Use of stm2457 in the preparation of a drug for preventing and treating nipah virus

Abstract

The application discloses application of STM2457 in preparation of medicines for preventing and treating nipah virus, and belongs to the technical field of medicines.The application mainly aims at m6A modification of a host, inhibits methyltransferase activity of METTL3 through STM2457, and then reduces m6A modification of the host to inhibit replication of the nipah virus, and has good effect.At present, most of the researches on virus medicines and vaccines are designed or modified according to the virus itself, the application provides a new idea for future drug research and development and vaccine research, and also provides a new use for STM2457.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of STM2457 in preparation of a medicine for preventing and treating a Nipah virus. BACKGROUND

[0002] Nipah virus (NiV) is a highly infectious zoonosis pathogen, which mainly causes acute fatal encephalitis, and the mortality rate is about 40%-92%.

[0003] NiV is mainly transmitted by contact, and the natural host is mainly a specific type of bat such as Pteropus vampyrus. The intermediate host is pig, dog, cat, horse and the like, and the terminal host is human, and NiV can also be transmitted between humans. NiV infection mainly causes damage to the nervous system and the respiratory system, which is called Nipah virus disease, and the typical symptoms are segmental myoclonus, hypertension, tachycardia, reflex loss and muscle tension reduction. The patient may die due to the symptoms of encephalitis, and the survivors also have permanent brain damage.

[0004] NiV belongs to the order of single-stranded negative strand RNA virus, the family of paramyxoviridae, the subfamily of paramyxoviridae, and the genus of henipavirus. The particle diameter is about 150-300 nm, which is spherical, polymorphic or filamentous, has a capsule membrane, and has a single-stranded negative strand RNA genome with a length of about 18.2 kb. At both ends of the gene open reading frame, there are UTRs, which contain gene-start (GS) sequences and gene-end (GE) sequences. The GS sequence can be used as a signal to start mRNA transcription and capping, and the GE can guide mRNA polyadenylation and transcription termination. The NiV genome encodes six structural proteins N, P, L, F, G and M and three non-structural proteins V, W and C. Among them, the N, P and L proteins form a complex and directly bind to the viral RNA to regulate the transcription and replication of the viral genome; F and G are glycoproteins, which form protrusions on the surface of NiV particles, participate in virus adsorption and membrane fusion to promote virus invasion, are the key determinants of infection and tropism, and can be used as the main object of vaccine strategy and antigen detection; the M protein participates in maintaining the morphology of the viral capsule membrane and regulating the NiV-induced antiviral innate immunity, and ubiquitination modification promotes the transport of the M protein out of the nucleus and promotes the budding of the virus. Although there are many studies on the infection and replication mechanism of NiV and the function of viral proteins in each replication link, the key host factors related to the pathogenic mechanism of NiV are still unclear. In addition, there is no effective antiviral drug for NiV, which causes a blank in the field of prevention and treatment of this type of virus.

[0005] At present, there are relatively mature pathogenic and serological detection and diagnosis techniques for NiV in the world, and the industry standard of "Nipah virus diagnosis technology" has been formulated in China. However, due to the need for high-level biosafety research conditions such as biosafety level 4 laboratory (BSL-4), the research on NiV is less and no antiviral drugs against NiV have been developed, and the main treatment method is still supportive care. Therefore, studying the replication mechanism of NiV and developing and stocking antiviral drugs against NiV are not only the basis for preventing and controlling the risk of NiV transmission, but also the strategic demand of the national health plan of our country.

