Application of small molecule compound SKLRI-73 in preparation of medicine for preventing or treating silkworm baculovirus infection

Abstract

The invention discloses application of a small molecule compound SKLRI-73 in preparation of a medicine for preventing or treating silkworm baculovirus infection. The small molecule compound is prepared based on a mainstream virtual screening technology and a high-throughput screening technology. The median poisoning concentration of cells is 8.657 [mu] M, the median effective concentration is 0.6924 [mu] M, and the virus titer is significantly reduced. When the final use concentration of the small molecule compound is 2.5 mu M, replication of silkworm baculovirus (MOI = 1) can be completely inhibited; when the final concentration is 10-100 [mu] M, proliferation and replication of baculovirus in bombyx mori can be effectively inhibited, and a prevention and control effect is achieved; when the final concentration is 100 [mu] M, an antiviral treatment effect is achieved; therefore, the method can be used for research and development of medicines for preventing and treating silkworm blood baculovirus and has application and popularization values.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a small molecule compound SKLRI-73 in the preparation of a drug for preventing or treating Bombyx mori baculovirus infection. Background Art

[0002] Silkworm blood-type purulent disease is caused by Bombyx mori nucleopolyhedrovirus (BmNPV). The etiology, infectiousness, and great harm pose a huge threat to the stable development of the sericulture industry. At present, the use of some chemical disinfectants (chlorine preparations, aldehyde preparations, surfactants, and lime) has a good preventive effect. However, these disinfectants are highly irritating to silkworm farmers and silkworms, which is not conducive to the normal growth of silkworms and will pollute the environment. Although chemicals such as β-propiolactone, alkylated 5'-guanine nucleotides, nalidixic acid, and imidazobactam have a certain preventive effect on BmNPV, the prevention, control, and treatment effects are not ideal. How to reduce the investment in drug research and development and obtain antiviral drugs with good effects for silkworms is of great significance. By combining virtual screening technology with high-throughput drug screening technology, the development and screening of anti-BmNPV drugs can be realized, the cost and cycle of drug research and development can be reduced, and a strong guarantee for virus prevention, control, and treatment in the silkworm industry can be provided. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a use of a small molecule compound SKLRI-73 in the preparation of a drug for preventing or treating Bombyx mori baculovirus infection.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] 1. Use of the small molecule compound SKLRI-73 in the preparation of a drug for preventing or treating Bombyx mori baculovirus infection, wherein the structural formula of the small molecule compound SKLRI-73 is shown in Formula I:

[0006]

[0007] Preferably, in the present invention, the effective concentration of the small molecule compound SKLRI-73 is 10 to 100 μM.

[0008] Preferably, the preventive concentration of the small molecule compound SKLRI-73 is 10 to 50 μM.

[0009] Preferably, the therapeutic concentration of the small molecule compound SKLRI-73 is 50-100 μM.

[0010] Preferably, the small molecule compound prevents or treats Bombyx mori baculovirus infection by inhibiting Bombyx mori baculovirus replication. Preferably, the small molecule compound is used at the individual level by spraying on clean mulberry leaves, drying and feeding.

[0011] The beneficial effects of the present invention are as follows: the small molecule compound obtained in the present invention is based on mainstream virtual screening technology and high-throughput screening technology, and the molecular formula is C 36 H 64 Cl 2 N 4 , the concentration of small molecules that is half cytotoxic (Concentration of cytotoxicity 50%; CC 50 ) is 8.657 μM, and the half effective concentration (Concentrationfor 50%of maximal effect; EC 50 ) was 0.6924 μM, and the virus significantly reduced the virus titer (Tissue culture infective dose 50%; TCID 50 ). When the final concentration of the small molecule compound is 2.5 μM, the replication of Bombyx mori baculovirus can be completely inhibited (MOI=1). When the final concentration of the small molecule compound on the silkworm body is 10-100 μM, the proliferation and replication of baculovirus in the silkworm body can be effectively inhibited, and the effect of prevention and control can be achieved. When the final concentration of the small molecule compound on the silkworm body is 10 μM-50 μM, the proliferation and replication of the virus in the silkworm body can be effectively prevented. At the same time, the small molecule compound has an antiviral therapeutic effect when the final concentration is 100 μM. The present invention can be used for the research and development and production of drugs for preventing and treating blood-type purulent disease in silkworms, and has application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0013] Figure 1 For the screening of anti-BmNPV drugs and analysis of the effectiveness of small molecules (A: drug (1μM) safety and effectiveness screening; B: analysis of small molecule drugs inhibiting viral fluorescent protein expression; C: CC 50 Analysis; D:EC 50 Analysis; E: Analysis of small molecules inhibiting the expression of viral nucleocapsid protein (VP39); F: Analysis of the inhibitory effect of small molecules at different concentrations on viral replication; G: Analysis of viral copy number; H: Analysis of the inhibitory effect of small molecules (2.5 μM) on viral replication (*p<0.05, **p<0.01)).

