Application of TXD-174 in preparation of product for treating and / or preventing flavivirus virus infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of TXD-174 in preparation of products for treating and / or preventing flavivirus infection. The invention provides a novel application of TXD-174 or a derivative thereof in preparation of a medicine for treating and / or preventing flavivirus virus infection. By taking dengue virus type 2 as an example, an inhibition experiment proves that the TXD-174 has a good inhibition effect on DENV2, not only has outstanding substantive features in the treatment aspect, but also can inhibit the virus activity when being infected with the dengue virus type 2 after being administered for 1 hour in advance, so that the TXD-174 also has a certain effect on preventing the dengue virus type 2 infection. The application essentially proves that TXD-174 has a wide application background in treatment and / or prevention of DENV2 infected diseases, a good candidate drug is provided for clinical treatment of diseases caused by DENV2, and the application has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of TXD-174 in preparing products for treating and / or preventing flavivirus infection. Background Art

[0002] Dengue virus belongs to a serotype subgroup in the Flaviviridae family. It is divided into four serotypes: 1, 2, 3, and 4 according to different antigenicity. Different strains of the same type also have antigenic differences. Type 2 is the most widely spread. There is cross-antigenicity between the various types of viruses. It also has some antigenic similarities with Japanese encephalitis virus and West Nile virus. The morphological structure of dengue virus is similar to that of Japanese encephalitis virus, but it is smaller in size, about 17 to 25nm, and is dumbbell-shaped, rod-shaped or spherical. It is wrapped with a layer of envelope protein on the surface. The outer layer of the envelope protein contains the envelope E protein, and the inner layer contains the membrane protein M (prM). The center is a 20-sided nucleocapsid structure composed of single-stranded positive-strand RNA and capsid protein C, encoding 3 structural proteins (C, prM / M, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The virus can proliferate in Aedes albopictus passage cells (C6 / 36 cells), monkey kidney, hamster kidney primary and passage cells, and produce obvious cytopathic effects. Dengue virus is mainly transmitted by vector insects such as Aedes aegypti and Aedes albopictus, causing dengue fever (DF) and dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with high morbidity and mortality. Dengue virus is a seasonal infectious disease prevalent in tropical and subtropical regions, seriously threatening public health security in tropical and subtropical regions. In recent years, dengue fever has been prevalent in Yunnan Province, Guangdong Province, Zhejiang Province, Taiwan Province, etc. in my country, mostly caused by imported cases. Faced with such a serious health threat, the necessity of dengue virus vaccine is becoming increasingly high. However, the pathogenic mechanism of dengue virus is not yet fully understood, which makes the development of vaccines slow. The only approved dengue vaccine (Dengvaxia) is the chimeric yellow fever-dengue quadrivalent vaccine (CYD-TDV), produced by Sanofi Pasteur in Paris, France. It is the first and only vaccine of its kind licensed for use in 19 countries. However, the vaccine cannot provide balanced protection against all serotypes, and due to special vaccination restrictions caused by Phase II and Phase III studies, Dengvaxia is only effective for those who have evidence of dengue virus infection. Therefore, it is of great significance to study the transmission, infection, pathogenic mechanism and anti-dengue virus drugs of dengue virus. The research and development of anti-dengue virus drugs is facing huge challenges, and the development of effective drugs that can be used to treat dengue fever has broad application prospects. Summary of the invention

[0003] The purpose of the first aspect of the present invention is to provide the use of TXD-174 or its derivatives in the preparation of products for treating and / or preventing flavivirus infection.

[0004] The second aspect of the present invention aims to provide a medicine.

[0005] The third aspect of the present invention aims to provide a method.

[0006] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of TXD-174 or its derivatives in the preparation of products for treating and / or preventing flavivirus infection.

[0008] The structural formula of TXD-174 is shown in Formula 1:

[0009]

[0010] In some embodiments of the invention, the Flavivirus comprises a dengue virus.

[0011] In some embodiments of the invention, the dengue virus comprises dengue virus type 2.

