阅读说明：本技术 Ml-60218用于预防或治疗非洲猪瘟的新用途 (New application of ML-60218 in preventing or treating African swine fever ) 是由 郑海学 李丹 吴盼雪 王延轶 冉勇 杨文萍 张敬 茹毅 �田宏 杨帆 张克山 于 2021-03-18 设计创作，主要内容包括：本发明属于非洲猪瘟治疗技术领域,具体涉及ML-60218用于预防或治疗非洲猪瘟的新用途。本发明意外发现,ML-60218能够显著抑制非洲猪瘟病毒蛋白p30的表达,阻止病毒入侵宿主细胞,可用于抑制ASFV的早期感染；并且ML-60218能够显著抑制非洲猪瘟病毒的复制,降低非洲猪瘟病毒感染后的病毒滴度,可作为非洲猪瘟病毒的抑制剂,用于预防或治疗非洲猪瘟。(The invention belongs to the technical field of African swine fever treatment, and particularly relates to a new application of ML-60218 in prevention or treatment of African swine fever. The invention unexpectedly discovers that ML-60218 can obviously inhibit the expression of African swine fever virus protein p30, prevent viruses from invading host cells and can be used for inhibiting the early infection of ASFV; and ML-60218 can obviously inhibit the replication of African swine fever virus, reduce the virus titer after the African swine fever virus infection, can be used as an inhibitor of the African swine fever virus, and is used for preventing or treating the African swine fever virus.)

The application of ML-60218 or a pharmaceutically acceptable salt thereof in preparing an African swine fever virus p30 protein expression inhibitor, wherein the ML-60218 has a structural formula shown as the following formula (I):

2. the use of claim 1, wherein the ML-60218 or the pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and/or adjuvant to prepare any one dosage form of tablet, spray, granule, capsule, oral liquid, injection, and suspension.

The use of ML-60218 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of African swine fever, said ML-60218 having the formula (I):

4. the use of claim 3, wherein the ML-60218 or the pharmaceutically acceptable salt thereof is incorporated with a pharmaceutically acceptable carrier and/or adjuvant and formulated into any one of a tablet, a spray, a granule, a capsule, an oral liquid, an injection, and a suspension.

The use of ML-60218 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention of African swine fever, wherein ML-60218 has the structural formula shown in formula (I):

6. the use of claim 5, wherein the ML-60218 or the pharmaceutically acceptable salt thereof is incorporated with a pharmaceutically acceptable carrier and/or adjuvant and formulated into any one of a tablet, a spray, a granule, a capsule, an oral liquid, an injection, and a suspension.

The use of ML-60218 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of early African swine fever virus infection, wherein ML-60218 has the structural formula shown in formula (I):

8. the use of claim 7, wherein the ML-60218 or a pharmaceutically acceptable salt thereof is incorporated with a pharmaceutically acceptable carrier and/or adjuvant and formulated into any one of a tablet, a spray, a granule, a capsule, an oral liquid, an injection, and a suspension.

Technical Field

The invention belongs to the technical field of African swine fever treatment, and particularly relates to a new application of ML-60218 in prevention or treatment of African swine fever.

Background

African Swine Fever (ASF) is an acute virulent infectious disease characterized by Fever of pigs and organ bleeding of the whole body of pigs caused by African Swine Fever Virus (ASFV), and the death rate of domestic pigs is as high as 100%. The disease first outbreaks in kenya 1921 and then is widely prevalent in domestic and wild pigs throughout africa. The 20 th century was introduced into europe in the 50 s, and the disease was cured for 40 years throughout europe. However, the disease was again introduced into grurgia from eastern africa in 2007, and then widely disseminated in eastern europe and introduced into elocusk, the far east russia, 2017. In 2019, at the beginning of 8 months, researchers of Hurongrong report the epidemic situation of the first African swine fever in China, and the disease spreads to 30 provinces and municipalities in China within a short time of one year, so that the disease continues to threaten the pig industry. As no effective vaccine or specific therapeutic medicine exists so far, once the epidemic situation of the African swine fever occurs, the epidemic situation can be controlled only by a killing means, but the mode not only causes economic loss, but also cannot meet the requirement of large-scale pig raising in China. Therefore, how to effectively control the ASF epidemic situation is one of the great challenges facing the pig industry in the world at present, and is also a major strategic subject to be urgently solved by ASF prevention and control in China.

p30 is a key structural protein in ASFV, is a main structural protein constituting virus particles, is also an important surface antigen, and is closely related to host cell tropism, pathogenicity and immunogenicity; p30 is expressed early in ASF infection, usually produced 2-4h post infection, and is expressed continuously throughout the infection, involved in virus internalization, and is involved in virus invasion into host cells.

