisting virus is different from a common design method of antisense oligonucleotide aiming at an AUG region of a virus mRNA translation initiation codon, and on the basis of a virus RNA secondary structure, a region which is not combined with nucleoprotein in an influenza virus segment 5 virus genome is selected as a target point, an antisense sequence is designed, and the series of phosphoramide morpholino oligonucleotides can be combined with a specific region in the influenza virus segment 5 virus RNA, block the transcription of the virus RNA through steric hindrance, and play a role in resisting virus. Meanwhile, the phosphoramide morpholino antisense oligonucleotide adopts a PMO structure with good nuclease stability, high antisense efficiency and good water solubility, and simultaneously, by introducing a piperazine group with positive charge under physiological conditions, the PMO compound can enter cells through a membrane, and the biological activity of the compound can be effectively enhanced.

Wherein, in the basic group, A represents adenine and the structural formula is

T represents thymine and has a structural formula

C represents cytosine and has a structural formula of->

G represents guanine and has a structural formula of->

In some embodiments, the base sequence of the antisense oligonucleotide is set forth in SEQ ID No. 1.

In some embodiments, the 5 '-terminus of the antisense oligonucleotide is-OH, and the-OH of the 5' -terminus is linked to any one of the structures of formulae 2-6 below:

the-OH at the 5' -end of the antisense oligonucleotide can be linked to the above structure to increase water solubility.

In some embodiments, all of the R of the phosphoramide morpholino nucleotide monomer with a base of C in the antisense oligonucleotide are N, N-dimethylamino; r of all the phosphoramide morpholino nucleotide monomers with the basic group of G is N, N-dimethylamino; r of the phosphoramide morpholino nucleotide monomer with the basic group of T is the same or different and is selected from N, N-dimethylamino and piperazinyl; the R of the phosphoramide morpholino nucleotide monomer with the basic group of A is the same or different and is selected from N, N-dimethylamino and piperazinyl. Namely, the R of the phosphoramide morpholino nucleotide monomer with the basic group of C, G is N, N-dimethylamino, and the R of the phosphoramide morpholino nucleotide monomer with the basic group of T, A can be N, N-dimethylamino or piperazinyl. Because the preparation cost of the piperazinyl base monomer is higher, in order to reduce the preparation cost, the piperazinyl is introduced by adopting the base A and the base T which are easy to prepare and high in coupling efficiency.

In some embodiments, the number of piperazinyl groups in the antisense oligonucleotide is 1-9; preferably, the number of piperazinyl groups is 3 to 6; further preferably, the number of piperazinyl groups is 5. The introduction of each piperazinyl can lead the oligonucleotide molecule to have corresponding positive charges, the proper number of the positive charges is beneficial to the molecule to enter cells, and experimental researches show that when the number of the bases containing the piperazinyl is 20-30% of the total number of the bases, a better transmembrane effect is achieved, so that when the number of the piperazinyl is within the range, the biological activity of the compound can be better enhanced.

In some embodiments, the antisense oligonucleotide has the structural formula shown below in formula 7:

in a second aspect, the present invention provides a pharmaceutical composition for anti-viral use, comprising the above antisense oligonucleotide and a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides the use of the antisense oligonucleotide described above in the preparation of a medicament for combating viruses.

In some embodiments, the virus is an influenza virus that is at least one of H1N1, H2N2, H3N2, H5N1, H7N7, H7N9, and H9N 2.

Examples

Preparation of phosphoramide morpholino antisense oligonucleotide:

the structural formula of PMO-1 is shown in the following formula 8:

the structural formula of PMO-2 is shown in the following formula 7:

the preparation method of PMO-2 comprises the following steps:

