Application of lurasidone in preparation of medicine for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection

Abstract

The invention provides application of lurasidone in preparation of a medicine for preventing and / or treating severe fever with thrombocytopenia syndrome virus, and belongs to the technical field of biological medicine. The method comprises the following steps: by taking nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) as a virtual screening target, carrying out molecular docking virtual screening on drug compounds approved by FDA in a small molecule database by utilizing AutoDock Vina to obtain delta Glt; according to the present invention, after the anti-SFTSV drug verification is performed, the lurasidone is screened to provide the strongest inhibition effect on the SFTSV, such that the lurasidone can achieve the anti-SFTSV infection purpose by inhibiting the duplication of the SFTSV;

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus. Background Art

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne zoonotic virus with high infectivity. The main clinical symptoms of SFTSV infection include fever, gastrointestinal symptoms, thrombocytopenia, elevated liver enzyme levels, etc. In severe cases, it can develop into multiple organ failure or death. At present, the treatment of fever with thrombocytopenia syndrome in clinical practice mainly focuses on symptomatic supportive treatment. Although SFTS has received great attention in clinical and public health, there is still no effective vaccine or specific drug, and there is an urgent need to develop effective specific antiviral drugs.

[0003] The replication process of the SFTSV genome depends on the viral ribonucleoprotein complex (RNP). The components of the SFTSV RNP consist of three parts: genomic RNA, RNA-dependent RNA polymerase (RDRP), and nucleoprotein (NP). Among them, NP is the scaffold protein of the viral RNP structure. The three-dimensional crystal structure shows that multiple nucleoproteins are connected by N-terminal extension arms to form a pentamer or hexamer structure. The nucleoprotein multimer structure plays an important role in the binding of viral genomic RNA (vRNA) and its own stability. Therefore, disrupting the activity of SFTSV-NP can effectively inhibit virus replication, thereby reducing the occurrence of this infectious disease.

[0004] In view of the high cost and long cycle of traditional methods for screening antiviral drugs, in order to quickly find effective candidate drugs to combat the urgency of emerging viral infectious diseases such as SFTSV, computer-aided molecular docking technology can be used to effectively screen targeted drugs. However, the antiviral activities of the screened drugs vary greatly, so it is difficult to predict the therapeutic effects of the screened drugs in clinical practice. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection.

[0006] The present invention provides the application of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection.

[0007] Preferably, the diseases related to severe fever with thrombocytopenia syndrome virus infection include severe fever with thrombocytopenia syndrome or complications of severe fever with thrombocytopenia syndrome.

[0008] Preferably, the complications of severe fever with thrombocytopenia syndrome include gastrointestinal bleeding and / or pulmonary hemorrhage.

[0009] Preferably, the lurasidone further includes at least one of the following forms: the salt form of lurasidone, the crystal form of lurasidone, and the solvate of lurasidone.

[0010] Preferably, the drug includes at least one of the following dosage forms: tablets, granules, capsules, powders, injections, and injection powders.

[0011] Preferably, the concentration of lurasidone in the drug is not less than 4.552 μM.

[0012] Preferably, the treatment of severe fever with thrombocytopenia syndrome virus infection includes inhibiting the replication ability of severe fever with thrombocytopenia syndrome virus.

[0013] Preferably, the lurasidone acts on the NP protein of severe fever with thrombocytopenia syndrome virus to inhibit virus replication.

[0014] The present invention provides a drug for treating severe fever with thrombocytopenia syndrome, and the active ingredients include lurasidone and other ingredients that inhibit severe fever with thrombocytopenia syndrome virus infection.

[0015] Preferably, other ingredients that inhibit severe fever with thrombocytopenia syndrome virus infection include antibodies that bind to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, polypeptide inhibitors that inhibit severe fever with thrombocytopenia syndrome virus infection, and compounds that inhibit severe fever with thrombocytopenia syndrome virus infection.