[0012] In some embodiments of the present invention, the TXD-174 or its derivative comprises at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

[0013] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0014] In some embodiments of the present invention, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0015] In some embodiments of the present invention, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0016] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0017] In some embodiments of the present invention, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0018] In some embodiments of the present invention, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0019] In some embodiments of the present invention, the salt formed with the organic acid includes a salt formed with the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, at least one of p-toluenesulfonic acid.

[0020] In some embodiments of the present invention, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine, and ornithine.

[0021] In some embodiments of the present invention, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0022] In some embodiments of the present invention, the pharmaceutically acceptable modification comprises at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0023] In some embodiments of the invention, the product comprises a drug.

[0024] In some embodiments of the present invention, the drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0026] Furthermore, for the convenience of medication, the active ingredient TXD-174 or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0027] In some embodiments of the present invention, the dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0028] In some embodiments of the present invention, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0029] Furthermore, the dosage forms for administration through the gastrointestinal tract include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0030] In some embodiments of the present invention, the non-gastrointestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0031] Furthermore, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0032] In some embodiments of the present invention, the product is administered to a mammal.

[0033] In some embodiments of the invention, the mammal comprises a human.

[0034] In the first aspect of the present invention, the present invention found that TXD-174 can inhibit the mRNA and protein expression of the key protein NS5 protein in dengue virus, and inhibit the synthesis of dengue virus NS5 protein and dsRNA after DENV2 infection. Not only does it have outstanding substantive characteristics in treatment, but it can also inhibit the activity of the virus when it is infected with dengue fever type 2 1 hour after the pre-administration treatment, proving that TXD-174 has a wide range of application backgrounds in the treatment of DENV2 infection diseases.

[0035] The second aspect of the present invention provides a drug comprising a therapeutically effective amount of TXD-174 or a derivative thereof.

[0036] In some embodiments of the present invention, the therapeutically effective amount at the cellular level is 5 to 20 μM; preferably, 7.5 to 15 μM.

[0037] In some embodiments of the present invention, the drug may further include other active ingredients, such as antipyretic drugs such as acetaminophen, ibuprofen, or broad-spectrum antiviral drugs such as interferon α.

[0038] The third aspect of the present invention provides any one of the methods 1) to 2), comprising the following steps: treating cells with TXD-174 or a derivative thereof;

[0039] 1) A method for inhibiting NS5 protein expression in vitro for non-therapeutic purposes;

[0040] 2) A method for inhibiting Flavivirus in vitro for non-therapeutic purposes.

[0041] In some embodiments of the present invention, the inhibition of NS5 protein expression includes inhibiting the mRNA and protein expression of NS5, a key protein in dengue virus, and inhibiting the synthesis of dengue virus NS5 protein and dsRNA.

[0042] The beneficial effects of the present invention are:

[0043] The present invention provides a new application of TXD-174 or its derivatives in the preparation of drugs for treating and / or preventing flavivirus infection. Taking dengue virus type 2 as an example, the present invention confirms that TXD-174 has a good inhibitory effect on DENV2 through the inhibition experiment of DENV2. It not only has outstanding substantial characteristics in treatment, but also can inhibit the activity of the virus when it is infected with dengue type 2 1 hour after the pre-administration treatment, indicating that TXD-174 also has a certain effect on preventing dengue virus type 2 infection. The present invention essentially proves that TXD-174 has a wide range of application backgrounds in the treatment and / or prevention of DENV2 infection diseases. The invention can provide a good candidate drug for the clinical treatment of diseases caused by DENV2, and has a very good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0045] Figure 1 This is the preparation reaction process of TXD-174.

[0046] Figure 2 This is the hydrogen spectrum result of TXD-174.

[0047] Figure 3 The carbon spectrum results of TXD-174 are shown below.

[0048] Figure 4 This is a graph showing the inhibitory effect of TXD-174 on viral pathology in BHK-21 cells infected with DENV2 and the results of cell viability.

[0049] Figure 5 This is a graph showing the results of TXD-174 inhibiting the mRNA and protein expression of viral NS5 protein in BHK-21 cells infected with DENV2.

