Application of compound for regulating and controlling METTL3 target spot in preparation of anti-enterovirus medicine

Abstract

The invention provides application of a compound for regulating and controlling an METTL3 target spot in preparation of an anti-enterovirus drug, and belongs to the field of biological medicine, the compound is AN-465 / 42162872, and the compound has a relatively strong anti-EV71 effect in both in-vitro and in-vivo infection models. An in-vitro cell experiment, EV71 infected C57BL / 6 suckling mouse model, m6A methylation sequencing and other methods are utilized to research the activity and potential action mechanism of anti-EV71, and the result shows that AN-465 / 42162872 can inhibit duplication of EV71 in a human RD cell strain in a dose-dependent manner under the condition of maintaining relatively good cell activity, so that the application of AN-465 / 42162872 in the preparation of the EV71 drug is developed. The action mechanism is mainly as follows: the methylation level of the 5002 adenine in the 2C region of EV71 nucleic acid is remarkably reduced by regulating the METTL3, so that virus replication is inhibited, and the METTL3 has relatively high anti-EV71 drug utilization value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an application of a compound for regulating a METTL3 target in the preparation of an anti-enteroviral drug. Background Art

[0002] In recent years, enterovirus 71 (EV71), as a highly pathogenic RNA virus, has attracted widespread attention worldwide. EV71 is mainly transmitted through the fecal-oral route, and is particularly susceptible to infants and young children under 5 years old. It can cause hand, foot and mouth disease (HFMD) and other serious neurological complications, and even lead to death. From 2008 to 2022, the number of reported cases of hand, foot and mouth disease in China has reached more than 40 million, and the cumulative number of deaths caused by EV71 has reached more than 3,000. HFMD caused by EV71 is posing a great threat to the health of infants and young children. Although there are some preventive vaccines for HFMD, there is no clinical drug that can completely cure the disease. Given 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 EV71 inhibitors in preclinical studies, and the main development strategies include screening for repurposed drugs, targeted structural design, and rational modification of previously effective drugs. The antiviral mechanisms of these drugs are mainly targeted at various stages of the EV71 virus life cycle, including viral inhibitors targeting key sites such as viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C protease (3Cpro), and 2A protease (2Apro). However, the EV71 virus has a high mutation rate, and these inhibitors that directly target specific sites of the virus are easily affected by viral mutations, resulting in drug failure; secondly, these inhibitors often require high doses to achieve effective antiviral effects, which may increase the toxic side effects of the drug and limit its 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 the use of etoposide in the preparation of EV71 virus inhibitors, which confirms that etoposide has a strong anti-EV71 virus effect at the cellular level and can be used to prepare EV71 virus inhibitors, and then used for the prevention and treatment of related diseases caused by EV71 virus.

[0005] In addition, Chinese patent CN116617226A (publication date: August 22, 2023) discloses the use of a novel indolequinoline compound as an enterovirus type 71 inhibitor. The novel indolequinoline derivatives are JL-70, JL-72 and JL-86. Through the anti-EV71 activity research experiment of the novel indolequinoline derivatives, it was found that the novel indolequinoline derivatives can inhibit the cytopathic effect (CPE) produced by EV71 on the host cell RD, enhance cell survival rate, and reduce the production of progeny viruses, and have the potential to be widely used in the preparation of anti-EV71 virus drugs.

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

[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 an antiviral effect, which is expected to overcome the problem of drug failure caused by virus mutation, reduce the toxic side effects of the drug, and provide new possibilities for the treatment of EV71 infection. Summary of the invention

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

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

[0010]

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

[0012] Furthermore, the enterovirus is EV71 virus.

[0013] Furthermore, the compounds are screened based on high-throughput virtual screening of Glide docking.

[0014] Compared with the prior art, the advantages and effects of this application are as follows:

[0015] 1. The present invention provides a compound for regulating the METTL3 target for use in the preparation of anti-enteroviral drugs. The compound AN-465 / 42162872 has strong affinity, strong binding ability and good interaction potential with METTL3.

[0016] 2. The present invention provides a compound for regulating the METTL3 target, and its use in the preparation of anti-enteroviral drugs. The compound AN-465 / 42162872 has a certain targeted METTL3 enzyme activity inhibition, can effectively inhibit EV71 virus-induced damage, and significantly reduce EV71 virus load.

[0017] 3. The present invention provides an application of a compound for regulating the METTL3 target in the preparation of anti-enteroviral drugs, which mainly regulates METTL3 to significantly downregulate the methylation level of adenine at position 5002 in the 2C region of EV71 nucleic acid, thereby inhibiting viral replication and achieving an antiviral effect.

[0018] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0019] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all 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 scale.