[0006] m6A modification is one of the most studied and widely used RNA modifications, and it is the first discovered reversible chemical modification. m6A modification is the methylation of the 6th N atom of adenine in RNA molecules, which widely exists in mRNA, miRNA, IncRNA, circRNA, tRNA and other protein-coding and non-coding RNAs, and its modification process is mainly completed by methyltransferase complex, demethylase and reading protein. Its methyltransferase includes METTL3, METTL14, WTAP and other auxiliary factors, demethylase mainly FTO, ALKBH5, and reading protein mainly YTH (IYT521-B homology) domain protein, nuclear heterogeneous ribonucleoprotein (hnRNP), insulin-like growth factor 2 mRNA binding protein (IGF2BP) and eukaryotic initiation factor (eIF). m6A modification is involved in almost all aspects of RNA metabolism, including splicing, nuclear export, translation and stability of mRNA precursors.

[0007] STM2457 is a selective inhibitor with oral activity, and the current research of STM2457 mainly focuses on cancer (such as leukemia, solid tumor) and metabolic diseases (such as fatty liver), such as Chinese invention patent with application number 202111543015.9 and Chinese invention patent application with application number 202310516010.X, but there are few people researching the role of STM2457 in preventing and treating viruses. SUMMARY

[0008] In view of the fact that there is no effective antiviral drug for NiV at present, the present application aims to provide a drug for preventing and treating Nipah virus, which has better broad-spectrum and can better avoid the problem of virus mutation.

[0009] To achieve the above-mentioned purpose, the technical scheme designed by the present application is as follows:

[0010] This invention provides an application of STM2457 in the preparation of drugs for the prevention and treatment of Nipah virus. This invention demonstrates that m6A modification can promote NiV virus replication, and developing anti-NiV drugs from the perspective of m6A modification is a novel approach. STM2457 binds to the catalytic site of METTL3, inhibiting the methyltransferase activity of METTL3, thereby reducing m6A modification in the host and thus inhibiting viral replication.

[0011] The STM2457 has a molecular weight of 444.53 and a molecular formula of C2457. 25 H 28 N6O2, the structural formula is shown below:

[0012] .

[0013] This invention primarily targets the m6A modification of the host. By inhibiting the methyltransferase activity of METTL3 through STM2457, it reduces the m6A modification of the host and thus inhibits the replication of Nipah virus, achieving good results.

[0014] Preferably, the Nipah virus is selected from the Malaysian lineage NiV-MY or the Bangladeshi lineage NiV-BD.

[0015] Preferably, the drug for preventing and treating Nipah virus includes STM2457 or a pharmaceutically acceptable salt of STM2457, and a pharmaceutically acceptable adjuvant.

[0016] Preferably, the pharmaceutically acceptable adjuvant includes one or more of a pharmaceutically acceptable carrier, excipient, or diluent.

[0017] Preferably, the dosage form of the drug for preventing and treating Nipah virus includes at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral solutions, injections, or powders.

[0018] Preferably, the drug for preventing and treating Nipah virus also includes at least one other active ingredient against Nipah virus.

[0019] Preferably, the method of administration of the drug includes at least one of oral, intratumoral, rectal, parenteral injection, and local administration.

[0020] The application avoids the conventional small molecule drugs that block the virus itself, and achieves an antiviral effect without relying on the viral gene sequence by changing the host factors, which can well avoid the problem of drug off-target caused by viral mutation. The application finds that m6A promotes viral replication by overexpressing METTL3 and ALKBH5 and knocking down ALKBH5. The m6A is reduced after the METTL3 activity is inhibited, thereby inhibiting the replication of the virus. The application finds that STM2457 can significantly reduce the yield of progeny viruses in the culture supernatant of infected cells; in the golden hamster infection model, STM2457 reduces the viral RNA level in the lung tissue and spleen of the golden hamster. The above can illustrate that STM2457 has a good inhibitory effect on NiV in cell and animal infection models.

[0021] Compared with the prior art, the application has the beneficial effects that:

[0022] (1) In view of the characteristics that RNA viruses are prone to mutation, the application focuses on the host factors rather than the virus itself, and inhibits the viral replication by changing the expression of the host factors without affecting the normal life activities of the cells, which has better broad-spectrum and better avoids the problem of viral mutation.

