Application of KU55933 or pharmaceutically acceptable salt thereof in preparation of drugs for resisting bovine nodular skin disease virus infection and treating bovine nodular skin disease virus infection
By using the specific targeted ATM kinase inhibitor KU55933, the replication and proliferation of LSDV is inhibited, and the problem of lack of effective treatment methods in the prior art has been solved, and the effect of significantly inhibiting viral proliferation and slowing down clinical symptoms has been achieved, providing a new method for the prevention and control of the nodular skin disease epidemic in bovine.
Patent Information
- Application Number
- CN202411688593.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-03-07
AI Technical Summary
The existing technology lacks effective treatment methods and specific vaccines to deal with the cattle nodule skin disease virus (LSDV) epidemic, which has led to the spread of the epidemic worldwide and seriously affecting the cattle breeding industry.
A specific targeting ATM kinase inhibitor KU55933 was used to significantly inhibit the replication and proliferation of LSDV by inhibiting the ATM kinase-dependent DNA damage response, thereby developing a new drug against bovine nodular skin disease virus.
It showed good inhibitory effect on LSDV proliferation at both the cellular and animal levels, effectively slowing down the clinical symptoms of the epidemic and viral load of skin tissues, and providing a new method for the prevention and control of the nodular skin disease epidemic in bovine.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of a specific targeted ATM kinase inhibitor KU55933 in the preparation of a drug for preventing or treating bovine nodular dermatosis virus. Background Art
[0002] Lumpy skin disease (LSD), also known as bovine nodular rash, bovine nodular dermatitis or bovine pimple skin disease, is an acute and subacute infectious disease caused by LSDV (Lumpy skin disease virus, LSDV), a member of the Poxviridae family and the Capripoxvirus genus. LSDV mainly causes fever, papules or skin nodules in cattle, such as on the head, neck, shoulders and udders. In severe cases, it presents pustular lesions and tissue necrotic scabs. When the host's immunity is low, LSDV-infected lesions may be secondary to bacterial infection, causing orchitis in bulls and bacterial or ulcerative mastitis in cows. The LSD epidemic has caused significant economic losses to the cattle industry and seriously affected the healthy development of the world's cattle industry.
[0003] The LSD epidemic was first discovered in the Republic of Zambia in 1929, and then the disease quickly spread to the Middle East, Eastern Europe, Central Asia and other regions, showing a global outbreak trend. LSD is an important newly imported animal disease in my country in recent years, which has caused significant economic losses to the cattle industry and seriously affected the healthy development of my country's cattle industry. However, due to the lack of effective treatments and specific vaccines for the disease, since 2019, LSD epidemics have been confirmed in 14 provinces, municipalities and autonomous regions across the country, and have shown a trend of gradual spread, and the prevention and control situation is becoming increasingly severe. The World Organization for Animal Health lists it as a notifiable animal disease. At present, my country now lists it as a Class II animal disease and controls it as a Class I pathogen.
[0004] The DNA damage response in cells that is dependent on Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) is crucial for the integrity of the genome in the "nucleus" and the normal function of cells. Studies have shown that most DNA viruses that replicate in the "nucleus" can activate the DNA damage response mediated by ATM and / or ATR after infecting target cells, thereby promoting efficient viral proliferation. Compared with other DNA viruses, members of the genus Poxvirus are the only enveloped DNA viruses that complete viral replication entirely in the "cytoplasm". A series of biochemical experiments have shown that bovine nodular dermatitis virus infection can activate the DNA damage repair response that is dependent on ATM and ATR kinases in host cells. The ATM kinase inhibitor KU55933 is highly selective for ATM and can effectively act on ATM-dependent phosphorylation. The inhibition of ATM-dependent phosphorylation is dose-dependent. KU55933 is highly selective and efficient for ATM, IC 50 and K i The values are 12.9nM and 2.2nM respectively. The specific inhibitor KU55933 targeting ATM kinase described in the present invention has a good effect of inhibiting the proliferation of bovine nodular dermatitis virus at both the cell and animal levels, and is expected to be developed into a new antiviral drug for preventing and treating bovine nodular dermatitis virus, with broad clinical application and market transformation potential. Summary of the invention
[0005] In view of the above technical problems, the present invention provides a use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for resisting bovine nodular dermatosis virus infection. The structural formula of KU55933 is shown in the following formula (I):
[0006]
[0007] Preferably, the KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0008] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.
[0009] Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing bovine nodular dermatosis virus, wherein the structural formula of KU55933 is shown in the following formula (I):
[0010]
[0011] Preferably, the KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0012] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.
[0013] Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating diseases caused by bovine nodular dermatosis virus, wherein the structural formula of KU55933 is shown in the following formula (I):
[0014]
[0015] Preferably, the KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0016] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.