weighing 0.050g (loading amount of 0.6 mmol/g) of resin modified molecule modified aminomethyl polystyrene solid-phase synthetic resin, adding 2.0mL of NMP into a solid-phase synthesis tube, swelling for 2.0h at room temperature, draining the solution, washing the resin for 3 times with 2.0mL of dichloromethane, performing solid-phase synthesis according to the following procedures and conditions, and performing morpholino nucleoside monomer molecule according to sequence base sequence (5' -TGGA) ⁺ AGA ⁺ AGAA ⁺ CAAGGA ⁺ GTT ⁺ GC-3') are attached to the solid phase resin one by one, wherein A ⁺ Indicating that the base is phosphorylpiperazinomolylamine adenosine, T ⁺ Indicating that the base is phosphorylpiperazinomorphymidine. And after the last monomer molecule is coupled, removing the terminal trityl, emptying the reaction solution, and washing with NMP. After the solid phase synthesis of the main chain base is finished, adding 2.0mL of lysate consisting of NMP solution containing 0.1M dithiothreitol and 0.73M triethylamine into the solid phase synthesis tube, reacting for 30.0min, collecting the lysate, continuously adding 1.0mL of lysate into the solid phase synthesis tube, reacting for 15.0min, collecting and combining the lysate. Transferring the lysate to a 50mL hydrothermal reaction kettle, and adding precooling15.0mL of concentrated ammonia water at (-20 ℃), and standing in an oil bath at 45 ℃ for reaction for 24 hours to remove the basic group and the main chain protecting group. After the reaction solution was cooled to room temperature, it was concentrated by centrifugation using Amicon Ultra-15 centrifugal filter (3 kDa, merck), and the concentrate was diluted with 0.28% aqueous ammonia and further concentrated by centrifugation 2 times. The concentrate was collected and the pH of the solution was adjusted with acetic acid =4.5, and further purified by Resources 15S (6 g, cytiva) cation exchange chromatography column. Eluent a phase was 20.0mM sodium acetate buffer (pH = 4.5) containing 25% acetonitrile, and eluent B was 20.0mM sodium acetate buffer (pH = 4.5) containing 25% acetonitrile and 0.5M NaCl. Eluting for 30min according to gradient of 0-50% of eluent B proportion and flow rate of 5.0mL/min, and collecting corresponding components. Collecting components, continuously centrifuging and concentrating by using an Amicon Ultra-15 centrifugal filter (3 kDa, merck), continuously washing and concentrating for 2 times by using distilled water, transferring the concentrated solution into a centrifugal tube, adding 1.0mL of distilled water, and freeze-drying to obtain a final product PMO-2 which is a white solid. HRMS (ESI) M/z [ M +11H ]] ¹¹⁺ ,calcd.for C ₂₈₃ H ₄₄₇ N ₁₄₈ O ₈₇ P ₂₂ ¹¹⁺ 726.538 and found. The mass spectrum is shown in FIG. 2.

The preparation method of PMO-1 is the same as that of PMO-2, only part of nucleotides are replaced conventionally, and the details are not repeated here. Mass spectrum data of the prepared product are as follows: HRMS (ESI) M/z [ M + H ]] ⁺ ,calcd.for C ₂₆₆ H ₄₁₂ N ₁₄₃ O ₈₂ P ₂₂ ⁺ 7602.7, found. The mass spectrum is shown in FIG. 1.

Example 1: cytotoxicity test of PMO-1 and PMO-2

MDCK cells were cultured at approximately 2X 10 ⁴ The density of cells/well was seeded into 96-well plates. In 5% of CO ₂ Incubation was carried out at 37 ℃ for about 24h, and when the cells grew to about 90% of the monolayer, the culture medium was discarded, each well was washed 2 times with PBS, 200. Mu.L of DMEM medium (containing 2% fetal bovine serum) containing 50.0. Mu.M PMO-1 (containing 6.0. Mu. MEndo-port) or 50.0. Mu.M PMO-2 was added to the experimental wells, and 3 replicate wells were set for each sample. And a blank control group and a normal cell control group are set up. Observing cell status every day, at 5% CO ₂ Culturing at constant temperature of 37 deg.C for 3 days, discarding culture solution, washing each well with PBS 2 times, adding neutral red DMEM 40 μ g/mL into each well, and culturingCells were stained with 100 μ L of medium (containing 2% fetal bovine serum). At 5% of CO ₂ After incubation for 4h at constant temperature of 37 ℃, the culture medium was discarded, each well was washed with PBS 2 times, and a 48% acidic ethanol aqueous solution containing 1% glacial acetic acid was added to each well. The Optical Density (OD) value at 540nm was measured with a spectrophotometer, and the cell viability was calculated according to the following equation. As shown in FIG. 3, the results indicate that PMO-1 and PMO-2 both showed significant cytotoxicity at the concentrations.

