Use of a compound for regulating mettl3 target in preparation of an antienterovirus drug

Abstract

The application provides an application of a compound for regulating a METTL3 target in preparation of an anti-enterovirus drug, belongs to the field of biological medicines, and the compound is AN-465 / 42162872, which presents strong anti-EV71 effects in an in-vitro and in-vivo infection model. In-vitro cell experiments, an EV71 infection C57BL / 6 mouse model, m 6 A methylation sequencing method and the like are used to carry out activity and potential mechanism research of the anti-EV71, and the results show that AN-465 / 42162872 can dose-dependently inhibit the replication of EV71 in a human RD cell strain under the condition of maintaining good cell activity, the mechanism is mainly that the methylation level of adenine at the 5002th position of the 2C region of the nucleic acid of EV71 is significantly lowered by regulating METTL3, so as to inhibit the virus replication, and the compound has strong anti-EV71 drug utilization value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to an application of a compound for regulating a METTL3 target in preparation of an anti-enterovirus drug. BACKGROUND

[0002] In recent years, Enterovirus 71 (EV71), as a strong pathogenic RNA virus, has attracted extensive attention worldwide. EV71 mainly spreads through the fecal-oral route, and is particularly susceptible to the population of infants under the age of 5, which can cause hand, foot and mouth disease (HFMD) and other severe nervous system complications, and even death. From 2008 to 2022, the cumulative number of reported HFMD cases nationwide reached more than 40 million, and the cumulative number of deaths caused by EV71 reached more than 3,000. HFMD caused by EV71 is posing a great threat to the health of the infant population. Although there are currently some preventive vaccines for HFMD, there is no clinical drug that can completely cure the disease. In view of the severity of EV71 infection and the limitations of current treatment methods, it is particularly important to find safer and more effective antiviral drugs.

[0003] Researchers have identified hundreds of potential inhibitors of EV71 in preclinical studies, and the main development strategies include screening and repurposing drugs, targeted structural design, and rational modification of previously effective drugs. The antiviral mechanisms of these drugs mainly target various stages of the EV71 viral life cycle, including viral inhibitors targeting viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C protease (3Cpro), and 2A protease (2Apro) and other key sites. However, EV71 virus has a high mutation rate, and these inhibitors directly targeting specific sites of the virus are easily affected by viral variation, leading to drug failure; secondly, these inhibitors often require high doses to achieve effective antiviral effects, which may increase the toxic side effects of drugs, limiting their clinical use in children. In addition, long-term use of direct viral inhibitors may also lead to the development of drug resistance, further limiting their clinical application.

[0004] For example, Chinese patent CN118717786A (publication date: October 1, 2024) discloses an application of etoposide in the preparation of an EV71 virus inhibitor, which confirms that etoposide has strong anti-EV71 virus effect at the cellular level and can be used for the preparation of an EV71 virus inhibitor, and further used for the prevention and treatment of diseases caused by EV71 virus.

[0005] And, Chinese patent CN116617226A (publication date: August 22, 2023) discloses the application of a new type of indole quinoline compound as an enterovirus 71 inhibitor. The new type of indole quinoline derivative is JL-70, JL-72 and JL-86. Through the new type of indole quinoline derivative anti-EV71 activity research experiment, it is found that the new type of indole quinoline derivative can inhibit the cytopathic effect (CPE) of EV71 produced on the host cell RD, enhance the cell survival rate, and reduce the progeny virus yield, and has the potential to be widely used in the preparation of anti-EV71 virus drugs.

[0006] The main reason for the above-mentioned shortcomings is that existing antiviral drugs mainly focus on directly inhibiting the replication and infection process of viruses, while ignoring the role of key enzymes in host cells in viral replication and pathogenic mechanisms. In the development of antiviral drugs, targeting specific key enzymes or proteins in the replication cycle of viruses has become an effective strategy. METTL3, as a key RNA methylation transferase, plays an important role in viral replication and pathogenic mechanisms. However, current research on antiviral drugs targeting METTL3 is still in its infancy, especially the specific drug screening and development for EV71 infection have not been fully developed.

[0007] Therefore, it is necessary to develop a broad-spectrum antiviral drug targeting the key molecule METTL3 in host cells to indirectly inhibit the replication and infection process of EV71 virus. By regulating the function of METTL3, the replication and pathogenicity of the virus are affected, thereby achieving the effect of antiviral, which is expected to overcome the problem of drug failure caused by viral variation, reduce the toxic side effects of drugs, and provide new possibilities for the treatment of EV71 infection. SUMMARY

[0008] In order to enhance the anti-EV71 virus activity by inhibiting the METTL3 target in host cells, the application provides the use of a compound for regulating the METTL3 target in the preparation of an antienterovirus drug.

[0009] Further, the compound is a compound AN-465 / 42162872 with the following chemical structure:

[0010]

[0011] Further, the molecular weight of the compound AN-465 / 42162872 is 433.29.

[0012] Further, the enterovirus is an EV71 virus.

[0013] Further, the compound is based on high-throughput virtual screening of Glide docking.

[0014] Compared with the prior art, the application has the following advantages and effects:

[0015] 1. The application provides application of the compound for regulating a METTL3 target point in preparation of an antienterovirus drug, the compound AN-465 / 42162872 has strong affinity, strong binding capacity and good interaction potential with the METTL3.

[0016] 2. The application provides application of the compound for regulating a METTL3 target point in preparation of an antienterovirus drug, the compound AN-465 / 42162872 has certain enzyme activity inhibition of targeting METTL3, can effectively inhibit EV71 virus-induced damage, and significantly reduces EV71 virus load.

[0017] 3. The application provides application of the compound for regulating a METTL3 target point in preparation of an antienterovirus drug, mainly by regulating METTL3, the methylation level of adenine at position 5002 in the 2C region of EV71 nucleic acid is significantly down-regulated, so that virus replication is inhibited, and an antiviral effect is achieved.

[0018] The above description is only a summary of the technical scheme of the application, in order to more clearly understand the technical means of the application, so as to implement the content of the specification, and in order to make the above and other purposes, characteristics and advantages of the application more obvious and easy to understand, the following will be described in detail with the preferred embodiments of the application and the accompanying drawings as follows.

[0019] According to the detailed description of the specific embodiments of the application in the following text combined with the drawings, those skilled in the art will more clearly understand the above and other purposes, advantages and characteristics of the application. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical scheme in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion.

[0021] Among them:

[0022] Figure 1 AN-465 / 42162872 and METTL3 binding mode diagram of an embodiment of the application;

[0023] Figure 2This is a graph showing the change of RMSD value of AN465 binding pocket amino acid residue Cα over simulation time in an embodiment of the present invention.

[0024] Figure 3 This is an SPR affinity detection diagram of AN465 targeting METTL3 & METTL14 according to an embodiment of the present invention.

