Application of STM2457 in preparation of medicine for preventing and treating Nipah virus

Abstract

The invention discloses application of STM2457 in preparation of a medicine for preventing and treating Nipah virus, and belongs to the technical field of medicine. The application mainly aims at m6A modification of a host, the activity of methyltransferase of METTL3 is inhibited through STM2457, then m6A modification of the host is reduced, replication of the Nipah virus is inhibited, and a good effect is achieved. At present, most of research and development of virus medicines and vaccines are designed or modified aiming at viruses, a new thought is provided for research and development of medicines and vaccines in the future, and a new application is also provided for STM2457.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically belongs to the application of STM2457 in preparing drugs for preventing and treating Nipah virus. Background Art

[0002] Nipah virus (NiV) is a highly contagious zoonotic pathogen that mainly causes acute fatal encephalitis with a mortality rate of approximately 40%-92%.

[0003] NiV is mainly transmitted by contact, and its natural hosts are mainly specific types of bats such as Pteropus fruit bats. The intermediate hosts include pigs, dogs, cats and horses, and the final host is humans, and it can be transmitted from person to person. NiV infection mainly causes damage to the nervous system and respiratory system, which is called Nipah virus disease. The typical symptoms are segmental myoclonus, hypertension, tachycardia, loss of reflexes and hypotonia. Patients with encephalitis symptoms may die, and survivors often suffer from permanent brain damage.

[0004] NiV belongs to the order Paramyxoviridae, the subfamily Paramyxovirinae, and the genus Henipavirus. Its particle diameter is about 150~300 nm, and it is spherical, pleomorphic or filamentous. It has an envelope and a single-stranded negative-sense RNA genome of about 18.2 kb. At both ends of the gene open reading frame are UTRs, which contain gene-start (GS) and gene-end (GE) sequences. The GS sequence can serve as a signal to start mRNA transcription and capping, while GE can guide mRNA polyadenylation and transcription termination. The NiV genome encodes six structural proteins, namely N, P, L, F, G and M, and three non-structural proteins, namely V, W and C. Among them, the N, P, and L proteins form a complex that directly binds to the viral RNA and regulates the transcription and replication of the viral genome; F and G are glycoproteins that form protrusions on the surface of NiV particles, participate in virus adsorption and membrane fusion, and thus promote virus invasion. They are key determinants of infection and tropism and can be used as the main target of vaccine strategies and antigen detection; M protein participates in maintaining the morphology of the viral envelope and regulating NiV-induced antiviral innate immunity. Ubiquitination modification promotes the transport of M protein out of the nucleus and promotes viral budding. Although there are many studies on the infection and replication mechanism of NiV and the functions of viral proteins in various replication links, the key host factors related to the pathogenesis of NiV are still unclear. In addition, there is no effective antiviral drug for NiV, which has created a gap that needs to be filled in the prevention and treatment of this type of virus.

[0005] Currently, there are relatively mature etiological and serological detection and diagnosis technologies for NiV internationally, and China has formulated an industry standard for "Nipah Virus Diagnostic Techniques". However, due to the need for high-level biosafety research conditions such as Biosafety Level 4 (BSL-4) laboratories, there is currently little research on NiV and no antiviral drugs against NiV have been developed. The main treatment method is still supportive care. Therefore, studying the replication mechanism of NiV and developing and stockpiling antiviral drugs against NiV are not only the basis for preventing and controlling the transmission risk of NiV, but also the strategic needs of China's national health plan.

[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 N atom at the 6th position of adenine in RNA molecules, which is widely present in protein-coding and non-coding RNAs such as mRNA, miRNA, IncRNA, circRNA, and tRNA. Its modification process is mainly completed by the methyltransferase complex, demethylase, and reader proteins. Its methyltransferases include METTL3, METTL14, WTAP, and some other cofactors. The demethylases are mainly FTO and ALKBH5, and the reader proteins are mainly YTH (IYT521-B homology) domain proteins, heterogeneous nuclear ribonucleoproteins (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 the splicing, nuclear export, translation, and stability of mRNA precursors.

[0007] STM2457 is an orally active selective inhibitor. Currently, the research on STM2457 mainly focuses on cancers (such as leukemia and solid tumors) and metabolic diseases (such as fatty liver), such as the Chinese invention patent with the application number 202111543015.9 and the Chinese patent application with the application number 202310516010.X. However, there is very little research on the role of STM2457 in preventing and treating viruses. Summary of the Invention

[0008] In view of the lack of effective antiviral drugs against NiV at present, the present invention aims to provide a drug for preventing and treating Nipah virus, which has better broad-spectrum properties and can better avoid the problem of virus mutation.