Example 2: test of antiviral Activity of PMO-1 and PMO-2

MDCK cells were cultured at approximately 2X 10 ⁴ The density of cells/well was seeded into 96-well plates. At 5% of CO ₂ Incubating at 37 deg.C for about 24 hr, removing culture medium when cell grows to about 90% of monolayer, washing each well with PBS for 2 times, and adding 100TCID ₅₀ Cells were infected with H1N1 virus solution (100. Mu.L/well in DMEM medium containing 2% fetal bovine serum) while 3 replicate wells were set for each sample with 100. Mu.L/well of DMEN medium containing 10.0. Mu.M PMO-1 (containing 12.0. Mu.M Endo-port) or 10.0. Mu.M PMO-2 (containing 2% fetal bovine serum). And an empty virus control and a normal cell control group were set up. In 5% of CO ₂ Incubation was performed at a constant temperature of 37 ℃ and cytopathic effect (CPE) was observed daily. After 3 days of culture, the culture medium was discarded, each well was washed 2 times with PBS, and cells were stained by adding 40g/mL of neutral red DMEM medium 100L (containing 2% fetal bovine serum) to each well. In 5% of CO ₂ After incubation for 4h at constant temperature of 37 ℃, the culture medium was discarded, each well was washed with PBS 2 times, and a 48% acidic ethanol aqueous solution containing 1% glacial acetic acid was added to each well. The Optical Density (OD) value at 540nm was measured with a spectrophotometer, and the cell viability was calculated according to the following equation. As shown in FIG. 4, the results of the study showed that the cell viability was 45.1% and 52.6% for PMO-1 and PMO-2, respectively, at a concentration of 5.0. Mu.M, showing anti-H1N 1 virus activity.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.

Claims (9)

1. A phosphoramide morpholino antisense oligonucleotide for antiviral use, comprising a base sequence shown in SEQ ID NO.1, wherein the structural formula of a phosphoramide morpholino monomer in the phosphoramide morpholino antisense oligonucleotide is shown in the following formula 1:

wherein Base is a basic group; r is N, N-dimethylamino or piperazinyl.

2. The phosphoramide morpholino antisense oligonucleotide of claim 1, wherein the base sequence of the antisense oligonucleotide is set forth in SEQ ID No. 1.

3. The phosphoramide morpholino antisense oligonucleotide of claim 1, wherein:

the 5 '-end of the antisense oligonucleotide is-OH, and the-OH of the 5' -end is linked to any one of the following formulas 2 to 6:

4. the phosphoramide morpholino antisense oligonucleotide of claim 1, wherein:

in the antisense oligonucleotide, R of all phosphoramide morpholino nucleotide monomers with C as basic group is N, N-dimethylamino; r of all the phosphoramide morpholino nucleotide monomers with the basic group of G is N, N-dimethylamino; r of the phosphoramide morpholino nucleotide monomer with the basic group of T is the same or different and is selected from N, N-dimethylamino and piperazinyl; the R of the phosphoramide morpholino nucleotide monomer with the basic group of A is the same or different and is selected from N, N-dimethylamino and piperazinyl.

5. The phosphoramide morpholino antisense oligonucleotide of claim 4, wherein:

in the antisense oligonucleotide, the number of the piperazinyl is 1 to 9.

6. The phosphoramide morpholino antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide has the formula 7 below:

7. a pharmaceutical composition for use in antiviral comprising the phosphoramide morpholino antisense oligonucleotide of any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. Use of a phosphoramide morpholino antisense oligonucleotide according to any one of claims 1-6 or a pharmaceutical composition according to claim 7 in the preparation of an antiviral medicament.

9. The use according to claim 8, wherein the virus is an influenza virus and the influenza virus is at least one of H1N1, H2N2, H3N2, H5N1, H7N7, H7N9 and H9N 2.

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