Application of pyrimethamine in preparation of antiviral preparation

Abstract

The invention discloses an application of pyrimethamine in preparation of an antiviral preparation. The invention provides application of pyrimethamine or pharmaceutically acceptable salt or ester or solvate thereof or a substance taking pyrimethamine as a main component in preparation of any one of the following products: an antiviral product; a product for treating and / or preventing diseases caused by viral infection; the invention relates to a product for improving symptoms caused by virus infection. Experiments prove that pyrimethamine has broad-spectrum anti-influenza A virus potential. Meanwhile, pyrimethamine has a remarkable inhibiting effect on Zika virus, dengue virus, novel coronavirus, Ebola replicable pseudovirus, herpes simplex virus, enterovirus and vaccinia virus, and it is preliminarily shown that pyrimethamine has broad-spectrum antiviral potential.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to the application of pyrimethamine in the preparation of antiviral preparations. Background Art

[0002] Over the past century, infectious diseases have emerged frequently. Infectious diseases such as the novel coronavirus, influenza, Ebola, and Zika have posed a serious threat to human health and have also had a significant impact on the global economy. These viruses often exhibit rapid onset, rapid spread, and mutability, posing a significant threat to global public health. Hundreds of viruses are known to cause human illness. Unfortunately, currently approved antiviral drugs can only treat infections with a few viruses, such as influenza. Effective treatments for the vast majority of viral diseases remain unavailable.

[0003] Viruses are highly genetically variable, which allows them to develop resistance to existing antiviral drugs. Drug-resistant viruses have been reported for influenza virus ion channel protein inhibitors such as amantadine and neuraminidase inhibitors such as oseltamivir. Broad-spectrum antiviral drugs, however, possess high antiviral activity against a wide range of viral strains and variants, thereby reducing viral transmission and prevalence. This is crucial for controlling the spread of epidemics and reducing the risk of viral infection, and has significant clinical demand and translational application value.

[0004] Pyrimethamine is an FDA-approved drug, and its structural formula is shown in Formula I (molecular formula is C 12 H 13 ClN4, molecular weight 248.71, CAS number 58-14-0), is mainly used for the prevention of malaria and can also be used to treat toxoplasmosis.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide the use of pyrimethamine in the preparation of antiviral preparations.

[0007] In a first aspect, the present invention claims the use of any of the following substances (1)-(3) in any of the following (A1)-(A3):

[0008] (1) Pyrimethamine;

[0009] (2) pharmaceutically acceptable salts, esters or solvates of pyrimethamine;

[0010] (3) Substances containing pyrimethamine or its pharmaceutically acceptable salts, esters or solvates as the main ingredient;

[0011] (A1) preparing antiviral products;

[0012] (A2) preparing products capable of treating and / or preventing diseases caused by viral infections;

[0013] (A3) Preparation of products capable of improving symptoms caused by viral infection.

[0014] In a second aspect, the present invention claims the use of any of the following substances (1)-(3) in the preparation of a product for inhibiting viruses at the cellular level:

[0015] (1) Pyrimethamine;

[0016] (2) pharmaceutically acceptable salts, esters or solvates of pyrimethamine;

[0017] (3) A substance containing pyrimethamine or a pharmaceutically acceptable salt, ester or solvate thereof as a main component.

[0018] In the above two aspects, the virus includes but is not limited to any one or more of the following: influenza virus, Zika virus, dengue virus, SARS-CoV-2, Ebola virus, herpes simplex virus type 1, enterovirus, and vaccinia virus.

[0019] Furthermore, the influenza virus may be influenza A virus.

[0020] Furthermore, the Zika virus may be ZIKA-SMGC-1.

[0021] Furthermore, the dengue virus may be dengue virus type 2.

[0022] Furthermore, the SARS-CoV-2 may be hCoV-19 / China / CAS-B001 / 2020.

[0023] Furthermore, the Ebola virus may be Zaire Ebola virus.

[0024] Furthermore, the herpes simplex virus may be herpes simplex virus type 1.

[0025] Furthermore, the enterovirus may be EV71.

[0026] Furthermore, the vaccinia virus may be VACV Western Reserve.

[0027] Furthermore, the influenza A virus may be an influenza virus of the H1N1, H3N2, H5N1, H5N6, H6N1, H7N9, H9N2 and / or H10N8 subtype.

[0028] In a specific embodiment of the present invention, the influenza A virus can be selected from any one or more of the following: A / California / 04 / 2009 (H1N1), A / Beijing / CAS0001 / 2007 (H3N2), A / duck / Guangdong / 04.22DGCP069-O / 2015 (H5N6, clade-2.3.4.4), A / pigeon / Sichuan / ncxn29 / 2014(H5N1,clade-2.3.4.4)、A / Taiwan / 2 / 2013(H6N1)、A / chicken / Guangdong / 04.22DGCP098-O / 2015 (H7N9), A / chicken / Guangdong / 04.15SZBAXQ005 / 2015(H9N2), A / chicken / Jiangxi / B18 / 2014(H10N8).

[0029] In a specific embodiment of the present invention, the Zika virus may be ZIKA-SMGC-1.

[0030] In a specific embodiment of the present invention, the dengue virus may be dengue virus type 2 strain 43.

[0031] In a specific embodiment of the present invention, the SARS-CoV-2 may be hCoV-19 / China / CAS-B001 / 2020.

[0032] In a specific embodiment of the present invention, the Ebola virus may be the Zaire Ebola virus Mayinga strain.

[0033] In a specific embodiment of the present invention, the herpes simplex virus may be HSV-1F strain.

[0034] In a specific embodiment of the present invention, the enterovirus is the EV71 BrCr strain.

[0035] In a specific embodiment of the present invention, the vaccinia virus may be VACV Western Reserve.

[0036] In both aspects above, the product may be a medicine.

[0037] Furthermore, the only active ingredient or one of the active ingredients of the drug is pyrimethamine (as shown in Formula I, the molecular formula is C 12 H 13 C l N4, molecular weight 248.71) or a pharmaceutically acceptable salt, ester or solvate thereof.

[0038] As required, the medicine may contain suitable carriers and / or excipients in addition to the active ingredients.

[0039] Experiments have shown that pyrimethamine has an inhibitory effect on influenza A virus. The EC value of the compound represented by Formula I against the H1N1 subtype influenza virus strain A / California / 04 / 2009 (H1N1) (abbreviated as CA04) (GISAID number: EPI175470) was determined. 50 The EC value for A / Beijing / CAS0001 / 2007(H3N2)(GISAID number:EPI1544268) is 1.670; 50 The EC value for A / duck / Guangdong / 04.22DGCP069-O / 2015 (H5N6, clade-2.3.4.4) (GD69) (GISAID number: EPI660071) is 1.583 μM. 50 The EC value for A / pigeon / Sichuan / NCXN29 / 2014 (H5N1, clade-2.3.4.4) (abbreviated as XN29) (GISAID number: EPI590898) is 1.926 μM. 50 The EC value for A / Taiwan / 2 / 2013(H6N1) (GISAID number: EPI459855) is 3.386μM. 50 The EC value for A / chicken / Guangdong / 04.22DGCP098-O / 2015(H7N9) (GISAID number: EPI666285) is 1.821μM. 50 The EC value for A / chicken / Guangdong / 04.15SZBAXQ005 / 2015(H9N2) (GISAID number: EPI661935) is 3.285μM. 50 The EC value for A / chicken / Jiangxi / B18 / 2014(H10N8) (GISAID number:EPI1544302) is 2.165μM. 50 It is 2.004μM.