[0016] The present invention provides the use of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection. First, the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus is used as the target for virtual screening, and the RNA binding site of this structure is selected as the active pocket. AutoDock Vina is used to perform molecular docking virtual screening on drug compounds in the FDA-approved small molecule database to obtain drug compounds with ΔG < -10 kcal / mol. Then, through verification of anti-SFTSV drugs, lurasidone is screened out to show the strongest inhibitory effect on SFTSV. It can be seen that the lurasidone can inhibit the replication ability of SFTSV by targeting the NP protein of SFTSV, achieving the drug activity against SFTSV. Description of the Drawings

[0017] Figure 1Identification results of compounds against SFTSV virus; where A is a schematic diagram of the research design; B is the result of collecting infected cells 48 h (hpi) after mixing the compound (10 μM) with SFTSV (MOI = 0.1) to infect Vero cells and detecting the viral genome by qRT-PCR; C is the DAPI staining result of mixing lurasidone with SFTSV (MOI = 0.1) to infect Huh-7 cells; the images were collected by a Zeiss Axio Observer microscope; scale bar: 100 μm;

[0018] Figure 2 Results of dose-dependent inhibitory effect and cell viability analysis of lurasidone in Vero cells, where A is the detection of viral infection by immunofluorescence after treating Vero cells with different concentrations of the drug; IC 50 : 50% inhibitory concentration; B is the result of measuring cell viability by CCK-8 method after treating Vero cells with different concentrations of the drug; CC 50 : 50% cytotoxic concentration. Detailed implementation manners

[0019] The present invention provides the use of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection.

[0020] In the present invention, the molecular formula of the lurasidone is C 28 H 36 N4O2S, the molecular weight is 492.68, the CAS number is 367514-87-2, and the structural formula is shown in Formula I. In the embodiments of the present invention, the lurasidone is purchased from MedChemExpress, and the product number is HY-B0032A.

[0021]

[0022] In the present invention, the lurasidone preferably further includes at least one of the following forms: the salt form of lurasidone, the crystal form of lurasidone, and the solvate of lurasidone. The salt form of lurasidone preferably includes lurasidone hydrochloride.

[0023] In the present invention, the severe fever with thrombocytopenia syndrome virus infection preferably includes severe fever with thrombocytopenia syndrome or complications of severe fever with thrombocytopenia syndrome. The severe fever with thrombocytopenia syndrome is an infectious case feature mainly manifested by fever with thrombocytopenia. A small number of severe patients have severe conditions and rapid progression, and can die due to multiple organ failure and ineffective treatment. Its main clinical manifestations are acute onset of fever, and most patients are accompanied by symptoms such as fatigue, anorexia, nausea, vomiting, abdominal pain, and diarrhea. Some patients have bleeding symptoms such as melena, gum bleeding, skin petechiae or ecchymoses, and conjunctival congestion. The complications of the severe fever with thrombocytopenia syndrome preferably include gastrointestinal bleeding and / or pulmonary hemorrhage.

[0024] In the present invention, the drug preferably includes at least one of the following dosage forms: tablets, granules, capsules, powders, injections, and injection powders. The concentration of lurasidone in the drug is preferably not less than 4.552 μM. In an embodiment of the present invention, the inhibitory activity of lurasidone at different concentrations against SFTSV was evaluated, and the IC 50 value of lurasidone was 4.552 μM. The CC 50 value of lurasidone for infected cells was greater than 80 μM, and the selectivity index (SI) was greater than 17. Therefore, lurasidone can be used as an active ingredient against SFTSV for the preparation of clinical drugs.

[0025] In the present invention, the treatment of severe fever with thrombocytopenia syndrome virus infection preferably includes inhibiting the replication ability of the severe fever with thrombocytopenia syndrome virus. Lurasidone preferably acts on the NP protein of the severe fever with thrombocytopenia syndrome virus to inhibit virus replication. In the embodiment of the present invention, in view of the fact that the nucleoprotein (NP) is one of the factors affecting the genome replication of SFTSV, the nucleoprotein (NP) was used as a virtual target, and the RNA binding site of this structure was selected as the active pocket for molecular docking virtual screening, and drug compounds with ΔG < -10 kcal / mol were selected for experimental verification. The immunofluorescence experiment was used to detect the proportion of infected cells and the qRT-PCR experiment was used to detect to reflect the antiviral activity of lurasidone, and the cell viability after lurasidone treatment characterized the cytotoxicity. The results showed that lurasidone could effectively reduce the SFTSV infection content in cells. After analysis, the selectivity index (SI) of lurasidone was greater than 17, indicating that lurasidone is a more potential candidate drug against SFTSV.