[0050] Figure 6 This is a graph showing the results of investigating the dosing pattern of TXD-174 on DENV2-infected BHK-21 cells.

[0051] Figure 7 This is a graph showing the viral inhibition results of TXD-174 on DENV2-infected BHK-21 cells treated at different dosing times.

[0052] Figure 8 This is the immunofluorescence result of TXD-174's inhibitory effect on NS5 protein and dsRNA synthesis in BHK-21 cells after DENV2 infection. DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0054] Example 1 Preparation and Characterization of TXD-174

[0055] 3-Indoleacetic acid (1.0 mmol, 1.0 equiv.), 5-aminopyrazole (2 mmol, 2.0 equiv.), Cu(OAc)2 . H2O (2mmol, 2.0equiv.) and DMF (10mL) were added to a 50mL round-bottom flask, evacuated, introduced with nitrogen, and placed in an oil bath at 100°C for heating and stirring for 24h. After the reaction was completed, the round-bottom flask was removed and cooled to room temperature, followed by addition of saturated sodium bicarbonate solution (20mL), and then ethyl acetate was added for extraction, 20mL was added each time, and extracted three times in total. The organic phase obtained by extraction was combined into a conical flask, dried with anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to remove the organic solvent, and purified and separated by thin layer chromatography to obtain 0.27g of the target product with a yield of 70%.

[0056] The reaction process is as follows Figure 1 shown.

[0057] The structural characterization of TXD-174 by hydrogen and carbon spectra was as follows Figure 2 , 3 shown.

[0058] 4-(4-((1H-Indol-3-yl)methyl)-5-amino-3-phenyl-1H-pyrazol-1-yl)-benzonitrile(174). 1 H NMR(400MHz, CDCl3)δ8.10(s,1H),7.94(d,J=7.8Hz,2H),7.75-7.69(m,4H),7.59(d,J=7.4Hz,1H),7 .38-7.37(m,4H),7.24-7.22(m,1H),7.14(t,J=6.8Hz,1H),6.94(s,1H),4.03(s,2H),3.65(brs,2H). 13C NMR (100MHz, CDCl3) δ152.6,143.8,142.7,136.7,133.4,133.2,128.6,128.2,127.8,127.0,122.6,122.4,122 .1,119.5,118.8,118.5,113.7,111.3,109.5,101.9,19.7.IR(KBr):3408,2227,1605,1513,1504,1380,743cm -1 .HRMS(ESI)m / z:[M+H] + Calcd for C 25 H 20 N5390.1713; found 390.1717.

[0059] Example 2 Safety Verification of TXD-174

[0060] 1. Experimental Materials

[0061] BHK-21, or baby hamster kidney cells, can be infected by type II dengue virus (DENV2) and is one of the most commonly used cells in dengue virus research. This embodiment first detects the cytotoxicity of TXD-174 to BHK-21 cells, and acts on BHK-21 cells with different concentrations of TXD-174 to understand the survival rate of cells at different TXD-174 concentrations, and to provide reference data for subsequent experiments on the inhibitory effect of TXD-174 on DENV2. BHK-21 cells (baby hamster kidney cells) are susceptible cells to DENV2. The BHK-21 cells in the experiment are owned by this department; MTT was purchased from Biyuntian Biotechnology Research Institute; fetal bovine serum was purchased from GIBICO, USA; DMEM was purchased from GIBICO, USA; and cell culture plates were purchased from Jiete.

[0062] 2. Experimental steps

[0063] 1) Inoculation of BHK-21 cells: Prepare a single cell suspension using DMEM medium containing 10% (V / V) fetal bovine serum, and inoculate 5,000 cells per well into a 96-well cell culture plate. The inoculation volume per well is 100 μL;

[0064] 2) Cultivate BHK-21 cells: culture at 37°C, 5% (V / V) CO2 for 24 h;

[0065] 3) Add TXD-174: Aspirate the medium in each well, add 100 μL of DMEM medium containing 2% (V / V) fetal bovine serum to each well to dilute the TXD-174 to the corresponding concentration, and add 100 μL of DMEM medium containing 2% (V / V) fetal bovine serum without drug to the control well;