[0025] Figure 4 This is an enzyme activity detection graph of a candidate compound targeting METTL3 according to an embodiment of the present invention;

[0026] Figure 5 Figure 200 shows the inhibitory effect of AN465 on EV71-induced CPE according to an embodiment of the present invention.

[0027] Figure 6 is a transmission electron microscopy observation of AN465 inhibiting human RD cell infection EV71 in an embodiment of the present invention.

[0028] Figure 7 This is a diagram illustrating the effect of AN465 on viral load in human RD cells and human PBMCs according to an embodiment of the present invention.

[0029] Figure 8 An embodiment of the present invention uses AN465 and STM2457 to monitor m during EV71 infection. 6 A modified image;

[0030] Figure 9 m, as an embodiment of the present invention 6 Correlation analysis diagram between changes in A modification and gene expression;

[0031] Figure 10 This is a gene pathway enrichment analysis diagram according to an embodiment of the present invention;

[0032] Figure 11 An embodiment of the present invention uses AN465 and STM2457 to target m on EV71 RNA. 6 A. Modification analysis diagram;

[0033] Figure 12 is a map of SELECT qPCR verification of EV71 methylation sites according to an embodiment of the present invention;

[0034] Figure 13 This is a clinical scoring chart of AN465 treatment for remission in suckling mice according to an embodiment of the present invention;

[0035] Figure 14 This is a graph illustrating the prolonged survival rate of infected suckling mice treated with AN465 according to an embodiment of the present invention.

[0036] Figure 15 shows the viral load and expression of key genes in the brain and skeletal muscle of EV71-infected suckling mice according to an embodiment of the present invention.

[0037] Figure 16 Figure 2 is a graph showing the alleviation of pathological lesions in the cerebellum of infected suckling rats by AN465 according to one embodiment of the application;

[0038] Figure 17 Figure 3 is a graph showing the alleviation of pathological lesions in the lung of infected suckling rats by AN465 according to one embodiment of the application;

[0039] Figure 18 Figure 4 is a graph showing the alleviation of pathological lesions in the muscle of infected suckling rats by AN465 according to one embodiment of the application. DETAILED DESCRIPTION

[0040] In order to make the purposes, technical solutions, and advantages of the embodiments of the present application clearer, the following will be combined with the accompanying drawings for the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. In the following description, specific details such as specific configurations and components are provided only for the purpose of helping to fully understand the embodiments of the present application. Therefore, one of ordinary skill in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, in order to be clear and concise, the description of known functions and structures is omitted in the embodiments.

[0041] It should be understood that the terms "one embodiment" or "the embodiment" mentioned throughout the specification mean that the specific features, structures or characteristics related to the embodiments are included in at least one embodiment of the present application. Therefore, "one embodiment" or "the embodiment" appearing throughout the specification does not necessarily mean the same embodiment. In addition, these specific features, structures or characteristics can be combined in one or more embodiments in any suitable manner.

[0042] In addition, reference numerals and / or letters can be repeated in different examples in the present application. Such repetition is for the purpose of simplification and clarity, and does not itself indicate a relationship between the various embodiments and / or arrangements discussed.

[0043] The term "and / or" herein is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that there are three cases of A alone, B alone, and A and B together. The term "and" herein is a description of another association relationship of the associated objects, which means that there can be two relationships, for example, A and B can mean that there are two cases of A alone and A and B together. In addition, the character " / " herein generally indicates that the associated objects before and after the " / " are in an "or" relationship.

[0044] The term "at least one", as used herein, is merely descriptive Borel correlation object, indicating that there may be three kinds of relationship, for example, A and B at least one, can indicate: the existence of A alone, A and B exist at the same time, B alone exist three cases.

[0045] It should also be noted that in this paper, such as the first and second relationship terms are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between the entities or operations. Moreover, the term "includes", "contains" or any other variant thereof is intended to cover non-exclusive inclusion.

[0046] Embodiment 1

[0047] This embodiment uses high-throughput virtual screening based on Glide docking to find specific targeting METTL3 anti-EV71 compound AN-465 / 42162872, including the following steps:

[0048] S1, select Chemdiv and Specs compound library (a total of 2,279,723 small molecules);

[0049] S2, QikProp filtering, Lipinski's five rules of drug filtering and Glide Dock filtering;

[0050] S3, cascade molecular docking by 50% HTVS, 15% SP and 15% XP mode;

[0051] S4, 25646 drug-like compounds are retained by binding free energy MM-GBSA;

[0052] S5, drug-like compounds are selected according to binding free energy and target binding mode: L088-1045, C781-0168, AN465 / 42162872, C191-0266;

[0053] S6, alanine scanning detection and molecular dynamics simulation, screening specific targeting METTL3 anti-EV71 compounds;

[0054] S7, affinity detection of AN-465 / 42162872 targeting METTL3;

[0055] S8, AN-465 / 42162872 targeting METTL3 enzyme activity inhibition detection;

[0056] Further, the step S6 is carried out by alanine scanning detection, and the four kinds of small molecules in the step S5 are subjected to molecular docking by using Glide docking, and the results are as follows Figure 1As shown, AN-465 / 42162872 docking results are better, the N-cyclohexylacetamide group of AN-465 / 42162872 and ASN549 form a hydrogen bond, the benzene ring in the compound and ARG536 form a π-cation bond, the morpholine of the morpholine ring and the propylamino group and ASP395 respectively form a salt bridge and a hydrogen bond.

[0057] The step S6 uses the Gromacs2022.3 version software to perform molecular dynamics simulation, including the following steps:

[0058] S61, pretreat the four types of small molecules in step S5, add GAFF force field to the small molecules by AmberTools22, and perform hydrogenation operation and calculate RESP potential by using Gaussian 16W software;

[0059] S62, add the calculated potential data to the topology file of the molecular dynamics system, and set the simulation conditions as static temperature 300K and normal pressure (1Bar), and use the tleap module of Amber18 to generate the topology and coordinate files of each system respectively;

[0060] S63, the parameters of the protein and the small molecule are described by Amber14SB and GAFF2 force field respectively, the solvent is selected as Tip3p water model, and the distance box edge of the protein complex is set as Add an appropriate number of Na+ions to neutralize the total charge of the simulation system;

[0061] Molecular dynamics simulation system runs:

[0062] S641, energy minimization is performed by the steepest descent method;

[0063] S642, isothermal-isochoric ensemble (NVT) equilibrium and isothermal-isobaric ensemble (NPT) equilibrium are respectively performed for 100000 steps, wherein the coupling constant is 0.1ps and the duration is 100ps;

[0064] S643, run free molecular dynamics simulation, the process is 5000000 steps, the step length is 2fs, and the total length is 100ns;

[0065] S644, after the simulation is completed, the trajectory is analyzed by using the self-contained tool of Gromacs software, and the root mean square deviation (RMSD), root mean square fluctuation value (RMSF), protein rotation radius (Rg), solvent accessible surface area (SASA), and hydrogen bond number (HBond) of the amino acid motion trajectory (Cpptraj program) are calculated.

