阅读说明：本技术 Gsk872在制备治疗肠道病毒71型感染导致的疾病的药物中的用途 (Application of GSK872 in preparation of medicines for treating diseases caused by enterovirus 71 infection ) 是由 许甜甜 王长兵 李英华 邝璐 郭敏 朱冰 陈海洋 于 2021-06-18 设计创作，主要内容包括：本发明公开了GSK872在制备治疗肠道病毒71型感染导致的疾病的药物中的用途,涉及制药领域。GSK872是蛋白激酶3抑制剂中的一种,本发明首次通过实验验证了蛋白激酶3抑制剂GSK872能够有效抑制肠道病毒71型引起的细胞病变,并结合Vero细胞存活实验,得到了可用于治疗肠道病毒71型感染导致的婴幼儿手足口病的GSK872的安全剂量,为进一步开发治疗婴幼儿手足口病药物提供了指导,同时,该化合物可接受进一步开发为治疗手足口病的药物,剂型和给药方式多样化,具有广泛的应用前景。(The invention discloses application of GSK872 in preparation of a medicine for treating diseases caused by enterovirus 71 infection, and relates to the field of pharmacy. The GSK872 is one of protein kinase 3 inhibitors, experiments verify that the protein kinase 3 inhibitor GSK872 can effectively inhibit cytopathic effect caused by enterovirus 71 for the first time, and the safe dose of the GSK872 for treating infant hand-foot-and-mouth disease caused by enterovirus 71 infection is obtained by combining Vero cell survival experiments, so that guidance is provided for further developing medicines for treating infant hand-foot-and-mouth disease, meanwhile, the compound can be further developed into medicines for treating hand-foot-and-mouth disease, and the preparation form and the administration mode are diversified, so that the compound has wide application prospect.)

Use of GSK872 in the manufacture of a medicament for the treatment or prevention of a disease caused by an enterovirus type 71 infection.

2. The use according to claim 1, wherein GSK872 has the molecular formula:

3. the use according to claim 1, wherein the disease caused by an enterovirus type 71 infection comprises infant hand-foot-and-mouth disease, herpangina, bronchitis, pneumonia, gastroenteritis, aseptic meningitis, acute flaccid paralysis or encephalitis.

4. The use according to claim 3, wherein the infant hand-foot-and-mouth disease is a disease caused by protein kinase 3 participating in replication of enterovirus 71 type, and/or is a disease caused by protein kinase 3 participating in cell necrosis caused by enterovirus 71 type.

5. A drug for treating or preventing a disease caused by enterovirus 71 infection, which comprises GSK872 as an antiviral ingredient;

preferably, the disease caused by the enterovirus 71 infection comprises hand-foot-and-mouth disease of infants.

6. The medicament of claim 5, comprising a pharmaceutically effective dose of GSK872 and a pharmaceutically acceptable pharmaceutical carrier.

7. The medicament according to claim 6, wherein the effective dose of GSK872 is 2.5 μ M or more, preferably 2.5 μ M.

8. The drug of claim 7, wherein the drug carrier comprises nanoseled particles loaded with a targeting agent;

preferably, the targeting agent comprises N epsilon carboxymethyllysine.

9. The medicament according to claim 8, wherein the molar ratio of GSK872 to Nepsilon-carboxymethyllysine is 1: 0.8-1.2.

10. A medicament as claimed in any one of claims 6 to 9, wherein the dosage form of the medicament comprises a granulate formulation and the mode of administration comprises oral administration.

Technical Field

The invention relates to the field of pharmacy, in particular to application of GSK872 in preparing a medicament for treating diseases caused by enterovirus 71 infection.

Background

Enterovirus 71 (EV 71) is a main pathogen causing Hand-foot-and-mouth disease (HFMD) of infants, and EV71 infection causes high proportion of severe cases and high fatality rate.

At present, the method is a prevention means mainly based on EV71 virus vaccine injection aiming at EV71 in China. The lack of safe and effective antiviral therapeutic drugs brings serious challenges to the prevention, control and treatment of EV 71. Therefore, effective medicaments for preventing and treating the hand-foot-and-mouth disease are problems to be solved urgently.