[0026] The present invention provides a drug for treating severe fever with thrombocytopenia syndrome, and the active ingredients include lurasidone and other ingredients that inhibit severe fever with thrombocytopenia syndrome virus infection.

[0027] In the present invention, other components for inhibiting severe fever with thrombocytopenia syndrome virus infection preferably include antibodies that bind to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, polypeptide inhibitors that inhibit severe fever with thrombocytopenia syndrome virus infection, and compounds that resist severe fever with thrombocytopenia syndrome virus infection.

[0028] The present invention has no special restrictions on other components for inhibiting severe fever with thrombocytopenia syndrome virus infection, and other components for inhibiting severe fever with thrombocytopenia syndrome virus infection well-known in the art can be used. For example, for the antibodies that bind to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, reference can be made to the antibodies that bind to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus recorded in the patent with the publication number CN109071637; for the compounds that inhibit severe fever with thrombocytopenia syndrome virus infection, reference can be made to the compounds that inhibit severe fever with thrombocytopenia syndrome virus infection recorded in the publication number CN118845751A; for the polypeptide inhibitors that inhibit severe fever with thrombocytopenia syndrome virus infection, reference can be made to the polypeptide inhibitors recorded in the patent with the publication number CN112759630A.

[0029] The following is a detailed description of the application of lurasidone provided by the present invention in the preparation of drugs for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection in conjunction with examples, but they cannot be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] Virtual screening based on molecular docking method

[0032] SFTSV NP was used as the target for virtual screening. The RNA-binding site of this structure was selected as the active pocket for molecular docking virtual screening. The receptor protein file was first retrieved from the Protein Data Bank (PDB; https: / / www.rcsb.org / search) for the structure of the SFTSV NP pentamer complexed with suramin (PDB: 4J4V); then all water molecules and suramin were removed from the structure using PyMOL (https: / / pymol.org). To dock the protein of interest with ligands using AutoDockVina, the AutoDockTools (v1.5.7) interface was used to prepare the input files. The protein structure was processed for hydrogen atom addition and charge assignment using AutoDockTools (v1.5.7) and saved in the PDBQT format. For ligand structure preparation, the three-dimensional structures of FDA-approved drug compounds were downloaded from the ZINC15 database in the format of Structure Data File (SDF). These molecules were then separated, hydrogenated, and the rotatable bonds were defined using OpenBabel (v3.1.1). The processed ligands were saved in the PDBQT format and used as the input for docking analysis. The center coordinates of the binding site for molecular docking were (67.44, 13.71, 17.20), and the grid size was For each ligand docked at the protein binding site, 9 different conformations were generated. The docking process was carried out using AutoDockVina. All visualization operations were performed using PyMOL (https: / / pymol.org / ) and BIOVIA Discovery Studio Visualizer.

[0033] Virtual screening results

[0034] The RNA-binding site of SFTSV NP is crucial for viral genome replication and transcription. Small molecule drugs can block the binding between NP and viral RNA by occupying this active site, thereby inhibiting viral infection. In this example, FDA-approved drugs in the ZINC15 database were selected as molecular docking ligands, and the RNA-binding site of NP was used as the molecular docking region. AutoDock Vina was used as the docking software to analyze the binding free energy (ΔG) of each drug to NP. The molecular docking results showed that there were 27 drug molecules with ΔG < -10 kcal / mol. At the same time, 8 FDA-approved drugs were selected for further study based on factors such as drug chemical structure, pharmacological properties, cytotoxicity, solubility, and potential side effects (Table 1).