[0066] Molecular dynamics simulation results, as shown in Figure 2 Figure 23, analyzed 100 ns simulation trajectories, in the second half of the time scale, i.e. from 60-100 ns, the RMSD fluctuation of AN-465 / 42162872 is less than that of the other three METTL3-targeting drug-like compounds, and the final average RMSD is stably between 0.25-0.30 nm, indicating that AN465 has favorable energy and thermodynamic stability in binding to METTL3.

[0067] At the same time, the data were analyzed in combination with the free energy (MM / PBSA) and free energy landscape. The MM / PBSA binding free energy showed that AN-465 / 42162872 exhibited a significant gas-phase free energy change of -94.87±0.44 kcal / mol, accompanied by a solvation free energy change (39.65±0.06 kcal / mol). The combined effect of the free energy change resulted in a relatively strong net binding affinity between the compound and METTL3, with a value of -55.22±0.45 kcal / mol, suggesting that AN-465 / 42162872 has good interaction potential with METTL3. The specific METTL3-targeting anti-EV71 compound AN-465 / 42162872 was screened out.

[0068] Further, the step S7 was performed on a Cytiva Biacore 8K instrument for surface plasmon resonance (SPR) experiments, including the following steps:

[0069] S71, using running buffer A (10 mM HBS-P+, pH 7.4) (Cytiva) to immobilize METTL3 & METTL14 (ICE Bioscience, InC.) on a CM5 sensor chip (Cytiva) at 25°C;

[0070] S72, using 250 mM EDC / 100 mM NHS (Cytiva) to activate the surface, with a contact time of 900 seconds and a flow rate of 10 μl / min;

[0071] S73, diluting METTL3 & METTL14 to a final concentration of 20 μg / mL in 10 mM acetate (pH 5.0) and injecting for 900 seconds;

[0072] S74, inactivating the surface by injecting 1M ethanolamine for 420 seconds until the immobilization density of METTL3 & METTL14 reached 10000-15000 RU.

[0073] S75, dilute the compound AN465 / 42162872 screened out in step S6 in running buffer B (10 mM HBS-P+, pH 7.4, 2% DMSO) at 25°C, and inject onto the immobilized target protein;

[0074] S76, the injection time of compound AN465 / 42162872 is 120 seconds, and the dissociation time is measured for 600 seconds at 25°C and 30 μL / min;

[0075] S77, the injection of the compound targeting METTL3 starts with the highest concentration of 100 μM, followed by 2-fold dilution, a total of 9 points;

[0076] S78, the injection of compound STM2457 starts with the highest concentration of 3 μM, followed by 3-fold dilution, a total of 9 points;

[0077] S79, subtract the sensorgrams of reference surface and blank injection from the raw data; determine the affinity and kinetic parameters by steady-state model and 1:1 binding model, respectively;

[0078] S710, pretreat the raw data by Biacore Insight software, subtract the sensorgrams of reference surface and blank injection to eliminate background interference;

[0079] S711, fit the processed data by steady-state model and 1:1 binding model to determine the affinity and kinetic parameters of the compound targeting METTL3 to the METTL3&METTL14 complex;

[0080] wherein the affinity constant (K D ) is calculated by the steady-state model, reflecting the binding strength of the compound to the target protein; while the kinetic parameters include the association rate constant (k a ) and the dissociation rate constant (k d ), which are calculated by the 1:1 binding model to obtain the affinity constant K D , and the relationship between the two rate constants at equilibrium can be represented by the affinity constant K D ,

[0081]

[0082] In the formula, the forward reaction rate constant (i.e. the association rate constant) is ka, and the reverse reaction rate constant (i.e. the dissociation rate constant) is kd.

[0083] Surface plasmon resonance (SPR) experiments further demonstrated that AN465 has strong affinity for METTL3 enzyme protein, with a binding constant (Ka) of 9.88 x 102 1 / Ms and a dissociation constant (Kd) of 8.65 x 10-3 1 / s, showing relatively strong binding capacity, and the affinity (K D ) reached 8.75 x 10 -6 M, indicating that it has high affinity with the METTL3&METTL14 complex. As shown in Figure 3 (a), the relative sensitivity time diagram of AN465 targeting METTL3&METTL14 plasmon resonance shows that the Rmax value of AN465 is 24.8, indicating that 24.8 response units can be generated per mole of ligand molecule at the saturation concentration; as shown in Figure 3 (b), the relative sensitivity volume molar concentration diagram of AN465 targeting METTL3&METTL14 plasmon resonance shows that the Chi 2 value is 7.86e+00, which is within an acceptable range, indicating good fitting degree.

[0084] Further, the step S8, METTL3 / 14 methyltransferase activity test experiment, uses the TR-FRET method to measure the methylation of METTL3 / 14 on RNA substrate, including the following steps:

[0085] S81, after adding the inhibitor containing 1% DMSO using Echo, 2.5 μL of METTL3 / 14 (ICE, S2206F-H01H) enzyme solution with a final concentration of 10 nM was added, centrifuged at 1000 rpm for 1 minute, and then reacted at 25°C for 10 minutes;

[0086] 2.5 μL of biotin-labeled METTL3-RNA (GenScript) and 0.5 μM SAM (promega, A120C) substrate reagent with a final concentration of 2 nM were added, centrifuged at 1000 rpm for 1 minute, and then reacted at 25°C for 60 minutes;

[0087] 2.5 μL of YTHDF1 (active motif, 31608) enzyme solution with a final concentration of 15 nM was added;

[0088] 2.5 μL of a mixed solution containing 700 nM Streptavidin-Tb (Perkin Elmer, 61SATLB) and 2600 nM Flag-m2d2 (Perkin Elmer, 61FG2DLA) was added;

[0089] After centrifugation at 1000 rpm for 1 minute, the reaction was carried out at 25°C for 1 hour;

[0090] The data was non-linearly fitted to a sigmoidal dose-response curve using GraphPad Prism 8 software, and the IC 50 value was calculated by the following formula:

[0091] The data was non-linearly fitted to a sigmoidal dose-response curve using GraphPad Prism 8 software, and the IC 50 value was calculated by the following formula:

[0092]

[0093] In the formula, Y represents the inhibition rate, X represents the concentration of the compound (in logarithmic form), IC 50 represents the half-inhibitory concentration, i.e. the concentration at which the compound inhibits enzyme activity by 50%, and n represents the slope of the curve.

[0094] The IC 50 value obtained by fitting was used to evaluate the inhibitory effect of the compound on METTL3 / 14 methyltransferase activity. As shown in Figure 4 , the AN465 targeted inhibition of METTL3 / 14 methyltransferase activity was detected using homogeneous time-resolved fluorescence technology (HTRF), and the test results showed that AN465 exhibited certain inhibitory activity, with an IC 50 of 1.782 μM.