[0021] in:

[0022] Figure 1 This is a diagram of the binding pattern of AN-465 / 42162872 and METTL3 according to one embodiment of the present invention;

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

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

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

[0026] Figure 5 This is a graph showing the inhibitory effect of AN465 on CPE caused by EV71 according to one embodiment of the present invention (×200);

[0027] FIG6 is a transmission electron microscopy observation of AN465 inhibiting the infection of human RD cells with EV71 according to an embodiment of the present invention;

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

[0029] Figure 8 The effects of AN465 and STM2457 on the EV71 infection period in one embodiment of the present invention 6 A modified picture;

[0030] Fig. 9 m is an embodiment of the present invention 6 A Correlation analysis diagram between modification changes and gene expression;

[0031] Fig.10 This is a gene pathway enrichment analysis diagram of an embodiment of the present invention;

[0032] Fig.11 The AN465 and STM2457 of one embodiment of the present invention are m-terminals on EV71 RNA. 6 A Modification analysis diagram;

[0033] FIG12 is a diagram of SELECT qPCR validation of EV71 methylation sites according to an embodiment of the present invention;

[0034] Fig.13 This is a clinical scoring chart of the remission of suckling mice treated with AN465 according to one embodiment of the present invention;

[0035] Fig.14 This is a graph showing the survival rate of infected suckling mice prolonged by AN465 treatment according to one embodiment of the present invention;

[0036] FIG15 is a diagram showing the viral load and expression of key genes in the brain and skeletal muscle of suckling mice infected with EV71 according to an embodiment of the present invention;

[0037] Fig.16 This is a diagram showing pathological changes in the cerebellum of infected suckling mice alleviated by AN465 according to an embodiment of the present invention;

[0038] Fig.17 This is a picture showing the pathological changes in the lung tissue of suckling mice in which AN465 alleviates infection according to one embodiment of the present invention;

[0039] Fig.18 This is a diagram showing that AN465 alleviates the pathological changes in the muscle tissue of infected suckling mice according to one embodiment of the present invention. Specific embodiments

[0040] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art 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, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0041] It should be understood that the references to "one embodiment" or "this embodiment" throughout the specification mean that the specific features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the references to "one embodiment" or "this embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0042] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity, and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0043] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0044] The term "at least one" in this article is merely a description of the association relationship of associated objects, indicating that there may be three relationships. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0045] It should also be noted that, in this article, relational terms such as first and second, etc. are only used 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 these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0046] Example 1

[0047] This example uses high-throughput virtual screening based on Glide docking to discover the anti-EV71 compound AN-465 / 42162872 that specifically targets METTL3, including the following steps:

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

[0049] S2, QikProp filtering, Lipinski's five-rule filtering, and Glide Dock filtering;

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

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

[0052] S5. Drug-like compounds were selected based on binding free energy and target binding mode: L088-1045, C781-0168, AN465 / 42162872, C191-0266;

[0053] S6, alanine scanning assay and molecular dynamics simulation to screen anti-EV71 compounds that specifically target METTL3;

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

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

[0056] Furthermore, in step S6, alanine scanning detection was performed, and Glide docking was used to perform molecular docking on the four types of small molecules in step S5. The results are as follows: Figure 1As shown in the figure, the docking result of AN-465 / 42162872 is good, and the N-cyclohexyl acetamide group of AN-465 / 42162872 is well connected with ASN549. Form hydrogen bonds between the benzene ring and ARG536 Forming a π-cationic bond, the morpholine and propylamino groups of the morpholine ring bind to ASP395 Form salt bridges and hydrogen bonds respectively.

[0057] In step S6, Gromacs 2022.3 version software is used to perform molecular dynamics simulation, including the following steps:

[0058] S61, pre-processing the four types of small molecules in step S5, adding GAFF force field to the small molecules using AmberTools22, and performing hydrogenation operation and calculating RESP potential using Gaussian 16W software;

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

[0060] The parameters of S63, protein and small molecule were described by Amber14SB and GAFF2 force fields, respectively. The solvent was selected as Tip3p water model, and the distance between the protein complex and the edge of the box was set to An appropriate number of Na+ ions were added to neutralize the total charge of the simulation system;

[0061] Molecular dynamics simulation system operation:

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

[0063] S642, 100,000 steps of isothermal and isochoric ensemble (NVT) equilibrium and isothermal and isobaric ensemble (NPT) equilibrium were performed, respectively, with a coupling constant of 0.1 ps and a duration of 100 ps;

[0064] S643, run free molecular dynamics simulation, the process has a total of 5000000 steps, the step size is 2fs, and the total duration is 100ns;

[0065] S644. After the simulation is completed, the trajectory is analyzed using the built-in tools of the Gromacs software to calculate the root mean square deviation (RMSD), root mean square fluctuation value (RMSF), protein gyration radius (Rg), solvent accessible surface area (SASA), and number of hydrogen bonds (HBond) of each amino acid motion trajectory (execute the Cpptraj program).