[0014] Figure 2Toxicological analysis of small molecule drugs at the individual level (A: appearance observation of silkworm cocoons and pupae after small molecule treatment; B: statistics of silkworm weight; C: statistics of silkworm survival rate; D: statistics of silkworm economic traits (*p<0.05, **p<0.01)).

[0015] Figure 3 For individual level analysis of small molecule drugs against viruses (1×10 6 The control effects of small molecules on silkworms (5th instar) and silkworms (4th instar) were analyzed (A: analysis of the effect of small molecules on inhibiting virus replication in silkworms (5th instar); B: analysis of the effect of small molecules on inhibiting virus replication in silkworms (4th instar); C: statistics of the survival rate of silkworms (5th instar) infected with polyhedron by small molecules; D: statistics of the survival rate of silkworms (4th instar) infected with polyhedron (*p<0.05, **p<0.01)).

[0016] Figure 4 For individual level analysis of small molecule drugs against viruses (1×10 5 / head) (A: prevention pattern diagram; B: analysis of the effect of small molecules on inhibiting virus replication; C: statistics on silkworm clustering rate; D: statistics on silkworm survival rate; E: statistics on silkworm total weight; F: statistics on silkworm cocoon weight; F: statistics on silkworm pupa weight (*p<0.05, **p<0.01)).

[0017] Figure 5 For individual level analysis of small molecule drugs against viruses (1×10 5 The therapeutic effect of the silkworm (A: treatment mode diagram; B: analysis of the effect of small molecules inhibiting viral replication; C: statistics of silkworm survival rate). DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0019] In the implementation case, the molecular formula of the small molecule compound is C 36 H 64 Cl 2 N 4 The structural formula is shown in Formula I. The Chinese name is octenidine. It is synthesized by pharmaceutical companies and chemical companies (such as Alfa Chemistry, LEAPCHEM CO., LTD., etc.). The small molecule compound is abbreviated as SKLRI-73.

[0020]

[0021] Example 1

[0022] The screening and research of anti-BmNPV drugs were carried out through virtual screening and related antiviral compound libraries. The vBm-EGFP virus recombinant bacmid constructed in the laboratory was used to purchase and analyze the effectiveness of candidate drugs. 50 ,EC 50 and TCID 50 The antiviral capacity was analyzed.

[0023] The specific steps for analyzing the effectiveness of candidate drugs are as follows:

[0024] (1) Inoculate approximately 1×10 4 TC-100 insect cell culture medium (0.6 ml) containing 10% fetal bovine serum (FBS) was added to each well.

[0025] (2) After culturing at 27°C for 48 h, the cells were used for subsequent drug efficacy analysis.

[0026] (3) All small molecule compounds SKLRI-73 were prepared in DMSO (10 mM) for subsequent experimental dilutions.

[0027] (4) The stock solution was diluted to a final concentration of 1 μM, mixed with budded virus (BV; MOI = 1), and added to a 48-well plate for analyzing the effectiveness of the candidate drug.

[0028] (5) After 24 hours, the well plate was placed under a fluorescence microscope to observe the expression of green fluorescent protein and measured by flow cytometry.

[0029] The results showed that the small molecule of the present invention could significantly inhibit the proliferation of the virus at 1 μM, with an inhibition rate of 45.37% ( Figure 1 , A).

[0030] The specific steps for the safety determination of candidate drugs are as follows:

[0031] (1) Inoculate approximately 1×10 4 TC-100 insect cell culture medium (0.6 ml) containing 10% FBS was added to each well.