[0095] Technical effects of the present embodiment: The present embodiment provides a method for obtaining a specific METTL3 targeting anti-EV71 compound through high-throughput virtual screening of Glide docking. The compound AN-465 / 42162872 has favorable energy and thermodynamic stability between METTL3, strong affinity, strong binding capacity, and good interaction potential. AN-465 / 42162872 has certain targeted inhibition of METTL3 / 14 methyltransferase activity.

[0096] Example 2

[0097] Based on the specific METTL3 targeting anti-EV71 compound AN-465 / 42162872 screened in Example 1, the present embodiment provides safety detection of AN-465 / 42162872 on human malignant embryonal rhabdomyosarcoma cells (human RD cells) and inhibition activity detection of AN-465 / 42162872 on EV71 virus.

[0098] Further, the safety detection of AN465 on human RD cells includes the following steps:

[0099] Three sample groups were set: experimental group (containing cells, culture medium, CCK-8 solution and AN465), control group (containing cells, culture medium, CCK-8 solution, without AN465), blank group (containing culture medium, CCK-8 solution, without cells and AN465), and the absorbance of each group was obtained:

[0100] The following steps were taken to obtain the absorbance of the experimental group:

[0101] 1) Prepare 100 μl of RD cell suspension in a 96-well plate, with a cell density of 5 x 10 5 cells / ml, and place the culture plate in a 37°C, 5% CO 2 incubator for 24 hours of pre-incubation;

[0102] 2) Add 10 μl of AN465 (working concentration 5-40 μM) to each well, and continue to culture for 12, 24, and 48 hours, respectively;

[0103] 3) After the incubation time, add 10 μl of CCK-8 solution to each well to avoid the formation of bubbles in the wells, and then incubate the culture plate in the incubator for 1-4 hours;

[0104] 4) Use a microplate reader to measure the absorbance value at 450 nm.

[0105] The following steps were taken to obtain the absorbance of the control group:

[0106] 1) Prepare 100 μl of RD cell suspension in a 96-well plate, with a cell density of 5 x 10 5 cells / ml, and place the culture plate in a 37°C, 5% CO 2 incubator for 24 hours of pre-incubation;

[0107] 2) After the incubation time, add 10 μl of CCK-8 solution to each well to avoid the formation of bubbles in the wells, and then incubate the culture plate in the incubator for 1-4 hours;

[0108] 3) Use a microplate reader to measure the absorbance value at 450 nm.

[0109] The following steps were taken to obtain the absorbance of the control group:

[0110] 1) Place the 96-well culture plate in a 37°C, 5% CO 2 incubator for 24 hours of pre-incubation;

[0111] 2) After the incubation time, add 10 μl of CCK-8 solution to each well to avoid the formation of bubbles in the wells, and then incubate the culture plate in the incubator for 1-4 hours;

[0112] 3) Using a microplate reader to determine the absorbance value at 450 nm.

[0113] Cell survival rate was calculated by the following formula:

[0114] Cell survival rate = [(As-Ab) / (Ac-Ab)]x100%;

[0115] In the formula: As: absorbance of the experimental group (containing cells, culture medium, CCK-8 solution and AN465)

[0116] Ac: absorbance of the control group (containing cells, culture medium, CCK-8 solution, without AN465)

[0117] Ab: absorbance of the blank group (containing culture medium, CCK-8 solution, without cells and AN465)

[0118] Further, the AN465 EV71 virus inhibition activity detection includes the following steps:

[0119] Set up three sample groups: experimental group (containing virus-infected cells, culture medium, CCK-8 solution and AN465), control group (containing virus-infected cells, culture medium, CCK-8 solution, without AN465), blank group (containing culture medium, CCK-8 solution, without cells and virus), and obtain the absorbance of each group:

[0120] Obtaining the absorbance of the experimental group includes the following steps:

[0121] 1) Prepare 100 μl of human RD cell or human peripheral blood mononuclear cell (PBMC) suspension in a 96-well plate, with a cell density of 5x10 5 6x10

[0122] 2) Prepare a virus suspension of 100 TCID50 / ml, then add 100 μl of virus suspension to each well, and place the culture plate in a 37°C, 5% CO2 incubator to allow the virus to fully adsorb to the cells;

[0123] 3) After 1 h, remove the virus-containing culture medium and gently wash the cells twice with serum-free medium to remove unadsorbed virus;

[0124] 4) Add 10 μl of AN465 solution at a predetermined concentration (working concentration 2.5-80 μM) to each well, and supplement with serum-free medium to a total volume of 100 μl;

[0125] 5) Continue to culture the culture plate under the same conditions for 24 h, and determine the cell survival rate or viral load;

[0126] 6) After incubation, 10 μΐ of CCK-8 solution was added to each well to avoid bubble formation in the well, and the plate was incubated in the incubator for 1-4 hours. Finally, the absorbance value at 450 nm was measured using a microplate reader.

[0127] Obtaining the absorbance of the control group includes the following steps:

[0128] 1) Prepare 100 μΐ of human RD cell or human peripheral blood mononuclear cell (PBMC) suspension in a 96-well plate, with a cell density of 5 x 10 5 cells / ml, and incubate the plate in a 37°C, 5% CO2 incubator for 24 hours;

[0129] 2) Prepare a virus suspension of 100 TCID50 / ml, then add 100 μΐ of virus suspension to each well, and incubate the plate in a 37°C, 5% CO2 incubator to allow the virus to fully adsorb to the cells;

[0130] 3) After 1 hour, remove the virus-containing medium and gently wash the cells twice with serum-free medium to remove unadsorbed virus;

[0131] 4) Continue to incubate the plate in a 37°C, 5% CO2 incubator for 24 hours to measure cell survival rate or viral load;

[0132] 5) After incubation, 10 μΐ of CCK-8 solution was added to each well to avoid bubble formation in the well, and the plate was incubated in the incubator for 1-4 hours. Finally, the absorbance value at 450 nm was measured using a microplate reader.

[0133] Obtaining the absorbance of the control group includes the following steps:

[0134] 1) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours;

[0135] 2) After incubation, 10 μΐ of CCK-8 solution was added to each well to avoid bubble formation in the well, and the plate was incubated in the incubator for 1-4 hours. Finally, the absorbance value at 450 nm was measured using a microplate reader.

[0136] Cell survival rate calculation formula:

[0137] Cell survival rate = [(As-Ab) / (Ac-Ab)] x 100%

[0138] Where:

[0139] As: absorbance of the experimental group (virus-infected cells, medium, CCK-8 solution, and AN465)

[0140] Ac: Absorbance of control group (containing virus-infected cells, culture medium, CCK-8 solution, not containing AN465)

[0141] Ab: Absorbance of blank group (containing only culture medium and CCK-8 solution, not containing cells and virus)

[0142] Cell inhibition rate = 100% - cell survival rate

[0143] Half maximal cytotoxicity concentration CC 50 (Half Maximal Cytotoxicity Concentration) definition:

[0144] CC 50 is the concentration of the drug that is able to cause toxicity in 50% of normal cells. The calculation logic is: treat cells with a series of different concentrations of drugs, determine the cell survival rate under each concentration, then use a nonlinear regression model to fit these data points, and find the drug concentration corresponding to the cell survival rate of 50% from the fitted curve, which is the CC 50 .

