Application of Compound SH in the Preparation of Drugs with Broad-spectrum Anti-coronavirus Activity

Abstract

The present invention provides the use of compound SH in the preparation of a drug with broad-spectrum anti-coronavirus activity, belonging to the field of biomedical technology. In the present invention, non-toxic concentration of compound SH is selected for the study of broad-spectrum anti-coronavirus activity. Compound SH can effectively inhibit the replication of beta-coronaviruses (SARS-CoV-2, HCoV-OC43) and alpha-coronaviruses (HCoV-NL63, HCoV-229E) in vitro. Moreover, compound SH has broad-spectrum anti-coronavirus activity, laying a foundation for the further development of broad-spectrum anti-coronavirus drugs, and having important development value and broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of compound SH in the preparation of broad-spectrum anti-coronavirus drugs. Background Art

[0002] Coronaviruses (CoV) are enveloped, single-stranded, positive-sense RNA viruses with the largest known genomes. They can cause infections in the respiratory, digestive, and nervous systems of animals and humans. The Ninth Report of the International Committee on Taxonomy of Viruses classifies them into four groups: α, β, γ, and δ. Alpha and β coronaviruses primarily infect mammals, while γ and δ coronaviruses primarily infect birds.

[0003] Coronaviruses can infect humans and a variety of animals, causing illnesses ranging from the common cold to severe acute respiratory syndrome. Before 2020, six coronaviruses were known to infect humans: human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), and OC43 (HCoV-OC43), which cause the common cold with upper respiratory tract symptoms, as well as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which can cause severe respiratory illness. The continued emergence of novel coronavirus mutants and the potential threat of unknown coronaviruses have created an urgent need for the development of drugs with broad-spectrum anti-coronavirus effects.

[0004] Current research on coronavirus drugs primarily targets highly pathogenic coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV). These drugs primarily include immunomodulators (interferon, ribavirin, etc.), viral entry inhibitors (monoclonal antibodies to the S protein, peptides targeting the key fusion region of the S protein, etc.), and viral replication inhibitors (such as viral polymerase-specific inhibitors and broad-spectrum protease inhibitors). However, most remain in the research phase. High-throughput screening of FDA-approved drug libraries or marketed small molecule compound libraries to explore new uses for existing drugs has become an important approach in drug development. "Drug repurposing" or "repurposing old drugs" offers numerous advantages, such as pre-existing data on pharmacological efficacy testing, functional targets, and clinical safety profiles for candidate drugs, which facilitates further toxicological and pharmacokinetic evaluations and formulation development. This can significantly reduce R&D risks, shorten development time, and reduce costs, making it an effective means of responding to sudden outbreaks of highly contagious diseases.

[0005] Through research on repurposing established drugs, several established drugs have been found to inhibit the replication of the novel coronavirus. Among them, remdesivir, favipiravir, chloroquine, and hydroxychloroquine have entered clinical trials. Remdesivir, in particular, became the first drug approved by the US FDA for the treatment of novel coronavirus infection. The main areas of research for innovative new drugs against novel coronavirus infection are the discovery of antibodies targeting the S protein of the novel coronavirus, which recognizes host cell receptors, and the development of small molecule inhibitors targeting key proteins involved in the novel coronavirus replication process, such as 3CL protease and RNA-dependent RNA polymerase. Furthermore, Traditional Chinese Medicine (TCM) has played a significant role in the prevention and treatment of novel coronavirus infection. Jinhua Qinggan Granules, Lianhua Qingwen Capsules, Xuebijing Injection, Shuanghuanglian Oral Liquid, Qingfei Paidu Decoction, Huashi Baidu Decoction, and Xuanfei Baidu Decoction have all entered clinical research and application for the treatment of novel coronavirus infection.

[0006] Compound SH, also known as "Si'aite San" in Chinese, is an oral antiviral compound traditional Chinese medicine. It is composed of five Chinese herbs: licorice, safflower, astragalus, mulberry bark, and artemisia capillaris. Based on the principles of traditional Chinese medicine (TCM), extracts of these five herbs are rationally combined with different solvents according to the principles of "monarch, minister, assistant, and envoy" (monarch, minister, and envoy). Compound SH is widely used in HIV / AIDS patients in China and Southeast Asian countries. Clinical trials have shown that SH is safe and effective, reducing HIV viral load by 14% to 35% in HIV-positive patients when used alone. Furthermore, combination therapy with nucleoside analog reverse transcriptase inhibitors (NRTIs) has shown stronger antiviral activity than NRTIs alone. There are also reports that combining SH with the protease inhibitor atazanavir can enhance its inhibitory activity. Currently, there are no reports of compound SH being used as an anti-coronavirus drug. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a new application of compound SH, specifically the application of compound SH in the preparation of broad-spectrum anti-coronavirus drugs.

