Application of phenazine-1-carboxylic acid or medicinal salt thereof in preparation of medicine for inhibiting African swine fever virus

Abstract

The invention discloses application of phenazine-1-carboxylic acid or medicinal salt thereof in preparation of drugs for inhibiting African swine fever virus. According to the invention, a double-report virus rASFV-Glucc / EGFP for co-expressing green fluorescent protein and Gaussian luciferase is used, and screening from 246 natural small molecule compounds finds that phenazine-1-carboxylic acid has a remarkable inhibition effect on the replication of ASFV; the inhibition effect of the PCA is further evaluated, and it is found that the half maximum cytotoxicity concentration of the PCA to primary porcine alveolar macrophages of target cells is 470.5 mu M, and the half maximum inhibition concentration of the PCA to ASFV in PAMs is 1.59 mu M. According to a dose-dependent inhibition experiment result, when the PCA concentration is 25 [mu] M, the ASFV inhibition effect can reach 100 times or more. The invention has application prospects in preparation of anti-ASFV drugs or preparations and the like.

Description

Technical Field

[0001] The present invention relates to the antiviral use of natural small molecule compounds, in particular to the use of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for inhibiting African swine fever virus, belonging to the field of antiviral application of phenazine small molecule compounds. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease caused by African swine fever virus (ASFV) infecting pigs (Galindo I, Alonso C. African swine fever virus: A review. Viruses. 2017; 9(5): 103), which has caused huge economic losses to the pig industry. The ASFV genome is 170 to 194 kb in length and can encode 68 structural proteins and more than 100 non-structural proteins (Wang Y, Kang W, Yang W, Zhang J, Li D, Zheng H. Structure of African swine fever virus and associated molecular mechanisms underlying infection and immunosuppression: a review. Front Immunol. 2021; 12: 715582). Since 2018, ASFV has gradually evolved from the initial genotype II virulent strain to a genotype I weak strain and a recombinant virulent strain of genotype II and genotype I (Zhang Y, Zhang Z, Zhang F, Zhang J, Jiao J, Hou M, Qian N, Zhao D, Zheng X, Tan X. ASFV transcription reporter screening system identifies ailanthone as abroad antiviral compound. Virol Sin. 2023; 38(3): 459-469). The huge genome and complex virus particle structure of ASFV, coupled with the constantly evolving genotype, have seriously hindered the development of ASF vaccines. At present, there is no safe and effective commercial vaccine or antiviral drug to prevent and control ASF. Therefore, it is particularly important to develop anti-ASFV drugs as a preventive strategy to replace vaccines.Existing studies have shown that certain compounds have good inhibitory effects on ASFV in vitro. Further discovery of new antiviral drugs will help provide new candidate compounds for future drug development (Li T, Zheng J, Huang T, Wang X, Li J, Jin F, Wei W, Chen X, Liu C, Bao M, Zhao G, Huang L, Zhao D, Chen J, Bu Z, Weng C. Identification of several African swine fever virus replication inhibitors by screening of a library of FDA-approved drugs. Virology. 2024; 593: 110014).

[0003] It has been found that some drugs or compounds can inhibit the replication of ASFV through different mechanisms. For example, ailanthone (AIL) can exert its antiviral effect by targeting prostaglandin E synthase 3 (p23) (Zhang Y, Zhang Z, Zhang F, Zhang J, Jiao J, Hou M, Qian N, Zhao D, ZhengX, Tan X. ASFV transcription reporter screening system identifies ailanthone as a broad antiviral compound. Virol Sin. 2023; 38(3): 459-469). The latest research found that bis-benzylisoquinoline alkaloids (BBAs) inhibit the internalization and replication of ASFV by destroying the late endosome / lysosome function, while other drugs or compounds such as triapine, cytarabine hydrochloride, dihydromyricetin, tetrandrine, berbamine, pentagastrin, cangrelor, fostamatinib and polygalic acid have also been shown to significantly inhibit the replication of ASFV in vitro. However, whether the above drugs or compounds can exert anti-ASFV effects in pigs needs further study. Therefore, it is of great significance to continue to discover more candidate anti-ASFV drugs and compounds and analyze their mechanisms of action for the formulation of effective ASFV prevention and control strategies in the future.

[0004] Phenazine-1-carboxylic acid (PCA) is a natural metabolite produced by microorganisms such as Pseudomonas and Streptomyces. It has strong antifungal activity against a variety of plant pathogens, such as Botrytis cinerea, Fusarium oxysporum, Pythium spp. and Xanthomonas oryzae, and has been registered as a broad-spectrum fungicide in China (Fang Yunling, Sun Shuang, Shen Yue, et al. Research and application of microbial pesticide phenazine [J]. Journal of Pesticide Science, 2014, 16 (4): 387-393). So far, no studies have shown that PCA has an inhibitory effect on ASFV or other viruses. Summary of the invention

[0005] The main purpose of the present invention is to use phenazine-1-carboxylic acid or its pharmaceutically acceptable salt in the preparation of drugs for inhibiting ASFV.