[0145] Half maximal inhibitory concentration IC 50 (Half Maximal Inhibitory Concentration) definition (for EV71 virus-infected cells):

[0146] IC 50 is the concentration of the drug that is able to inhibit 50% of EV71 virus-infected cells. The calculation logic is: in the presence of virus-infected cells, treat them with a series of different concentrations of drugs, determine the cell inhibition rate (or virus inhibition rate) under each concentration, then use a nonlinear regression model (such as Log(inhibitor) vs. response-Variable slope model) to fit these data points, and find the drug concentration corresponding to the inhibition rate of 50% from the fitted curve, which is the IC 50 . The results are shown in Figure 5 , Figure 5 (a) is the half maximal cytotoxicity concentration graph, the half maximal cytotoxicity concentration CC 50 of AN465 to human RD cells is 147.30 μM; Figure 5 (b) is the half maximal effect concentration graph, the half maximal effect concentration IC 50 of AN465 to inhibit EV71-infected cells is 18.48 μM, and the treatment index TI = 7.97, Figure 5 (c) is the blank group mock, control group EV71 infection, and experimental group (5, 10, 20 μM AN465) in the detection of AN465 inhibition activity against EV71 virus.

[0147] The technical effect of the present embodiment: when the dosage of AN465 to human RD cells reaches 147.3 μM, toxicity to normal cells occurs, which is much higher than the half-effect concentration of AN465 in inhibiting EV71 infected cells, indicating that AN465 has less toxicity to human RD cells.

[0148] Example 3

[0149] This example is based on Examples 1 and 2 to observe the effect of AN-465 / 42162872 in inhibiting EV71 infection of human RD cells using transmission electron microscopy. Three sample groups are set up, with normal RD cells as the Mock group of the control group, EV71 virus infected RD cells as the EV71 infection group of the virus model group, and 20 μM AN465 treated EV71 virus infected RD cells as the AN465 (20 μM) group of the treatment group. Fresh cells of the three sample groups are observed by transmission electron microscopy and images are captured.

[0150] The transmission electron microscopy observation includes the following steps:

[0151] 1) Cell sampling and fixation: carefully cut the fresh cells to minimize mechanical damage; within 1-3 minutes after the cells are taken out, take about 1 mm 3 of the sample; immediately place the cell cluster into a culture dish containing electron microscope fixative, and use a scalpel to cut it into 1 mm 3 pieces; transfer these small pieces to an EP tube containing fresh fixative and store at 4°C; rinse the sample with 0.1M phosphate buffered saline (PBS, pH 7.4) three times, 15 minutes each time;

[0152] 2) Post-fixation: further fix the sample in 1% osmium tetroxide in 0.1M PBS (pH 7.4) for 2 hours at room temperature, and note to avoid light; rinse again with 0.1M PBS three times, 15 minutes each time;

[0153] 3) Dehydration: dehydrate the cells in a series of ethanol solutions with concentrations from 30% to 100%, 20 minutes for each step; rinse with 100% acetone twice, 15 minutes each time;

[0154] 4) Penetration and embedding: penetrate the cells in a mixture of acetone and 812 embedding agent (1:1) for 2-4 hours at 37°C, then in a 1:2 mixture overnight; treat with pure 812 embedding agent for 5-8 hours; embed the sample in pure 812 embedding agent in an embedding plate and place it in a 37°C oven overnight;

[0155] 5) Polymerization: place the embedding plate in a 60°C oven for 48 hours to polymerize the resin;

[0156] 6) Ultra-thin sectioning: The polymerized resin block was removed and ultra-thin sections (60-80 nm) were cut from the resin block using an ultramicrotome and collected on 150 mesh Formvar coated copper grids;

[0157] 7) Staining: The copper grids were stained in 2% uranyl acetate ethanol solution for 8 minutes in the dark, then rinsed with 70% ethanol three times, and then with ultrapure water three times; stained in 2.6% lead citrate solution for 8 minutes, taking care to avoid exposure to CO2, and then rinsed with ultrapure water three times; excess liquid was absorbed with filter paper, and dried at room temperature overnight;

[0158] 8) Imaging and analysis: The prepared sample was observed under a transmission electron microscope.

[0159] Further analysis was performed on the images captured by transmission electron microscopy of the three control groups, as shown in Figure 6, under a transmission electron microscope (TEM), Figure 6(a) is the Mock group, the RD cells are spindle-shaped, the nucleus is obvious, the cytoplasm is arranged in order, and no virus particles are observed under 20,000 times magnification; Figure 6(b) is the RD cell infected with EV71 virus EV71 infection group, the group is morphologically distorted, the cell membrane is damaged, the nucleus is condensed, the cytoplasm is arranged in disorder, and virus particles can be clearly observed under 20,000 times magnification; Figure 6(c) is the RD cell infected with EV71 virus treated with 20 μM AN465 AN465 (20 μM) group, the pathological changes of the group are reduced, indicating that the virus-induced damage is effectively inhibited.

[0160] As shown in Figure 6(d),

[0161] Data analysis and plotting were performed using Graphpad prism 7.0 software, and statistical analysis was performed using one-way ANOVA. # indicates the significance of whether the virus model group is successfully infected with the virus compared with the normal control group, # indicates p≤0.05, ## indicates p≤0.01, ### indicates p≤0.001, and the smaller the p value, the higher the significance of the virus infection; * indicates the significance of whether the treatment group is effective against the virus compared with the virus model group, * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001, and the smaller the p value, the higher the significance of the virus. It can be seen that the AN465 (20 μM) group has a significance of ≤0.001 compared with the Mock group, the AN465 (20 μM) group has a significance of ≤0.001 compared with the EV71 infection group, and the number of virus particles under 20,000 times magnification of the RD cell infected with EV71 virus treated with 20 μM AN465 AN465 (20 μM) group is significantly reduced.

[0162] The technical effect of the embodiment: AN465 can effectively inhibit EV71 virus-induced damage and reduce the number of viruses.

[0163] Example 4

[0164] The present embodiment is based on Examples 1-3 to detect the viral load (qPCR) of EV71 in human cell samples by using METTL3 classic inhibitor STM2457 and AN465.