[0023] (2) STM2457 is currently under clinical trials, so the probability of practical application is large; at the same time, the application also provides a new application approach for STM2457.

[0024] (3) At present, most of the researches on viral drug development and vaccine research are designed or modified based on the virus itself, and the application provides a new idea for future drug development and vaccine research. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is a graph showing that overexpression of METTL3 significantly increases the viral RNA abundance in NiV-infected Vero cells.

[0026] Figure 2 Figure 2 is a graph showing that knocking down ALKBH5 significantly increases the viral RNA abundance in NiV-infected Vero cells.

[0027] Figure 3 Figure 3 is a graph showing that STM2457 reduces the yield of NiV-MY progeny viruses in the culture supernatant.

[0028] Figure 4 Figure 4 is a graph showing that STM2457 reduces the yield of NiV-BD progeny viruses in the culture supernatant.

[0029] Figure 5 Figure 5 is a graph showing the drug concentration of STM2457 for inhibiting half of the viral replication.

[0030] Figure 6 Figure 6 is a graph showing that STM2457 reduces viral RNA levels in the lung of a golden hamster infection model.

[0031] Figure 7 Figure 7 is a graph showing that STM2457 reduces viral RNA levels in the spleen of a golden hamster infection model. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further described and explained by the following examples. The raw materials used in the examples are commercially available or prepared by conventional methods.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0034] NiV is mainly transmitted by contact, and the natural host is mainly a specific type of bat such as Pteropus vampyrus. Intermediate hosts include pigs, dogs, cats and horses, and the final host is humans, and it can be transmitted from person to person. The genome of NiV belongs to the order of single-stranded negative strand RNA virus, the family of paramyxovirus, the subfamily of paramyxovirus, and the genus of henipavirus. The NiV outbreak in Southeast Asia can be divided into at least two lineages: NiV-MY and NiV-BD, but the pathogenicity difference between NiV-MY and NiV-BD is less studied and the specific molecular mechanism is unclear. The particle diameter of NiV is about 150-300 nm, which is spherical, polymorphic or filamentous, has a capsule membrane, and has a single-stranded negative strand RNA genome with a length of about 18.2 kb, wherein the 3' leader sequence and the 5' tail region can be used as a genome replication and transcription element. The UTR is at both ends of the gene open reading frame, which contains a gene-start (GS) sequence and a gene-end (GE) sequence in the UTR, the GS sequence can be used as a signal to start mRNA transcription and capping, and the GE can guide mRNA polyadenylation and transcription termination. The NiV genome encodes six structural proteins N, P, L, F, G and M and three non-structural proteins V, W and C. Among them, N, P and L proteins form a complex and directly bind to viral RNA to regulate viral genome transcription and replication; F and G are glycoproteins that form protrusions on the surface of NiV particles, participate in virus adsorption and membrane fusion to promote virus invasion, are the key determinants of infection and tropism, and can be used as the main object of vaccine strategy and antigen detection; M protein is involved in maintaining the morphology of viral membrane and regulating NiV-induced antiviral innate immunity, ubiquitination modification promotes the transport of M protein out of the nucleus and promotes virus budding. Although there are many studies on the mechanism of NiV infection and replication and the function of viral proteins in each replication step, the key host factors related to the pathogenesis of NiV are still unclear. In addition, there is no effective antiviral drug for NiV, which causes a gap in the field of prevention and treatment of this type of virus.

[0035] To solve the above technical problems, the present application provides an application of STM2457 in anti-NiV drugs with the following structure:

[0036] .

[0037] The STM2457 provided by the present application can inhibit the replication of Nipah virus by inhibiting the methyltransferase activity of METTL3, and then reducing the m6A modification of the host. In subsequent studies, the present application found that STM2457 can significantly reduce the yield of progeny virus in the culture supernatant after Nipah virus infection. In the golden hamster infection model, STM2457 reduces the viral RNA level in the lung and spleen of golden hamsters. These results show that STM2457 has good inhibitory effect on NiV in cell and animal infection models, and has important significance for the prevention and treatment of Nipah virus.