[0066] 4) Color development: After 48 hours of culture, add 10 μL of 5 mg / ml MTT solution to each well, continue to culture for 4 hours at 37°C, 5% (V / V) CO2, then add 150 μL DMSO and shake on a plate shaker for 15 minutes until the Formazan is completely dissolved under ordinary optical microscope;

[0067] 5) Measurement and calculation: The absorbance value was measured at 570 nm. The survival rate of cells at different concentrations of TXD-174 was calculated by dividing the absorbance value of the cells at that concentration by the absorbance value of the control well, multiplied by 100%.

[0068] 3. Experimental results

[0069] The results are shown in Table 1.

[0070] Table 1 Toxicity experiment of different concentrations of TXD-174 on BHK-21 cells

[0071]

[0072] The survival rate of TXD-174 at a concentration of 80 μM was approximately 88%, and at concentrations of 40 μM and below, the cell survival rate was greater than 99%, with no obvious toxicity to cells.

[0073] Example 3 TXD-174 inhibits DENV2 proliferation in BHK-21 cells

[0074] Different concentrations of TXD-174 were used on BHK-21 cells infected with DENV2 to obtain the inhibitory efficiency of TXD-174 on the virus from three aspects: cell survival rate, viral protein level, and mRNA level. The concentration of TXD-174 used in this experiment was within the range of concentrations that made the BHK-21 cells survive at more than 80%, which can rule out the influence of TXD-174 concentration on the analysis of experimental data.

[0075] 1. Experimental steps

[0076] 1) BHK-21 was inoculated into the cell culture plate. After 24 hours, the cells grew to a monolayer, and the area of ​​the bottom of the well covered by the cells was about 80% to 90%. The culture medium was aspirated, washed once with PBS, and 100 μL of the virus sample was inoculated and adsorbed at 37°C for 1 hour. After the adsorption was completed, the virus solution in each well was aspirated and washed once with PBS. TXD-174 at a specified concentration diluted with DMEM culture medium containing 2% (V / V) fetal bovine serum was added, and cultured at 37°C under 5% (V / V) CO2 culture conditions. After 96 hours, when the cells showed obvious cytopathic effects, they were photographed and the cell viability was detected by MTT; or after 48 hours of culture, the total protein and total RNA of the cells were collected and the content of viral protein and viral RNA in the cells were detected by protein immunoblotting and real-time fluorescence quantitative PCR, respectively.

[0077] 2) Color development: After 48 hours of culture, add 10 μL of 5 mg / mL MTT solution to each well, continue to culture for 4 hours at 37°C, 5% (V / V) CO2, then add 150 μL DMSO until the Formazan is completely dissolved under ordinary optical microscope.

[0078] 3) Measurement and calculation: The absorption value was measured at 570 nm. The virus inhibition rate of TXD-174 at different concentrations was calculated as follows: 1-(OD value of the experimental group-OD value of the control group) / (OD value of the dengue group-OD value of the control group) at that concentration, multiplied by 100%.

[0079] 2. Experimental results

[0080] The results are shown in Table 2 and Figure 4 shown.

[0081] Table 2 Virus inhibition rate of different concentrations of TXD-174 on DENV2-infected BHK-21 cells

[0082]

[0083] Table 2 and Figure 4 The data showed that the inhibition rate of TXD-174 on DENV2 reached 69% at 5μM and 97% at 10μM, showing a good effect in inhibiting DENV2-infected cells.

[0084] Example 4 TXD-174 inhibits mRNA translation of DENV2 key protein NS5 protein

[0085] 1. Experimental steps

[0086] 1) Collecting total cell RNA: Add 500 μL Buffer RL reagent (Vazyme) to each well of a 24-well cell culture plate. After standing at room temperature for 5 minutes, aspirate the supernatant and extract RNA according to the kit protocol. Finally, add 20 μL of autoclaved water to dissolve the RNA and store it at -80°C for later use. Use a UV spectrophotometer to detect the 260 / 280 absorbance ratio and calculate the RNA concentration.