[0035] Table 1 Information on compounds selected by molecular docking in the FDA-approved drug library against SFTSV nucleoprotein

[0036] Ranking Name Docking Score (kcal / mol) 1 Ergot Alkaloids -11 2 Daclatasvir -10.7 3 Lumacaftor -10.3 4 Grazoprevir -10.3 5 Lurasidone -10.2 6 Saquinavir -10.1 7 Tadalafil -10.1 8 Isavuconazole -10.1

[0037] Example 2

[0038] 1. Virus amplification and culture

[0039] Digest Vero cells with trypsin, count them and evenly seed them into a T75 cell culture flask for overnight culture. The next day, discard the medium in the cell culture flask. After washing the residual medium with PBS, add the virus diluted with serum-free medium into the cell well plate. After 2 h of infection, replace it with medium containing 2% serum and continue to culture until the virus is harvested after a predetermined time.

[0040] 2. Virus titer determination

[0041] 1) Seed 4×10 5 Vero cells (per well) into a 6-well plate and then place it in a cell culture incubator at 37 °C and 5% CO2 for overnight culture.

[0042] 2) Dilute the virus 10-fold serially and inoculate it into Vero cells, and incubate for 1 h.

[0043] 3) Discard the virus supernatant in the 6-well plate and wash it twice with PBS. Then add DMEM medium containing 1% low melting point agar and 2% fetal bovine serum preheated at 37 °C and let it stand at room temperature for about 20 minutes until the medium solidifies.

[0044] 4) Return the cell plate to the cell culture incubator at 37 °C and 5% CO2 and continue to culture. After about 4 - 6 days, when obvious plaques appear on the cell plate, add 4% paraformaldehyde solution to fix for 2 h.

[0045] 5) Discard the paraformaldehyde solution, add 1% crystal violet staining solution and stain for 15 minutes.

[0046] 6) Rinse off the crystal violet staining solution with running water. After air-drying the cell plate, count the number of plaques.

[0047] 3. Cytotoxicity determination of the drug

[0048] 1) Seed Vero cells into a 96-well plate and culture them overnight in a constant temperature incubator at 5% CO2 and 37 °C. Add different concentrations of the drug to be tested (8 drugs screened in Example 1) to Vero cells and treat for 48 h. A group containing an equal amount of DMSO is used as a control.

[0049] 2) Detect cell viability by CCK-8 method: After the drug treatment is completed, discard the medium. After washing with PBS, add 100 μl of fresh medium to each well and add 10 μl of CCK-8 detection reagent. Read the absorbance value at 450 nm with an enzyme-linked immunosorbent assay reader after 2 h.

[0050] 3) Calculation of survival rate: Formula I is as follows

[0051] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100% Formula I;

[0052] As = Absorbance of the experimental well (absorbance of the well containing cells, medium, CCK-8 and the test compound).

[0053] Ab = Absorbance of the blank well (absorbance of the well containing medium and CCK-8).

[0054] Ac = Absorbance of the control well (absorbance of the well containing cells, medium, CCK-8 and DMSO)

[0055] 4. Immunofluorescence assay

[0056] Cells were co-infected with the candidate drug and SFTSV at a multiplicity of infection (MOI) of 0.1. After incubation at 37 °C for 1 h, the medium was discarded and the cells were washed three times with PBS. Subsequently, fresh DMEM medium containing 2% fetal bovine serum with the candidate drug was added for continued culture. After 48 h of virus infection, the cells were washed three times with PBS and then fixed and permeabilized with pre-cooled methanol for 15 minutes. Then the methanol was discarded, and the cells were blocked with PBS buffer containing 2% BSA for 1 h. Subsequently, the cells were incubated with the NP antibody overnight. The next day, after washing with PBS, the cells were stained with a fluorescently labeled secondary antibody for 1 h in the dark. Then the cell nuclei were stained with DAPI. Finally, the ratio of infected cells was analyzed using the ImageXpress Micro high-content system (Molecular Devices).