[0038] The STM2457 with the structure of the above formula in the present application can significantly inhibit the replication of NiV virus in Vero cells.

[0039] The present application evaluates the inhibitory activity of STM2457 against Nipah virus infected cells by culturing Nipah virus. The inhibitory activity of STM2457 against Nipah virus replication in the golden hamster infection model is evaluated by intraperitoneal injection.

[0040] In some embodiments, the Nipah virus is selected from the Malaysian lineage NiV-MY or the Bangladesh lineage NiV-BD.

[0041] The present application provides a medicine comprising a compound or a pharmaceutically acceptable salt of the compound used in the above application, and a pharmaceutically acceptable auxiliary.

[0042] Specifically, "pharmaceutically acceptable" refers to those ligands, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for administration to patients and are commensurate with a reasonable benefit / risk ratio.

[0043] A "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulation material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted beta -cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0044] The dosage form of the drug includes at least one of a tablet, a capsule, an aqueous or oily suspension, a granule, an emulsion, an oral liquid, an injection liquid or a powder, according to different usage modes of the adjuvant.

[0045] The dosage form and administration mode of the compound of the present application or its pharmaceutical composition are not particularly limited. For different dosage forms of the drug, a suitable administration mode can be selected for administration.

[0046] Representative administration modes include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection and topical administration.

[0047] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such other ingredients as binders, (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) humectants, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) moisturizing agents, e.g., glycerol, (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, e.g., paraffin, (f) absoφtion accelerators, e.g., quaternary ammonium compounds, (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate, (h) absorbents, e.g., kaolin, and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms also can comprise buffering agents. Solid compositions such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, e.g., enteric coatings and other coatings of

[0048] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and the like, as well as mixtures thereof. In addition, the liquid dosage forms can contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and

[0049] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0050] Dosage forms for topical administration of a compound of this application include ointments, powders, sprays, and inhalers. The active component is admixed with a pharmaceutical carrier, e.g., sterile, fixed oils, or water, and any of a variety of other pharmaceutically acceptable carriers, e.g., preservatives, buffers, or pushers, as can be required.

[0051] Embodiments of the application will be described in detail below with reference to examples.

[0052] Example 1

[0053] Study on m6A promoting NiV replication:

[0054] (1) One day before transfection, prepare Vero cells (ATCC, CCL-81, African green monkey kidney cells) in a 24-well plate, and when the cells grow to 80%, transfect pFlag-METTL3 and siALKBH5 into the Vero cells using Transit-X2 (Mirus, MIR 6004), and culture at 37 ℃, 5% CO2 for 24 h;

[0055] (2) On the day of infection, replace the culture medium with 2% FBS (Celligent)-containing DMEM (Gibco, C11995500BT);

[0056] (3) Infect (MOI=0.01) with NiV-MY Malaysia strain Nipah virus (provided by the Microorganism and Virus Culture Preservation Center of the Wuhan Institute of Microbiology, Chinese Academy of Sciences), and collect total RNA samples from the cells 48 h later, and detect the virus copy number by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction).

[0057] As can be seen from Figure 1 , the viral RNA content increases after overexpression of METTL3; as can be seen from Figure 2 , the viral RNA content increases after knockdown of ALKBH5; overexpression of METTL3 or knockdown of ALKBH5 can increase the level of m6A modification, and the increase in the level of m6A modification can promote the increase in the viral RNA content, so the above results show that m6A promotes NiV replication.