[0040] Experiments have shown that pyrimethamine has an EC 50 It is 6.388μM.

[0041] Experiments have shown that pyrimethamine has an EC value of 100 for SARS-CoV-2. 50 It is 2.043μM.

[0042] Experiments have shown that pyrimethamine has an effect on the EC of Ebola replicable pseudovirus. 50 It is 7.253μM.

[0043] Experiments have shown that pyrimethamine has an EC 50 It is 1.928μM.

[0044] Experiments have shown that pyrimethamine has an EC 50 It is 1.641μM.

[0045] Experiments have shown that pyrimethamine has an effect on the EC of enterovirus EV71. 50 It is 16.33μM.

[0046] Experiments have shown that the EC of pyrimethamine against vaccinia virus (VACV) 50 It is 20.9μM.

[0047] Experiments have shown that pyrimethamine has low toxicity to cells and has a negative effect on CC of MDCK cells. 50 The concentration of the compound is >100μM, and it can be used as a safe and effective anti-influenza virus drug.

[0048] Experiments have shown that pyrimethamine has a low toxicity to Vero cells and has a negative effect on the CC of Vero cells (48h). 50 >100μM, CC of Vero cells (72h) 50 The concentration of vaccinia virus is 66.82 μM, which can be used as a safe and effective drug against Zika virus, herpes simplex virus and vaccinia virus.

[0049] Experiments have shown that pyrimethamine has little toxicity to BHK-21 cells and has a negative effect on CC 50 The concentration of the compound is 100 μM, which can be used as a safe and effective anti-dengue virus drug.

[0050] Experiments have shown that pyrimethamine has little toxicity to Vero-E6 cells and has a negative effect on CC 50 The concentration of 54.99 μM can be used as a safe and effective anti-SARS-CoV-2 novel coronavirus drug.

[0051] Experiments have shown that pyrimethamine has little toxicity to Huh7.5.1 cells and has no significant effect on CC 50 The concentration of the compound was >100 μM, indicating that it can be used as a safe and effective anti-Ebola virus drug.

[0052] Experiments have shown that pyrimethamine has less toxicity to RD cells and has a negative effect on CC 50 The concentration of β-actin is >100μM, which can be used as a safe and effective anti-enteroviral drug.

[0053] In summary, pyrimethamine has a significant inhibitory effect on influenza A viruses of the H1N1, H3N2, H5N1, H5N6, H6N1, H7N9, H9N2, and H10N8 subtypes, preliminarily showing that pyrimethamine has a broad-spectrum anti-influenza A virus potential. At the same time, pyrimethamine has a significant inhibitory effect on Zika virus, dengue virus, SARS-CoV-2, Ebola replication-competent pseudovirus, herpes simplex virus, enterovirus, and vaccinia virus, preliminarily showing that pyrimethamine has a broad-spectrum antiviral potential. And the present invention verifies its safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The inhibitory activity of pyrimethamine against influenza A virus.

[0055] Figure 2 The inhibitory activity of pyrimethamine against Zika virus (ZIKV).

[0056] Figure 3 The inhibitory activity of pyrimethamine against dengue virus (DENV-2).

[0057] Figure 4 The inhibitory activity of pyrimethamine against the SARS-CoV-2 novel coronavirus.

[0058] Figure 5 The inhibitory activity of pyrimethamine against Ebola replication-competent pseudovirus.

[0059] Figure 6 The inhibitory activity of pyrimethamine against herpes simplex virus (HSV-1).

[0060] Figure 7 The inhibitory activity of pyrimethamine against enterovirus EV71.

[0061] Figure 8 The inhibitory activity of pyrimethamine against vaccinia virus (VACV).

[0062] Figure 9 The inhibitory activity of pyrimethamine on the cell viability of MDCK cells.

[0063] Figure 10 The inhibitory activity of pyrimethamine on Vero cells.

[0064] Figure 11 is the cell viability inhibitory activity of pyrimethamine on Vero-E6 cells.

[0065] Figure 12 The inhibitory activity of pyrimethamine on the cell viability of BHK-21 cells.

[0066] Figure 13 The inhibitory activity of pyrimethamine on the cell viability of Huh7.5.1 cells.

[0067] Figure 14 is the cell viability inhibitory activity of pyrimethamine on RD cells.

[0068] Figure 15 This is the weight change curve of mice in the experiment of protecting mice from influenza virus challenge with pyrimethamine (oral administration).

[0069] Figure 16 This is the mouse death and survival curve (oral gavage) in the experiment of protecting mice from influenza virus challenge with pyrimethamine.

[0070] Figure 17 This is the curve of mouse body weight changes in the experiment of protecting mice against influenza virus challenge with pyrimethamine (nasal drops).

[0071] Figure 18 This is the mouse death and survival curve in the experiment of protecting mice from influenza virus challenge with pyrimethamine (nasal drops). DETAILED DESCRIPTION

[0072] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0073] The experimental methods in the following examples, unless otherwise specified, are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, are all commercially available.

[0074] The H1N1 subtype influenza virus strains involved in the following examples are:

[0075] A / California / 04 / 2009(H1N1) (abbreviated as CA04): recorded in the article “Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261”. The public can obtain this information from the applicant for use only in repeating the experiments of the present invention and for no other use.

[0076] The H3N2 subtype influenza virus strains involved in the following examples are:

[0077] A / Beijing / CAS0001 / 2007(H3N2): described in “Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261”. The public can obtain the data from the applicant for use only in repeating the experiments of the present invention and shall not use it for any other purpose.

[0078] The H5N6 subtype influenza virus strains involved in the following examples are:

[0079] A / duck / Guangdong / 04.22DGCP069-O / 2015 (H5N6, clade-2.3.4.4) (abbreviated as GD69): recorded in the article "Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261". The public can obtain it from the applicant for use only in repeating the experiments of the present invention and shall not use it for any other purpose.

[0080] The H5N1 subtype influenza virus strains involved in the following examples are:

[0081] A / pigeon / Sichuan / NCXN29 / 2014 (H5N1, clade-2.3.4.4) (abbreviated as XN29): recorded in the article "Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261". The public can obtain the data from the applicant for use only in repeating the experiments of the present invention and shall not use it for any other purpose.

[0082] The H6N1 subtype influenza virus strains involved in the following examples are:

[0083] A / Taiwan / 2 / 2013(H6N1): described in the article "Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261". The public can obtain this information from the applicant for use only in repeating the experiments of the present invention and for no other use.