[0032] (2) After incubation at 27°C for 48 h, the cells were used for subsequent drug safety analysis.

[0033] (3) Dilute the above stock solution to a final concentration of 1 μM, add it to a 48-well plate, and mix well.

[0034] (4) After 24 h, CCK-8 was added to the well plate, incubated at 27°C for 2 h, and then detected using an ELISA reader at a wavelength of 450 nm.

[0035] The results showed that the safety of the small molecule compound SKLRI-73 of the present invention at 1 μM was 76.5%, and the cytotoxicity at 5 μM was 56%. The fluorescence results showed that the candidate drug significantly inhibited the expression of the fluorescent protein of the virus ( Figure 1 , A and B).

[0036] CC of small molecule compound SKLRI-73 50 Analysis, the specific steps are as follows:

[0037] (1) Inoculate approximately 1×10 4 TC-100 insect cell culture medium (1 ml) containing 10% FBS was added to each well.

[0038] (2) After culturing at 27°C for 48 h, small molecule drugs were added to the cells at final concentrations of 0, 0.01, 0.1, 1, 2.5, 5, and 10 μM, respectively.

[0039] (3) After 24 h of culture, CCK-8 was added to the well plate and incubated at 27°C for 2 h. The plate was then detected using an ELISA reader at a wavelength of 450 nm and analyzed using GraphPad. 50 The experimental results show that the small molecule of the present invention is CC 50 8.657μM( Figure 1 , C).

[0040] EC of small molecule drugs 50 Analysis, the specific steps are as follows:

[0041] (1) Inoculate approximately 1×10 4 TC-100 insect cell culture medium (1 ml) containing 10% FBS was added to each well.

[0042] (2) After culturing at 27°C for 48 h, small molecule drugs and BV (MOI = 1) were added to the cells at final concentrations of 0 μM, 0.1 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM, respectively.

[0043] (3) After culturing for 24 h and 48 h, the expression of EGFP fluorescent protein, BmNPV proliferation and replication-related genes (gp41), and VP39 was analyzed.

[0044] The experimental results show that the small molecule of the present invention is EC 50 At 10 μM, the expression of gp41 and VP39 was significantly reduced ( Figure 1, D and E).

[0045] TCID of small molecule drugs 50 Analysis, the specific steps are as follows:

[0046] (1) Inoculate approximately 1×10 4 TC-100 insect cell culture medium (1 ml) containing 10% FBS was added to each well.

[0047] (2) After culturing at 27°C for 48 h, small molecule drugs and BV (MOI = 1) were added to the cells at a final concentration of 2.5 μM.

[0048] (3) After 24 h, 48 h, 72 h, and 96 h of culture, samples were collected and the viral titer was determined by the endpoint method and the gp41 expression was analyzed.

[0049] The experimental results showed that small molecule drugs significantly reduced the infectivity of the virus and reduced the replication of the virus ( Figure 1 , F and G). Therefore, the drug small molecule of the present invention can significantly inhibit the proliferation and replication of the virus, and has good cell safety, and the performance of silkworm antiviral drugs can be explored at the individual level.

[0050] Example 2

[0051] The maximum final concentration of small molecules is 500 μM. To explore the safety of drugs in individuals, the specific steps are as follows:

[0052] (1) The small molecule drug at the above final concentration was sprayed on the leaves, and after drying naturally, it was fed to silkworms (Dazao strain, 4th instar and above) twice a day. The weight change and mortality of the silkworms were counted daily, and the changes in economic traits such as cocoons were observed.

[0053] (2) The experimental results showed that 500 μM small molecules did not significantly change the appearance and economic characteristics of silk cocoons and pupae ( Figure 2 , A, D). At the same time, the weight and survival rate of silkworms did not decrease ( Figure 2 , B~C).

[0054] Example 3

[0055] The above small molecule stock solution was diluted to a final concentration of 10 μM and 100 μM to analyze the effectiveness of the small molecule in preventing and controlling viruses in vivo. The specific steps are as follows:

[0056] (1) The above small molecules are mixed with viral inclusion bodies (10 6 After mixing, silkworms (Dazao strain, 5th instar and above) were added.