[0165] The viral load (qPCR) of EV71 in human cell samples includes the following steps:

[0166] 1) Four sample groups were set up for human RD cells and human PBMC cells respectively;

[0167] The human RD cell sample group includes: normal RD cells as the normal control group of the Mock group, EV71 virus-infected RD cells as the virus model group of the EV71 infection group, 20 μM AN465-treated EV71 virus-infected RD cells as the treatment group 1 of the AN465 (20 μM) group, and 20 μM STM2457-treated EV71 virus-infected RD cells as the treatment group 2 of the STM2457 (20 μM) group;

[0168] The human PBMC cell sample group includes: normal PBMC cells as the normal control group of the Mock group, EV71 virus-infected PBMC cells as the virus model group of the EV71 infection group, 20 μM AN465-treated EV71 virus-infected PBMC cells as the treatment group 1 of the AN465 (20 μM) group, and 20 μM STM2457-treated EV71 virus-infected PBMC cells as the treatment group 2 of the STM2457 (20 μM) group;

[0169] 1) Extract RNA from the four sample groups of human RD cells and the four sample groups of human PBMC cells:

[0170] Centrifuge at 4°C at 12000 rpm for 10 minutes, and take the supernatant;

[0171] Add 100 μl of chloroform, invert the centrifuge tube for 15 seconds to mix thoroughly, and stand for 3 minutes;

[0172] Centrifuge again at 4°C at 12000 rpm for 10 minutes;

[0173] Transfer 400 μl of the supernatant to a new centrifuge tube, add 550 μl of isopropanol, invert to mix, and stand at -20°C for 15 minutes;

[0174] Centrifuge at 12000 rpm for 10 minutes at 4℃, the white precipitate at the bottom of the tube is the RNA;

[0175] After the liquid is absorbed, 1 ml of 75% ethanol is added, and the precipitate is washed by inverting and mixing, and centrifuged at 12000 rpm for 5 minutes at 4℃;

[0176] After repeating the washing step once, the liquid is completely absorbed, and the centrifuge tube is placed on the clean bench and blown for 3-5 minutes;

[0177] Add 15 μl of RNA dissolving solution to dissolve the RNA;

[0178] Use UV5NANO to detect the concentration and purity of RNA: first, zero the instrument, then take 2.5 μl of the RNA solution to be tested and place it on the detection base, lower the sample arm, and start the absorbance detection using the software on the computer. If the RNA concentration is too high, dilute it appropriately to a final concentration of 200 ng / μl.

[0179] 2) Synthesis of cDNA: qPCR premix solution 5×SweScript All-in-One SuperMix 4 μL, gDNA remover 1 μL, total RNA*10 μL, nuclease-free water 5 μL were used to prepare the reverse transcription reaction system;

[0180] 3) Mix gently and centrifuge;

[0181] 4) On a general PCR instrument, sequentially perform 25℃ reaction for 5 min, 42℃ reaction for 30 min, 85℃ reaction for 5 sec, and heat cover 105℃ to complete reverse transcription;

[0182] 5) Prepare the following mixture in a qPCR tube: template (cDNA) 1 μL, qPCR premix 2×ChamQ Universal SYBR reaction solution 5 μL, specific reverse primer for target gene Gene specific primer F (10 μM) 0.2 μL, Gene specific primer R (10 μM) 0.2 μL, RNase-free water 3.6 μL;

[0183] The EV71 virus amplification primer sequence is as follows:

[0184]

[0185] 6) Perform qPCR reaction: Cycle the reaction 40 times at 95℃ within 5 seconds; cycle the reaction 40 times at 60℃ within 30 seconds; perform a melting curve test once at 95℃ within 15 seconds; perform a melting curve test once at 60℃ within 60 seconds; and perform a melting curve test once at 95℃ within 15 seconds.

[0186] 7) Result Data Processing:

[0187] Relative quantitative analysis was performed using the ΔΔCT method. The virus model group and treatment groups 1 and 2 were used as test samples, and the control group was used as the control sample. The fluorescence signal intensity during the PCR reaction of the test and control samples was monitored in real time. When the signal reached a preset threshold, the corresponding cycle number was recorded as the CT value. The CT value (Cycle Threshold) represents the number of cycles required for the fluorescence signal to reach the set threshold during the PCR reaction.

[0188] The ΔΔCT method includes the following formulas:

[0189] A = CT(target gene, sample to be tested) - CT(internal standard gene, sample to be tested)

[0190] B = CT(target gene, control sample) - CT(internal standard gene, control sample)

[0191] K = AB

[0192] Expression multiple = 2 -K

[0193] In the formula: A is the difference in CT values ​​between the target gene and the internal control gene in the test sample (ΔCT); A' is the difference in CT values ​​between the target gene and the internal control gene in the control sample (ΔCT); K is the difference in ΔCT values ​​between the EV71 virus gene and the internal reference gene GAPDH (ΔΔCT); and the fold change is the fold change in the expression level of the target gene in the treatment group relative to the internal reference gene GAPDH.

[0194] Data analysis and graphing were performed using Graphpad Prism 7.0 software, and one-way ANOVA was used for statistical analysis. # indicates a difference between the virus model group and the normal control group; # indicates p ≤ 0.05, ## indicates p ≤ 0.01, and ### indicates p ≤ 0.001, reflecting whether the model successfully infected the virus. * indicates a difference between the treatment group and the virus model group; * indicates p ≤ 0.05, ** indicates p ≤ 0.01, and *** indicates p ≤ 0.001, reflecting whether the drug was effective against the virus.

[0195] like Figure 7 As shown, Figure 7(a) EV71 viral load in four sample groups of human RD cells infected with EV71 virus, Figure 7 (b) EV71 viral load in four sample groups of human PBMC cells infected with EV71 virus. Compared with the classic METTL3 inhibitor STM2457 (20 μM), AN465 (20 μM) significantly reduced the EV71 viral load in human RD cells and human PBMC cells infected with EV71 virus.

[0196] Technical effects of this embodiment: AN465 has high biocompatibility in human PBMC and human RD cells, and can significantly reduce the EV71 viral load.

[0197] Example 5

[0198] This embodiment is based on Examples 1-4 to verify the inhibition of AN-465 / 42162872 METTL3-targeting compounds on EV71 infection in m 6 A and transcriptome characteristics and methylation modification sites SELLECT qPCR influence verification.

[0199] Three groups were set up to m 6 A sequencing and SELLECT qPCR influence verification: no drug group: cells infected with EV71, AN465 drug group: cells treated with AN465 after EV71 infection, and STM2457 drug group: cells treated with STM2457 after EV71 infection. The treatment concentration of AN465 and STM2457 was 20 μM.