[0066] The molecular dynamics simulation results are shown in Figure 2 As shown, analyzing the 100ns simulation trajectory, in the second half of the time scale, i.e., from 60-100ns, the RMSD fluctuation of AN-465 / 42162872 is smaller than that of the other three METTL3-targeting drug compounds, and the final average RMSD is stable between 0.25-0.30nm, indicating the energy favorableness and thermodynamic stability of AN465 binding to METTL3.

[0067] At the same time, data such as binding free energy (MM / PBSA) and free energy profile were analyzed. MM / PBSA binding free energy showed that AN-465 / 42162872 exhibited a significant gas phase free energy change of -94.87±0.44kcal / mol, accompanied by a change in solvation free energy (39.65±0.06kcal / 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±0.45kcal / mol, indicating that AN-465 / 42162872 and METTL3 have good interaction potential. The anti-EV71 compound AN-465 / 42162872 specifically targeting METTL3 was screened out.

[0068] Furthermore, step S7 is performed on a Cytiva Biacore 8K instrument to perform a surface plasmon resonance (SPR) experiment, comprising the following steps:

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

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

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

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

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

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

[0075] S77, the compound targeting METTL3 was injected at the highest concentration of 100 μM, followed by 2-fold dilutions for a total of 9 points;

[0076] S78, compound STM2457, was injected initially at the highest concentration of 3 μM, followed by 3-fold dilutions for a total of 9 points;

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

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

[0079] S711. Fit the processed data using a steady-state model and a 1:1 binding model to determine the affinity and kinetic parameters of the METTL3-targeted compound and the METTL3&METTL14 complex;

[0080] Among them, the affinity constant (K D ) is calculated by the steady-state model, which reflects the binding strength between the compound and the target protein; while the kinetic parameters include the binding rate constant (k a ) and the dissociation rate constant (k d ), the affinity constant K was calculated by the 1:1 binding model D , at equilibrium, the relationship between these two rate constants can be expressed by the affinity constant K D To express,

[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 had a strong affinity for the METTL3 enzyme protein, with a binding constant (Ka) of 9.88×102 1 / Ms and a dissociation constant (Kd) of 8.65×10-3 1 / s, showing a relatively strong binding ability and affinity (K D ) reaches 8.75×10 -6 M, indicating that it has a high affinity with the METTL3&METTL14 complex. Figure 3 (a) shows the relative sensitivity time diagram of the plasma resonance of AN465 targeting METTL3 & METTL14. The Rmax value of AN465 is 24.8, indicating that at saturation concentration, each mole of ligand molecule can produce 24.8 response units; Figure 3 (b) shows the relative sensitivity of plasma resonance of AN465 targeting METTL3 & METTL14 in terms of volume mole concentration. 2 The value is 7.86e+00, which is within the acceptable range, indicating a good fit.

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

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

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

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

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

[0089] After centrifugation at 1000 rpm for 1 min, the mixture was reacted at 25 °C for 1 h;

[0090] The Ratio 665 / 620 signal value was read using a BMG instrument, the reading value of the negative control (1% DMSO well) was set to 0% inhibition, and the reading value of the positive control (highest concentration well of the control compound) was set to 100% inhibition. The data were nonlinearly fitted to a S-shaped dose-response curve using GraphPadPrism8 software to determine the IC 50 value.

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

[0092]

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

[0094] IC obtained by fitting 50 The inhibitory effect of the compound on METTL3 / 14 methyltransferase activity was evaluated by Figure 4 As shown in Figure 2, AN465 was used to detect the targeted inhibition of METTL3 / 14 methyltransferase activity using homogeneous time-resolved fluorescence technology (HTRF). The test results showed that AN465 exhibited certain inhibitory activity, and its IC 50 It is 1.782μM.

[0095] Technical effect of this embodiment: This embodiment provides a method for obtaining a specific targeted METTL3 anti-EV71 compound by means of high-throughput virtual screening of Glide docking. The compound AN-465 / 42162872 has energy favorableness and thermodynamic stability in binding with METTL3, strong affinity, strong binding ability, and good interaction potential. AN-465 / 42162872 has a certain targeted inhibition of METTL3 / 14 methyltransferase activity.