[0084] The H7N9 subtype influenza virus strains involved in the following examples are:

[0085] A / chicken / Guangdong / 04.22DGCP098-O / 2015(H7N9): described in “Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261”. The public can obtain this information from the applicant for use only in repeating the experiments described in this invention and for no other use.

[0086] The H9N2 subtype influenza virus strains involved in the following examples are:

[0087] A / chicken / Guangdong / 04.15SZBAXQ005 / 2015(H9N2): described in “Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261”. The public can obtain this information from the applicant for use only in repeating the experiments of the present invention and for no other use.

[0088] The H10N8 subtype influenza virus strains involved in the following examples are:

[0089] A / chicken / Jiangxi / B18 / 2014(H10N8): described in “Quan C, Wang Q, Zhang J, Zhao M, Dai Q, Huang T, et al. Avian Influenza A Viruses among Occupationally Exposed Populations, China, 2014–2016. Emerg Infect Dis. 2019; 25(12): 2215-2225. https: / / dx.doi.org / 10.3201 / eid2512.190261”. The public can obtain the data from the applicant for use only in repeating the experiments of the present invention and shall not use it for any other purpose.

[0090] The Zika virus strains involved in the following examples are:

[0091] ZIKA-SMGC-1, GENBANK: KX266255, recorded in “Ma W, Li S, Ma S, et al. Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. [J]. Cell, 2017, 167(6): 1511. DOI: 10.1016 / j.cell.2016.11.016.”, is available to the public from the applicant and may only be used to repeat the experiments of the present invention and may not be used for any other purpose.

[0092] The SARS-CoV-2 strains involved in the following examples are:

[0093] SARS-CoV-2, hCoV-19 / China / CAS-B001 / 2020, National Microbiology DataCenter NMDCN0000102-3, GISAID databases EPI_ISL_514256-7: recorded in the article "An engineered bispecific human monoclonal antibody against SARS-CoV-2[J]. Nature Immunology, 2022, 23(3): 423-430. DOI: 10.1038 / s41590-022-01138-w.", which is available to the public from the applicant and may only be used to repeat the experiments of the present invention and may not be used for other purposes.

[0094] The Ebola pseudovirus strains involved in the following examples are:

[0095] The replication-competent pseudovirus of the Mayinga strain of Zaire EBOV (with GFP green fluorescence) is recorded in the article "Gong M, Yang Y, Huang Y, et al. et al. Novel quinolone derivatives targeting humandihydroorotate dehydrogenase suppress Ebola virus infection invitro. Antiviral Research. 2021; 194: 105161." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention and may not be used for other purposes.

[0096] The dengue virus (DENV-2) strains involved in the following examples are:

[0097] DENV-2 strain 43 is described in the article “Liu J, Liu Y, Nie K, et al. Flavivirus NS1 protein infected host sera enhances viral acquisition by mosquitoes. NatMicrobiol. 2016Jun 20;1(9):16087.doi:10.1038 / nmicrobiol.2016.87.PMID:27562253;PMCID:PMC5003325.” and is available to the public from the applicant for use only in repeating the experiments of the present invention and for no other use.

[0098] The herpes simplex virus (HSV-1) strains involved in the following examples are:

[0099] The HSV-1F strain is recorded in the article "Maertzdorf J, Remeijer L, Van Der Lelij A, Buitenwerf J, Niesters HG, Osterhaus AD, Verjans GM. Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates. J Clin Microbiol. 1999 Nov; 37(11): 3518-23. doi: 10.1128 / JCM.37.11.3518-3523.1999. PMID: 10523545; PMCID: PMC85683." and is available to the public from the applicant for use only in repeating the experiments of the present invention and not for any other use.

[0100] The vaccinia virus (VACV Western Reserve) strains involved in the following examples are:

[0101] The VACV Western Reserve strain is described in the article "Zeng J, Li Y, Jiang L, Luo L, Wang Y, Wang H, Han X, Zhao J, Gu G, Fang M, Huang Q, Yan J. Mpox multi-antigen mRNA vaccine candidates by a simplified manufacturing strategy afford efficient protection against lethal orthopoxvirus challenge. Emerg Microbes Infect. 2023 Dec; 12(1): 2204151. doi: 10.1080 / 22221751.2023.2204151. PMID: 37070521; PMCID: PMC10167873." and is available to the public from the applicant for use only in repeating the experiments of the present invention and not for any other use.

[0102] Enterovirus EV71 (EV71 BrCr strain) strain involved in the following examples:

[0103] The EV71 BrCr strain is described in the article "Qin Y, Lin L, Chen Y, Wu S, Si X, Wu H, Zhai X, Wang Y, Tong L, Pan B, Zhong X, Wang T, Zhao W, Zhong Z. Curcumin inhibits the replication of enterovirus 71 in vitro. Acta Pharm Sin B. 2014 Aug; 4(4): 284-94. doi: 10.1016 / j.apsb.2014.06.006. Epub 2014 Jul 24. PMID: 26579397; PMCID: PMC4629085." The public can obtain it from the applicant for use only in repeating the experiments of the present invention and may not use it for any other purpose.

[0104] Example 1: Evaluation of the inhibitory efficacy of pyrimethamine against various viruses

[0105] 1. Evaluation of the inhibitory effect of pyrimethamine on influenza A virus using a multi-concentration gradient experiment at the cellular level

[0106] 1. Add double antibiotics (penicillin and streptomycin) and trypsin (T9935, Trypsin from bovine pancreas, SIGMA / final concentration of 2μg / ml) to DMEM culture medium to prepare maintenance solution, and use this maintenance solution to dilute the influenza virus to make the influenza virus titer 100TCID 50 / 50μl maintenance solution.

[0107] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution 5-fold in a stepwise gradient to prepare test solutions of varying concentrations (100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, and 0.0064 μM). Each concentration was diluted in a 50 μl volume. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0108] 3. MDCK cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When the cells grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0109] 4. Take the 96-well plate prepared in step 3, add the system prepared in step 1 to the wells, place it in a 37°C, 5% CO2 incubator for incubation for 1 hour, then discard it. Add the system prepared in step 2 to the wells, and place it in a 37°C, 5% CO2 incubator for incubation for 24 hours.

