Phosphoramidite morpholino antisense oligonucleotides for use against viruses and uses thereof

Abstract

The application provides a phosphoramidate morpholino antisense oligonucleotide for antiviral, which comprises a base sequence shown as SEQ ID NO. 1, the antisense oligonucleotide is a phosphoramidate morpholino antisense oligonucleotide, the structural formula of the phosphoramidate morpholino nucleotide monomer in the antisense oligonucleotide is shown in the following formula, Base is a base; R is N, N-dimethylamine or piperazine. The antisense oligonucleotide can be combined with a specific region in the fifth segment of viral RNA of an influenza virus, block the transcription of the viral RNA through steric hindrance, play an antiviral role, and use a PMO structure which is good in nuclease stability, high in antisense efficiency and good in water solubility, and at the same time, by introducing a piperazine group which is positively charged under physiological conditions, the PMO compound can enter the cell across the membrane, and the biological activity of the compound can be effectively enhanced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a phosphoramidone morpholine antisense oligonucleotide for antiviral purposes and its applications. Background Technology

[0002] Influenza viruses cause global pandemics every year, with seasonal influenza potentially causing up to 650,000 deaths annually. Currently, there are relatively few drugs available for treating influenza, primarily neuraminidase inhibitors such as oseltamivir and peramivir; ion channel M2 blockers such as amantadine; and RNA-dependent RNA polymerase inhibitors such as favipiravir. Furthermore, drug resistance to these small-molecule drugs is becoming increasingly serious, making the development of novel antiviral drugs of significant practical value.

[0003] Oligonucleotide therapy is considered a new generation of effective drug discovery technology platform following small molecule drugs and protein drugs. PMO refers to phosphoramide morpholine antisense oligonucleotides. In the PMO structure, a morpholine ring replaces the five-membered sugar ring in the nucleic acid structure, and an electrically neutral dimethylphosphoramide replaces the negatively charged phosphate ester group. It has advantages such as good nuclease stability, high water solubility, and low toxicity. In particular, replacing some of the dimethylamine groups in the PMO backbone with positively charged piperazine groups under physiological conditions, resulting in positively charged piperazine-modified PMOs (PMOplus), can effectively promote the entry of target sequences into cells. Based on this technology, Sarepta Therapeutics in the United States has already approved three drugs for the treatment of Duchenne muscular dystrophy, demonstrating good efficacy and safety. Specific PMO antisense sequences can specifically bind to corresponding nucleic acid fragments on RNA through base complementarity pairing, blocking protein synthesis through steric hindrance and producing corresponding biological effects. This characteristic makes PMO technology particularly suitable for antiviral therapy. Several antiviral drugs developed using PMO technology are already undergoing preclinical or clinical trials, such as AVI-7537 for Ebola virus infection, AVI-7288 for Marburg virus infection, and AVI-7100 for influenza. These drugs primarily exert their antiviral effects by blocking the AUG promoter codon region in the mRNA of specific viral proteins, thereby inhibiting protein translation. A study has reported a method for designing antisense sequences directly based on the influenza virus segment 5 genome sequence. By studying the secondary structure of the influenza virus segment 5 genome, corresponding antisense sequences were designed targeting the RNA secondary structure in regions not bound to nucleoproteins. Among these, 2'-methoxy-modified antisense oligonucleotides and 2'-methoxy-modified antisense delimited nucleosides targeting the 878–888 region exhibited the best antiviral activity, indicating that this region is a potential target for antisense oligonucleotide drug design. However, the antisense oligonucleotide sequence designed in the paper is relatively short, containing only 11 bases, and the 2'-methoxy-modified antisense oligonucleotides and 2'-methoxy-modified antisense locked nucleosides used require transfection reagents to promote cell uptake. These issues limit its further research as an anti-influenza virus drug. Summary of the Invention

[0004] This invention addresses the serious problem of drug resistance to small molecule drugs. Currently available oligonucleotide therapies often suffer from difficulties in cell absorption of antisense oligonucleotide sequences. Therefore, this invention provides a phosphoramidone morpholine-based antisense oligonucleotide for antiviral use and its application. This phosphoramidone morpholine-based antisense oligonucleotide can bind to a specific region in the fifth segment of influenza virus RNA, blocking viral RNA transcription through steric hindrance and exerting an antiviral effect. It utilizes a PMO structure, which offers good nuclease stability, high antisense efficiency, and good water solubility. Furthermore, the introduction of a positively charged piperazine group under physiological conditions facilitates the transmembrane entry of the PMO compound into cells, effectively enhancing the compound's biological activity.