Disclosure of Invention

The invention aims to provide a novel application of a protein kinase 3 inhibitor GSK872 and a medicament for realizing the application.

In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the invention provides the use of the protein kinase 3 inhibitor GSK872 in the manufacture of a medicament for the treatment or prevention of a disease caused by an enterovirus type 71 infection, which was discovered unintentionally by the inventors and verified experimentally.

The GSK872 provided by the invention is an effective and specific protein kinase 3 inhibitor, is combined with a RIP3 kinase region with high affinity (IC50 is 1.8nM), has an enzyme activity inhibiting IC50 of 1.3nM, only has weak cross reaction, and has a molecular structural formula as follows:

the diseases caused by the enterovirus 71 infection comprise infant hand-foot-and-mouth disease, herpangina, bronchitis, pneumonia, gastroenteritis, aseptic meningitis, acute flaccid paralysis and encephalitis.

The infant hand-foot-and-mouth disease is a disease caused by the protein kinase 3 participating in replication of enterovirus 71, and/or the infant hand-foot-and-mouth disease is a disease caused by the protein kinase 3 participating in cell necrosis caused by enterovirus 71.

In a second aspect, the present invention provides a drug for treating or preventing a disease caused by enterovirus 71 infection, which comprises GSK872 as an antiviral ingredient, wherein the drug exerts its pharmacological effect in a cell matrix.

Preferably, the disease caused by the enterovirus 71 infection comprises hand-foot-and-mouth disease of infants.

In an alternative embodiment, the medicament provided by the present invention comprises a pharmaceutically effective dose of GSK872 and a pharmaceutically acceptable pharmaceutical carrier. The "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the vector and GSK872 are combined without significantly reducing the efficacy of GSK 872. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifying agents and wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

In an alternative embodiment, the effective dose of GSK872 in the medicament is 2.5 μ M or more, preferably 2.5 μ M, which means that the GSK872 is used in an amount sufficient to significantly improve the condition without causing serious side effects.

In an alternative embodiment, the drug carrier comprises nanoseled particles loaded with a targeting agent.

Preferably, the targeting agent comprises N epsilon carboxymethyllysine.

In an alternative embodiment, the molar ratio of GSK872 to N epsilon carboxymethyllysine is 1:0.8 to 1.2.

In an alternative embodiment, the above-mentioned medicament is administered orally, and the medicament for oral administration includes solid dosage forms such as capsules, tablets, pills, powders and granules. In these solid dosage forms, GSK872 is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Such solid dosage forms as tablets, dragees, capsules, pills and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

The GSK872 with pharmaceutically effective dose and the pharmaceutically applicable nano selenium particle carrier loaded with the targeting agent are prepared into the formula granules, the formula granules are taken orally, so that the medicine is fully absorbed in small intestines, then under the guidance of the targeting agent, the nano selenium particles deliver the GSK872 with the effective dose to cell matrixes of cells infected with pathogens, and the treatment effect on the infant hand-foot-and-mouth disease caused by the enterovirus 71 infection is realized by blocking the replication of the enterovirus 71 and/or preventing normal Vero necrosis.

The invention verifies that the protein kinase 3 inhibitor GSK872 can effectively inhibit cytopathic effect caused by enterovirus 71 for the first time through experiments, and obtains the safe dose of the GSK872 for treating the infant hand-foot-and-mouth disease caused by enterovirus 71 infection by combining Vero cell survival experiments, thereby providing guidance for further developing the medicine for treating the infant hand-foot-and-mouth disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the toxicity microscopic examination results of different concentrations of GSK872 on Vero cells in example 1 of the present invention;

FIG. 2 shows the results of CCK8 assay of Vero cytotoxicity of GSK872 in different concentrations in example 1;

FIG. 3 shows microscopic examination results of the protective effect of GSK872 on Vero cells at different concentrations in example 1 of the present invention;