[0110] 5. Take the 96-well plate from step 4, aspirate the culture supernatant, add 100 μl / well of fixative (3 parts by volume ethanol + 2 parts by volume acetone, stored in a -20°C refrigerator), fix the cells at room temperature for 15 minutes, aspirate the fixative, wash three times with PBST, add 100 μl of PBST containing 5% (mass percentage) skim milk powder to each well, incubate at 37°C for 1 hour. Discard the supernatant, add 100 μl of anti-influenza A virus NP protein antibody (100083-A, Influenza A 7307, R&D) diluted 1:5000, and incubate at 37°C for 1 hour. Wash the wells five times with PBST (5 min each), then add 100 μl of horseradish enzyme-conjugated goat anti-mouse IgG secondary antibody (BE0102-100, Goat Anti-Mouse IgG (H&L)-HRP Conjugated Goat Anti-Mouse Secondary Antibody, EASYBIO) diluted 1:5000 to each well and incubate at 37°C for 1 hour. Wash the wells five times with PBST (5 min each), then add 100 μl of TMB color development solution. After 10 min of color development, add 100 μl of 2M HCl. Take the test samples and read the OD value on a Multiskan FC microplate reader. 450 The data were processed using EXCEL and the EC of the inhibitor was calculated using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0111] The results are as follows Figure 1 As shown, the EC value of pyrimethamine against A / California / 04 / 2009(H1N1) (abbreviated as CA04) 50 The EC value for A / Beijing / CAS0001 / 2007(H3N2) was 1.670μM. 50The EC50 for A / duck / Guangdong / 04.22DGCP069-O / 2015 (H5N6, clade-2.3.4.4) (abbreviated as GD69) was 1.926 μM; the EC50 for A / pigeon / Sichuan / NCXN29 / 2014 (H5N1, clade 2.3.4.4) (abbreviated as XN29) was 3.386 μM; the EC50 for A / Taiwan / 2 / 2013 (H6N1) was 1.821 μM; the EC50 for A / chicken / Guangdong / 04.22DGCP098-O / 2015 (H7N9) was 1. 50 The EC value for A / chicken / Guangdong / 04.15SZBAXQ005 / 2015(H9N2) was 3.285μM. 50 The EC value for A / chicken / Jiangxi / B18 / 2014(H10N8) was 2.165μM. 50 It is 2.004μM.

[0112] II. Evaluation of the inhibitory effect of pyrimethamine on Zika virus (ZIKV) using a multi-concentration gradient assay at the cellular level

[0113] 1. Add double antibiotics (penicillin and streptomycin) and 2% fetal bovine serum (FBS) to DMEM culture medium to prepare a maintenance solution, and use this maintenance solution to dilute the Zika virus (ZIKA-SMGC-1) to make the Zika virus titer 100 TCID 50 / 50μl maintenance solution.

[0114] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution 5-fold in a stepwise gradient to prepare test solutions of varying concentrations (100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, and 0.0064 μM). Each concentration was diluted in a 50 μl volume. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0115] 3. Vero cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0116] 4. Incubate the system prepared in step 1 and step 2 together at room temperature for 30 minutes. Then, add the cells to the 96-well plate prepared in step 3 and incubate at 37°C, 5% CO2 for 1 hour. After 1 hour, discard the cell culture supernatant and add the system prepared in step 2 to the wells again. Incubate at 37°C, 5% CO2 for 72 hours.

[0117] 5. Remove the 96-well plate from step 4, aspirate the culture supernatant, add 100 μl / well of fixative (4% paraformaldehyde), fix the cells at room temperature for 20 min, aspirate the fixative, wash three times with PBST, add 100 μl of PBST containing 5% (mass percentage) skim milk powder to each well, incubate at 37°C for 1 h. Discard the supernatant, and add 100 μl of a 1:1000 dilution of anti-ZIKV E protein primary antibody (40543-R033-H-100, ZIKV-E Antibody, Sino Biological), incubate at 37°C for 1 h. Wash the wells five times with PBST for 5 minutes each. Add 100 μl of horseradish enzyme-conjugated goat anti-rabbit IgG secondary antibody (BE0101-100, Goat Anti-Rabbit IgG (H&L)-HRP Conjugated Goat Anti-Rabbit Secondary Antibody, EASYBIO) diluted 1:2500 to each well and incubate at 37°C for 1 hour. Wash the wells five times with PBST for 5 minutes each, then add 100 μl of TMB color development solution. After color development for 10 minutes, add 100 μl of 2M HCl. Take the test sample and detect it on a Multiskan FC microplate reader, and read the OD value. 450 The data were processed using EXCEL and the EC of the inhibitor was calculated using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0118] The results are as follows Figure 2 As shown, the EC of pyrimethamine against Zika virus (ZIKV) 50 It is 6.388μM.

[0119] 3. Evaluation of the inhibitory effect of pyrimethamine on dengue virus (DENV-2) using a multi-concentration gradient assay at the cellular level

[0120] 1. Add double antibiotics (penicillin and streptomycin) and 2% fetal bovine serum (FBS) to DMEM culture medium to prepare a maintenance solution, and use this maintenance solution to dilute dengue virus (dengue virus type 2 strain 43) to a dengue virus titer of 1000 PFU / 500 μl maintenance solution.

[0121] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution two-fold in a stepwise gradient to prepare test solutions of varying concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, and 0.390625 μM). Each concentration was diluted in 800 μl. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0122] 3. BHK-21 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 12-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0123] 4. Add the system prepared in step 1 to the 12-well plate prepared in step 3 and incubate in a 37°C, 5% CO2 incubator for 2 hours. After 2 hours, discard the cell culture supernatant and add the system prepared in step 2 to the cell wells again. Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0124] 5. Add low melting point agarose (V2111, Agarose, Low Melting Point, Promega) to PBS solution, place in a pressure cooker, and heat at 121°C for 20 minutes to sterilize to prepare a 4% low melting point agarose solution (the volume ratio of low melting point agarose to PBS solution is 4%).

[0125] 6. Add double antibodies (penicillin and streptomycin) and 2% fetal bovine serum (FBS) to DMEM culture medium to prepare maintenance solution, and add 4% low melting point agarose solution to this maintenance solution (the volume ratio of maintenance solution to 4% low melting point agarose solution is 3:1)

[0126] 7. Take the 12-well plate from step 4, aspirate the culture supernatant, make up a volume of 500 μl, and add it to the 12-well plate from step 3. Incubate at 37°C, 5% CO2 for 2 hours. After 2 hours, discard the cell culture supernatant and add 1 ml of the system prepared in step 6 to each well. Incubate at 37°C, 5% CO2 for 5-7 days.

[0127] 8. After clear viral plaques can be seen under light, take the 12-well plate from step 7, discard the culture supernatant, add 2 ml of fixative (4% paraformaldehyde solution) to each well, and fix at 37°C for 1 hour. Discard the fixative and wash with deionized water 2-3 times. Add 1 ml of crystal violet staining solution (C0121, Beyotime) and stain at 37°C for 1 hour. Discard the crystal violet staining solution and wash with deionized water 2-3 times. Count the viral plaques in each well of the 12-well plate, process the data with EXCEL, and calculate the EC value of the inhibitor using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0128] The results are as follows Figure 3 As shown, the EC of pyrimethamine against dengue virus (DENV-2) 50 It is 1.928μM.

[0129] IV. Evaluation of the inhibitory effect of pyrimethamine on SARS-CoV-2 using a multi-concentration gradient experiment at the cellular level

[0130] 1. Add double antibodies (penicillin and streptomycin) to DMEM culture medium to prepare a maintenance solution, and use this maintenance solution to dilute the SARS-CoV-2 new coronavirus hCoV-19 / China / CAS-B001 / 2020 to make the SARS-CoV-2 new coronavirus titer 50TCID 50 / 50μl maintenance solution.