[0006] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0007] One aspect of the present invention is to use phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof for preparing drugs for inhibiting animal viruses.

[0008] In a preferred embodiment of the present invention, the animal virus is African swine fever virus.

[0009] In a preferred embodiment of the present invention, the concentration of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof is 25 μM.

[0010] The base addition salts, solvates or prodrugs of phenazine-1-carboxylic acid of the present invention are also included in the present invention.

[0011] Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of the invention by reacting the carboxylic acid-containing moiety with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, alkali metal or alkaline earth metal-based cations, such as lithium, sodium, potassium, calcium, magnesium and aluminum salts, and the like, as well as non-toxic quaternary ammonium salts and amine cations, including ammonium, tetramethylammonium, tetraethylammonium methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium and ethylammonium, and the like. Other representative organic amines that can be used to form basic addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0012] The phenazine-1-carboxylic acid of the present invention can also be a solvate, and the solvent can be ethanol, water, etc., which can contain different amounts of water, such as a monohydrate, a hemihydrate, a hemihydrate, a dihydrate or a trihydrate, etc.

[0013] The present invention also includes prodrugs of phenazine-1-carboxylic acids. According to the present invention, prodrugs are derivatives of the above compounds, which themselves may have weak or no activity, but after administration, are converted into the corresponding biologically active form under physiological conditions (e.g., by metabolism, solvolysis or other means).

[0014] Another aspect of the present invention is to provide a pharmaceutical composition for inhibiting animal viruses and to apply the pharmaceutical composition to the preparation of drugs for inhibiting animal viruses.

[0015] A preferred specific embodiment of the present invention is a pharmaceutical composition for inhibiting animal viruses, comprising: an active ingredient for inhibiting animal viruses and a carrier or excipient acceptable to pharmaceutical preparations; wherein the active ingredient for inhibiting animal viruses is phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof.

[0016] In a preferred embodiment of the present invention, the animal virus is African swine fever virus.

[0017] In a preferred embodiment of the present invention, the concentration of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof is 25 μM.

[0018] In a preferred specific embodiment of the present invention, the pharmaceutically acceptable carrier is one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinyl pyrrolidone, low molecular weight dextran, sodium chloride, calcium gluconate or calcium phosphate; the excipient may be an antioxidant complexing agent, a filler, a skeleton material, etc.

[0019] In a preferred embodiment of the present invention, the pharmaceutical composition is prepared as an oral preparation or an injection preparation; preferably, the oral preparation includes a powder, a granule, or an oral liquid.

[0020] The compositions of the present invention will generally be administered orally, parenterally, intravenously, intramuscularly, subcutaneously or by other injection routes. The compositions may be displayed in discrete dosage units where appropriate, and may be administered by any method well known in the pharmaceutical field. Pharmaceutical compositions suitable for oral administration are listed as individually packaged units, such as powders, granules, or oral liquids, each containing a predetermined amount of active substance. Oral tablets may contain conventional pharmaceutical excipients, such as binders, fillers, lubricants, disintegrants or wetting agents. The compositions may be formulated for in vitro administration, such as by injection (bolus or continuous infusion), and may be infused in unit dosage form in small dose ampoules or in multi-dose containers with added preservatives. The compositions may be suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulating agents, such as suspending agents, stabilizers and / or dispersants.

[0021] The present invention uses a dual reporter virus rASFV-Gluc / EGFP that co-expresses green fluorescent protein and Gaussia luciferase, and screens 246 natural small molecule compounds to find that PCA has a significant inhibitory effect on the replication of ASFV. After evaluating its cytotoxicity, it was found that PCA has a very low toxicity to PAMs, CC 50 The anti-ASFV activity of PCA in PAMs was further evaluated within the safe concentration range. The results showed that PCA had a significant inhibitory effect on ASFV, and its IC 501.59 μM, using CC 50 and IC 50 The calculated SI reached 295.9; when the PCA concentration reached 25 μM, the inhibitory effect on ASFV could be as high as 100 times or more. These data show that PCA has high use value and application potential. Therefore, the present invention reveals that PCA has a significant inhibitory effect on ASFV in PAMs, provides a new target for the formulation of ASF prevention and control strategies, and is expected to develop efficient and low-cost anti-ASFV drugs based on PCA through strategies such as combined drug use or organic synthesis.

[0022] The following examples are provided to illustrate the present invention and are not intended to limit the present invention. The meanings of the abbreviations are as follows:

[0023] As used herein, "salts" refers to those salts that retain the biological effectiveness and properties of the parent compound and are not biologically harmful or otherwise deleterious at the dosages administered. Salts of the compounds of the present invention may be prepared from inorganic or organic bases.

[0024] The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic bases. The term "pharmaceutically acceptable salts" refers to salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., within the scope of reasonable medical judgment, and are comparable to salts. Reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. described pharmaceutically acceptable salts in detail in "Pharmaceutical Science" in 1977.

[0025] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, ie, does not cause unacceptable levels of undesired biological effects or interact in a deleterious manner.