FIG. 4 shows the results of CCK8 assay of Vero cells protected by GSK872 at different concentrations in example 1;

FIG. 5 shows the flow cytometer test results of example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to determine that the GSK872 and the medicine taking the GSK872 as the main component have the effect of treating the infant hand-foot-and-mouth disease caused by the enterovirus 71 type infection, the invention provides the following detection method:

1. a method for determining that GSK872 has an anti-enterovirus 71 effect, comprising the steps of:

(1) preparation of GSK872 solution

GSK872 was configured to four concentrations of 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M using DMSO for use;

(2) enterovirus 71 virulence assay

Separately using DME for enterovirus type 71M gradient dilution to 10^-1、10^-2、10^-3、10^-4、10^-5、 10^-6、10^-7And 10^-8Adding virus diluent with 8 concentrations into cell culture plate according to 100 μ L per well, wherein Vero cells are contained in each well to be tested and have concentration of 4 × 10⁴Setting a normal control group only added with DMEM (dimethyl ether), placing a cell culture plate in a cell culture box for culture, after the culture is finished, removing supernatant, washing with PBS (phosphate buffered saline), adding 200 mu L of DMEM into each hole to be tested, continuing the culture, observing the cell state under a microscope, calculating the virus TCID50 by using a Reed-Muench method, repeating the experiment for three times, calculating the TCID50 of the enterovirus 71 type, and recording;

(3) toxicity detection of GSK872 solution on Vero cells

Vero cells at 4X 10⁴One/well was plated on cell culture plates at 37 ℃ with 5% CO₂Culturing in an incubator, then respectively adding GSK872 with the concentrations of 10 MuM, 5 MuM, 2.5 MuM, 1.25 MuM and 0.625 MuM into the first group of wells to be detected to process Vero cells, adding DMEM (namely a control group) into the second group of wells to be detected, continuously culturing, then carrying out cell survival rate detection, determining the OD570 value of each well, and recording;

(4) determination of anti-enterovirus 71 type effect of GSK872 solution

Cultured Vero cells were digested with 0.25% trypsin at 4X 10⁴One/well plated in cell plates at 37 ℃ with 5% CO₂The culture is carried out in an incubator, then 100TCID50 doses of enterovirus 71 are added for 2h, the supernatant is discarded and washed, then the first group is added with GSK872 treatment with the concentration of 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M, the second group is added with DMEM (namely a control group), the culture is continued, then the cell survival experiment is carried out, and the OD570 value of each well is determined and recorded.

2. The method for judging that the medicine for treating the infant hand-foot-and-mouth disease caused by the enterovirus 71 infection by taking GSK872 as a main medicinal component has the effect of resisting the enterovirus 71 comprises the following steps:

(1) preparation of drug sample to be tested and GSK872 solution

GSK872 was configured to four concentrations of 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M using DMSO for use;

grinding and dissolving a drug sample to be detected, dissolving the drug sample by using DMSO (dimethyl sulfoxide), determining the concentration of GSK872 in the solution, and taking the ground solution of the drug sample to be detected with the corresponding concentration of the GSK872 of 10 mu M, 5 mu M, 2.5 mu M, 1.25 mu M and 0.625 mu M for later use;

the preparation method of the drug to be detected comprises the steps of dissolving N epsilon carboxymethyl lysine in DMSO, adding methanol to dilute the solution into four concentrations of 10 mu M, 5 mu M, 2.5 mu M, 1.25 mu M and 0.625 mu M, mixing the GSK872 solution and the N epsilon carboxymethyl lysine solution in an equimolar manner, adding hydroxylation-treated nano selenium particles, stirring, and carrying out spray drying to obtain a drug sample to be detected.