[0131] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution 5-fold in a stepwise gradient to prepare test solutions of varying concentrations (100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, and 0.0064 μM). Each concentration was diluted in 100 μl. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0132] 3. Vero-E6 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0133] 4. Take the 96-well plate from step 3, add the system prepared in step 2 to the wells, incubate in a 37°C, 5% CO2 incubator for 1 hour, then discard. Add the system prepared in step 1 to the wells, incubate in a 37°C, 5% CO2 incubator for 1 hour, then discard. Add the system prepared in step 2 to the wells again, and incubate in a 37°C, 5% CO2 incubator for 48 hours.

[0134] 5. Take the 96-well plate from step 4, aspirate the culture supernatant, and soak the plate in fixative (4% paraformaldehyde). Fix the cells at room temperature for 3-4 hours, then discard the fixative. Wash the plate three times with PBST for 5 minutes each. Add 100 μl of 0.3% Triton-X-100 (C03-03002, 0.3% Triton-X-100, Bioss) to each well and incubate at room temperature for 30 minutes. Wash the plate three times with PBST for 5 minutes each. Add 100 μl of PBST containing 5% (mass percentage) skim milk powder to each well and incubate at 37°C for 1 hour. Discard the supernatant and add 100 μl of 1:7500 diluted SARS-CoV-2 Nucleocapsid rabbit monoclonal antibody (40588-R0002, SARS-CoV2 (2019-nCoV) Nucleocapsid Antibody, Sino Biological) and incubate at 37°C for 1 hour. Wash with PBST five times, 5 minutes each time, and add 100 μl of horseradish enzyme-conjugated goat anti-rabbit IgG secondary antibody (BE0101-100, Goat Anti-Rabbit IgG (H&L)-HRPConjugated goat anti-rabbit secondary antibody, EASYBIO) diluted 1:2500 to each well and incubate at 37°C for 1 hour. Wash with PBST five times, 5 minutes each time, and then add 100 μl of TMB color development solution. After color development for 10 minutes, add 100 μl of 2M HCl. Take the test sample and detect it on a Multiskan FC microplate reader, and read the OD 450 The data were processed using EXCEL and the EC of the inhibitor was calculated using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0135] The results are as follows Figure 4 As shown, the EC value of pyrimethamine against SARS-CoV-2 novel coronavirus is 50 It is 2.043μM.

[0136] 5. Evaluation of the Inhibitory Effect of Pyrimethamine on Ebola Replication-Competent Pseudoviruses Using Cell-Based Multi-Concentration Gradient Experiments

[0137] 1. Add double antibiotics (penicillin and streptomycin) to DMEM culture medium to prepare a maintenance solution, and use this maintenance solution to dilute the Ebola (Mayinga strain of Zaire EBOV) replication-competent pseudovirus (with GFP green fluorescence) to make the Ebola replication-competent pseudovirus titer 1000 TCID 50 / 50μl maintenance solution.

[0138] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution 5-fold in a stepwise gradient to prepare test solutions of varying concentrations (100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, and 0.0064 μM). Each concentration was diluted in 100 μl. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0139] 3. Huh7.5.1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When the cells grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0140] 4. Mix the system prepared in step 1 and the system prepared in step 2 and incubate in a 37° C., 5% CO 2 incubator for 1 hour.

[0141] 5. Take the 96-well plate from step 3, add the system from step 4 into the 96-well plate at 100 μl / well, and incubate in a 37°C, 5% CO2 incubator for 72 h.

[0142] 6. Use the high-content cell imaging analysis system CQ1 to count the green fluorescence value in each well, process the data using EXCEL, and calculate the EC of the inhibitor using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0143] The results are as follows Figure 5As shown, the EC value of pyrimethamine against Ebola replication-competent pseudovirus is 50 It is 7.253μM.

[0144] VI. Evaluation of the Inhibitory Effect of Pyrimethamine on Herpes Simplex Virus (HSV-1) Using a Cell-Based Multi-Concentration Gradient Experiment

[0145] 1. Add double antibiotics (penicillin and streptomycin) to DMEM culture medium to prepare maintenance solution, and use this maintenance solution to dilute the herpes simplex virus HSV-1F strain to make the herpes simplex virus HSV-1 titer 50TCID 50 / 50μl maintenance solution.

[0146] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the maintenance solution prepared in step 1, dilute the stock solution two-fold in a stepwise gradient to prepare test solutions of varying concentrations (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM). Each concentration was diluted in a 50 μl volume. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0147] 3. Vero cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0148] 4. Take the 96-well plate prepared in step 3, add the system prepared in step 1 to the wells, place in a 37°C, 5% CO2 incubator for incubation for 1 hour, then add the system prepared in step 2 to the wells, place in a 37°C, 5% CO2 incubator for incubation for 48 hours.

[0149] 5. Remove the 96-well plate from step 4, aspirate the culture supernatant, and soak the plate in fixative (4% paraformaldehyde). Fix the cells at room temperature for 3-4 hours, then discard the fixative. Wash the plate three times with PBST for 5 minutes each. Add 100 μl of 0.3% Triton-X-100 (C03-03002, 0.3% Triton-X-100, Bioss) to each well and incubate at room temperature for 30 minutes. Wash the plate three times with PBST for 5 minutes each. Add 100 μl of PBST containing 10% goat serum (C0005, Normal Goat Serum, Bioss) to each well and incubate at 37°C for 1 hour. Discard the plate and add 50 μl of a 1:5000 dilution of anti-HSV1+HSV2 Gd antibody (ab6507, Anti-HSV1+HSV2 Gd antibody, abcam) and incubate at 37°C for 1 hour. Wash 5 times with PBST, 5 min each time, and add 50 μl of 1:500 diluted Goat anti-mouse secondary antibody (Y6104L, Goat Anti-Mouse IgG (H&L, BIOLEADER; secondary antibody with green fluorescence) was incubated at 37°C in the dark for 1 hour. Washed five times with PBST, 5 minutes each time. 30 μl of DAPI staining solution (C02-04002, DAPI solution (Nuclear Labeling), Bioss) was added to each well and incubated at room temperature in the dark for 10 minutes. Washed three times with PBST, 5 minutes each time.

[0150] 6. Use the high-content cell imaging analysis system CQ1 to count the green fluorescence value in each well, process the data using EXCEL, and calculate the EC of the inhibitor using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0151] The results are as follows Figure 6 As shown, the EC of pyrimethamine against herpes simplex virus (HSV-1) 50 It is 1.641μM.

[0152] VII. Evaluation of the Inhibitory Effect of Pyrimethamine on Enterovirus EV71 BrCr Strain Using a Cell-Based Multi-Concentration Gradient Experiment

[0153] 1. Add double antibiotics (penicillin and streptomycin) and 10% fetal bovine serum (FBS) to DMEM culture medium to prepare a maintenance solution, and use this maintenance solution to dilute the enterovirus EV71 BrCr strain to a titer of 100 TCID 50 / 50μl maintenance solution.