ML-60218, 2-chloro-N- [3- (5-chloro-3-methylbenzo [ b ] thiophen-2-yl) -1-methyl-1H-pyrazol-5-yl ] -benzenesulfonamide, is a small molecule inhibitor of Saccharomyces cerevisiae and human RNA polymerase III. At 200. mu.M, there was 88%, 94% and 90% inhibition of the in vitro transcription activity of Saccharomyces cerevisiae, Candida albicans and human RNA polymerase III, respectively. Among them, the literature (Melchjorsen J, et al, early Innate Recognition of Herpes Simplex Virus in Human Primary macromolecules Is meditated via the MDA 5/MAVS-dependents and MDA5/MAVS/RNA Polymerase III-Independent Pathways.) discloses that RNA Polymerase III inhibitor ML60218 does not inhibit IFN-. beta.and TNF-. alpha.levels after HSV-1 infection, i.e., ML60218 does not inhibit replication of HSV-1 Virus.

The invention unexpectedly discovers that ML-60218 can obviously inhibit the expression of African swine fever virus protein p30, prevent viruses from invading host cells and can be used for inhibiting the early infection of ASFV; and ML-60218 can obviously inhibit the replication of African swine fever virus, reduce the virus titer after the African swine fever virus infection, can be used as an inhibitor of the African swine fever virus, and is used for preventing or treating the African swine fever virus.

Disclosure of Invention

Aiming at the technical problems, the invention provides a new application of ML-60218 in inhibiting African swine fever virus infection, which specifically comprises the following contents:

in a first aspect, the invention provides an application of ML-60218 or a pharmaceutically acceptable salt thereof in preparing an African swine fever virus p30 protein expression inhibitor, wherein the ML-60218 has a structural formula shown as the following formula (I):

preferably, the ML-60218 or the pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any one dosage form of tablets, sprays, granules, capsules, oral liquid, injections and suspensions.

In a second aspect, the invention provides an application of ML-60218 or a pharmaceutically acceptable salt thereof in preparing a medicament for treating African swine fever, wherein the structural formula of ML-60218 is shown as the following formula (I):

preferably, the ML-60218 or the pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any one dosage form of tablets, sprays, granules, capsules, oral liquid, injections and suspensions.

In a third aspect, the invention provides an application of ML-60218 or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing African swine fever, wherein the structural formula of ML-60218 is shown as the following formula (I):

preferably, the ML-60218 or the pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any one dosage form of tablets, sprays, granules, capsules, oral liquid, injections and suspensions.

In a fourth aspect, the invention provides an application of ML-60218 or a pharmaceutically acceptable salt thereof in preparing a medicament for treating early infection of African swine fever virus, wherein the structural formula of ML-60218 is shown as the following formula (I):

preferably, the ML-60218 or the pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any one dosage form of tablets, sprays, granules, capsules, oral liquid, injections and suspensions.

The invention has the beneficial effects that: the invention unexpectedly discovers that ML-60218 can obviously inhibit the expression of African swine fever virus protein p30, prevent viruses from invading host cells and can be used for inhibiting the early infection of ASFV; and ML-60218 can obviously inhibit the replication of African swine fever virus, reduce the virus titer after the African swine fever virus infection, can be used as an inhibitor of the African swine fever virus, and is used for preventing or treating the African swine fever virus.

Drawings

FIG. 1 Virus titre following African swine fever virus infection;

FIG. 2 African swine fever virus infection gene copy number;

FIG. 3 is a graph showing the results of the expression level of p30 protein;

FIG. 4 is a graph showing cytotoxicity results of ML-60218.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.