[0057] (2) After 6 hours, spray the small molecules on the leaves, let them dry naturally, and then feed them twice a day.

[0058] (3) Count the mortality rate of silkworms and analyze the expression level of gp41 in the silkworms.

[0059] The experimental results showed that the small molecule significantly reduced the expression of gp41 ( Figure 3 , A and B). It also significantly reduced the mortality of silkworms, and the effect of 100 μM was the best ( Figure 3 , C and D). This indicates that small molecules (100 μM) have a good preventive and control effect on viruses.

[0060] Example 4

[0061] The above small molecule stock solution was diluted to a final concentration of 10 μM and 50 μM, and the preventive effect of the small molecule on the virus was analyzed. The prevention process is as follows: Figure 4 As shown in A.

[0062] (1) The above small molecules were diluted to a final concentration of 10 μM and 50 μM, and then fed to silkworms (Dazao strain, 5th instar).

[0063] (2) 24 hours later, the silkworms were given the virus inclusion bodies to lick (10 5 The corresponding small molecule drugs were sprayed on mulberry leaves twice a day, and the leaves were naturally dried and fed to silkworms. After 4 days, the mortality rate, HE staining, gp41 expression and changes in the final economic traits were counted.

[0064] The experimental results showed that the small molecule drug (10μM and 50μM) had a good preventive effect. The small molecule inhibited the expression of viral gp41 and increased the clustering rate and survival rate of silkworms ( Figure 4 , BD). At the same time, the small molecule significantly increased the weight of silkworm pupae and cocoons ( Figure 4 , EG).

[0065] Example 5

[0066] The above small molecule stock solution was diluted to a final concentration of 50 μM and 100 μM to analyze the therapeutic effect of the small molecule on the virus. The experimental process is as follows: Figure 5 As shown in A.

[0067] (1) The 5th instar silkworms were fed with virus inclusion bodies (10 5 / head), and feed mulberry leaves once normally after 6 hours.

[0068] (2) After 24 hours, the above small molecules were diluted to a final concentration of 50 μM and 100 μM, and then sprayed on mulberry leaves. After drying naturally, the small molecule drugs were fed to the silkworms. The small molecule drugs were sprayed on mulberry leaves twice a day to feed the silkworms.

[0069] (3) After 4 days, the mortality rate was counted and the expression level of gp41 was analyzed.

[0070] The experimental results showed that the small molecule drug (100 μM) had a good therapeutic effect. The small molecule drug inhibited the expression of viral gp41 and increased the survival rate of silkworms by 22% ( Figure 5 , B and C).

[0071] In vivo administration method recommendation: The target small molecule of the present invention and related excipients are diluted to 10μM-100μM with pure water, sprayed on clean mulberry leaves, and naturally dried. Then, they can be fed to 4- or 5-year-old silkworms 1-2 times a day. For the purpose of prevention, low-concentration small molecules can be appropriately selected for spraying, which can achieve the effects of prevention and control, and can effectively inhibit the proliferation of baculovirus in silkworms. For the purpose of treatment, high-concentration small molecules can be appropriately selected, sprayed and fed twice a day to reduce the death of silkworms caused by pus disease. The small molecules of the present invention can be mainly used for the treatment of blood-type pus disease in silkworms.

[0072] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of the small molecule compound SKLRI-73 in the preparation of a drug for preventing or treating Bombyx mori baculovirus infection, characterized in that: The structural formula of the small molecule compound SKLRI-73 is shown in Formula I:

2. The use according to claim 1, characterized in that: The effective concentration of the small molecule compound SKLRI-73 is 10 to 100 μM.

3. The use according to claim 1, characterized in that: The preventive concentration of the small molecule compound SKLRI-73 is 10 to 50 μM.

4. The use according to claim 1, characterized in that: The therapeutic concentration of the small molecule compound SKLRI-73 is 50 to 100 μM.

5. The use according to claim 1, characterized in that: The small molecule compound prevents or treats Bombyx mori baculovirus infection by inhibiting the replication of Bombyx mori baculovirus.

6. The use according to claim 1, characterized in that: The small molecule compound is used at the individual level by spraying on clean mulberry leaves and then drying them for feeding.

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