Application of polyketides in the preparation of EV71 virus inhibitors

Abstract

The present invention discloses the use of polyketide compounds in the preparation of EV71 virus inhibitors, belonging to the technical field of antiviral drugs. The present invention finds that the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have anti-EV71 virus activity, can inhibit the cytopathic effect caused by the EV71 virus, enhance the survival rate of infected cells, inhibit the replication and proliferation of the EV71 virus in cells, and reduce the production of progeny viruses. This shows that the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have the potential to be used in the preparation of specific therapeutic drugs for anti-EV71 infection and have broad clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral drugs, and in particular to the application of polyketide compounds in the preparation of EV71 virus inhibitors. Background Art

[0002] Enterovirus 71 (EV71), a member of the genus Enterovirus in the family Picornaviridae, is one of the most common pathogens causing hand, foot, and mouth disease (HFMD) in infants and young children. This disease is sometimes accompanied by severe central nervous system complications, including aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurological cardiopulmonary failure, and even death. Since its first report in 1974, EV71 has caused numerous outbreaks and epidemics worldwide, with a particularly severe situation in the Asia-Pacific region, and in my country. In view of the significant harm HFMD poses to the lives and health of the Chinese people, the Chinese government designated HFMD a Category C infectious disease in 2008 and has enacted a series of relevant laws and regulations to strictly control its spread. Currently, there are no specific treatments for EV71-related diseases, and a vaccine was only launched in 2015, and its preventive efficacy remains to be determined. Therefore, the development of specific and effective anti-EV71 drugs is imperative.

[0003] Polyketides are a class of secondary metabolites produced by plants or microorganisms. They are widely found in nature, with over 10,000 species discovered to date. Polyketides have complex structures and possess diverse biological activities, including antimicrobial, antitumor, and antioxidant properties, making them widely used in agriculture, the food industry, and healthcare. (3S,4S)-Sclerotinin A is a natural isocoumarin product first isolated from Penicillium citrinum and Sclerotinia sclerotiorum. As a plant growth regulator, (3S,4S)-sclerotinin A promotes the growth of rice seedlings at a concentration of 5 ppm. Ligustrone B is a rare fungal metabolite whose skeleton contains chromone and indone structures. As the primary product of the plant pathogen Cercospora ligustrina, the biological activities of Ligustrone B are rarely reported. However, the antiviral activity of the above compounds, especially their inhibitory activity against enterovirus 71 (EV71), has not been reported yet. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of polyketide compounds in the preparation of EV71 virus inhibitors to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a polyketide compound in preparing an EV71 virus inhibitor.

[0007] The present invention also provides the use of polyketide compounds in preparing medicines for treating diseases caused by EV71 virus.

[0008] The present invention also provides a pharmaceutical composition for treating diseases caused by EV71 virus, comprising at least one of the (3S, 4S)-sclerotinin A and ligustrone B.

[0009] The structural formula of the (3S,4S)-sclerotinin A is shown in formula (I):

[0010] Formula (I).

[0011] The structural formula of the ligustrone B is shown in formula (II):

[0012] Formula (II).

[0013] The present invention also provides a pharmaceutical preparation for treating diseases caused by EV71 virus, comprising the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention discovered that the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have anti-EV71 virus activity, inhibiting the cytopathic effect caused by the EV71 virus, enhancing the survival rate of infected cells, inhibiting the replication and proliferation of the EV71 virus within cells, and reducing the production of progeny viruses. This suggests that the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have the potential to be used in the preparation of specific therapeutic drugs against EV71 infection and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1The results show that the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have inhibitory effects on EV71-induced CPE in RD cells.

[0018] Figure 2 The effects of polyketide compounds (3S,4S)-sclerotinin A and ligustrone B on the survival rate of RD cells infected with EV71.

[0019] Figure 3 The inhibitory effect of polyketide compounds (3S,4S)-sclerotinin A and ligustrone B on EV71 progeny virus production. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0026] The embodiments of the present invention provide the use of polyketide compounds in the preparation of EV71 virus inhibitors.

[0027] In some specific embodiments, the polyketide compound is at least one of (3S, 4S)-sclerotinin A or ligustrone B, and its structural formula is as follows: Figure 1 shown.

[0028] The embodiments of the present invention provide the use of polyketide compounds in the preparation of drugs for treating diseases caused by EV71 virus.

[0029] In some specific embodiments, the polyketide compound is at least one of (3S, 4S)-sclerotinin A or ligustrone B.

[0030] The structural formula of the (3S,4S)-sclerotinin A is shown in formula (I):

[0031] Formula (I).

[0032] The structural formula of the ligustrone B is shown in formula (II):

[0033] Formula (II).

[0034] An embodiment of the present invention provides a pharmaceutical composition for treating diseases caused by EV71 virus, comprising at least one of (3S, 4S)-sclerotinin A or ligustrone B.

[0035] The compounds (3S, 4S)-sclerotinin A and ligustrone B used in the present invention were isolated and obtained by our laboratory from the rice solid fermentation product of Talaromyces funiculosus.

[0036] An embodiment of the present invention provides a pharmaceutical preparation for treating diseases caused by EV71 virus, comprising the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0037] In some specific embodiments, the pharmaceutically acceptable carrier is suitable for granules, tablets, pills, capsules, injections or dispersions.