[0009] To achieve the above object, the technical solution designed by the present invention is as follows: The present invention provides an application of STM2457 in the preparation of a drug for preventing and treating Nipah virus. The present invention proves that m6A modification can promote the replication of NiV virus, and developing drugs against NiV from the perspective of m6A modification is a new idea. STM2457 binds to the catalytic site of METTL3, inhibits the methyltransferase activity of METTL3, thereby reducing the m6A modification of the host and then inhibiting the replication of the virus.

[0010] The molecular weight of the said STM2457 is 444.53, and the molecular formula is C 25 H 28 N6O2, and the structural formula is shown as follows: 。

[0011] The present invention mainly aims at the m6A modification of the host, inhibits the methyltransferase activity of METTL3 through STM2457, and then reduces the m6A modification of the host to inhibit the replication of Nipah virus, with good effects.

[0012] Preferably, the Nipah virus is selected from the Malaysian lineage NiV-MY or the Bangladeshi lineage NiV-BD.

[0013] Preferably, the drug for preventing and treating Nipah virus comprises STM2457 or a pharmaceutically acceptable salt of STM2457, and a pharmaceutically acceptable adjuvant.

[0014] Preferably, the said pharmaceutically acceptable adjuvant comprises one or more of a pharmaceutically acceptable carrier, excipient or diluent.

[0015] Preferably, the dosage form of the drug for preventing and treating Nipah virus comprises at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral liquids, injections or powders.

[0016] Preferably, the drug for preventing and treating Nipah virus further comprises at least one other anti-Nipah virus active ingredient.

[0017] Preferably, the administration mode of the said drug comprises at least one of oral, intratumoral, rectal, parenteral injection and topical administration.

[0018] The present invention avoids the conventional small molecule drugs that block the virus itself, starts from changing the host factors, achieves antiviral effects without relying on the viral gene sequence, and can well avoid the problem of drug off-target caused by viral mutations. The present invention uses overexpression of METTL3 and ALKBH5, knocks down ALKBH5 and finds that m6A promotes viral replication. After the activity of METTL3 is inhibited, m6A decreases, thereby inhibiting the replication of the virus. The present invention has found through research 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 level of viral RNA in the lung tissue and spleen of the golden hamster. The above can illustrate that STM2457 has a good inhibitory effect on NiV in both cell and animal infection models.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) In view of the fact that RNA viruses are prone to mutation, the present invention focuses on the host factors rather than the virus itself in drug development. Without affecting the normal life activities of cells, the virus replication is inhibited by changing the expression of host factors. This has better broad-spectrum and better avoids the problem of virus mutation.

[0020] (2) Studies have shown that STM2457 is currently in clinical trials, so the probability of practical application is very high; at the same time, the present invention also provides a new application approach for STM2457.

[0021] (3) Currently, most of the research on viral drug development and vaccine research is aimed at designing or modifying the virus itself. This invention provides new ideas for future drug development and vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 3 Overexpression of METTL3 significantly increased viral RNA abundance in Nipah virus-infected Vero cells.

[0023] Figure 2 Knockdown of ALKBH5 significantly increased viral RNA abundance in Nipah virus-infected Vero cells.

[0024] Figure 3 This is a graph showing the yield of Nipah virus progeny viruses in the culture supernatant after STM2457 reduced NiV-MY.

[0025] Figure 4 This is a graph showing the yield of Nipah virus progeny viruses in the culture supernatant after STM2457 reduced NiV-BD.

[0026] Figure 5 It is the drug concentration of STM2457 that inhibits half of the viral replication.

[0027] Figure 6 In the golden hamster infection model, the figure showing that STM2457 reduces the abundance of viral RNA in the lung tissue of golden hamsters.

[0028] Figure 7 In the golden hamster infection model, the figure showing that STM2457 reduces the level of viral RNA in the spleen of golden hamsters. Detailed implementation manners