[0005] To address the aforementioned problems, this invention provides a phosphoramidoxolino antisense oligonucleotide for antiviral purposes, wherein the phosphoramidoxolino antisense oligonucleotide comprises the base sequence shown in SEQ ID NO.1, and the antisense oligonucleotide is a phosphoramidoxolino antisense oligonucleotide. The structural formula of the phosphoramidoxolino nucleotide monomer in the antisense oligonucleotide is shown in Formula 1 below:

[0006]

[0007] Where Base is a base; R is N,N-dimethylamino or piperazine.

[0008] Preferably, the base sequence of the antisense oligonucleotide is as shown in SEQ ID NO.1.

[0009] Preferably, the 5'-terminus of the antisense oligonucleotide is -OH, and the -OH at the 5'-terminus is linked to any one of the structures in Formulas 2-6 below:

[0010]

[0011] Preferably, in the antisense oligonucleotides, the R of all phosphoramidimolecular morpholine nucleotide monomers with a C base is N,N-dimethylamino; the R of all phosphoramidimolecular morpholine nucleotide monomers with a G base is N,N-dimethylamino; the R of phosphoramidimolecular morpholine nucleotide monomers with a T base is selected from N,N-dimethylamino and piperazine; and the R of phosphoramidimolecular morpholine nucleotide monomers with an A base is selected from N,N-dimethylamino and piperazine.

[0012] Preferably, the number of piperazine groups in the antisense oligonucleotide is 1-9.

[0013] Preferably, the structural formula of the antisense oligonucleotide is shown in Formula 7 below:

[0014]

[0015] A second aspect of the present invention provides a pharmaceutical composition for antiviral use, the pharmaceutical composition comprising the above-described antisense oligonucleotide and a pharmaceutically acceptable carrier.

[0016] A third aspect of the present invention provides the use of the above-described antisense oligonucleotide in the preparation of antiviral drugs.

[0017] Preferably, the virus is an influenza virus, and the influenza virus is at least one of H1N1, H2N2, H3N2, H5N1, H7N7, H7N9 and H9N2.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The antiviral phosphoramidolino antisense oligonucleotide of the present invention can bind to a specific region in the fifth segment of influenza virus RNA, and block viral RNA transcription through steric hindrance to exert an antiviral effect. Furthermore, it uses a PMO structure with good nuclease stability, high antisense efficiency, and good water solubility. At the same time, by introducing a piperazine group that carries a positive charge under physiological conditions, it is beneficial for the PMO compound to enter the cell across the membrane, which can effectively enhance the biological activity of the compound. Attached Figure Description

[0020] Figure 1 This is the mass spectrum of PMO-1 in an embodiment of the present invention;

[0021] Figure 2 This is the mass spectrum of PMO-2 in an embodiment of the present invention;

[0022] Figure 3 This is a graph showing the results of the PMO-1 and PMO-2 cytotoxicity experiments in Example 1 of this invention;

[0023] Figure 4 This is a graph showing the experimental results of the anti-influenza virus activity of PMO-1 and PMO-2 in Example 2 of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] A first aspect of this invention provides a phosphoramidoxolino antisense oligonucleotide for antiviral purposes, the phosphoramidoxolino antisense oligonucleotide comprising the base sequence shown in SEQ ID NO.1, wherein the antisense oligonucleotide is a phosphoramidoxolino antisense oligonucleotide, and the structural formula of the phosphoramidoxolino nucleotide monomer in the antisense oligonucleotide is shown in Formula 1 below:

[0026]

[0027] Where Base is a base; R is N,N-dimethylamino or piperazine.

[0028] The sequence structure of SEQ ID NO.1 is as follows:

[0029] 5'-TGGAAGAAGAACAAGGAGTTGC-3'.

[0030] The antiviral phosphoramidolin-based antisense oligonucleotides of this invention differ from commonly used antisense oligonucleotide design methods that target the AUG region of the viral mRNA translation start codon. Based on the secondary structure of viral RNA, this invention selects the region in influenza virus segment 5 of the viral genome that does not bind to nucleoproteins as the target site and designs an antisense sequence. This series of phosphoramidolin-based oligonucleotides can bind to specific regions in influenza virus segment 5 viral RNA, blocking viral RNA transcription through steric hindrance and exerting an antiviral effect. Simultaneously, this phosphoramidolin-based antisense oligonucleotide uses a PMO structure, which exhibits good nuclease stability, high antisense efficiency, and good water solubility. Furthermore, the introduction of a positively charged piperazine group under physiological conditions facilitates the transmembrane entry of PMO compounds into cells, effectively enhancing the compound's biological activity.

[0031] In this structure, A represents adenine, and its structural formula is: T represents thymine, with the structural formula as follows: C represents cytosine, and its structural formula is: G represents guanine, with the structural formula as follows:

[0032] In some embodiments, the base sequence of the antisense oligonucleotide is shown in SEQ ID NO.1.