[0038] Example 1

[0039] 1. Test content:

[0040] Analysis of the anti-EV71 activity of compounds: The anti-EV71 activity of polyketide compounds (3S,4S)-sclerotinin A and ligustrone B was evaluated by combining cytopathic effect analysis and MTT cell viability assay.

[0041] 2. Test method:

[0042] 2.1 Toxicity of compounds to host RD cells

[0043] RD cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator until a confluent monolayer was formed. The cell culture medium was discarded and cell maintenance medium containing the compounds (3S,4S)-sclerotinin A and ligustrone B was added at concentrations of 200 μM, 175 μM, 150 μM, 125 μM, 100 μM, and 75 μM, respectively. After 48 hours, the cytotoxicity of the cells was observed and recorded. The cell survival rate was determined by the MTT assay. The median toxic concentration (CC) of the drugs for the cells was calculated using SPSS 11.5 software. 50 ).

[0044] Cell viability = (mean OD of drug group 492 Value / average OD of cell control group 492 value) × 100%.

[0045] 2.2 Inhibitory activity of compounds against EV71

[0046] RD cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator until the monolayer was full. The culture medium was discarded and 100 TCID 50 Cells were infected with EV71 virus solution for 1.5 hours. (3S,4S)-sclerotinin A and ligustrone B were added at concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, respectively, and ribavirin (500 μM, 250 μM, 125 μM, 62.5 μM, and 31.25 μM) was used as a positive control. After incubation for approximately 48 hours, when approximately 90% of the virus control wells showed CPE, the cells were observed under a microscope. Figure 1As shown in Figure 2, RD cells typically appear oval or polygonal and adhere to the culture dish. Viral infection can cause significant cytopathological effects in RD cells, with the cells becoming rounded and detaching from the culture dish. CPE is recorded as follows: no cytopathic effect is scored as -, less than 25% cytopathic effect is scored as +, 25%–50% cytopathic effect is scored as ++, 50%–75% cytopathic effect is scored as +++, and greater than 75% cytopathic effect is scored as ++++.

[0047] After the CPE observation was completed, the MTT method was used to detect the inhibition rate of the drug on EV71. The specific steps were as follows: 50 μL of MTT (5 mg mL –1 After incubation for 3–4 h, the supernatant was removed and an equal volume of DMSO was added to dissolve the precipitate. The corresponding absorbance (OD492 value) was read at 492 nm using a microplate reader. The inhibition rate of the drug on EV71 was calculated using the following formula. The half-maximal effective concentration (EC) of the drug was calculated using SPSS 11.5 software. 50 ).

[0048] Virus inhibition rate = [OD of drug-treated group (drug + virus) 492 - Virus control group OD 492 ] / (OD of cell control group 492 - Virus control group OD 492 ) × 100%.

[0049] 2.3 Therapeutic Index (TI) of a Drug

[0050] TI = CC 50 / EC 50 .

[0051] The higher the therapeutic index, the greater the antiviral potential.

[0052] 3. Experimental results

[0053] Table 1 Cytotoxicity and anti-EV71 activity of polyketides

[0054]

[0055] The results of the cytotoxicity and anti-EV71 activity tests of polyketide compounds are shown in Table 1. Compounds (3S,4S)-sclerotinin A and ligustrone B have significant inhibitory activity against EV71, and their therapeutic index exceeds that of the positive control drug ribavirin, indicating that they have great antiviral potential. Figure 2 As shown, (3S,4S)-sclerotinin A inhibited the cell cycle by 76% at 25 μM, and Ligustrone B inhibited the cell cycle by 65% ​​at 12.5 μM.

[0056] Example 2

[0057] The inhibitory effect of polyketide compounds (3S,4S)-sclerotinin A and ligustrone B on EV71 progeny virus production was tested. The test results are as follows:

[0058] 1. Test content

[0059] The inhibitory effect of the compounds on the production of EV71 progeny viruses after EV71 infection of RD cells was detected.

[0060] 2. Test methods

[0061] RD cells in the logarithmic growth phase were plated in 24-well plates, and 100 TCID 50 Cells were infected with EV71 and incubated at 37°C for 1.5 hours. The virus solution was removed, the cells were washed three times with PBS, and cell maintenance medium containing 100 µM (3S,4S)-sclerotinin A and ligustrone B was added. After 48 hours, the cells and supernatant were collected and lysed by freeze-thawing at –20°C and 37°C three times. EV71 virus titers were determined by the TCID50 method.

[0062] 3. Test results

[0063] like Figure 3 As shown, the virus titers of RD cells treated with polyketide compounds (3S,4S)-sclerotinin A and ligustrone B were significantly reduced compared with the virus control group, with a decrease of 2.7 log and 2.9 log respectively.

[0064] In summary, the polyketide compounds (3S,4S)-sclerotinin A and ligustrone B have strong inhibitory activity against EV71, can inhibit RD cell death caused by EV71 virus, and can be prepared into clinically effective drugs against EV71 infection.

[0065] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of polyketide compounds in the preparation of EV71 virus inhibitors, characterized in that: The polyketide compound is (3S, 4S)-sclerotinin A or ligustrone B; The structural formula of the (3S,4S)-sclerotinin A is shown in formula (I): Formula (I); The structural formula of the ligustrone B is shown in formula (II): Formula (II).

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