[0096] Example 2

[0097] This example is based on the specific METTL3-targeted anti-EV71 compound AN-465 / 42162872 screened in Example 1, and provides a safety test of AN-465 / 42162872 on human malignant embryonic rhabdoid tumor cells (human RD cells) and an inhibitory activity test on EV71 virus.

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

[0099] Three sample groups were set up: experimental group (containing cells, culture medium, CCK-8 solution and AN465), control group (containing cells, culture medium, CCK-8 solution, but not AN465), blank group (containing culture medium, CCK-8 solution, but not cells and AN465), and the absorbance of each group was obtained:

[0100] Obtaining the absorbance of the experimental group includes the following steps:

[0101] 1) Prepare 100 μl of RD cell suspension in a 96-well plate with a cell density of 5×10 5 The culture plates were placed at 37°C with 5% CO 2 Pre-culture in an incubator for 24 hours;

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

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

[0104] 4) Use an ELISA reader to measure the absorbance at 450 nm.

[0105] Obtaining the absorbance of the control group includes the following steps:

[0106] 1) Prepare 100 μl of RD cell suspension in a 96-well plate with a cell density of 5×10 5 The culture plates were placed at 37°C with 5% CO 2 Pre-culture in an incubator for 24 hours;

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

[0108] 3) Use an ELISA reader to measure the absorbance at 450 nm.

[0109] Obtaining the absorbance of the control group includes the following steps:

[0110] 1) Place the 96-well culture plate at 37°C and 5% CO 2 Pre-culture in an incubator for 24 hours;

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

[0112] 3) Use an ELISA reader to measure the absorbance at 450 nm.

[0113] The cell viability was calculated using the following formula:

[0114] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%;

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

[0116] Ac: absorbance of control group (containing cells, culture medium, CCK-8 solution, but not AN465)

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

[0118] Furthermore, the detection of the inhibitory activity of AN465 against EV71 virus includes the following steps:

[0119] Three sample groups were set up: experimental group (containing virus-infected cells, culture medium, CCK-8 solution and AN465), control group (containing virus-infected cells, culture medium, CCK-8 solution, but not AN465), blank group (containing culture medium, CCK-8 solution, but not cells and viruses), and the absorbance of each group was obtained:

[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 at a cell density of 5×10 5 cells / ml, and the culture plate was placed in an incubator at 37°C, 5% CO2 for 24 hours;

[0122] 2) Prepare a 100 TCID50 / ml virus suspension, then add 100 μl of the virus suspension to each well, and incubate 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 medium and gently wash the cells twice with serum-free medium to remove unadsorbed viruses;

[0124] 4) Add 10 μl of AN465 solution of 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) The culture plate was cultured under the same conditions for 24 h, and the cell survival rate or viral load was measured;

[0126] 6) After the incubation, add 10 μl of CCK-8 solution to each well, avoid forming bubbles in the wells, and then incubate the culture plate in an incubator for 1-4 hours. Finally, use an ELISA reader to measure the absorbance value at 450 nm.

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

[0128] 1) Prepare 100 μl of human RD cell or human peripheral blood mononuclear cell (PBMC) suspension in a 96-well plate at a cell density of 5×10 5 cells / ml, and the culture plate was placed in an incubator at 37°C, 5% CO2 for 24 hours;

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

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

[0131] 4) The culture plate was placed in an incubator at 37°C and 5% CO2 for 24 h, and the cell survival rate or viral load was measured;

[0132] 5) After the incubation, add 10 μl of CCK-8 solution to each well, avoid forming bubbles in the wells, and then incubate the culture plate in an incubator for 1-4 hours. Finally, use an ELISA reader to measure the absorbance value at 450 nm.

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

[0134] 1) Place the 96-well culture plate in an incubator at 37°C, 5% CO2 for 24 hours;

[0135] 2) After the incubation, add 10 μl of CCK-8 solution to each well, avoid forming bubbles in the wells, and then incubate the culture plate in an incubator for 1-4 hours. Finally, use an ELISA reader to measure the absorbance value at 450 nm.

[0136] The cell survival rate calculation formula is:

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

[0138] in:

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

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

[0141] Ab: absorbance of blank group (containing only culture medium and CCK-8 solution, without cells and viruses)

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

[0143] The cytotoxic concentration (CC) 50 (Half Maximal Cytotoxicity Concentration) definition:

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

[0145] IC50 cellular effect concentration 50 (Half Maximal Inhibitory Concentration) definition (for EV71 virus infected cells):