[0008] The present invention provides the use of compound SH in preparing a broad-spectrum anti-coronavirus drug.

[0009] The present invention provides the use of compound SH in preparing a broad-spectrum coronavirus replication inhibitor.

[0010] Preferably, the coronavirus includes α-group coronavirus and / or β-group coronavirus.

[0011] Preferably, the α-group coronavirus includes HCoV-NL63 and / or HCoV-229E.

[0012] Preferably, the cell used to inhibit the replication of HCoV-NL63 is LLC-MK2;

[0013] The cells used to inhibit the replication of HCoV-229E were MRC-5 cells.

[0014] Preferably, when the viral infection multiplicity of HCoV-NL63 is 0.01, the EC of compound SH is 50 It is 1.87μg / ml.

[0015] Preferably, when the viral infection multiplicity of HCoV-229E is 0.01, the EC of compound SH is 50 It is 10.22μg / ml.

[0016] Preferably, the β-group coronavirus includes SARS-CoV-2 and / or HCoV-OC43.

[0017] Preferably, the cells inhibiting SARS-CoV-2 replication are Vero;

[0018] The cell used to inhibit HCoV-OC43 replication is BHK-21.

[0019] Preferably, when the multiplicity of infection of the SARS-CoV-2 virus is 0.01, the EC of the compound SH is 50 7.3 μg / ml;

[0020] When the viral infection multiplicity of HCoV-OC43 was 0.01, the EC 50 It is 4.4μg / ml.

[0021] The present invention provides the use of compound SH in the preparation of a broad-spectrum anti-coronavirus drug. The present invention conducted an in vitro anti-coronavirus activity test, and the results showed that compound SH can effectively inhibit the replication of SARS-CoV-2 in Vero cells, and the half effective concentration (EC) of the drug that inhibits the replication efficiency of SARS-CoV-2 by 50% at an MOI of 0.01 is 1. 50 The compound SH can effectively inhibit the replication of HCoV-OC43 in BHK-21 cells, and the half effective concentration EC of the drug that inhibits the replication efficiency of HCoV-OC43 by 50% at MOI = 0.01 is 7.3 μg / ml. 50 The compound SH can effectively inhibit the replication of HCoV-NL63 in LLC-MK2 cells, and the half effective concentration EC of the drug that inhibits the replication efficiency of HCoV-NL63 by 50% at 0.01 MOI is 4.4 μg / ml. 50 The compound SH can effectively inhibit the replication of HCoV-229E in MRC5 cells, and the half effective concentration EC of the drug that inhibits the replication of HCoV-229E by 50% at MOI = 0.01 is 1.87 μg / ml. 50 The results of the cytotoxicity test of the compound SH of the present invention showed that the CC of the compound SH in Vero, BHK-21, LLC-MK2 and MRC-5 cells was 10.22 μg / ml. 50 The selectivity index (SI) of the SH compound against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E in Vero, BHK-21, LLC-MK2, and MRC-5 cells was 10.9, 30.33, 12.83, and 5.81, respectively. This indicates that the SH compound exhibits excellent antiviral efficacy against a variety of coronaviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The inhibitory effect of different concentrations of compound SH on SARS-CoV-2 virus replication; the horizontal axis is the drug concentration of compound SH, and the vertical axis is the inhibition rate. The curve fitted by the dark points in the box represents the inhibition efficiency of SARS-CoV-2 virus replication;

[0023] Figure 2 The inhibitory effect of different concentrations of compound SH on HCoV-OC43 virus replication; the horizontal axis is the drug concentration of compound SH, and the vertical axis is the inhibition rate. The curve fitted by the dark points in the box represents the inhibition efficiency of HCoV-OC43 virus replication;

[0024] Figure 3 The inhibitory effect of different concentrations of compound SH on HCoV-NL63 virus replication; the horizontal axis is the drug concentration of compound SH, and the vertical axis is the inhibition rate. The curve fitted by the dark points in the box represents the inhibition efficiency of HCoV-NL63 virus replication;

[0025] Figure 4 The inhibitory effect of different concentrations of compound SH on HCoV-229E virus replication; the horizontal axis is the drug concentration of compound SH, and the vertical axis is the inhibition rate. The curve fitted by the dark points in the box represents the inhibition efficiency of HCoV-229E virus replication. DETAILED DESCRIPTION

[0026] The present invention provides the use of compound SH in preparing a broad-spectrum anti-coronavirus drug.