[0017] The present invention proves from both the cell and animal levels that after LSDV infects susceptible cells, it can activate the double-strand DNA damage response (DNA double-strand breakage, DSB) dependent on ATM kinase and the single-strand DNA damage response (DNA single-strand breakage, SSB) dependent on ATR kinase. In the present invention, the specific ATM kinase drug inhibition experiment shows that the activation of the DSB response that ATM kinase depends on, rather than the SSR response that ATR kinase depends on, can promote the replication and proliferation of LSDV, and the use of specific targeted ATM kinase drugs to treat LSDV-infected susceptible cells can significantly inhibit the proliferation of LSDV inside and outside the cells. At the same time, drug treatment of infected animals can also effectively slow down the clinical symptoms of the disease and the viral load of skin tissue. The ATM kinase-specific inhibitor KU55933 is expected to be used in the development of drugs for preventing or treating LSDV, providing a new method for the effective prevention and control of the epidemic of bovine nodular skin disease, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0019] Figure 1 The ATM kinase inhibitor KU55933 in Example 1 of the present invention inhibits the replication and proliferation of LSDV in three susceptible cells in a drug concentration-dependent and time point-dependent manner; wherein (A, E and I) the virus content TCID in the supernatant of MDBK, Vero and hTERT-CSF three-degree cell culture after KU55933 drug treatment on the third day 50 Results; (B, F and G) Western blotting analysis of MDBK, Vero and hTERT-CSF cells on the third day after KU55933 treatment; (C, G and K) TCID in the supernatant of MDBK, Vero and hTERT-CSF cells on the second and fourth days after KU55933 treatment 50 Assay; (D, H and L) Western blotting analysis in MDBK, Vero and hTERT-CSF cells on the second and fourth days after KU55933 drug treatment.
[0020] Figure 2 In Example 2 of the present invention, KU55933 drug treatment significantly inhibited the replication and proliferation of fluorescently labeled LSDV-EGFP in three cells: MDBK, Vero, and hTERT-CSF; the KU55933 drug treatment concentrations of different cells were marked in white. The green fluorescently labeled LSDV strain was labeled as LSDV-EGFP, and the EGFP fluorescence emphasized the difference in the proliferation of the virus in MDBK, Vero, and hTERT-CSF.
[0021] Figure 3 The ATM kinase inhibitor KU55933 in Example 3 of the present invention significantly inhibits the clinical signs of LSDV-infected cattle and the replication and proliferation of the virus in the skin tissue. (A) Schematic diagram of the animal experiment grouping treated with KU55933 drug; (B) Statistical results of the anal temperature measurement of cattle in different experimental groups treated with KU55933 drug within 28 days; (C) Statistical results of the clinical symptom scoring of different experimental groups treated with KU55933 drug within 28 days; (D) Typical clinical symptom pictures of different experimental groups treated with KU55933 drug; (E) Schematic diagram of the qPCR measurement of the virus content in skin tissue samples; (F) qPCR measurement results of the virus content in skin tissue in different experimental groups treated with KU55933 drug. DETAILED DESCRIPTION
[0022] The technical solution of the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.
[0023] Example 1
[0024] Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing bovine nodular dermatosis virus infection, wherein the structural formula of KU55933 is as shown in the following formula (I):
[0025]
[0026] The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0027] The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
[0028] Example 2
[0029] Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing bovine nodular dermatosis virus, wherein the structural formula of KU55933 is shown in the following formula (I):
[0030]
[0031] The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0032] The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
[0033] Example 3
[0034] Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating diseases caused by bovine nodular dermatosis virus, wherein the structural formula of KU55933 is shown in the following formula (I):
[0035]
[0036] The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0037] The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
[0038] Example 4
[0039] The ATM kinase inhibitor KU55933 inhibited the replication and proliferation of LSDV in three susceptible cell lines in a drug concentration-dependent and time-point-dependent manner:
[0040] 1. Configuration of ATM kinase inhibitor preparations:
[0041] KU55933 is in powder form at room temperature, so use a pipette to extract 1.2643 mL of DMSO, add it to 5 mg of KU55933 powder, mix thoroughly to make a 10 mM mother solution, and store it in a -80°C refrigerator. When used, take the mother solution and dilute it into the cell culture medium to make working solutions of different concentrations.
[0042] 2. Cell inoculation and use of KU55933 preparations:
[0043] 1) Plate laying: Take Vero, MDBK and hTERT-CSF cells in good condition and plate them at 1×10 6 The cells were plated at a density of 10 cells / mL in a 6-well cell plate, with 2 mL per well, and cultured in a cell culture incubator at 37°C containing 5% carbon dioxide.