[0057] 5. Reverse transcription quantitative real-time fluorescence analysis (qRT-PCR)

[0058] Total cellular RNA was extracted from cells using the RNeasy Mini Kit (Catalog No.: 17921KD1, Axygen), and after concentration determination, it was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Catalog No.: R323-01, Vazyme). The qRT-PCR reaction was performed on a Bio-Rad CFX96 Touch real-time detection system. The qRT-PCR primers used were as follows:

[0059] SFTSV-F: 5'-CTGGGCAATGGAAACCGGAAG-3' (SEQ ID NO:1);

[0060] SFTSV-R: 5'-CAATGAGGAAGAAGTGAACAAGT-3' (SEQ ID NO:2);

[0061] GAPDH-F: 5'-CAAGAAGGTGGTGAAGCA-3' (SEQ ID NO:3);

[0062] GAPDH-R: 5'-AAGGTGGAAGAGTGGGTG-3' (SEQ ID NO:4);

[0063] The qRT-PCR reaction system is shown in Table 2 below:

[0064] Table 2 qRT-PCR reaction system

[0065] Component Volume SYBR Green Master Mix (2×) 10 μl Forward Primer (10 μM) 0.4 μl Reverse Primer (10 μM) 0.4 μl Sterile Ultra-pure Water 8.2 μl cDNA 1 μl Total Volume 20 μl

[0066] The qRT-PCR reaction program is shown in Table 3 below:

[0067] Table 3 qRT-PCR reaction program

[0068]

[0069] 6. Experimental results

[0070] 6.1 Results of anti-SFTSV drug verification

[0071] After mixing these 8 drugs with SFTSV respectively and infecting Vero cells, the intracellular viral RNA expression levels were detected after 48 h. Four drugs, saquinavir, daclatasvir, lurasidone, and tadalafil, showed obvious antiviral activities, among which lurasidone had the strongest inhibitory effect ( Figure 2 in B). The results of immunofluorescence experiments further proved that lurasidone could significantly inhibit the infection of SFTSV.

[0072] 6.2 Analysis of drug antiviral efficiency and cytotoxicity

[0073] To further evaluate the antiviral efficiency and cytotoxicity analysis of the candidate drug lurasidone, the half-maximal inhibitory concentration (IC 50 ) and the half-maximal cytotoxic concentration (CC 50 ) of the drug were detected. As Figure 2 shown, the IC 50 value of lurasidone was 4.552 μM; the CC 50 value of lurasidone was greater than 80 μM. The selectivity index (SI) is a key index for evaluating the therapeutic potential of a drug. The selectivity index (SI) of lurasidone was greater than 17 (see Table 4), indicating that lurasidone is a more promising anti-SFTSV candidate drug.

[0074] Table 4 Half inhibitory concentration (IC 50 ) and half cytotoxicity (CC 50 ) evaluation

[0075] Drug <![CDATA[IC 50 (μM)]]> <![CDATA[CC 50 (μM)]]> SI Lurasidone 4.552 ＞80 ＞17

[0076] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. The use of lurasidone in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome virus infection.

2. The application according to claim 1, characterized in that: The disease associated with severe fever with thrombocytopenia syndrome virus infection includes severe fever with thrombocytopenia syndrome or complications of severe fever with thrombocytopenia syndrome.

3. The application according to claim 2, characterized in that: Complications of the fever with thrombocytopenia syndrome include gastrointestinal bleeding and / or pulmonary bleeding.

4. The use according to claim 1, characterized in that: The lurasidone also includes at least one of the following forms: a salt form of lurasidone, a crystal form of lurasidone, and a solvent mixture of lurasidone.

5. The use according to claim 1, characterized in that: The medicine comprises at least one of the following dosage forms: tablets, granules, capsules, powders, injections and injection powders.

6. The use according to claim 1, characterized in that: The concentration of lurasidone in the drug is not less than 4.552 μM.

7. The use according to claim 1, characterized in that: The treatment of severe fever with thrombocytopenia syndrome virus infection includes inhibiting the replication ability of severe fever with thrombocytopenia syndrome virus.

8. The use according to claim 7, characterized in that: The lurasidone acts on the NP protein of severe fever with thrombocytopenia syndrome virus to inhibit the replication of the virus.

9. A drug for treating severe fever with thrombocytopenia syndrome, characterized in that: The active ingredients include lurasidone and other ingredients that inhibit SFTS virus infection.

10. The drug according to claim 9, characterized in that: Other components that inhibit severe fever with thrombocytopenia syndrome virus infection include antibodies that bind to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, polypeptide inhibitors that inhibit severe fever with thrombocytopenia syndrome virus infection, and compounds that inhibit severe fever with thrombocytopenia syndrome virus infection.

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