The experiments described in the following examples obtain biosafety permits and african swine fever laboratory activity permits:

according to the related requirements of biosafety of a Lanzhou veterinary research institute of the Chinese agricultural academy of sciences, a biological safety 3-level laboratory (BSL-3) and related biological safety of African swine fever, the Lanzhou veterinary research institute biological safety committee, the laboratory animal ethics committee, the Chinese agricultural academy of sciences biological safety committee, the Lanzhou veterinary research institute experimental animal ethics committee and the Lanzhou veterinary research institute biological safety committee report step by step, the permission of developing highly pathogenic ASFV pathogens and animal research is obtained by the agricultural department, and the permission is recorded by the agricultural rural department and meets the requirements of national biological safety level.

Experimental cells, viral sources as described in the examples below:

primary Porcine Alveolar Macrophages (PAM) and primary bone marrow macrophages (BMDM) were taken from healthy SPF Bama minipigs aged 2-4 months, aseptically harvested, lysed with erythrocyte lysate (Biosharp Co.), removed of erythrocytes, centrifuged at low speed, discarded supernatant, and the cell pellet resuspended in 10% FBS (Biosharp Co.)From PAN) in RPMI 1640 complete medium (purchased from Gibco), placed at 37 ℃ in 5% CO₂Culturing in an incubator.

The ASFV CN/GS/2018 isolate comes from the national African swine fever regional laboratory (Lanzhou), belongs to genotype II, and has the virus titer of 5 multiplied by 10⁷TCID₅₀The strain/mL is the 4 th generation strain after PAM cell propagation, is preserved in China center for type culture Collection in 12 months and 30 days in 2020, and has the preservation number of CCTCC NO: v202096; and (4) storage address: wuhan, Wuhan university, China; and (4) contacting the telephone: 027-68752319.

ML-60218(C₁₉H₁₅Cl₂N₃O₂S₂) Purchased from MedChemExpress corporation.

Other reagents in the experiment are common commercial reagents unless otherwise specified; the procedures in the experiments are those known in the art unless otherwise specified.

The African swine fever virus p30 protein expression inhibitor refers to an experimental reagent for scientific research. The compound ML-60218 can inhibit the expression of African swine fever virus p30 protein, and can be used as an experimental reagent for scientific research to research the related mechanism of the African swine fever virus p30 protein.

Example 1 Effect of ML-60218 on replication of African Swine fever Virus infection and Gene transcription expression

1. Changes in African Swine fever Virus infection and replication

Culture of porcine alveolar macrophages (PAM, 1X 10) in 12-well plates with RPMI 1640+ 10% FBS medium⁶Per well), experimental groups infected with ASFV CN/GS/2018 strain (0.1MOI) for 6h, 12h, 24h after being treated with ML-60218 (50. mu.M) for 2 h; after the control group was treated with DMSO (1%) for 2h, the ASFV CN/GS/2018 strain (0.1MOI) was infected for 6h, 12h, and 24 h. Collecting cells and supernatant at different treatment and different time points, repeatedly freezing and thawing at-80 ℃ for 3 times, and using the cells and supernatant as samples, continuously diluting the samples by 10 times by serum-free RPMI 1640 respectively, making 7 dilutions, repeating 8 holes for each dilution, inoculating the samples to PAM cells for culture, and adding pig red blood cells; place the cell plate at 37 ℃ in 5% CO₂Conditioned for 7 days, and the cell cultures were observed dailyErythrocyte adsorption reaction (HAD) in the wells. Calculation of HAD50, the calculation method of which refers to TCID₅₀The method of (3).

The erythrocyte adsorption reaction (HAD) is based on the phenomenon that porcine red blood cells are adsorbed around mononuclear macrophages infected with african swine fever virus, thereby producing erythrocyte adsorption. The results are shown in FIG. 1, which infected HAD in control group (DMSO)₅₀Higher value, and HAD after treatment with ML-60218₅₀The value decreases. The results show that the compound ML-60218 can obviously inhibit the virus titer of ASFV.