[0146] IC 50 Refers to the concentration of the drug that can inhibit EV71 virus infection of cells by 50%. The calculation logic is: in the presence of virus-infected cells, a series of different concentrations of drugs are used for treatment, and the cell inhibition rate (or virus inhibition rate) at each concentration is measured. Then, a nonlinear regression model (such as Log (inhibitor) vs. response-Variable slope model) is used to fit these data points. From the fitted curve, the drug concentration corresponding to the inhibition rate of 50% is found, which is the IC 50 The results are as follows Figure 5 As shown, Figure 5 (a) is the half cytotoxic concentration diagram, the half cytotoxic concentration CC of AN465 on human RD cells 50 147.30 μM; Figure 5 (b) is the cell half effective concentration diagram, AN465 inhibits EV71 infection of cells half effective concentration IC 50 is 18.48μM, therapeutic index TI=7.97, Figure 5 (c) The blank group mock, control group EV71 infection, and experimental group (5, 10, 20 μM AN465) in the detection of AN465's inhibitory activity against EV71 virus.

[0147] Technical effect of this embodiment: AN465 will only be toxic to normal cells when the dosage of human RD cells reaches 147.3 μM, which is much higher than the half-effective concentration of AN465 for inhibiting EV71-infected cells. It can be seen that AN465 has less toxicity to human RD cells.

[0148] Example 3

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

[0150] The transmission electron microscope observation comprises the following steps:

[0151] 1) Cell sampling and fixation: Carefully cut fresh cells to minimize mechanical damage; within 1-3 minutes after the cells are taken out, take a sample of about 1mm 3 The cell clusters were immediately placed in a culture dish containing electron microscopy fixative and cut into 1 mm 3 Transfer the pieces to EP tubes containing fresh fixative and store at 4°C; Rinse the samples three times with 0.1 M phosphate buffered saline (PBS, pH 7.4), 15 minutes each time;

[0152] 2) Post-fixation: The samples were further fixed in 1% osmium tetroxide in 0.1 M PBS (pH 7.4) for 2 hours at room temperature and protected from light; then rinsed three times with 0.1 M PBS, 15 minutes each time;

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

[0154] 4) Permeabilization and embedding: Permeabilize cells in a mixture of acetone and 812 embedding medium (1:1) for 2-4 hours at 37°C, then overnight in a 1:2 mixture; treat with pure 812 embedding medium for 5-8 hours; embed the samples in pure 812 embedding medium in embedding plates and place in a 37°C oven overnight;

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

[0156] 6) Ultrathin sectioning: Take out the polymerized resin block, cut ultrathin sections (60-80 nm) from the resin block using an ultrathin microtome, and collect them on a 150-mesh Formvar-coated copper grid;

[0157] 7) Staining: Stain the copper grid in 2% uranyl acetate ethanol solution for 8 minutes in the dark, then rinse three times with 70% ethanol and then rinse three times with ultrapure water; stain in 2.6% lead citrate solution for 8 minutes, avoid exposure to CO2, and rinse three times with ultrapure water; remove excess liquid with filter paper and dry overnight at room temperature;

[0158] 8) Imaging and analysis: Observe the prepared samples under a transmission electron microscope.

[0159] The images captured by transmission electron microscopy of the three control groups were further analyzed, as shown in Figure 6. Under transmission electron microscopy (TEM), Figure 6(a) is the Mock group, in which the RD cells are spindle-shaped, with obvious nuclei and orderly arranged cytoplasm, and no virus particles are observed at 20,000 times magnification; Figure 6(b) is the EV71infection group of RD cells infected with EV71 virus, in which the morphology is distorted, the cell membrane is damaged, the nucleus is condensed, and the cytoplasm is disordered. Virus particles can be clearly observed at 20,000 times magnification; Figure 6(c) is the AN465 (20μM) group of RD cells infected with EV71 virus treated with 20μM AN465, in which the pathological changes are alleviated, indicating that the virus-induced damage is effectively inhibited.

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

[0161] Graphpad prism 7.0 software was used for data analysis and graphing, and one-way ANOVA was used for statistical analysis. # indicates the significance of whether the virus model group was successfully infected with the virus compared with the normal control group, # indicates p≤0.05, ## indicates p≤0.01, and ### indicates p≤0.001. The smaller the p, the higher the significance of 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. The smaller the p, the higher the significance of virus effectiveness. It can be seen that the significance of the AN465 (20μM) group compared with the Mock group is ≤0.001, and the significance of the AN465 (20μM) group compared with the EV71 infection group is ≤0.001. The number of virus particles in the RD cells infected with EV71 virus in the AN465 (20μM) group treated with 20μM AN465 was significantly reduced under 20,000 times magnification.

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

[0163] Example 4

[0164] This example is based on Examples 1-3, and the viral load (qPCR) of EV71 in human cell samples was detected by using the METTL3 classical inhibitors STM2457 and AN465.