[0027] The present invention provides the use of compound SH in preparing a broad-spectrum coronavirus replication inhibitor.

[0028] The present invention has no particular limitation on the compound SH, and conventional sources of compound SH known in the art can be used. In the examples of the present invention, the compound SH was purchased from Thailand Zetai Co., Ltd.

[0029] In the present invention, the coronavirus preferably includes α-group coronavirus and / or β-group coronavirus. The α-group coronavirus preferably includes HCoV-NL63 and / or HCoV-229E. The cell used to inhibit the replication of HCoV-NL63 is preferably LLC-MK2. When the multiplicity of infection of the HCoV-NL63 virus is 0.01, the EC of the compound SH is 50 The preferred concentration is 1.87 μg / ml. In the cytotoxicity experiment, the CC of HCoV-NL63 50 The selectivity index SI of HCoV-NL63 is 30.33. The cell that inhibits the replication of HCoV-229E is preferably MRC-5. When the multiplicity of infection of HCoV-229E is 0.01, the EC of compound SH is 50 The preferred concentration is 10.22 μg / ml. In the cytotoxicity experiment, CC of HCoV-229E 50 The selectivity index SI of HCoV-229E was 5.81.

[0030] In the present invention, the beta group coronavirus preferably includes SARS-CoV-2 and / or HCoV-OC43. The cells used to inhibit SARS-CoV-2 replication are preferably Vero; when the multiplicity of infection of the SARS-CoV-2 virus is 0.01, the EC of the compound SH is 50The preferred concentration is 7.3 μg / ml. In the cytotoxicity experiment, the CC of SARS-CoV-2 50 The selectivity index SI of SARS-CoV-2 is 10.91. The cell for inhibiting HCoV-OC43 replication is preferably BHK-21. When the multiplicity of infection of HCoV-OC43 is 0.01, the EC of compound SH is 50 The preferred concentration is 4.4 μg / ml. In the cytotoxicity experiment, the CC of HCoV-OC43 50 The selectivity index SI of HCoV-OC43 was 12.83.

[0031] The present invention has no particular limitation on the dosage form of the drug or inhibitor, and any dosage form of the drug or inhibitor known in the art may be used.

[0032] The application of the compound SH provided by the present invention in the preparation of a broad-spectrum anti-coronavirus drug is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] Cytotoxicity assay of compound SH

[0035] This example involves four coronaviruses, namely SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, and their sensitive cell lines are Vero, BHK-21, LLC-MK2, and MRC-5, respectively. To test the safe dosage concentration of compound SH, this example uses the CCK-8 method to detect the toxicity of compound SH in these four sensitive cell lines. The detection principle is as follows: Cell Counting Kit-8 (abbreviated as CCK-8) is a WST-8-based detection reagent widely used for cell proliferation and cytotoxicity. WST-8 chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazolium monosodium salt, a compound similar to MTT. In the presence of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate, it is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of viable cells, so this characteristic can be used to directly analyze cell proliferation and toxicity. Use a microplate reader to measure the light absorption value at the corresponding wavelength. Within a certain cell count range, the depth of the color is proportional to the number of viable cells. The number of viable cells is determined based on the measured absorbance value (OD value). The larger the OD value, the stronger the cell activity, which indicates the lower the drug toxicity when measuring drug toxicity.

[0036] The specific operation is as follows: Vero, BHK-21, LLC-MK2 and MRC-5 cells were cultured at 3×10 4 Cells were seeded into 96-well plates and cultured in DMEM supplemented with 10% fetal bovine serum for 16 hours until the cell density reached 80%. The culture medium was then aspirated and replaced with DMEM supplemented with 2% fetal bovine serum. 100 mg of Siatesan (Compound SH, purchased from Zetai Co., Ltd., Thailand) was prepared, added to 10 ml of DMSO, vortexed, and dissolved to a 10 mg / ml stock solution. The solution was then refrigerated at 4°C until ready for use. Upon use, the cells were added to working solutions diluted in DMEM supplemented with 2% fetal bovine serum to concentrations of 100, 20, 4, 0.8, 0.16, and 0.032 μg / ml. Four replicates were performed for each drug concentration, along with a blank control group (no drug or cells) and a cell control group (no drug). Place in a 37°C, 5% CO2 incubator and continue culturing for 72 hours. Discard the supernatant and add CCK-8 working solution to each well. Incubate at 37°C for 1-4 hours. Finally, measure the absorbance at 450 nm using a multifunctional microplate reader. Calculate the cell activity inhibition rate according to formula I:

[0037] Cell activity inhibition rate (%) = (drug group - blank control group) / (cell control group - blank control group) × 100% Formula I

[0038] The drug concentration of compound SH was used as the horizontal axis and the cell proliferation inhibition rate was used as the vertical axis. The mean value and standard deviation were calculated by GraphpadPrism 8 software to fit the curve. The cytotoxic CC of compound SH was calculated after the drug concentration was converted into a logarithmic value. 50 .