[0033] In some embodiments, the 5'-terminus of the antisense oligonucleotide is -OH, and the -OH at the 5'-terminus is linked to any of the structures in Formulas 2-6 below:

[0034]

[0035] When the -OH at the 5' end of the antisense oligonucleotide is linked to the above structure, it can increase the water solubility.

[0036] In some embodiments, in the antisense oligonucleotides, the R group of all C-based phosphoramidoxomorphonucleotide monomers is N,N-dimethylamino; the R group of all G-based phosphoramidoxomorphonucleotide monomers is N,N-dimethylamino; the R group of T-based phosphoramidoxomorphonucleotide monomers is selected from N,N-dimethylamino and piperazine groups (either the same or different); and the R group of A-based phosphoramidoxomorphonucleotide monomers is selected from N,N-dimethylamino and piperazine groups (either the same or different). That is, the R group of C and G-based phosphoramidoxomorphonucleotide monomers is N,N-dimethylamino, while the R group of T and A-based phosphoramidoxomorphonucleotide monomers can be either N,N-dimethylamino or piperazine. Because the preparation cost of piperazine-based monomers is high, to reduce the preparation cost, easily prepared and highly efficient bases A and T are used to introduce the piperazine group.

[0037] In some embodiments, the number of piperazine groups in the antisense oligonucleotide is 1-9; preferably, the number of piperazine groups is 3-6; more preferably, the number of piperazine groups is 5. The introduction of each piperazine group imparts a corresponding positive charge to the oligonucleotide molecule. An appropriate number of positive charges facilitates molecule entry into cells. Experimental studies have shown that when the number of piperazine bases is 20%-30% of the total bases, it exhibits better transmembrane activity. Therefore, using the number of piperazine groups within the above range can better enhance the biological activity of the compound.

[0038] In some embodiments, the structural formula of the antisense oligonucleotide is shown in Formula 7 below:

[0039]

[0040] A second aspect of the present invention provides a pharmaceutical composition for antiviral use, the pharmaceutical composition comprising the above-described antisense oligonucleotide and a pharmaceutically acceptable carrier.

[0041] A third aspect of the present invention provides the use of the above-described antisense oligonucleotide in the preparation of antiviral drugs.

[0042] In some embodiments, the virus is an influenza virus, and the influenza virus is at least one of H1N1, H2N2, H3N2, H5N1, H7N7, H7N9 and H9N2.

[0043] Example

[0044] Preparation of phosphoramidolino antisense oligonucleotides:

[0045] The structural formula of PMO-1 is shown in Equation 8 below:

[0046]

[0047] The structural formula of PMO-2 is shown in Equation 7 below:

[0048]

[0049] The preparation method of PMO-2 is as follows:

[0050] Weigh 0.050 g (0.6 mmol / g) of resin-modified aminomethyl polystyrene solid-phase synthesis resin, add 2.0 mL of NMP to the solid-phase synthesis tube, allow it to swell at room temperature for 2.0 h, then drain the solution. Wash the resin three times with 2.0 mL of dichloromethane. Perform the solid-phase synthesis process according to the procedure and conditions in the table below. The morpholinonucleotide monomer molecules are arranged according to the sequence base order (5'-TGGA). + AGA + AGAA + CAAGGA + GTT + GC-3') are sequentially attached to the solid resin, wherein A + This indicates that the base is phosphorylpiperazine morpholinoadenosine, T + This indicates that the base is phosphorylpiperazinylmorpholinothymidine. After the coupling of the last monomer molecule is completed, the terminal triphenylmethyl group is removed, the reaction solution is emptied, and the mixture is washed with NMP. After the solid-phase synthesis of the main chain base is completed, 2.0 mL of lysis buffer consisting of NMP solution containing 0.1 M dithiothreitol and 0.73 M triethylamine is added to the solid-phase synthesis tube. After reacting for 30.0 min, the lysis buffer is collected. Another 1.0 mL of lysis buffer is added to the solid-phase synthesis tube and reacted for 15.0 min. The lysis buffers are collected and combined. The lysis buffer is transferred to a 50 mL hydrothermal reactor, and 15.0 mL of pre-cooled (-20℃) concentrated ammonia is added. The mixture is allowed to stand in a 45℃ oil bath for 24 h to remove the base and main chain protecting group. After the reaction solution is cooled to room temperature, it is concentrated by centrifugation using an Amicon Ultra-15 centrifuge filter (3 kDa, Merck). The concentrate is diluted with 0.28% ammonia and concentrated twice more by centrifugation. The concentrated solution was collected and the pH was adjusted to 4.5 with acetic acid. Further purification was performed using a Resources 15S (6g, Cytiva) cation exchange chromatography column. Elution buffer A was 20.0 mM sodium acetate buffer (pH = 4.5) containing 25% acetonitrile, and elution buffer B was 20.0 mM sodium acetate buffer (pH = 4.5) containing 25% acetonitrile and 0.5 M NaCl. Elution was carried out at a gradient of 0-50% using elution buffer B, at a flow rate of 5.0 mL / min, for 30 min. The collected fractions were then collected and concentrated by centrifugation using an Amicon Ultra-15 centrifuge filter (3 kDa, Merck). The solution was washed twice with distilled water. The concentrate was transferred to a centrifuge tube, 1.0 mL of distilled water was added, and the solution was freeze-dried to obtain the final product PMO-2, 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, found:726.519. Mass spectrum as follows: Figure 2 As shown.