(2) Enterovirus 71 virulence assay

Enterovirus 71 was diluted to 10 with DMEM gradient^-1、10^-2、10^-3、10^-4、10^-5、 10^-6、10^-7And 10^-8Adding virus diluent with 8 concentrations into a cell culture plate according to 100 mu L of virus diluent per well, wherein each well to be tested of the cell culture plate contains Vero cells with the concentration of 4 multiplied by 10⁴Setting a normal control group only added with DMEM (dimethyl ether), placing a cell culture plate in a cell culture box for culture, after the culture is finished, removing supernatant, washing with PBS (phosphate buffered saline), adding 200 mu L of DMEM into each hole to be detected, continuing the culture, observing the cell state under a microscope, calculating the virus TCID50 by using a Reed-Muench method, repeating the experiment for three times, calculating the TCID50 of the enterovirus 71 type, and recording;

(3) toxicity detection of Vero cells of drug test sample to be detected

Vero cells at 4X 10⁴One/well was plated on cell culture plates at 37 ℃ with 5% CO₂Culturing in an incubator, adding GSK872 with concentration of 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M into the first group of wells to be tested to treat Vero cells, adding corresponding concentration of drug sample solution into the second group of wells to be tested, adding DMEM (i.e. control group) into the third group of wells to be tested, culturing, detecting cell survival rate, and determining each cell survival rateWell OD570 values, and recorded;

(4) determination of anti-enterovirus 71 action of drug sample to be tested

Cultured Vero cells were digested with 0.25% trypsin at 4X 10⁴One/well plated in cell plates at 37 ℃ with 5% CO₂Culturing in an incubator, adding 100TCID50 dosage of enterovirus 71, incubating for 2h, discarding supernatant and washing, adding GSK872 with concentration of 10 μm/μ l, 5 μm/μ l, 2.5 μm/μ l, 1.25 μm/μ l and 0.625 μm/μ l to the first group, adding corresponding concentration of test drug sample solution to the second group, adding DMEM (namely control group) to the third group, continuing culturing, performing cell viability experiment, determining OD570 value of each well, and recording.

The detection results of the method for judging that the GSK872 has the effect of resisting the enterovirus 71 and the method for judging that the medicine for treating the infant hand-foot-and-mouth disease caused by the enterovirus 71 has the effect of resisting the enterovirus 71 prove that the GSK872 has the effect of resisting the enterovirus 71, and the dosage of the GSK872 in the medicine for treating the infant hand-foot-and-mouth disease caused by the GSK872 enterovirus 71 can be better determined by comparing the effect of resisting the enterovirus 71 containing effective dose of the medicine with the effect of resisting the enterovirus 71 of a GSK872 solution, so that a basis is provided for developing the medicine taking the GSK872 as a main antiviral component.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 use of GSK872 in the preparation of an anti-Enterovirus 71 drug

Reagents and materials

Enterovirus type 71 (GenBank No. FJ439769.1) used in this example was obtained from a central laboratory clinical isolate from the women's medical center, guangzhou city. (Zhongjiayu, Zhubing, Hualiang, Wangzhong, Kuang xylol, Xijiahui, Chenassist in 2008 and 2010 Enterovirus 71 Guangzhou isolate genome-wide sequence analysis of the journal of Chinese epidemiology [ J ]. 2011,32(7): 700-.

The Vero cells used in this example were purchased from the shanghai life science research institute cell bank of the chinese academy of sciences, catalog No.: GNO10 are provided.

GSK872 used in this example was obtained from MCE corporation, Cat.No. HY-101872.

Second, enterovirus 71 virulence determination

Enterovirus 71 was diluted to 10 with DMEM gradient^-1、10^-2、10^-3、10^-4、10^-5、 10^-6、10^-7And 10^-8Adding the virus liquid into Vero cells according to 100 mu L per well to obtain 8 concentrations in total, setting a normal Vero cell control group, placing a 96-well plate into a cell culture box for culturing for 2h, removing supernatant, washing 3 times with PBS, adding 200 mu L DMEM per well, continuing culturing, counting the CPE number as shown in table 1, calculating the virus TCID50 by using a Reed-Muench method, repeating the experiment for three times, and calculating the TCID50 of EV71 to be 1 x 10^-5.11The calculation process is as follows:

TABLE 1 CPE Numbers statistics

CCID50 was calculated according to Reed and Muench two's method. The formula is as follows:

distance ratio (percentage above 50% rate of illness-50%)/(percentage above 50% rate of illness-percentage below 50% rate of illness)

＝(55-50)/(55-8)

＝0.11

IgTCID50 difference between log dilution in distance proportion + log dilution above 50% disease rate

＝0.11*(-1)+(-5)

＝-5.11

TCID50＝10^-5.11I.e. diluting the virus 10^-5.1150% of the cells were diseased by inoculating 100. mu.l.