[0044] 2) KU55933 treatment: When the cell confluence reaches about 80-100%, take out the 6-well plate, discard the 10% DMEM, use DMEM medium with 10% serum content, dilute the 10mM KU55933 stock solution to different working concentrations according to different cell types, mix well and inoculate into the 6-well plate, place in a cell culture incubator at 37°C containing 5% carbon dioxide, and incubate for 1 hour in advance.
[0045] 3) Inoculation: After 1 hour of drug pretreatment, LSDV / FJ / CHA / 2021 strain was inoculated at a dose of 1 MOI, and the LSDV virus solution was diluted in serum-free DMEM medium (TCID 50 =1×10 6.23 ) and inoculated into a 6-well plate, placed in a cell culture incubator at 37°C with 5% carbon dioxide to culture the virus for 4 hours. Place in the incubator until the experimental point, collect the culture supernatant, and measure the virus titer in the cell culture supernatant and the intracellular virus content. The collected cells and cell culture supernatant samples were placed in a -80°C refrigerator for later use.
[0046] 4)TCID 50 Determination:
[0047] To determine the virus content in the culture supernatant of the cells to be tested, the cell culture supernatant collected at different time points was serially diluted (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 and 10 -11), then inoculate a 96-well plate, repeat 8 wells for each dilution, and use the last column as a negative control. After 4 hours of virus adsorption, wash with PBS three times, add 200μL of 10% FBS DMEM, place in a 37℃ incubator, observe daily, and continue to culture for 4 days. Calculate the virus titer in the cell culture supernatant according to the Reed-Muench method. Figure 1 As shown in A, 1E and 1I, TCID 50 The results showed that KU55933 treatment could significantly inhibit the proliferation of LSDV in the culture supernatants of MDBK, Vero and hTERT-CSF cells in a dose-dependent manner. Figure 1 C, 1G, and 1K, TCID 50 The results showed that KU55933 drug treatment could significantly inhibit the replication and proliferation of LSDV in the culture supernatants of MDBK, Vero and hTERT-CSF cells in a time-point-dependent manner.
[0048] 5) Western Blotting (WB) assay:
[0049] To determine the intracellular viral content of the detection band, add 200 μL of cell lysis solution to the 6-well plate cell samples at different time points, lyse in an ice bath for 15 minutes, collect the mixed cell samples, centrifuge at 12000 rpm / min for 10 minutes, collect the cell lysate in a new 1.5 mL EP tube, add 2× protein loading buffer, boil in a 100°C metal bath for 10 minutes, and perform protein immunoblotting analysis. According to the LSDV-specific protein antibody LSDV ORF29 and LSDV ORF123 protein bands, the intracellular LSDV virus content in the cell sample to be detected is determined. Figure 1 As shown in B, 1F, and 1J, Western blotting analysis results showed that KU55933 drug treatment could significantly inhibit the proliferation of LSDV in MDBK, Vero, and hTERT-CSF cells in a dose-dependent manner. Figure 1 As shown in D, 1H and 1L, the results of Western blotting analysis showed that KU55933 drug treatment could significantly inhibit the proliferation of LSDV in three types of cells, MDBK, Vero and hTERT-CSF, in a time point-dependent manner.
[0050] Example 5
[0051] KU55933 drug treatment significantly inhibited the replication and proliferation of fluorescently labeled LSDV-EGFP in Vero, MDBK and hTERT-CSF cells.
[0052] 1. Plate: Take Vero, MDBK and hTERT-CSF cells in good condition and plate them at 1×10 6 The cells were plated at a density of 10 cells / mL in a 6-well cell plate, with 2 mL per well, and cultured in a cell culture incubator at 37°C containing 5% carbon dioxide.
[0053] 2. KU55933 treatment: When the cell confluence reaches about 80-100%, take out the 6-well plate, discard the 10% DMEM, use DMEM medium with 10% serum content, dilute the 10mM KU55933 stock solution to different working concentrations according to different cell types, mix well and inoculate into the 6-well plate, place in a cell culture incubator at 37°C containing 5% carbon dioxide, and incubate for 1 hour in advance.
[0054] 3. Virus inoculation and photography: After drug pretreatment for 1 hour, fluorescently labeled LSDV-EGFP was inoculated at a dose of 1 MOI, and the LSDV-EGFP virus solution was diluted with serum-free DMEM medium (10 5.5 TCID 50 ) and inoculated into a 6-well plate, and placed in a cell culture incubator at 37°C with 5% carbon dioxide for 4 hours to allow virus adsorption. Place in the incubator until the test point, and observe and take pictures using the EVOS M5000 microscope imaging system. Figure 2 Compared with the positive control (vertical first column), KU55933 treatment can significantly inhibit the fluorescence intensity of green fluorescence in MDBK (horizontal first row), Vero (horizontal second row) and hTERT-CSF (horizontal third row) cells, showing a dose-dependent trend, indicating that the ATM kinase-specific inhibitory drug KU55933 can significantly inhibit the proliferation and replication of LSDV-EGFP in susceptible host cells at the cellular level.