[0058] Example 2

[0059] Cell experiment study on STM2457 inhibiting Nipah virus replication:

[0060] (1), using dimethyl sulfoxide (DMSO, sigma, item number: 67-68-5) to configure STM2457 (MCE, HY-134836) mother liquor, using DMEM medium to dilute STM2457, to obtain working concentration of 0 μM, 5 μM, 20 μM STM2457 working solution;

[0061] (2), using STM2457 mixed solution with concentration of 0 μM, 5 μM, 20 μM to treat Vero cells respectively;

[0062] (3), by NiV-MY Malaysia strain Nipah virus (provided by Microorganism and Virus Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences) and NiV-BD Bangladesh Nipah virus (Microorganism and Virus Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences) infection (MOI=0.01), 48 h after collecting supernatant virus content, through RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) to detect virus copy number.

[0063] It can be seen from Figure 3 that after treated by 5 μM and 20 μM STM2457, the copy number of NiV-MY virus is significantly reduced. It can be seen from Figure 4 that after treated by 5 μM and 20 μM STM2457, the copy number of NiV-BD virus is significantly reduced. It shows that STM2457 can effectively inhibit the replication of Nipah virus at 5~20 μM concentration.

[0064] Example 3

[0065] STM2457 inhibits half of the drug concentration of virus replication determination experiment:

[0066] (1), according to the change of virus copy number, using dimethyl sulfoxide (DMSO) to configure STM2457 mother liquor, using DMEM medium to dilute STM2457, to obtain working concentration of 0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM;

[0067] (2) After treating Vero cells (African green monkey kidney cells) with diluted STM2457, infecting them with the Malaysia strain and the Bangladesh strain of Nipah virus, and collecting the supernatant virus content 48 hours later, the virus copy number was detected by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction).

[0068] From Figure 5 It can be seen that the STM2457 concentration that inhibits half of the NiV-MY replication is 9.112 μM, and the STM2457 concentration that inhibits half of the NiV-BD replication is 8.959 μM.

[0069] Example 4

[0070] Animal experiment of STM2457 inhibiting Nipah virus replication:

[0071] Healthy experimental golden hamsters with similar body weights were respectively given intraperitoneal administration (dosing concentration of 20 mg / kg according to the body weight of the golden hamster) on the day of infection with Nipah virus (intraperitoneal infection, 500 LD50 / golden hamster) and the day after infection with Nipah virus, and were dissected on the fourth day after infection with Nipah virus, and the virus load in the lung tissue and the spleen was respectively detected (RT-qPCR). Figure 6 Figure 7

[0072] From Figures 6~7 It can be seen that, compared with the control group, the virus load in the lung tissue and the spleen of the golden hamsters in the STM2457 treatment group decreased significantly, which means that STM2457 can inhibit the replication and spread of the virus in the host cells.

[0073] It should be understood that the above examples are only used to illustrate the content of the present application and are not used to limit the protection scope of the present application. In addition, it should be understood that, after reading the content taught by the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.​​

Claims

1. The use of an STM2457 or a pharmaceutically acceptable salt of STM2457 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The molecular structure of the STM2457 is shown in the following formula: 。 2. The use of STM2457 or a pharmaceutically acceptable salt of STM2457 according to claim 1 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The Nipah virus mentioned is selected from the Malaysian lineage NiV-MY or the Bangladeshi lineage NiV-BD.

3. The use of STM2457 or a pharmaceutically acceptable salt of STM2457 according to claim 1 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The aforementioned drugs for the prevention and treatment of Nipah virus include pharmaceutically acceptable adjuvants.

4. The use of STM2457 or a pharmaceutically acceptable salt of STM2457 according to claim 1 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The dosage forms of the drugs for preventing and treating Nipah virus include at least one of the following: tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral solutions, injections, or powders.

5. The use of STM2457 or a pharmaceutically acceptable salt of STM2457 according to claim 1 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The drug for preventing and treating Nipah virus also includes at least one other active ingredient against Nipah virus.

6. The use of STM2457 or a pharmaceutically acceptable salt of STM2457 according to claim 1 in the preparation of drugs for the prevention and treatment of Nipah virus, characterized in that, The medication can be administered orally or via parenteral injection.

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