Third, toxicity detection of GSK872 to Vero cells

Vero cells at 4X 10⁴One/well in 96-well plates at 37 ℃ with 5% CO₂After 24h of incubation in the incubator, Vero cells were treated with GSK872 at concentrations of 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M and 0.625. mu.M, respectively. After 48h of culture, the Vero cells were observed for damage by GSK872 under an optical microscope, and as a result, as shown in FIG. 1, it can be seen that no cytotoxicity was observed at the concentrations of GSK872 of 0.625. mu.M and 1.25. mu.M, and the cytotoxicity became more and more significant with the increase in the concentration of GSK872 when the concentration of GSK872 was 2.5. mu.M to 10. mu.M. After the total culture time is 48h, the cell survival rate is detected by a CCK8 method, CCK8 reagent with the concentration of 5mg/mL is added into a culture plate at 10 mu L/hole, the mixture is fully mixed, the mixture is incubated at 37 ℃ for 30min, then the liquid in the culture plate is sucked away, and the OD570 value of each hole is determined. The OD570 value of the Vero cell control group was set to 100%, and the cell survival rate (%) was determined as (OD)₅₇₀Experimental group/OD₅₇₀Control group) x 100% was calculated and the results are presented as a bar graph, see fig. 2, where: p < 0.05, x: p < 0.01, it can be seen that the survival rate of Vero cells treated with GSK at 0.625. mu.M and 1.25. mu.M was not different from that of control group cells, and the survival rate of Vero cells treated with GSK at 2.5. mu.M, 5. mu.M and 10. mu.M decreased with the increase in GSK concentration. Indicating that GSK concentrations of 0.625. mu.M and 1.25. mu.M are not toxic to Vero cells.

Detection of protective effect of GSK872 anti-enterovirus 71 on Vero cells

Cultured Vero cells were digested with 0.25% trypsin at 4X 10⁴One/well in 96-well plates at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24 hours, the enterovirus 71 was incubated for 2 hours with a dose of 100TCID50 the following day, the supernatant was discarded and washed with PBS 3 times, and then GSK872 at concentrations of 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M and 0.625. mu.M was added to the supernatant. After the culture was continued for 48 hours, the damage of the Vero cells by GSK872 was observed under a light microscope, and the results are shown in FIG. 3, in which it can be seen that the GSK872 at concentrations of 0.625. mu.M and 1.25. mu.M had no effect on the treatment of EV71 virus, and the cytopathic effect was evident. When the concentration of GSK872 is 2.5 μ M, the therapeutic effect isObviously, cytopathic effects are reduced. When the concentration of GSK872 is 5 μ M and 10 μ M, the cell toxicity is high and the cytopathic effect is obvious. Then, a cell survival rate CCK8 experiment is carried out, CCK8 reagent with the concentration of 5mg/mL is added into each well, the mixture is fully mixed and incubated for 30min at 37 ℃, then liquid in the culture plate is sucked away, and the OD570 value of each well is measured. The OD570 value of the Vero cell control group was set to 100%, and the cell survival rate (%) was determined as (OD)₅₇₀Experimental group/OD₅₇₀Control group) x 100% was calculated and the results are presented as a bar graph, see fig. 4, where: p < 0.05, x: p is less than 0.01, and as can be seen from the figure, the survival rate of Vero cells treated by GSK872 of 10 mu M and 5 mu M is not different from that of cells in a control group, and the survival rate of Vero cells treated by GSK872 of 2.5 mu M, 1.25 mu M and 0.625 mu M is reduced along with the reduction of the concentration of GSK872, which indicates that the GSK872 has certain protective effect on Vero cells infected by viruses and the effect of GSK872 in inhibiting the viruses is concentration-dependent. GSK872 is not toxic to Vero cells at 2.5. mu.M and is protective against virus infected Vero cells.