[0154] 2. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, use the maintenance solution prepared in step 1 to dilute the stock solution 2-fold in a stepwise gradient to prepare test solutions of different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.1953125 μM), with a volume of 100 μl for each concentration. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also set up using 0.25% DMSO as a substitute for the test solution.

[0155] 3. RD cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0156] 4. Take the 96-well plate from step 3, add the system prepared in step 2 to the wells, incubate in a 37°C, 5% CO2 incubator for 1 hour, then discard. Add the system prepared in step 1 to the wells, incubate in a 37°C, 5% CO2 incubator for 1 hour, then discard. Add the system prepared in step 2 to the wells again, and incubate in a 37°C, 5% CO2 incubator for 48 hours.

[0157] 5. Take the 96-well plate from step 4, aspirate the culture supernatant, add 100 μl / well of fixative (3 parts by volume ethanol + 2 parts by volume acetone, stored in a -20°C refrigerator), fix the cells at room temperature for 15 minutes, aspirate the fixative, wash three times with PBST for 5 minutes each, add 100 μl of PBST containing 5% (mass percentage) skim milk powder to each well, incubate at 37°C for 1 hour. Discard the supernatant, add 100 μl of a 1:2500 dilution of Enterovirus 71VP1 antibody (GTX637688-S, Enterovirus 71VP1 antibody, Genetex), and incubate at 37°C for 1 hour. Wash the wells five times with PBST (5 min each time). Add 100 μl of horseradish enzyme-conjugated goat anti-rabbit IgG secondary antibody (BE0101-100, Goat Anti-Rabbit IgG (H&L)-HRP Conjugated, EASYBIO) at a dilution of 1:2500 to each well and incubate at 37°C for 1 h. Wash the wells five times with PBST (5 min each time), then add 100 μl of TMB color development solution. After developing in the dark for 10 min, add 100 μl of 2 M HCl.

[0158] 6. Take the test sample and test it on Multiskan FC microplate reader to read the OD 450 The data were processed using EXCEL and the EC of the inhibitor was calculated using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0159] The results are as follows Figure 7 As shown, the EC of pyrimethamine against enterovirus EV71 BrCr strain 50 It is 16.33μM.

[0160] 8. Evaluation of the Inhibitory Effect of Pyrimethamine on Vaccinia Virus (VACV) Using a Cell-Based Multi-Concentration Gradient Assay

[0161] 1. Add double antibiotics (penicillin and streptomycin) and 2% fetal bovine serum (FBS) to DMEM culture medium to prepare a maintenance solution. Dilute vaccinia virus (Western Reserve strain) with this maintenance solution to a titer of 200 pfu / 500 μl of maintenance solution.

[0162] 2. Add 10 g of high-viscosity sodium carboxymethylcellulose (C5013, Carboxymethylcellulose sodium salt, Sigma-Aldrich) and 10 g of low-viscosity sodium carboxymethylcellulose (C5678, Carboxymethylcellulose sodium salt, Sigma-Aldrich) to 1 L of double-distilled water, place in a sterilizer at 121°C, and sterilize at high temperature and high pressure for 30 min to prepare a 2% sodium carboxymethylcellulose solution.

[0163] 3. Prepare the covering solution by mixing DMEM high glucose culture medium (2X) and the system prepared in step 2 in a volume ratio of 1:1 and keep warm at 37°C until use.

[0164] 4. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mM stock solution. Then, using the overlay solution prepared in step 3, dilute the stock solution two-fold in a series of dilutions to produce test solutions of varying concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM). Each concentration was diluted in 1000 μl / well. The final DMSO concentration in the test solution was 0.25% (volume percentage). A negative control group was also established using 0.25% DMSO as a substitute for the test solution.

[0165] 5. Vero cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 12-well plates and incubated in a 37°C, 5% CO2 incubator overnight. When they grew to 90%-100% density, the culture supernatant was discarded and the cells were washed twice with PBS.

[0166] 6. Take the 12-well plate from step 3, add the system prepared in step 1 to the wells, place in a 37°C, 5% CO2 incubator for incubation for 1 hour, then discard, add the system prepared in step 4 to the wells, and place in a 37°C, 5% CO2 incubator for incubation for 48 hours.

[0167] 7. Take the 96-well plate from step 6, discard the culture supernatant, add 2 ml of fixative (4% paraformaldehyde solution) to each well, and fix at 37°C for 1 hour. Discard the fixative and wash with deionized water 2-3 times. Add 1 ml of crystal violet staining solution (C0121, Beyotime) and stain at 37°C for 1 hour. Discard the crystal violet staining solution and wash with deionized water 2-3 times. Count the viral plaques in each well of the 12-well plate, process the data with EXCEL, and calculate the EC value of the inhibitor using Graphpad software. 50 (half-maximal effect concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in the efficacy experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogEC 50)))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0168] The results are as follows Figure 8 As shown, the EC of pyrimethamine against vaccinia virus (VACV) 50 It is 20.9μM.

[0169] IX. Cytotoxicity evaluation of inhibitors on MDCK cells

[0170] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0171] 2. MDCK cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When they grew to a density of 70%-90%, the culture supernatant was discarded and the plates were washed twice with PBS.

[0172] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0173] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Discard the culture supernatant and wash twice with PBS. Add 100 μl of DMEM culture medium and 10 μl of CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3 hours. Take the test sample and detect it on a Multiskan FC microplate reader to read the OD 450 The data were processed using GraphPad Prism5 software to draw the drug-to-MDCK cell viability evaluation curve and calculate the CC of the inhibitor. 50 (Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50)))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0174] The results are as follows Figure 9 As shown, the CC of MDCK cells was affected by pyrimethamine. 50 is >100 μM.

[0175] 10. Cytotoxicity evaluation of inhibitors on Vero cells

[0176] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0177] 2. Vero cells (from ATCC) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to a density of 70%-90%, the culture supernatant was discarded and the cells were washed twice with PBS.

[0178] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0179] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 48h / 72h. Discard the culture supernatant and wash twice with PBS. Add 100μl DMEM culture medium and 10μl CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3h. Take the test sample and detect it on a Multiskan FC microplate reader to read the OD 450 The data were processed using GraphPad Prism5 software to draw the cell viability evaluation curve of the drug on Vero cells and calculate the CC of the inhibitor. 50 (Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50)))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0180] The results are as follows Figure 10 As shown, the CC of Vero cells was affected by pyrimethamine for 48 h. 50 CC>100μM, 72h 50 It is 66.82μM.

[0181] 11. Cytotoxicity evaluation of inhibitors on Vero-E6 cells

[0182] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0183] 2. Vero-E6 cells (from ATCC) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to a density of 70%-90%, the culture supernatant was discarded and the cells were washed twice with PBS.

[0184] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0185] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 48 hours. Discard the culture supernatant and wash twice with PBS. Add 100 μl of DMEM culture medium and 10 μl of CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3 hours. Take the test sample and detect it on a Multiskan FC microplate reader to read the OD 450 The data were processed using GraphPad Prism5 software to draw the cell viability evaluation curve of the drug on Vero-E6 cells and calculate the CC of the inhibitor. 50(Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0186] The results are as follows Figure 11 As shown, the CC of Vero-E6 cells was affected by pyrimethamine. 50 It is 54.99μM.