2. Copy number of gene

Culture of porcine alveolar macrophages (PAM, 1X 10) in 12-well plates with RPMI 1640+ 10% FBS medium⁶Per well), experimental groups infected with ASFV CN/GS/2018 strain (0.1MOI) for 6h, 12h, 24h after being treated with ML-60218 (50. mu.M) for 2 h; after the control group was treated with DMSO (1%) for 2h, the ASFV CN/GS/2018 strain (0.1MOI) was infected for 6h, 12h, and 24 h. Collecting cells and supernatant at different treatment and different time points, and repeatedly freezing and thawing at-80 deg.C for 3 times to obtain samples. The Ct values of different processed samples are directly detected by using a Pro Taq HS premixed probe method qPCR kit (ACCURATE BIOLOGY), and the change of the gene copy number is obtained according to a standard curve.

The total volume of the Q-PCR reaction system is 25 mu L, and the Q-PCR reaction system comprises: 12.5 mu L of 2x pro taq HS probe premix, 2.5 mu L of upstream and downstream primers, 1 mu L of probe and 2.5ul of inactivated venom are used as templates, and sterile deionized water is supplemented to 25 mu L;

the reaction conditions are as follows: at 95 ℃ for 2 min; 95 ℃, 7s, 60 ℃, 12s, 3 cycles; 95 ℃, 6s,58 ℃,11s,40 cycles;

a forward primer 5'-GATACCACAAGATCAGCCGT-3'; a reverse primer 5'-CTGCTCATGGTATCAATCTTATCGA-3'; probe FAM-CCACGGGAGGAATACCAACCCAGTG-TAMRA.

The gene copy number detection result is shown in figure 2, the gene copy number of ASFV in the PAM cell added with ML-60218 is obviously lower than that of the cell added with DMSO, and the compound ML-60218 can inhibit the expression and the replication of the African swine fever virus.

P30 protein expression levels

Overnight incubation in 12-well plates with RPMI 1640+ 10% FBS mediumPorcine alveolar macrophage (PAM, 10X 10)⁶) (ii) a Experimental groups cells were treated with ML-60218(50 μ M) for 2h before infection with ASFV CN/GS/2018 strain (MOI ═ 0.1); uninfected control group infected ASFV CN/GS/2018 strain after treating cells with DMSO (1%) for 2h (MOI ═ 0.1); and continuously culturing the treated cells for 6h, 12h and 24h respectively, collecting cell cultures, washing the cells once by PBS, centrifuging, and removing supernatant. Extracting total protein, and detecting p30 protein expression difference by using a western-blotting method.

p30 protein expression level test results as shown in FIG. 3, the ASFV p30 protein expression level was increased in the infected control group (DMSO + ASFV) and the test group (ML-60218+ ASFV) compared to the uninfected control group (control group); however, the expression level of ASFV p30 protein in the experimental group (ML-60218+ ASFV) was significantly decreased relative to the infection control group (DMSO + ASFV). The result shows that ML-60218 can obviously inhibit the protein expression level of p30 in the African swine fever virus gene, prevent the virus from invading host cells and can be used for inhibiting the early infection of ASFV.

Example 2 cytotoxicity of ML-60218

And (3) carrying out cytotoxicity detection on the small molecular compound ML-60218 by using the constructed stable in-vitro cell screening system and a CCK-8 method. Culture of porcine alveolar macrophages (PAM, 2X 10) in RPMI 1640+ 10% FBS medium in 96-well plates⁵Wells) overnight, different concentrations of ML-60218(12.5 μ M25 μ M50 μ M100 μ M200 μ M) were added to the wells, while blank wells (containing medium only), control wells (containing cells and medium) were set, 10 μ L of CCK-8 solution was added to each well of the plate after incubating the plate in the incubator for 2h, the plate was incubated in the incubator for 1-4h, and gentle mixing on a shaker was possible before reading the plate. And reading the absorbance at 450nm by a microplate reader, and calculating the cell survival rate.

As shown in FIG. 4, ML-60218 shows less toxicity to cells and better safety.

In conclusion, the ML-60218 can obviously inhibit the expression of African swine fever virus protein p30, prevent viruses from invading host cells, and can be used for inhibiting the early infection of ASFV; moreover, ML-60218 can obviously inhibit the replication of African swine fever virus, reduce the virus titer after the African swine fever virus infection, can be used as an inhibitor of the African swine fever virus, and is used for preventing or treating the African swine fever virus

The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.