[0165] The viral load of EV71 in human cell samples (qPCR) 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 groups included: the Mock group with normal RD cells as the normal control group, the EV71 infection group with RD cells infected with EV71 virus as the virus model group, the AN465 (20 μM) group with RD cells infected with EV71 virus treated with 20 μM AN465 as treatment group 1, and the STM2457 (20 μM) group with RD cells infected with EV71 virus treated with 20 μM STM2457 as treatment group 2;

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

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

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

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

[0172] Centrifuge again at 12,000 rpm for 10 min at 4°C;

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

[0174] Centrifuge at 12,000 rpm for 10 minutes at 4°C. The white precipitate at the bottom of the tube is RNA.

[0175] After removing the liquid, add 1 ml of 75% ethanol, invert and mix to wash the precipitate, and centrifuge at 12,000 rpm for 5 minutes at 4°C;

[0176] After repeating the washing step once, remove the liquid and place the centrifuge tube on a clean bench for 3-5 minutes;

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

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

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

[0180] 3) Mix gently and centrifuge;

[0181] 4) On a common PCR instrument, react at 25°C for 5 min, 42°C for 30 min, 85°C for 5 sec, and heat the cover at 105°C to complete reverse transcription;

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

[0183] The EV71 virus amplification primer sequences are as follows:

[0184]

[0185] 6) Perform qPCR reaction: perform the following reactions in sequence: 40 cycles at 95°C within 5 seconds; 40 cycles at 60°C within 30 seconds; melting curve once at 95°C within 15 seconds; melting curve once at 60°C within 60 seconds; melting curve once at 95°C within 15 seconds, perform qPCR reaction;

[0186] 7) Result data processing:

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

[0188] The ΔΔCT method includes the following formula:

[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 value between the target gene and the internal standard gene in the test sample (ΔCT); A is the difference in CT value between the target gene and the internal standard gene in the control sample (ΔCT); K is the difference in ΔCT value between the EV71 virus gene and the internal reference gene GAPDH (ΔΔCT); the expression fold is the change in the expression level of the target gene in the treatment group relative to the internal reference gene GAPDH

[0194] Graphpad prism 7.0 software was used for data analysis and graphing, and one-way ANOVA was used for statistical analysis. # indicates that the virus model group is compared with the normal control group, # indicates p≤0.05, ## indicates p≤0.01, and ### indicates p≤0.001, reflecting whether the model is successfully infected with the virus; * indicates that the treatment group is compared with the virus model group, * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001, reflecting whether the drug is effective against the virus.

[0195] like Figure 7 As shown, Figure 7(a) EV71 virus 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 both human RD cells and human PBMC cells infected with EV71 virus.

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

[0197] Example 5

[0198] This example is based on Examples 1-4 to investigate the effects of AN-465 / 42162872 targeting METTL3 compounds on EV71 infection 6 A and transcriptome characteristics and methylation modification site SELLECT qPCR impact verification.

[0199] Set up three groups to perform m 6 A Sequencing and SELLECT qPCR validation: No drug group: cells infected with EV71, AN465 drug group: cells infected with EV71 and then treated with AN465, and STM2457 drug group: cells infected with EV71 and then treated with STM2457. The treatment concentrations of AN465 and STM2457 were both 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 assessed by Agilent Bioanalyzer, with a RIN value greater than 7.0;

[0202] Isolate mRNA, fragment it and 6 A-specific antibody incubation;

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

[0204] The 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 the human hg38 genome using HISAT2 (v2.1.0), and transcript levels of Ensembl annotated genes were quantified using StringTie (v1.3.4d) to calculate transcripts per million (TPM);

[0206] TPMs were quantile normalized for all samples. m was identified by creating 100 bp sliding windows (50 bp overlap) in the exonic region and calculating the RPKM for each window according to previously published studies and the method of Dominissini et al. 6 A peak; windows with an enrichment score (winscore) higher than 2 were identified as m 6 Peak A;

[0207] HOMER software was used to analyze motif enrichment, and random shuffled sequences were used as controls. 6 The distribution of peak A is shown in ten intervals on the “mega gene” within the 5’UTR, CDS, and 3’UTR.

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

[0209] Using Epi-SELECT m 6 A site identification kit (R202106M-01-100T, Guangzhou Abbio Biotechnology Co., Ltd.) was used for SELECT fluorescence quantitative qPCR detection;

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

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

[0212] Quantitative PCR (qPCR) was performed on a CFX96TM Real-Time System (BIO-RAD) using ChamQ Universal SYBR qPCR Master Mix (Vazyme), with OR800939.1 representing the complete genome of enterovirus A71 strain 560;

[0213] The SELECT assay was used to assess the differential methylation levels at positions 5002 and 5040 within the 2C protein region (encoded by nucleotides 4058 to 5044), which were specifically targeted by designed primer sequences.