[0087] 2) Reverse transcription of mRNA into cDNA: Reaction system: RNA 1mg, 5× PrimeScript RT Master Mix (TAKARA) 4μL, autoclaved water to a total volume of 20μL. Carefully mix and incubate at 37℃ for 15min, 85℃ for 5sec.

[0088] 3) Real-time fluorescence quantitative PCR detection of the mRNA level of the viral non-structural protein NS5 in each sample: Reaction system (10 μL): cDNA template 1 μL, qPCR Master Mix (Vazyme) 5 μL, forward and reverse primers 0.4 μL, autoclaved water 3.2 μL. After mixing, pre-denaturation at 95°C for 10 min, 95°C for 15 sec, 60°C for 1 min (40 cycles).

[0089] Table 3 Forward and reverse primers for each target RNA

[0090]

[0091] 4) Calculation: Based on the CT value of each sample, use 2 -ΔΔCт The RNA level of each sample relative to the control group was calculated by Figure 5 shown.

[0092] 2. Experimental results

[0093] Figure 5 As the concentration of TXD-174 increased, the mRNA level of the viral nonstructural protein NS5 gradually decreased, and 10 μM almost completely inhibited it. This shows that TXD-174 can inhibit the mRNA level of the key protein NS5 in dengue virus.

[0094] Example 5 TXD-174 inhibits protein expression of DENV2 virus key protein NS5

[0095] 1. Experimental steps

[0096] 1) Collect total cell protein: BHK-21 cells were treated with drugs and dengue virus (DENV2), and protein was extracted 48 hours later: cells were washed with cold PBS, RIPA lysis buffer (Keygen Biotech) was added to each well of a 24-well plate, and phosphatase inhibitors (Keygen Biotech) and protease inhibitors (Keygen Biotech) were added at 50 μL per well, cells were scraped and collected in a 1.5 mL Eppendorf tube, centrifuged at 12000 rcf at 4°C for 15 min, and the supernatant was transferred to a new Eppendorf tube. 50 μL of 2× loading buffer solution was added to make the protein concentration of each sample consistent. Denature at 100°C for 10 min, and store at -20°C for later use.

[0097] 2) Protein immunoblotting to detect the expression difference of dengue virus protein after treatment with gradient concentration of TXD-174: 10% SDS polyacrylamide gel configuration: Add various reagents in order, configure 10% separation gel and quickly pour it into the glass gap, leaving the distance required for pouring the concentrated gel, quickly cover the separation gel with a layer of anhydrous ethanol, after the separation gel is polymerized, pour out the anhydrous ethanol layer, dry it with filter paper, continue to pour the concentrated gel, and insert the comb. Electrophoresis: Prepare electrophoresis buffer, 1L per sample tank, load the sample, 10μL sample per well, turn on the power, start with 80V constant voltage for 25min, after the dye front enters the separation gel, change to 120V, determine the electrophoresis time according to the separation degree of the pre-stained protein marker and the position of bromophenol blue, generally run about 60min.

[0098] 3) Transfer: Remove the gel from the glass plate, cut the PVDF membrane of the gel size, and soak it in methanol for 1 minute. Soak the gel, filter paper, and PVDF membrane in the transfer buffer. Install the transfer device in the following order: negative electrode-cotton pad-2 layers of filter paper-gel-PVDF membrane-3 layers of filter paper-cotton pad-positive electrode. Stack the above items one by one and align them accurately. Place the transfer device in the transfer box, confirm that the electrodes are correct, and wet transfer at 100V for 90 minutes. After blocking with 5% skim milk at room temperature for 1 hour, incubate with the target primary antibody at 4°C overnight, and develop after secondary antibody binding the next day.

[0099] 4) Protein blocking: The PVDF membrane was blocked in 5% skim milk at room temperature for 1 hour. After blocking, the membrane was washed 3 times on a shaker at room temperature for 10 minutes each time.

[0100] 5) Primary antibody hybridization: Place the PVDF membrane with the protein side facing up in a small box, add the primary antibody containing NS5 (GeneTex) prepared in antibody diluent, and incubate at 4°C on a shaker overnight. Wash the membrane with membrane wash solution on a shaker at room temperature for 3 times, 10 minutes each time.