[0039] The results are shown in Table 1. The CC of compound SH in Vero, BHK-21, LLC-MK2 and MRC-5 cells 50 They were >80μg / ml, 54.06μg / ml, 56.71μg / ml and 59.41μg / ml respectively.

[0040] Table 1 Cytotoxicity of compound SH in different cells

[0041]

[0042] Example 2

[0043] In vitro anti-coronavirus activity test of compound SH

[0044] 1. Viral infection and drug effects

[0045] Vero, BHK-21, LLC-MK2 and MRC-5 cells were cultured at 1×104 Cells / well were seeded in a 96-well culture plate and cultured in DMEM containing 10% fetal bovine serum for 16 hours until the cell density reached 80%. The cell culture medium was then aspirated and replaced with DMEM containing 2% fetal bovine serum. To evaluate the anti-SARS-CoV-2 effect of compound SH, compound SH was added to the corresponding Vero cell wells at final concentrations of 80, 20, 5, 1.25, 0.31, and 0.08 μg / ml. The anti-HCoV-OC43, HCoV-NL63, and HCoV-2 effects of Siatesan were evaluated. When the 29E virus was effective, compound SH was added to the corresponding cell wells at final concentrations of 100, 20, 4, 0.8, 0.16, and 0.032 μg / ml, respectively. At the same time, corresponding cell control wells without drug and virus control wells with virus alone were set up; within 1 hour after the addition of the drug, SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E were added to the corresponding sensitive cell wells at a volume of 10 μl per well to make the virus infection multiplicity of infection (MOI=0.01). The cells were placed in a 37°C, 5% CO2 incubator and cultured for 48 hours, and the supernatant was collected.

[0046] 2. Fluorescence quantitative RT-PCR detection of the inhibitory effect of compound SH on SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E

[0047] The absolute fluorescence quantitative RT-PCR method was used to detect the corresponding viral target genes of SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E to reflect the viral replication level.

[0048] Viral RNA was extracted according to the instructions of the Xi'an Tianlong Viral RNA Nucleic Acid Extraction Kit, and RT-PCR detection was performed using the One Step PrimeScript RT-PCR Kit (RR064A). The primer and probe sequences for detecting each virus are as follows:

[0049] The primer and probe sequences for detecting SARS-CoV-2 are as follows:

[0050] The upstream primer sequence (q-SARS-CoV-2-F) is:

[0051] 5'-CCCTGTGGGTTTTACACTTAA-3' (SEQ ID NO: 1);

[0052] The downstream primer sequence (q-SARS-CoV-2-R) is:

[0053] 5'-ACGATTGTGCATCAGCTGA-3'(SEQ ID NO:2),

[0054] The probe sequence (q SARS-CoV-2-probe) is:

[0055] 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3' (SEQ ID NO: 3).

[0056] The primer and probe sequences for detecting HCoV-OC43 are as follows:

[0057] The upstream primer sequence (q-OC43-F) is:

[0058] 5'-GCTCAGGAAGGTCTGCTCC-3' (SEQ ID NO: 4);

[0059] The downstream primer sequence (q-OC43-R) is:

[0060] 5'-TCCTGCACTAGAGGCTCTGC-3' (SEQ ID NO:5),

[0061] The probe sequence (q-OC43-probe) is:

[0062] 5'-FAM-TTCCAGATCTACTTCGCGCACATCC-TAMRA-3' (SEQ ID NO: 6);

[0063] The primer and probe sequences for detecting HCoV-NL63 are as follows:

[0064] The upstream primer sequence (q-NL63-F) is:

[0065] 5'-AGGACCTTAAATTCAGACAACGTTCT-3' (SEQ ID NO:7),

[0066] The downstream primer sequence (q-NL63-R) is:

[0067] 5'-GATTACGTTTGCGATTACCAAGACT-3' (SEQ ID NO:8),

[0068] The probe sequence (q-NL63-probe) is:

[0069] 5'-FAM-TAACAGTTTTAGCACCTTCCTTAGCAACCCAAACA-TAMR A-3' (SEQ ID NO:9).