[0214] 3) Data statistical analysis:

[0215] Graphpad prism 7.0 software was used for data analysis and graphing, and one-way ANOVA was used for statistical analysis. # indicates that the virus model group is compared with the normal control group, # indicates p≤0.05, ## indicates p≤0.01, and ### indicates p≤0.001, reflecting whether the model is successfully infected with the virus; * indicates that the treatment group is compared with the virus model group, * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001, reflecting whether the drug is effective against the virus.

[0216] The results showed that in cells treated with AN465 and STM2457, m 6 The number of peak A has decreased. Figure 8 As shown in (a), a total of 8641 methylated genes were identified in cells infected with EV71, which is the largest number; 8119 methylated genes were identified in cells treated with AN465, which is the second largest number; 5736 methylated genes were identified in the STM2457 treatment group, which is the smallest number; Figure 8 (b) shows that for m 6 A site conservation analysis showed that AN465 and STM2457 had no significant effect on m 6 A distribution has little impact; Figure 8 (c) shows that for m 6 Analysis of the A site sequence pattern showed that AN465 and STM2457 had little effect on the pattern.

[0217] like Fig. 9 As shown, Fig. 9 (a) is AN465 for m 6 A influence diagram of motifs and distributions, Fig. 9 (b) STM2457 for m 6 A motif and distribution influence diagram. 6 Analysis of conservation and sequence patterns at the A site showed that AN465 and STM2457 had a significant effect on m6 The effects on A motifs and distribution were weak, indicating that AN465 and STM2457 do not broadly affect all m 6 A modification, but mainly acts on specific m 6 A site.

[0218] like Fig.10 As shown, Fig.10 (a) is to convert m 6 After combining A-seq with RNA-seq, AN465 had a significant effect on m 6 A correlation effect diagram between modification changes and gene expression, Fig.10 (b) is to convert m 6 After combining A-seq with RNA-seq, STM2457 6 A correlation diagram between changes in modification and gene expression. 6 A-seq combined with RNA-seq, STM2457 and AN465 treated m 6 There was a significant correlation between changes in A modifications and gene expression: the genes altered by STM2457 were mainly related to oncogenic pathways, while the genes affected by AN465 were related to immune regulation.

[0219] like Fig.11 As shown, m 6 A modification, AN465 is more effective in regulating the mRNA of EV71 RNA overall 6 A level, compared with the non-drug treatment group, the AN465-treated group significantly downregulated 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 μM) on the methylation level of adenine at position 5002 in the 2C region of EV712C nucleic acid in the SELECT detection test; Figure 12(a) is a graph showing the effect of AN465 (20 μM) on the methylation level of adenine at position 5040 in the EV712C nucleic acid in the SELECT detection test; Figure 12(c) is a graph showing the effect of STM2457 (20 μM) on the methylation level of adenine at position 5002 in the 2C region of EV712C nucleic acid in the SELECT detection test; Figure 12(d) is a graph showing the effect of STM2457 (20 μM) on the methylation level of adenine at position 5040 in the EV712C nucleic acid in the SELECT detection test. The SELECT detection experiment showed that AN465 (20 μM) significantly downregulated the methylation level of adenine at position 5002 in the 2C region of EV71 nucleic acid, but had 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 adenine methylation site at position 5002; in contrast, STM2457 (20 μM) had no significant regulatory effect on the methylation of adenine in the 2C region of EV71 nucleic acid.

[0221] Technical effect of this embodiment: By comparing the mRNA of STM2457 and AN465 on EV71 virus RNA 6 A modification, AN465 has a specific inhibitory effect on the 5002nd adenine methylation site, and AN465 is more effective in regulating m on EV71 viral RNA overall. 6 A level.

[0222] Example 6

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

[0224] This example uses newborn C57BL / 6 mice to establish an animal model of EV71 infection, which has been approved by the Experimental Animal Ethics Committee of Guangxi Medical University (approval number: 202312121). Newborn mice were raised under specific pathogen-free (SPF) conditions to ensure their welfare and reduce pain. The experiment started on the second day after the mice were born, and they were randomly divided into four groups (8 mice in each group): control group (normal mice); virus model group (mice infected with EV71 virus (800,000TCID50)); treatment group 1 (mice infected with EV71 virus (800,000TCID50) injected with 5mg / kg AN465); treatment group 2 (mice infected with EV71 virus (800,000TCID50) injected with 10mg / kg AN465).

[0225] Each mouse in the virus model group and the treatment group was intracranially injected with live EV71 virus (800,000 TCID50) dissolved in sterile phosphate buffered saline (PBS).