RIPK3 is widely expressed in embryos and in a number of mature tissues, and RIPK3 expressing tissues have been reported to be: liver, kidney, brain, lung, heart, testis, spleen, prostate, lymphocytes, placenta, pancreas, skeletal muscle, colon, etc. RIPK3 has serine/threonine kinase activity, can sense the change of the intracellular environment, and is a key protein for regulating and controlling cell necrosis, in addition, RIPK3 plays an important role in infectious diseases and aseptic inflammatory diseases, and researches report that RIPK3 also participates in the activation of inflammasome and the regulation and control of various inflammatory signals, but how RIPK3 determines and regulates different signal mechanisms under different conditions is not clear, particularly whether RIPK3 plays a role in the signal conduction path of the hand-foot-and-mouth disease process caused by enterovirus 71 infection or not, and no report is found on the fact that a safer and more effective anti-enterovirus 71 medicament can be developed.

Example 2 preparation of GSK872 anti-Enterovirus 71 drug

The embodiment provides a granular medicine for oral administration, which is prepared by dissolving N epsilon carboxymethyl lysine in DMSO, adding methanol to dilute the solution to 1.25 mu M, mixing a GSK872 solution and the N epsilon carboxymethyl lysine solution in an equimolar manner, adding hydroxylated nano selenium particles, stirring, and performing spray drying to obtain a medicine sample to be tested.

Example 3 detection of anti-Enterovirus 71 Activity Using GSK872 as the Main pharmaceutical ingredient

Reagents and materials

Enterovirus type 71 (GenBank accession number FJ439769.1) used in this example was obtained from a Central laboratory clinical isolate from the Children's medical center for women in Guangzhou, City, as in example 1. (Zhongjiayu, Zhubing, Hualiang, Wangzhong, Kuang xylol, Xijiahui, Chenassist in 2008 and 2010 Enterovirus 71 Guangzhou isolate genome-wide sequence analysis of the journal of Chinese epidemiology [ J ]. 2011,32(7): 700-.

The Vero cells used in this example were the same as in example 1, purchased from shanghai life science research institute cell bank, chinese academy of sciences, catalog No.: GNO10 are provided.

GSK872 used in this example was the same as in example 1 and was obtained from MCE, Cat.No. HY-101872.

The drug used in this example was the anti-enterovirus 71 drug containing GSK872 as a main ingredient, which was prepared in example 2.

Preparation of drug sample to be detected and GSK872 solution

The GSK872 was configured to four concentrations of 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M using DMSO for use;

flow cytometry detection

Culturing Vero cells at a cell density of 4X 10⁴Adding EV71 virus for incubation for 2h during one/hole, adding GSK872 drug groups with different concentrations, culturing for 48h, collecting cells, and adjusting the cell number to 1-5 × 10⁶Centrifuge at 1000r/min for 5 min. The cell pellet was gently resuspended in 0.2ml of pre-cooled PBS and centrifuged at 1000r/min at room temperature for 5 min. The cell pellet was gently resuspended in 0.2ml of pre-cooled binding buffer. Adding 5 mul Annexin V-FITC and 5 mul PI for staining, mixing evenly, incubating for 10-15 min at room temperature in a dark place, and detecting by a flow cytometer.

The detection result is shown in fig. 5, and it can be seen from fig. 5 that the apoptosis of the virus group is high, and the apoptosis of the GSK872 drug group is reduced along with the increase of the concentration (0.625 μ M, 1.25 μ M and 2.5 μ M), which indicates that the GSK872 can effectively inhibit the apoptosis caused by EV 71. When the concentration of GSK872 is 5. mu.M, apoptosis is increased due to toxicity to cells.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.