[0101] 6) Secondary antibody hybridization: Place the PVDF membrane protein side up in a small box, add 5% (w / v) skim milk buffer containing secondary antibody (rabbit antibody or mouse antibody, Proteintech), shake at room temperature for 1 hour. Wash the membrane 3 times with washing solution on a shaker at room temperature for 10 minutes each time

[0102] 7) Chemiluminescence development: Add ECL developer A / B (Xinsaime) evenly in proportion, drop the luminescent liquid on the PVDF membrane, shake well, expose and develop, and save the image.

[0103] 2. Experimental results

[0104] The results are as follows Figure 5 As shown, as the concentration of TXD-174 increases, the protein level of the viral non-structural protein NS5 gradually decreases, and 10 μM almost completely inhibits the expression of related proteins. This shows that TXD-174 can inhibit the protein expression level of NS5, a key protein in dengue virus.

[0105] Example 6 TXD-174 administration mode exploration test on DENV2 infected BHK-21 cells

[0106] After confirming that TXD-174 has a good antiviral effect, we further explored the stage of viral infection that TXD-174 acts on, which can be judged by staged drug addition. Therefore, a pre-treatment group was set up, that is, TXD-174 was used to treat cells for 1 hour before infecting DENV2 to observe the preventive effect of TXD-174 on DENV2 infection; a co-treatment group, that is, TXD-174 and DENV2 were incubated into cells at the same time to observe whether TXD-174 had an inhibitory effect on the virus entry stage; a post-treatment group, that is, after infecting DENV2 to allow the virus to enter the cells, TXD-174 was added to treat the cells, so that the cells were cultured and grown under the action of TXD-174. The co-treatment + post-treatment group is a combination of the two incubation methods.

[0107] 1. Experimental steps

[0108] 1) BHK-21 cells were inoculated into the cell culture plate. After 24 hours, the cells grew into a monolayer with the cells covering about 80% to 90% of the bottom of the well. The culture medium was aspirated, and the cells were washed once with PBS. The cells were infected and treated with TXD-174 according to the pre-treatment group, co-treatment group, post-treatment group, and co-treatment + post-treatment group, and cultured at 37°C with 5% (V / V) CO2.

[0109] 2) After culturing for 48 hours, the total protein and total RNA of the cells were collected and the content of viral protein and the content of viral RNA in the cells were detected by western blotting and real-time fluorescence quantitative PCR, respectively. The specific methods were the same as those in Example 4.

[0110] 2. Experimental results

[0111] The results are as follows Figure 6 shown. Figure 3 The antiviral mode of TXD-174 was observed at both the transcription level and the expression level. The experimental results showed that TXD-174 also had a weak inhibitory effect on the entry of the virus, and the most obvious effect was in the post-treatment group, indicating that TXD-174 played an anti-viral infection role after the virus entered the host cells.

[0112] Example 7 Virus inhibition test of TXD-174 on BHK-21 cells infected with DENV2 at different administration times

[0113] Since the virus replicates differently at different time periods during the DENV2 virus entry into cells, the time point at which TXD-174 effectively works after DENV2 entry is explored by adding TXD-174 treatment at different times after virus infection. That is, TXD-174 is added to cells at 0, 2, 4, 8, 12, and 24 hpi after DENV2 infection.

[0114] 1. Experimental methods

[0115] 1) BHK-21 was inoculated into the cell culture plate. After 24 hours, the cells grew to a monolayer, and the cells covered about 80% to 90% of the bottom of the well. The culture medium was aspirated, washed once with PBS, and 500 μl of the virus sample was inoculated and adsorbed at 37°C for 1 hour. After adsorption, the virus solution in each well was aspirated at 0, 2, 4, 8, 12 and 24 hours after DENV2 infection, and washed once with PBS. TXD-174 at a specified concentration diluted with DMEM medium containing 2% (V / V) fetal bovine serum was added, and cultured at 37°C under 5% (V / V) CO2 culture conditions.