[0200] 1) Cell m 6 A sequencing and RNA sequencing include the following steps:

[0201] Total RNA was extracted using Trizol reagent, and its quality and quantity were evaluated by Agilent Bioanalyzer, with an RIN value greater than 7.0;

[0202] mRNA was isolated, fragmented and hybridized to m 6 A specific antibodies were incubated;

[0203] After purification and precipitation, m 6 A-enriched fragments were used to construct strand-specific cDNA libraries, and 2x100 bp double-end sequencing was performed on Illumina HiSeq2500;

[0204] Raw sequencing data were uploaded to Genome Sequence Archive (https: / / ngdc.cncb.ac.cn / gsa-human, accession number HRA007624) (PMID=34400360, PMID=34718731);

[0205] Sequencing reads were aligned to human hg38 genome using HISAT2 (v2.1.0) and StringTie (v1.3.4d) was used to quantify the transcript level of Ensembl annotated genes, calculating transcripts per million (TPM);

[0206] TPM of all samples were quantile-normalized. According to previously published studies and the method of Dominissini et al., m 6 A peaks were identified by creating 100bp sliding windows (50bp overlap) in the exonic regions and calculating RPKM for each window. Windows with winscore higher than 2 were identified as m 6 A peaks;

[0207] Motif enrichment was analyzed using HOMER software, with randomized sequences as control, m 6 A peaks were shown on the distribution of "mega genes" within 5'UTR, CDS and 3'UTR, divided into ten intervals.

[0208] 2) SELLECT qPCR validation includes the following steps:

[0209] SELECT fluorescent quantitative qPCR detection was performed using Epi-SELECT m 6 A site identification kit (R202106M-01-100T, Guangzhou Aibio Biotechnology Co., Ltd.);

[0210] 1 μg of total RNA extracted from RD cells was mixed with 1 μM Up Primer, 1 μM Down Primer and 5 μM dNTP in 17 μL of 1x CutSmart buffer, and these primers and probes were specifically designed to target the 2C region of EV71;

[0211] 3 μL of a mixture consisting of 0.3 μL SELECT TM DNA polymerase, 0.47 μL SELECT TM ligase and 2.23 μL ATP was added to the RNA mixture to make the total volume 20 μL;

[0212] Quantitative PCR (qPCR) was performed on a CFX96™ real-time system (BIO-RAD) using ChamQ Universal SYBR qPCR Master Mix (Vazyme). OR800939.1 represents the complete genome of enterovirus A71 strain 560.

[0213] The differential methylation levels at sites 5002 and 5040 within the 2C protein region (encoded by nucleotides 4058 to 5044) were assessed using the SELECT assay, with these sites specifically targeted by designed primer sequences.

[0214] 3) Data statistical analysis:

[0215] Data analysis and graphing were performed using Graphpad Prism 7.0 software, and one-way ANOVA was used for statistical analysis. # indicates a difference between the virus model group and the normal control group; # indicates p ≤ 0.05, ## indicates p ≤ 0.01, and ### indicates p ≤ 0.001, reflecting whether the model successfully infected the virus. * indicates a difference between the treatment group and the virus model group; * indicates p ≤ 0.05, ** indicates p ≤ 0.01, and *** indicates p ≤ 0.001, reflecting whether the drug was effective against the virus.

[0216] The results showed that in cells treated with AN465 and STM2457, m 6 The number of peaks A has decreased. For example... Figure 8 As shown in (a), 8641 methylated genes were identified in cells infected with EV71, the highest number; followed by cells treated with AN465 with 8119 methylated genes; and the STM2457-treated group had the fewest methylated genes with 5736. Figure 8 As shown in (b), for m 6 Site A conservation analysis showed that AN465 and STM2457 conserved m 6 Distribution A has a smaller impact; such as Figure 8 As shown in (c), for m 6 A-site sequence pattern analysis showed that AN465 and STM2457 had little impact on the pattern.

[0217] like Figure 9 As shown, Figure 9 (a) is AN465 for m 6 A diagram illustrating the influence of motifs and distribution. Figure 9 (b) is for STM2457 paired with m 6 A diagram illustrating the influence of motif and distribution. For m 6 Analysis of site A conservation and sequence pattern showed that AN465 and STM2457 conserved m6 The A motif and distribution have weak effects, indicating that AN465 and STM2457 do not broadly affect all m 6 A modifications, but mainly act on specific m 6 A sites.

[0218] As Figure 10 shown, Figure 10 (a) is the correlation between the changes of m 6 A-seq and gene expression after combining m 6 A modification changes and gene expression after AN465 treatment, and Figure 10 (b) is the correlation between the changes of m 6 A-seq and gene expression after combining m 6 A modification changes and gene expression after STM2457 treatment. By combining m 6 A-seq and RNA-seq, there is a significant correlation between the changes of m 6 A modification and gene expression after STM2457 and AN465 treatment: the genes changed by STM2457 are mainly related to oncogenic pathways, while the genes affected by AN465 are related to immune regulation.

[0219] As Figure 11 shown, m 6 A modification on EV71 viral RNA, AN465 is more effective in regulating the m 6 A level of EV71 RNA as a whole, compared with the untreated control group, the AN465 administration group significantly down-regulates the methylation level of the 2C region (4500-5200bp) of EV71 RNA.

[0220] As shown in Figure 12, Figure 12(a) is a graph showing the effect of AN465 (20 mM) on the methylation level of adenine at position 5002 in the 2C region of EV712C nucleic acid in a SELECT detection test; Figure 12(b) is a graph showing the effect of AN465 (20 mM) on the methylation level of adenine at position 5040 in EV712C nucleic acid in a SELECT detection test; Figure 12(c) is a graph showing the effect of STM2457 (20 mM) on the methylation level of adenine at position 5002 in the 2C region of EV712C nucleic acid in a SELECT detection test; Figure 12(d) is a graph showing the effect of STM2457 (20 mM) on the methylation level of adenine at position 5040 in EV712C nucleic acid in a SELECT detection test. The SELECT detection test shows that AN465 (20 mM) significantly down-regulates the methylation level of adenine at position 5002 in the 2C region of EV71 nucleic acid, but has no effect on the methylation of other sites, such as the methylation of adenine at position 5040, indicating that AN465 has a specific inhibitory effect on the methylation site of adenine at position 5002; in contrast, STM2457 (20 mM) has no significant regulatory effect on the methylation of adenine in the 2C region of EV71 nucleic acid.

[0221] Technical effects of this embodiment: By comparing the effects of STM2457 and AN465 on the methylation of EV71 viral RNA, it is found that AN465 has a specific inhibitory effect on the methylation site of adenine at position 5002 in the 2C region of EV71 nucleic acid, and AN465 is more effective in regulating the methylation level of EV71 viral RNA as a whole. 6 A modification, AN465 has a specific inhibitory effect on the methylation site of adenine at position 5002, and AN465 is more effective in regulating the methylation level of EV71 viral RNA as a whole. 6 A level.

[0222] Example 6

[0223] This example is based on Examples 1-5 to study the protective effect of AN-465 / 42162872 targeting METTL3 in a mouse model of EV71 infection.