[0070] The primer and probe sequences for detecting HCoV-229E are as follows:

[0071] The upstream primer sequence (q-229E-F) is:

[0072] 5'-CGCAAGAATTCAGAACCAGAG-3'(SEQ ID NO:10),

[0073] The downstream primer sequence (q-229E-R) is:

[0074] 5'-GGCAGTCAGGTCTTCAACAA-3' (SEQ ID NO: 11),

[0075] The probe sequence (q-229E-probe) is:

[0076] 5'-HEX-CCACACTTCAATCAAAAGCTCCCAAATG-TAMRA-3' (SEQ ID NO: 12).

[0077] The reaction system was: 10 μL 2×One Step SYBR RT-PCR Buffer III, 0.5 μL Takara Ex TaqHS, 0.5 μL PrimeScript RT Enzyme Mix II, 0.5 μL upstream primer, 0.5 μL downstream primer, 5 μL RNA template, and made up to 25 μL with sterile double-distilled water.

[0078] The reaction parameters were: 42°C for 5 minutes, 95°C for 10 seconds per cycle; 95°C for 5 seconds, 60°C for 30 seconds, for 40 cycles. Fluorescence signals were collected after extension. Four replicates were performed for each sample. The CT values ​​of the samples were calculated and the viral copy number in the sample was calculated by substituting the measured CT values ​​into the standard curve. The viral replication inhibition rate was calculated according to Formula II:

[0079] Virus replication inhibition rate (%) = (virus control group - drug control group) / virus control group × 100% Formula II.

[0080] 3. Curve Fitting and EC Calculation 50

[0081] The concentration of compound SH was used as the horizontal axis and the viral replication inhibition rate was used as the vertical axis. The mean and standard deviation of the inhibition efficiency were calculated using Graphpad Prism 8 software to fit the curve. The EC value of compound SH was calculated after converting the drug concentration into logarithm. 50 The inhibitory effects of compound SH on SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E are shown in Figure 1 、 Figure 2 、 Figure 3 and Figure 4 .like Figure 1 As shown in the figure, compound SH can effectively inhibit the replication of SARS-CoV-2 in Vero cells, and the half effective concentration of the drug EC that inhibits the replication efficiency of SARS-CoV-2 by 50% at MOI = 0.01 is 50 is 7.3 μg / ml; Figure 2 As shown in the figure, compound SH can effectively inhibit the replication of HCoV-OC43 in BHK-21 cells, and the half effective concentration of the drug EC that inhibits the replication efficiency of HCoV-OC43 by 50% at MOI = 0.01 is 50 is 4.4 μg / ml; Figure 3 As shown in Figure 2, compound SH can effectively inhibit the replication of HCoV-NL63 in LLC-MK2 cells, and the half effective concentration of the drug EC that inhibits the replication efficiency of HCoV-NL63 by 50% at 0.01 MOI is 50 is 1.87 μg / ml; Figure 4 As shown in the figure, compound SH can effectively inhibit the replication of HCoV-229E in MRC5 cells, and the half effective concentration of the drug EC that inhibits 50% of HCoV-229E replication at MOI = 0.01 is 50 It is 10.22μg / ml.

[0082] 4. Calculation of the selection index SI

[0083] CC by drug toxicity 50 and the effective concentration EC 50 The ratio of can be calculated to obtain the selectivity index SI (Selective Index, SI), which is an important indicator for judging the effect of drugs. Among them, the selectivity index> 1 indicates that the drug is effective and safe. The larger the SI value, the safer the drug and the more significant the effect. According to the cytotoxicity test results CC obtained in Example 1 50 The half effective concentration EC of the drug for viral inhibition obtained in Example 2 is 50 , and further calculate the selection index SI according to formula III.

[0084] Selection index SI = CC 50 / EC 50 Formula III

[0085] The selectivity index (SI) of compound SH against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E in Vero, BHK-21, LLC-MK2, and MRC-5 cells was 10.9, 30.33, 12.83, and 5.81, respectively. This result indicates that compound SH has a good antiviral effect against the four human coronaviruses: SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E.

[0086] Table 2 Study on the broad-spectrum anti-coronavirus effect of compound SH

[0087]

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Siatensan in the preparation of a drug with broad-spectrum anti-coronavirus activity, wherein the coronavirus is an alpha-group coronavirus and / or a beta-group coronavirus; The alpha-group coronavirus is HCoV-NL63; The beta-group coronavirus is SARS-CoV-2 and / or HCoV-OC43.

2. Application of Siatensan in the preparation of an inhibitor of broad-spectrum coronavirus replication, wherein the coronavirus is an alpha-group coronavirus and / or a beta-group coronavirus; The alpha-group coronavirus is HCoV-NL63; The beta-group coronavirus is SARS-CoV-2 and / or HCoV-OC43.

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