[0187] 12. Cytotoxicity evaluation of inhibitors on BHK-21 cells

[0188] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0189] 2. BHK-21 cells (from ATCC) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to a density of 70%-90%, the culture supernatant was discarded and the cells were washed twice with PBS.

[0190] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0191] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Discard the culture supernatant and wash twice with PBS. Add 100 μl of DMEM culture medium and 10 μl of CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3 hours. Take the test sample and detect it on a Multiskan FC microplate reader to read the OD 450 The data were processed using GraphPad Prism5 software to draw the cell viability evaluation curve of the drug on BHK-21 cells and calculate the CC of the inhibitor. 50(Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0192] The results are as follows Figure 12 As shown, pyrimethamine has an inhibitory effect on CC of BHK-21 cells. 50 is 100 μM.

[0193] 13. Cytotoxicity evaluation of inhibitors on Huh7.5.1 cells

[0194] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0195] 2. Huh7.5.1 cells (from ATCC) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to a density of 70%-90%, the culture supernatant was discarded and the cells were washed twice with PBS.

[0196] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0197] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 72 hours. Discard the culture supernatant and wash twice with PBS. Add 100 μl of DMEM culture medium and 10 μl of CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3 hours. Take the test sample and detect it on a Multiskan FC microplate reader. Read the OD value. 450 The data were processed using GraphPad Prism5 software to draw the cell viability evaluation curve of the drug on Huh7.5.1 cells and calculate the CC of the inhibitor. 50(Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0198] The results are as follows Figure 13 As shown, pyrimethamine has an inhibitory effect on CC of Huh7.5.1 cells. 50 is >100 μM.

[0199] 14. Cytotoxicity evaluation of inhibitors on RD cells

[0200] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40mM stock solution, then dilute it 5-fold with DMEM culture medium containing double antibodies (penicillin and streptomycin) to test solutions with different final concentrations (100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, 0.00128μM, 0.000256μM, 0.0000512μM). The final DMSO concentration in the test solution is 0.25% (volume percentage).

[0201] 2. RD cells (from ATCC) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to a density of 70%-90%, the culture supernatant was discarded and the cells were washed twice with PBS.

[0202] 3. Prepare the reaction system: Add different concentrations of the test solution to 100 μl of DMEM culture medium, setting up three replicate wells for each concentration. Also, set up a negative control group using 0.25% DMSO instead of the test solution, and a blank control group using DMEM culture medium instead of the test solution.

[0203] 4. Take the 96-well plate prepared in step 2 and add the system prepared in step 3 to the wells. Incubate in a 37°C, 5% CO2 incubator for 48 hours. Discard the culture supernatant and wash twice with PBS. Add 100 μl of DMEM culture medium and 10 μl of CCK-8 reagent (Dojin Co., Ltd., Japan) to each well and incubate in a 37°C, 5% CO2 incubator for 1-3 hours. Take the test sample and detect it on a Multiskan FC microplate reader to read the OD 450 The data were processed using GraphPad Prism5 software to draw the cell viability evaluation curve of the drug on RD cells and calculate the CC of the inhibitor. 50(Medium cytotoxic concentration, i.e., the concentration of the test substance that causes 50% of cells to become cytotoxic in a cytotoxic experiment), calculated as follows: Y = Bottom + (Top-Bottom) / (1 + 10^((X-LogCC 50 )))(X is the Log value corresponding to the drug concentration, Y is the drug inhibition rate, Top and Bottom are the maximum and minimum inhibition rates corresponding to the drug inhibition curve).

[0204] The results are as follows Figure 14 As shown, pyrimethamine has an effect on CC of RD cells. 50 is >100 μM.

[0205] 15. Experimental study on protection of mice against influenza virus challenge by inhibitors (oral administration)

[0206] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mg / ml stock solution. Then dilute with 0.5% sodium carboxymethyl cellulose (BD105513, Carboxymethyl cellulose, sodium salt, Bidex Pharmaceuticals) to a test solution with a final concentration of 4 mg / ml. The final concentrations of DMSO and sodium carboxymethyl cellulose in the test solution are 10% (volume percentage) and 0.5% (mass percentage). This test solution is the inhibitor described below.

[0207] 2. Prepare the virus (A / California / 07 / 2009(H1N1) mouse-adapted strain (abbreviated as CA07): as described in "Jiang H, Peng W, Qi J, Chai Y, Song H, Bi Y, Rijal P, Wang H, Oladejo BO, Liu J, Shi Y, Gao GF, Townsend AR, Wu Y. Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus. mBio. 2020 Oct. 6; 11(5):e02315-20.doi:10.1128 / mBio.02315-20.PMID:33024040;PMCID:PMC7542365." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes). The virus was diluted to 5LD with PBS. 50 .

[0208] 3. Prepare BALB / c mice. All female BALB / c mice were purchased from Vital River Pharmaceuticals and were 6-8 weeks old. Information such as mouse grouping, inhibitor dosage, and dosing frequency is shown in Table 1.

[0209] Table 1. Oral administration, groups and dosages of inhibitors in the challenge and protection experiments on mice

[0210]

[0211]

[0212] The preventive dosing group (5 mice) was first given the inhibitor by gavage (see step 1), 270 μl per mouse. 24 hours later, influenza virus was challenged by nasal drops (see step 2), 50 μl per mouse. Starting from the first dose, the drug was administered every 48 hours, for a total of 5 doses. The weight of the mice was monitored and recorded every day. In accordance with the "Guidelines for the Evaluation of Humane Endpoints of Experimental Animals" (RB / T173-2018) of the Certification and Accreditation Administration of the People's Republic of China, the humane endpoint of the experimental animals was reached when the weight of the mice dropped by 20%, and they were euthanized.

[0213] The therapeutic administration group (5 mice) was first challenged with influenza virus via intranasal drops (see step 2), with 50 μl per mouse. Immediately following the challenge, the mice were given the inhibitor (see step 1) via oral gavage, with 270 μl per mouse. Starting with the first dose, the mice were administered every 48 hours for a total of five doses. Mouse weights were monitored and recorded daily. Mice with a 20% weight loss were considered dead and euthanized.

[0214] The data were processed using GraphPad Prism5 software to draw the mouse weight change curve and mouse death and survival curve in the inhibitor's protection experiment against influenza virus in mice (oral gavage).

[0215] The results are as follows Figure 15 and Figure 16 As shown, the protection rate of the mice with 60 mg / kg of the inhibitor (preventive administration group) was 100%, and the protection rate of the mice with 60 mg / kg of the inhibitor (therapeutic administration group) was 60%.