[0224] This example uses neonatal C57BL / 6 mice to establish an EV71 infected animal model, which has been approved by the Experimental Animal Ethics Committee of Guangxi Medical University (Approval No: 202312121). The neonatal mice are raised under specific pathogen-free (SPF) conditions to ensure their welfare and reduce suffering. The experiment starts from the second day after the birth of the mice, which are randomly divided into four groups (8 mice in each group): control group (normal mice); virus model group (mice infected with EV71 virus (800,000 TCID50)); treatment group 1 (mice infected with EV71 virus (800,000 TCID50) injected with 5 mg / kg of AN465); treatment group 2 (mice infected with EV71 virus (800,000 TCID50) injected with 10 mg / kg of AN465).

[0225] The virus model group and the treatment group were intracranially injected with live EV71 virus (800,000 TCID50) dissolved in sterile phosphate buffer solution (PBS) per mouse.

[0226] The treatment group was intraperitoneally injected with AN465 (5 or 10 mg / kg) daily from the second to the sixth day after infection. The four groups were monitored daily for clinical symptoms, survival rate and body weight until the 16th day after infection. Disease progression was assessed using a scoring system of 0 (healthy) to 5 (death). On the seventh day after infection, the mice were sacrificed and brain, heart, lung and muscle tissues were collected for hematoxylin-eosin (H&E) staining pathological analysis.

[0227] Data analysis and plotting were performed using Graphpad prism 7.0 software, and statistical analysis was performed using one-way ANOVA. Compared with the control group, # represents p≤0.05, ## represents p≤0.01, and ### represents p≤0.001; compared with the virus model group, * represents P≤0.05, ** represents P≤0.01, and *** represents P≤0.001.

[0228] Based on the established neonatal C57BL / 6 mouse EV71 infection model, this embodiment used a inoculation amount of 800,000 TCID50 for in vivo experiments. As the infection time increased, the clinical symptom score of the virus-infected mice gradually increased; on the eighth day after infection, the clinical symptom score of the virus control group mice reached 4 points, showing obvious clinical symptoms such as bilateral limb paralysis.

[0229] As shown in Figure 13 , the clinical symptom score of the 10 mg / kg AN465 treatment group mice decreased to less than 2 points, showing hind limb weakness or single limb paralysis, indicating that the clinical symptoms were significantly alleviated; in contrast, the clinical symptoms of the 5 mg / kg AN465 treatment group mice did not significantly decrease.

[0230] As shown in Figure 14 , as the number of infection days increased, the survival rate of EV71 virus-infected suckling mice gradually decreased; on the eighth day after infection, all mice in the virus control group died, while the 16-day survival rate of the 10 mg / kg AN465 treatment group mice was more than 70%. The survival rate trend after AN465 treatment was consistent with the clinical symptom results.

[0231] As shown in Figure 15, Figure 15(a) shows the viral load in the skeletal muscle of the control group, virus model group, and treatment group 2 suckling mice on day 7 post-infection; Figure 15(b) shows the expression of the METTL3 gene in the brain and skeletal muscle of the control group, virus model group, and treatment group 2 suckling mice on day 7 post-infection; Figure 15(c) shows the expression of the NFKB1A gene in the brain and skeletal muscle of the control group, virus model group, and treatment group 2 suckling mice on day 7 post-infection; and Figure 15(d) shows the expression of the JUN gene in the brain and skeletal muscle of the control group, virus model group, and treatment group 2 suckling mice on day 7 post-infection. On day 7 of EV71 infection, the viral load in the skeletal muscle of suckling mice was higher than that in the brain tissue. AN465 (10 mg / kg) significantly reduced the viral load in both tissues. The expression of the METTL3 gene was significantly increased in the skeletal muscle of infected suckling mice, while AN465 (10 mg / kg) significantly downregulated the expression of the METTL3 gene in both brain and skeletal muscle tissues. Similarly, the expression of the NFKB1A gene was also increased in the brain and skeletal muscle after infection, while AN465 (10 mg / kg) significantly downregulated the expression of both the NFKB1A and JUN genes in both tissues.

[0232] Following H&E staining of brain, lung, and muscle tissues, EV71 infection resulted in significant pathological changes in brain tissue, such as... Figure 16 As shown in Figures 15(a), 15(b), and 15(c), these are neuronal cell diagrams of the cerebellar region in the brain tissue of the control group, the virus model group, and the treatment group 2, respectively. Figure 15(d) shows the histological scores of the brain tissue of the control group, the virus model group, and the treatment group 2. In the virus model group, the neurons in the cerebellar region of the brain tissue showed swelling, nuclear pyknosis, accompanied by edema, vasodilation, inflammatory cell infiltration, and erythrocyte diffusion. AN465 (10 mg / kg) significantly reduced the brain tissue score of EV71 infection.

[0233] EV71 infection leads to significant pathological changes in lung tissue, such as Figure 17 As shown, Figure 17 (a), (b), and (c) are lung tissue cell images of two suckling mice in the control group, virus model group, and treatment group, respectively. Figure 17 (d) shows the lung tissue scores of the control group, virus model group, and treatment group of two suckling mice. The lung tissue of the virus model group showed alveolar wall thickening, inflammatory cell infiltration, and alveolar septal congestion, while AN465 (10 mg / kg) significantly reduced the lung tissue score of EV71 infection.

[0234] EV71 infection leads to significant pathological changes in muscle tissue, such as Figure 18 As shown, Figure 18 (a), (b), and (c) are images of muscle tissue cells from two suckling mice in the control group, virus model group, and treatment group, respectively. Figure 18(d) The histological scores of the muscle of the suckling rats in the control group, the virus model group and the treatment group 2. Muscle fiber degeneration and necrosis were observed in the muscle tissue of the virus model group, accompanied by inflammatory cell infiltration and interstitial edema, while AN465 (10 mg / kg) significantly reduced the muscle tissue score of EV71 infection.

[0235] These findings indicate that EV71 infection caused significant damage to multiple organ systems. After treatment with 10 mg / kg of AN465, the pathological changes of the above-mentioned tissues and organs of the suckling rats were significantly reduced.

[0236] Technical effects of the present embodiment: AN465 significantly down-regulated the expression of NFKB1A gene and JUN gene in the brain and skeletal muscle tissue of the suckling rats.

[0237] The above only describes the preferred embodiments of the present application and is not intended to limit the protection scope of the present application. For those skilled in the art, the present application can have various changes and variations. Any changes, modifications, replacements, integrations and parameter changes to these embodiments within the spirit and principles of the present application, which achieve the same functions by conventional substitutions or without departing from the principles and spirit of the present application, fall within the protection scope of the present application.

Claims

1. The use of a compound for regulating a METTL3 target in the preparation of an antienterovirus drug, characterized in that, The compound is a compound AN-465 / 42162872 with the following chemical structural formula: ； The compound belongs to an acetamide derivative; and the enterovirus is an EV71 virus. 2.The use of the compound for regulating the METTL3 target point in the preparation of an antienterovirus drug according to claim 1, characterized in that, The compound AN-465 / 42162872 has a molecular weight of 433.29.

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