[0029] The technical solutions of the present invention will be further described and illustrated below through examples. All raw materials used in the examples can be purchased commercially or prepared by conventional methods.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] NiV is mainly transmitted by contact, and its natural hosts are mainly specific types of bats such as fruit bats of the genus Pteropus. The intermediate hosts include pigs, dogs, cats and horses, and the final host is humans, and it can be transmitted from person to person. The NiV genome belongs to the order of single-stranded negative-sense RNA viruses, the family of Paramyxoviridae, the subfamily of Paramyxovirinae, and the genus Henipavirus. The NiV that broke out in Southeast Asia can be divided into at least two lineages: the Malaysian lineage NiV-MY and the Bangladeshi lineage NiV-BD, but the difference in pathogenicity between NiV-MY and NiV-BD has been less studied and the specific molecular mechanism is unclear. NiV particles are about 150~300 nm in diameter, spherical, polymorphic or filamentous, with an envelope, and have a single-stranded negative-sense RNA genome of about 18.2 kb, of which the 3' leader sequence and 5' tail region can serve as the original for genome replication and transcription. At both ends of the gene open reading frame are UTRs, which contain gene-start (GS) and gene-end (GE) sequences. The GS sequence can serve as a signal to initiate mRNA transcription and capping, while GE can guide mRNA polyadenylation and transcription termination. The NiV genome encodes six structural proteins, namely N, P, L, F, G and M, and three non-structural proteins, namely V, W and C. Among them, the N, P and L proteins form a complex that directly binds to the viral RNA and regulates the transcription and replication of the viral genome; F and G are glycoproteins that form protrusions on the surface of NiV particles, participate in virus adsorption and membrane fusion, thereby promoting viral invasion, and are key determinants of infection and tropism. They can be used as the main targets of vaccine strategies and antigen detection; M protein participates in maintaining the morphology of the viral envelope and regulating NiV-induced antiviral innate immunity. Ubiquitination modification promotes the transport of M protein out of the nucleus and promotes viral budding. Although there are many studies on the infection and replication mechanism of NiV and the functions of viral proteins in various replication links, the key host factors related to the pathogenesis of NiV are still unclear. In addition, there is no effective antiviral drug for NiV, which creates a gap that needs to be filled in the prevention and treatment of this type of virus.

[0032] In order to solve the above technical problems, the present invention provides an application of STM2457 having the following structure in an anti-Nipah virus drug: .

[0033] The STM2457 provided by the present invention inhibits the replication of Nipah virus by inhibiting the methyltransferase activity of METTL3, thereby reducing the m6A modification of the host. In subsequent studies, the present invention found that STM2457 can significantly reduce the yield of progeny virus in the culture supernatant after Nipah virus infection. In a golden hamster infection model, STM2457 reduces the viral RNA levels in the lungs and spleens of golden hamsters. These results indicate that STM2457 has good inhibitory effects on NiV in both cell and animal infection models, and is of great significance for the prevention and treatment of Nipah virus.

[0034] In the present invention, STM2457 with the above structure significantly inhibits the replication of NiV virus in Vero cells.

[0035] In the present invention, the inhibitory activity of STM2457 against Nipah virus-infected cells was evaluated through the culture of Nipah virus. And the activity of STM2457 in inhibiting the replication of Nipah virus in a golden hamster infection model was evaluated by intraperitoneal injection.

[0036] In some embodiments, the Nipah virus is selected from the Malaysian lineage NiV-MY or the Bangladeshi lineage NiV-BD.

[0037] The present invention provides a drug, comprising the compound or a pharmaceutically acceptable salt thereof used in the above application, and a pharmaceutically acceptable adjuvant.

[0038] Specifically, "pharmaceutically acceptable" refers to those ligands, materials, compositions and / or dosage forms that are suitable for administration to a patient within the scope of reasonable medical judgment and are commensurate with a reasonable benefit / risk ratio.

[0039] "Pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions or vehicles such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. 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 that are compatible with the administration of the drug. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in 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 β-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 glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0040] Depending on the different usage modes of the adjuvants, the dosage forms of the drug include at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral liquids, injections or powders.

[0041] There are no particular limitations on the dosage forms and administration modes of the compounds or their pharmaceutical compositions of the present invention. For drugs of different dosage forms, suitable administration modes can be selected for administration.

[0042] Representative administration modes include but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection and topical administration.

[0043] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as sodium citrate or calcium phosphate dibasic, or with the following components: (a) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxypropylmethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrants, e.g., agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) adsorbents, e.g., kaolin; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and release of the active compound or compounds in such compositions may be delayed in a portion of the digestive tract in a delayed manner. Examples of embedding components that may be used are polymeric and wax-like substances. If necessary, the active compound may also be in the form of microcapsules with one or more of the above excipients.

[0044] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. Besides the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifying agents, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances. Besides these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and perfumes. For example, suspensions may contain suspending agents, specifically, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar, or mixtures of these substances.

[0045] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0046] Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants. They are prepared by mixing the active ingredient with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required under aseptic conditions.

[0047] The embodiments of the present invention will be described in detail below in conjunction with the examples.

[0048] Example 1 Research on the promotion of NiV replication by m6A: (1) One day before transfection, prepare Vero cells (ATCC, CCL-81, African green monkey kidney cells) in a 24-well plate. When the cells reach 80% confluence, transfect pFlag-METTL3 and siALKBH5 into the Vero cells using Transit-X2 (Mirus, MIR 6004), and culture them at 37 °C and 5% CO2 for 24 h; (2) On the day of infection, change to a 2% DMEM (Gibco, C11995500BT) medium containing FBS (Celligent); (3) Infect with NiV-MY Malaysian strain Nipah virus (provided by the Microorganism and Virus Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences) (MOI = 0.01). After 48 h, collect the total RNA samples of the cells and detect the virus copy number by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction).