[0216] 16. Inhibitor protection against influenza virus in mice (nasal drops)

[0217] 1. Dissolve pyrimethamine in 100% DMSO to prepare a 40 mg / ml stock solution. Then dilute with 0.5% sodium carboxymethyl cellulose (BD105513, Carboxymethyl cellulose, sodium salt, Bidex Pharmaceuticals) to prepare test solutions with final concentrations of 4 mg / ml, 2 mg / ml, 0.4 mg / ml, and 0.2 mg / ml, respectively. The final concentration of DMSO in the test solutions was 10% (volume percentage), and the final concentration of sodium carboxymethyl cellulose was 0.5% (mass percentage). The same solvent was used as a control. These test solutions are the inhibitors described below.

[0218] 2. Prepare the virus (A / California / 07 / 2009(H1N1) mouse-adapted strain (abbreviated as CA07): as described in "Jiang H, Peng W, Qi J, Chai Y, Song H, Bi Y, Rijal P, Wang H, Oladejo BO, Liu J, Shi Y, Gao GF, Townsend AR, Wu Y. Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus. mBio. 2020 Oct. 6; 11(5):e02315-20.doi:10.1128 / mBio.02315-20.PMID:33024040;PMCID:PMC7542365." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes). The virus was diluted to 2LD with PBS. 50 .

[0219] 3. Prepare BALB / c mice. All BALB / c mice were purchased from Vital River Pharmaceuticals and were female, 6-8 weeks old. Information such as mouse grouping, inhibitor dosage, and dosing frequency is shown in Table 2.

[0220] Table 2. Intranasal administration, groups and dosages of inhibitors in the challenge and protection experiment on mice

[0221]

[0222] For the prophylactic treatment group (5 mice), 50 μl of the inhibitor (see step 1) was administered intranasally 3 hours before challenge. Three hours later, the influenza virus challenge (see step 2) was administered intranasally 50 μl per mouse. Mouse weights were monitored and recorded daily. Mice were considered dead if their body weight decreased by 20% and were euthanized.

[0223] The therapeutic administration group (5 mice) was first challenged with influenza virus by intranasal drops (see step 2), 50 μl per mouse. Then, 3 hours after the challenge, the mice were given inhibitors by intranasal drops (see step 1), 50 μl per mouse. The weight of the mice was monitored and recorded daily. In accordance with the "Guidelines for the Evaluation of Humane Endpoints of Laboratory Animals" (RB / T173-2018) of the China National Certification and Accreditation Administration, when the weight loss of mice was 20%, the humane endpoint of the experimental animals was reached and euthanasia was performed.

[0224] The data were processed using GraphPad Prism5 software to plot the mouse weight change curve and mouse death and survival curve in the inhibitor's protection experiment against influenza virus in mice (nasal drops).

[0225] The results are as follows Figure 17 and Figure 18 As shown, the protection rate of the inhibitor 10 mg / kg (preventive administration group) and 10 mg / kg (therapeutic administration group) for mice was 100%; the protection rate of the inhibitor 5 mg / kg (preventive administration group) and 5 mg / kg (therapeutic administration group) for mice was 100%; the protection rate of the inhibitor 1 mg / kg (preventive administration group) for mice was 100%, and the protection rate of 1 mg / kg (therapeutic administration group) for mice was 40%; the protection rate of the inhibitor 0.5 mg / kg (preventive administration group) for mice was 80%, and the protection rate of 0.5 mg / kg (therapeutic administration group) for mice was 40%.

[0226] 17. Results Analysis

[0227] EC 50 It refers to the drug concentration that can effectively inhibit 50% of cells from being infected with the virus. The smaller the value, the better the inhibitory effect on the virus.

[0228] CC 50 It is the drug concentration that causes 50% of cells to become diseased. The higher the value, the lower the toxicity to cells.

[0229] from Figures 9 to 14 It can be seen that pyrimethamine has less toxicity to different cells, and has a negative effect on CC of MDCK cells, Vero cells (48h), Huh7.5.1 cells and RD cells. 50 Greater than 100 μM, CC of BHK-21 cells50 At 100 μM, the CC of Vero cells (72h) 50 The CC of Vero-E6 cells was 66.82 μM. 50 The concentration of pyrimethamine was 54.99 μM, indicating that pyrimethamine has an inhibitory effect on influenza virus without harming cells and can be used as a safe and effective broad-spectrum antiviral drug.

[0230] Animal challenge protection experiments showed that pyrimethamine could protect mice from influenza virus infection after oral administration or nasal administration.

[0231] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of any of the substances shown in (1)-(3) below in any of (A1)-(A3) below: (1) Pyrimethamine; (2) A pharmaceutically acceptable salt, ester or solvate of pyrimethamine; (3) A substance having pyrimethamine or a pharmaceutically acceptable salt, ester or solvate thereof as the main component; (A1) Preparation of an antiviral product; (A2) Preparation of a product capable of treating and / or preventing diseases caused by viral infections; (A3) Preparation of a product capable of improving symptoms caused by viral infections.

2. Use of any of the substances shown in (1)-(3) below in the preparation of a product for inhibiting viruses at the cellular level: (1) Pyrimethamine; (2) A pharmaceutically acceptable salt, ester or solvate of pyrimethamine; (3) A substance having pyrimethamine or a pharmaceutically acceptable salt, ester or solvate thereof as the main component.

3. The use according to claim 1 or 2, characterized in that: the virus is selected from any one or more of the following: influenza virus, Zika virus, dengue virus, SARS-CoV-2, Ebola virus, herpes simplex virus, enterovirus, vaccinia virus.

4. The use according to claim 3, characterized in that: the influenza virus is influenza A virus; and / or the Zika virus is ZIKA-SMGC-1; and / or the dengue virus is dengue virus type 2; and / or the SARS-CoV-2 is hCoV-19 / China / CAS-B001 / 2020; and / or the Ebola virus is Ebola virus Zaire strain; and / or the herpes simplex virus is herpes simplex virus type 1; and / or the enterovirus is EV71; and / or the vaccinia virus is VACV Western Reserve.

5. The use according to claim 4, characterized in that: the influenza A virus is influenza virus subtypes H1N1, H3N2, H5N1, H5N6, H6N1, H7N9, H9N2 and / or H10N8; and / or the dengue virus is dengue virus type 2 strain 43; and / or the Ebola virus is Ebola virus Zaire strain Mayinga; and / or the herpes simplex virus is HSV-1F strain; and / or the enterovirus is EV71 BrCr strain.

6. The use according to claim 5, characterized in that: The influenza A virus is selected from any one or more of the following: A / California / 04 / 2009, A / Beijing / CAS0001 / 2007, A / duck / Guangdong / 04.22DGCP069-O / 2015, A / pigeon / Sichuan / ncxn29 / 2014, A / Taiwan / 2 / 2013, A / chicken / Guangdong / 04.22DGCP098-O / 2015, A / chicken / Guangdong / 04.15SZBAXQ005 / 2015, A / chicken / Jiangxi / B18 / 2014.

7. The application according to any one of claims 1-6, characterized in that: the product is a drug.

8. The application according to claim 7, characterized in that: the only active ingredient or one of the active ingredients of the drug is pyrimethamine or a pharmaceutically acceptable salt or ester or solvate thereof.

Images