[0055] Example 6
[0056] The ATM kinase-specific inhibitor KU55933 significantly inhibited the clinical signs and viral proliferation in the skin tissue of LSDV-infected cattle.
[0057] 1. Drug treatment and poison challenge experiment of cattle:
[0058] Under the experimental conditions, 6 cows weighing 120-150 kg were selected as experimental hosts. Four of the 6 cows were randomly divided into 2 experimental groups (Group 1 and Group 2), and the remaining 2 cows were used as the negative group (Group 3, PBS). Two cows in Group 1 were infected with LSDV (LSDV / FJ / CHA / 2021) intradermally. The diluted LSDV (6.23log TCID 50 / mL) was injected intradermally at 16 sites on the back of the experimental cattle. At the same time, two cattle in two groups were injected intradermally with an equal amount of LSDV mixed with KU55933 (50mM / mL) (LSDV+KU55933, Group 2), and 0.2mL (50mM / mL) was injected intramuscularly on days 1, 2, 3, 4, 5, 7, 9, 11, and 13 to maintain the effective drug concentration. The third group (Group 3) was inoculated with an equal amount of PBS as a negative control (such as Figure 3 A).
[0059] 2. Clinical observation and record:
[0060] Six experimental animal cows were housed in two rooms and fed by professionals in the isolated animal facility of the large animal P3 animal room of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The overall breeding environment was comfortable, with a temperature of 24°C and a humidity of 50% to 60%. The clinical signs of the three groups of cows were monitored in real time for 28 days, and the rectal temperature was recorded daily. In the skin biopsy, electric scissors were used to remove hair from the skin site, and photos were taken with a camera after the operation. Figure 3 As shown in B and 3C, the rectal body temperature of the positive control group Group 1 (LSDV group) and the KU55933 drug treatment group Group 2 (LSDV+KU55933 group) was slightly lower, and the clinical symptoms were all low scores. Figure 3 As shown in clinical picture D, the ATM kinase specific inhibitor KU55933 drug treatment can effectively reduce the size of skin nodules caused by LSDV infection. The above results show that the targeted ATM kinase specific inhibitor KU55933 can effectively reduce the clinical symptoms and clinical manifestations of cattle infected with LSDV.
[0061] 3. Absolute fluorescence quantitative determination (Quantitative polymerase chain reaction, qPCR):
[0062] Weigh 0.3 mg of the collected skin tissue, add 750 μL PBS, grind with a tissue grinder, and use a pipette to take 100 μL of the supernatant of the tissue grinding solution. Use a viral genome extraction kit to extract the viral genome in the skin tissue, and use the absolute fluorescence quantitative method to determine the viral content in the skin tissue. Among them, the absolute fluorescence quantitative primers are:
[0063] qPCR-ORF61-Forward,5′CAACCGGAACGACAAAC-3′;
[0064] qPCR-ORF61-Reverse, 5′ACTCGCCTTTGTGGTTACAT-3′. Then according to the standard curve, y=-3.0502x+45.97 (R2 =0.9977) to calculate the virus content (copies / μL). Figure 3 E and 3F, absolute fluorescence quantitative qPCR results show that the ATM kinase-specific inhibitor KU55933 can effectively inhibit the viral replication and proliferation of LSDV in skin tissue.
[0065] The above results show that the host ATM kinase-specific inhibitor described in the present invention can significantly inhibit the proliferation of LSDV wild strains (LSDV / FJ / CHA / 2021 strains) at the cellular level and animal level, and can be used to prepare drugs for anti-LSDV infection or treatment of LSDV, providing a new treatment option for the treatment and prevention of LSDV, and has broad clinical application prospects.
[0066] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing bovine nodular dermatosis virus infection, characterized in that: The structural formula of KU55933 is shown in the following formula (I):
2. The use according to claim 1, characterized in that The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
3. The use according to claim 2, characterized in that The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
4. Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing bovine nodular dermatosis virus, characterized in that: The structural formula of KU55933 is shown in the following formula (I):
5. The use according to claim 4, characterized in that The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
6. The use according to claim 5, characterized in that The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.
7. Use of KU55933 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating diseases caused by bovine nodular dermatosis virus, characterized in that: The structural formula of KU55933 is shown in the following formula (I):
8. The use according to claim 7, characterized in that The KU55933 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
9. The use according to claim 8, characterized in that The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.