[0051] The preparation method for PMO-1 is the same as that for PMO-2, except for the routine replacement of some nucleotides, which will not be elaborated here. The mass spectrometry data of the prepared product are: HRMS(ESI) m / z [M+H] + ,calcd.for C 266 H 412 N 143 O 82 P 22 + :7602.7, found:7603.9. Mass spectrum as follows: Figure 1 As shown.

[0052] Example 1: PMO-1 and PMO-2 Cytotoxicity Assay

[0053] MDCK cells were loaded at approximately 2 × 10 4 Cells were seeded at a density of 1:1 in 96-well plates. Incubation was performed at 37°C (5% CO2) for approximately 24 hours. When the cells reached approximately 90% confluence with a monolayer, the culture medium was discarded. Each well was washed twice with PBS. 200 μL / well of DMEM medium (containing 2% fetal bovine serum) containing 50.0 μM PMO-1 (containing 6.0 μM Mendo-Porter) or 50.0 μM PMO-2 was added to each well. Three replicates were prepared for each sample. A blank control and a normal cell control group were also included. Cell status was observed daily. After culturing at 37°C (5% CO2) for 3 days, the culture medium was discarded. Each well was washed twice with PBS, and 100 μL / well of DMEM medium (containing 2% fetal bovine serum) containing 40 μg / mL neutral red was added to each well for staining. After incubation at a constant temperature of 37°C and 5% CO2 for 4 hours, the culture medium was discarded, and each well was washed twice with PBS. A 48% acidic ethanol solution containing 1% glacial acetic acid was then added to each well. The optical density (OD) at 540 nm was measured using a spectrophotometer, and cell viability was calculated using the following formula. Figure 3 As shown, the results indicate that, at the specified concentrations, neither PMO-1 nor PMO-2 exhibited significant cytotoxicity.

[0054]

[0055] Example 2: Antiviral activity experiments of PMO-1 and PMO-2

[0056] MDCK cells were loaded at approximately 2 × 10 4 Cells were seeded at a density of approximately 96 cells / well into 96-well plates. The plates were incubated at 37°C (5% CO2) for approximately 24 hours. Once the cells had formed a monolayer of approximately 90%, the culture medium was discarded, and each well was washed twice with PBS and then inoculated with 100 TCID50 solution. 50 Cells were infected with H1N1 virus solution (100 μL / well, prepared in DMEM medium containing 2% fetal bovine serum). Simultaneously, 100 μL / well of DMEN medium (containing 2% fetal bovine serum) containing 10.0 μM PMO-1 (containing 12.0 μM Endo-porter) or 10.0 μM PMO-2 was added to each well. Three replicates were set up for each sample. Empty virus control and normal cell control groups were also included. Cells were incubated at 37°C with 5% CO2, and cytopathic effect (CPE) was observed daily. After 3 days of culture, the culture medium was discarded, and each well was washed twice with PBS. 100 μL of DMEM medium (containing 2% fetal bovine serum) containing 40 g / mL neutral red was added to each well for staining. After incubation at 37°C with 5% CO2 for 4 hours, the culture medium was discarded, and each well was washed twice with PBS. 48% acidic ethanol aqueous solution containing 1% glacial acetic acid was added to each well. The optical density (OD) value at 540 nm was measured using a spectrophotometer, and cell viability was calculated using the following formula. Figure 4 As shown, the results indicate that at a concentration of 5.0 μM, the cell viability rates of PMO-1 and PMO-2 were 45.1% and 52.6%, respectively, demonstrating anti-H1N1 virus activity.

[0057]

[0058] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A phosphoramidone morpholino antisense oligonucleotide for antiviral use, characterized in that, The structural formula of the antisense oligonucleotide is shown in Formula 1 or Formula 2 below:

2. A pharmaceutical composition for antiviral use, said pharmaceutical composition comprising the phosphoramidomorpholine antisense oligonucleotide as described in claim 1 and a pharmaceutically acceptable carrier.

3. The use of the phosphoramidone morpholino antisense oligonucleotide of claim 1 or the pharmaceutical composition of claim 2 in the preparation of an antiviral drug, wherein the virus is an influenza virus, and the influenza virus is H1N1.

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