[0049] It can be seen from Figure 1 that the viral RNA content increases after overexpression of METTL3; it can be seen from Figure 2 that the viral RNA content increases after knockdown of ALKBH5; overexpression of METTL3 or knockdown of ALKBH5 can increase the m6A modification level, and the increase in the m6A modification level can promote the increase in the viral RNA content. Therefore, the above results indicate that m6A promotes NiV replication.

[0050] Example 2 Cell experiment study on the inhibition of Nipah virus replication by STM2457: (1) Prepare a stock solution of STM2457 (MCE, HY-134836) using dimethyl sulfoxide (DMSO, sigma, product number: 67-68-5), and dilute STM2457 with DMEM medium to obtain STM2457 working solutions with working concentrations of 0 μM, 5 μM, and 20 μM; (2) Treat Vero cells with STM2457 mixtures at concentrations of 0 μM, 5 μM, and 20 μM, respectively. (3) Infect with NiV-MY Malaysian strain Nipah virus (provided by the Microbial and Viral Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences) and NiV-BD Bangladesh Nipah virus (Microbial and Viral Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences) at a multiplicity of infection (MOI) of 0.01. After 48 h, collect the supernatant virus content and detect the virus copy number by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction).

[0051] It can be seen from Figure 3 that after treatment with 5 μM and 20 μM of STM2457, the copy number of NiV-MY virus decreased significantly. It can be seen from Figure 4 that after the cells were treated with 5 μM and 20 μM of STM2457, the copy number of NiV-BD virus decreased significantly. This indicates that STM2457 can effectively inhibit the replication of Nipah virus at a concentration of 5 - 20 μM.

[0052] Example 3 Experiment for determining the drug concentration of STM2457 that inhibits half of the virus replication: (1) Prepare the mother solution of STM2457 with dimethyl sulfoxide (DMSO) according to the change of virus copy number, and dilute STM2457 with DMEM medium to obtain working concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 40 μM. (2) After treating Vero cells (African green monkey kidney cells) with the diluted STM2457, infect them with Nipah virus of Malaysian strain and Nipah virus of Bangladesh strain. After 48 h, collect the supernatant virus content and detect the virus copy number by RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction).

[0053] It can be seen from Figure 5 that the concentration of STM2457 that inhibits half of the replication of NiV-MY is 9.112 μM, and the concentration of STM2457 that inhibits half of the replication of NiV-BD is 8.959 μM.

[0054] Example 4 Animal experiment on STM2457 inhibiting the replication of Nipah virus: Healthy experimental golden hamsters with similar body weights were intraperitoneally administered (at a dosage concentration of 20 mg / kg according to the body weight of the golden hamster) on the day of infection with Nipah virus (intraperitoneal injection for virus challenge, 500 LD50 / golden hamster) and on the second day after infection with Nipah virus. They were dissected on the fourth day after infection with Nipah virus, and the viral loads in the lung tissue and spleen were detected respectively ( Figure 6 、 Figure 7 ).

[0055] From Figures 6 - 7 It can be seen that compared with the control group, the viral loads in the lung tissue and spleen of golden hamsters in the STM2457 treatment group decreased significantly, which means that STM2457 can inhibit the replication and spread of the virus in host cells.

[0056] It should be understood that the above embodiments are only used to illustrate the content of the present invention and not to limit the protection scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of STM2457 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The molecular structure of the STM2457 is shown as follows: 。 2. Use of STM2457 according to claim 1 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The Nipah virus is selected from the Malaysian lineage NiV-MY or the Bangladeshi lineage NiV-BD.

3. Use of STM2457 according to claim 1 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The drug for preventing and treating Nipah virus includes STM2457 or a pharmaceutically acceptable salt of STM2457, and a pharmaceutically acceptable adjuvant.

4. Use of STM2457 according to claim 3 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The pharmaceutically acceptable adjuvant includes one or more of a pharmaceutically acceptable carrier, excipient or diluent.

5. Use of STM2457 according to claim 1 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, 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 liquids, injections or powders.

6. Use of STM2457 according to claim 1 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The drug for preventing and treating Nipah virus further includes at least one other anti-Nipah virus active ingredient.

7. Use of STM2457 according to claim 1 in the preparation of a drug for preventing and treating Nipah virus, characterized in that, The administration method of the drug includes at least one of oral, intratumoral, rectal, parenteral injection and topical administration.

Images