[0226] The treatment group received daily intraperitoneal injections of AN465 (5 or 10 mg / kg) from day 2 to day 6 after infection. Clinical symptoms, survival rate, and body weight of the four groups were monitored daily until day 16 after infection. Disease progression was assessed using a scoring system ranging from 0 (healthy) to 5 (death). On day 7 after infection, mice were sacrificed, and brain, heart, lung, and muscle tissues were collected for pathological analysis by hematoxylin-eosin (H&E) staining.

[0227] Graphpad prism 7.0 software was used for data analysis and graphing, and one-way ANOVA was used for statistical analysis. Compared with the control group, # indicates p≤0.05, ## indicates p≤0.01, and ### indicates p≤0.001; compared with the virus model group, * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001.

[0228] Based on the established newborn C57BL / 6 mouse EV71 infection model, this example used an inoculum of 800,000 TCID50 for in vivo experiments. As the infection time increased, the clinical symptom scores of virus-infected mice gradually increased; on the 8th day after infection, the clinical symptom score of the virus control group mice reached 4 points, showing obvious clinical symptoms, such as paralysis of both limbs.

[0229] like Fig.13 As shown, the clinical symptom scores of mice in the 10 mg / kg AN465 treatment group dropped below 2 points, manifested as hind limb weakness or monoplegia, indicating that the clinical symptoms were significantly alleviated; in contrast, the clinical symptoms of mice in the 5 mg / kg AN465 treatment group did not show significant relief.

[0230] like Fig.14 As shown in the figure, as the number of days of infection increases, the survival rate of suckling mice infected with EV71 virus gradually decreases; on the 8th day after infection, all mice in the virus control group died, while the 16-day survival rate of mice in the 10mg / kg AN465 treatment group exceeded 70%. The survival rate trend after AN465 treatment is consistent with the clinical symptom results.

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

[0232] After H&E staining of brain, lung and muscle tissues, EV71 infection caused obvious brain tissue pathological changes such as Fig.16 As shown, Figure 15 (a), (b), and (c) are images of neurons in the cerebellum region of the brain tissue of the control group, the virus model group, and the treatment group 2, respectively, and Figure 15 (d) is the histological score of the brain of the control group, the virus model group, and the treatment group 2. The neurons in the cerebellum region of the brain tissue of the virus model group showed swelling and nuclear condensation, accompanied by edema, vasodilation, inflammatory cell infiltration, and red blood cell diffusion, while AN465 (10 mg / kg) significantly reduced the brain tissue score infected with EV71.

[0233] EV71 infection causes obvious pathological changes in lung tissue such as Fig.17 As shown, Fig.17 (a)(b)(c) are lung tissue cell images of two newborn mice in the control group, virus model group, and treatment group, respectively. Fig.17 (d) Lung histological scores of the control group, virus model group, and treatment group 2 neonatal 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 causes obvious pathological changes in muscle tissue such as Fig.18 As shown, Fig.18 (a)(b)(c) are the muscle tissue cell images of the control group, virus model group and treatment group 2. Fig.18(d) Histological scores of the muscles of the control group, virus model group, and treatment group 2. Myofiber 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 infected by EV71.

[0235] These findings suggest that EV71 infection causes significant damage to multiple organ systems. After treatment with 10 mg / kg AN465, the pathological changes in the above-mentioned tissues and organs in suckling mice were significantly alleviated.

[0236] Technical effect of this embodiment: AN465 significantly down-regulated the expression of NFKB1A gene and JUN gene in the brain and skeletal muscle tissue of neonatal mice.

[0237] The above description is only the preferred embodiment of the present invention, which does not limit the protection scope of the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Within the spirit and principle of the present invention, any change, modification, replacement, integration and parameter change of these embodiments by conventional substitution or capable of achieving the same function without departing from the principle and spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of a compound that regulates the METTL3 target in the preparation of anti-enteroviral drugs.

2. The use of a compound for regulating the METTL3 target according to claim 1 in the preparation of an anti-enteroviral drug, characterized in that: The compound is compound AN-465 / 42162872 having the following chemical structural formula:

3. Use of a compound for regulating the METTL3 target according to claim 1 in the preparation of an anti-enteroviral drug, characterized in that: The molecular weight of the compound AN-465 / 42162872 is 433.

29.

4. Use of a compound for regulating the METTL3 target according to claim 1 in the preparation of an anti-enteroviral drug, wherein the enterovirus is EV71 virus.

5. Use of a compound for regulating the METTL3 target according to claim 1 in the preparation of an anti-enteroviral drug, wherein the compound is based on high-throughput virtual screening of Glide docking.

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