[0116] 2) After culturing for a specified time, the total protein and total RNA of the cells were collected and the content of viral protein and viral RNA in the cells were detected by western blotting and real-time fluorescence quantitative PCR, respectively, using the same method as above.

[0117] 2. Experimental results

[0118] Figure 7 The results showed that in the early stages after viral infection (e.g., 0-8 hpi), the addition of TXD-174 reduced viral protein by more than 99%. Although the addition of TXD-174 at 12 and 24 hpi also reduced viral protein levels, the therapeutic effect was greatly reduced. At the same time, similar results were shown at the transcriptional level as at the protein level. This further illustrates that TXD-174 plays a significant antiviral role in the early stages of viral infection.

[0119] Experimental Example 8 Immunofluorescence test of the inhibitory effect of TXD-174 on NS5 protein and dsRNA synthesis in BHK-21 cells infected with DENV2

[0120] 1. Experimental methods

[0121] 1) Cell treatment: 24 hours in advance, inoculate cells in a specific Confocal dish. When the cell growth area is about 80% to 90%, wash once with PBS, add 1 ml of virus, and adsorb for 1 hour at 37°C. Aspirate and discard the virus solution in each well, wash once with PBS. Add TXD-174 diluted in DMEM medium containing 2% (V / V) fetal bovine serum, and culture at 37°C under 5% CO2 culture conditions;

[0122] 2) Sample processing:

[0123] a. After 48 h of culture, the medium was discarded and the cells were washed with PBS three times, 10 min each time, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 20 min.

[0124] b. Blocking: Block with PBGB (PBST containing 3% BSA) for 1 h at room temperature;

[0125] c. Primary antibody hybridization: cells were combined with primary antibodies (NS5 and J2 antibodies) in PBGB overnight at 4°C;

[0126] d. Secondary antibody hybridization and nuclear staining: Wash with PBS 3 times, 10 min / time, incubate the cells with FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine isothiocyanate) secondary antibodies at room temperature for 1 hour, protect from light, wash with PBS 3 times, 10 min / time. Finally, stain the cell nuclei with DAPI for 10 min.

[0127] 3) Images were taken and processed using a confocal microscope (Nikon).

[0128] 2. Experimental results

[0129] like Figure 8 As shown, it can be observed that within the concentration range of 5 to 20 μM, TXD-174 can effectively inhibit the synthesis of double-stranded RNA (dsRNA) of DENV2 and the expression of non-structural protein NS5. dsRNA is produced during the replication cycle of various viruses and can trigger innate immune responses. If the cell is infected with a dsRNA virus, dsRNA is present, indicating that TXD-174 can significantly inhibit the replication behavior of DENV2 after DENV2 infection.

[0130] In summary, the present invention discloses for the first time the use of TXD-174 in the preparation of drugs for the treatment and / or prevention of flavivirus infection, which has a good effect on the inhibitory activity of dengue virus type 2. It not only has outstanding substantial characteristics in treatment, but also can inhibit the activity of the virus when the patient is infected with dengue virus type 2 1 hour after administration in advance. This shows that TXD-174 also has a certain effect on the prevention of dengue virus type 2 infection, and is very likely to be developed into a new drug for anti-dengue infection.

Claims

1. Use of TXD-174 and its derivatives in the preparation of products for treating and / or preventing flavivirus infection.

2. The use according to claim 1, characterized in that: The Flavivirus genus includes dengue virus.

3. The use according to claim 2, characterized in that: The TXD-174 or its derivatives include at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

5. The use according to claim 3, characterized in that: The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

6. The use according to claim 1, characterized in that: The products include pharmaceuticals.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 6, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. The use according to any one of claims 2 to 8, characterized in that: The dengue virus includes dengue virus type 2.

10. Any of the methods 1) to 2) comprises the following steps: Treating cells with TXD-174 or its derivatives; 1) A method for inhibiting NS5 protein expression in vitro for non-therapeutic purposes; 2) A method for inhibiting Flavivirus in vitro for non-therapeutic purposes.

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