[0026] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a PCA result diagram for screening 246 natural small molecule compounds; among them, Figure 1 -A is a schematic diagram of screening of small molecule compounds against ASFV; Figure 1 -B is a graph showing the inhibition rate of small molecule compounds on ASFV based on luciferase activity after rASFV-Gluc / EGFP infected PAMs for 36 hours;

[0028] Figure 1-C is the inhibitory effect of PCA on green fluorescence expression after rASFV-Gluc / EGFP infected PAMs for 36 hours; Figure 1 -D is the chemical structural formula of PCA; Figure 1 -E is the half toxic concentration of PCA to PAMs; Figure 1 -F is the half-maximal inhibitory concentration diagram of PCA against ASFV in PAMs;

[0029] Figure 2 This is a graph showing the results of different doses of PCA inhibiting ASFV replication; Figure 2 -A is the inhibitory effect of PCA on green fluorescence expression after rASFV-Gluc / EGFP infected PAMs and treated with different doses (5, 10 or 25 μM) of PCA or DMSO (0 μM PCA treatment) 48 h after infection. The scale bar is 400 μm; Figure 2 -B is a graph showing the effect of PCA (0, 5, 10 or 25 μM) on the luciferase produced by rASFV-Gluc / EGFP infected PAMs; Figure 2 -C is a graph showing the inhibitory effect of PCA (0, 5, 10 or 25 μM) on ASFV genome copies; Figure 2 -D is the effect of PCA (0, 5, 10 or 25 μM) on ASFV virus titer (HAD 50 )’s inhibitory effect diagram; Figure 2 -E is a graph showing the inhibitory effect of PCA on ASFV p72 and A137R proteins 48 h after infection with ASFV-WT (ASFV HLJ / 18) and PAMs treated with PCA (0, 5, 10 or 25 μM). DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0031] Experimental Example 1 Screening of compounds that inhibit African swine fever virus from 246 natural small molecule compounds and experiments on the inhibition of African swine fever virus replication by compounds at different concentrations

[0032] 1 Test method

[0033] 1.1 Gaussia luciferase (Gluc) activity detection

[0034] rASFV-Gluc / EGFP (MOI = 0.2) was mixed with PCA (0, 5, 10 or 25 μM) and then infected with PAMs. After 48 h of infection, the cell culture supernatant was aspirated and inactivated at 70°C. The inactivated culture supernatant was added to an opaque 96-well plate (20 μL / well), and 30 μL of Gluc substrate was added to each well for 10 min. After the fluorescence signal expression was stable, Gluc activity was detected in a focused multifunctional fluorescence analyzer.

[0035] 1.2 EGFP detection

[0036] rASFV-Gluc / EGFP (MOI = 0.2) was mixed with PCA (0, 5, 10 or 25 μM) and then infected PAMs (10 5 cells / well), and 48 h after infection, the expression of green fluorescence was observed under an inverted fluorescence microscope.

[0037] 1.3 Screening of anti-ASFV compounds

[0038] rASFV-Gluc / EGFP (MOI = 0.2) was mixed with the natural small molecule compound to be screened (10 μM) or dimethyl sulfoxide (DMSO) and then infected PAMs. After 36 hours of infection, the cell culture supernatant was aspirated and inactivated at 70°C. The inactivated culture supernatant was added to a light-proof 96-well plate (20 μL / well), and 30 μL of Gluc substrate was added to each well for 10 minutes. After the fluorescence signal expression was stable, Gluc activity was detected in a focused multifunctional fluorescence analyzer. DMSO was used as a negative control and the culture medium was used as a blank control.

[0039] The ASFV inhibition rate of natural small molecule compounds was calculated based on Gluc activity: ASFV inhibition rate = [(control group) - (experimental group)] / [(control group) - (blank group)] × 100%.

[0040] 1.4 Cytotoxicity assay

[0041] PAMs were plated in 96-well cell culture plates (10 5 After the cells adhered to the wall, different concentrations of PCA were added and incubated at 37°C, 5% CO 2Incubate under the conditions of . After 48 hours, CTG reagent is added to the cells to lyse the cells. The firefly luciferase (Fluc) contained in the CTG reagent will undergo a redox reaction with the ATP produced by cell lysis to cause bioluminescence. DMSO was used as a negative control and the culture medium was used as a blank control. Three replicates were performed for each concentration of the compound. The relative light units (RLUs) generated by bioluminescence were detected by a focused multifunctional fluorescence analyzer to calculate cell viability.

[0042] Cell viability = [(experimental group) - (blank group)] / [(control group) - (blank group)] × 100%.

[0043] 1.5qPCR detection

[0044] PAMs were plated on 24-well cell culture plates. After the cells adhered to the wall, rASFV-Gluc / EGFP (MOI = 0.2) was mixed with PCA (5, 10 or 25 μM) and infected with PAMs. The supernatant was discarded after 2 hours and replaced with fresh medium containing only PCA for continued culture. After 48 hours of infection, the cells and the supernatant of the culture medium were collected. The sample was added to a nucleic acid extraction or purification kit, and the ASFV genome was extracted using an automatic nucleic acid extractor. The extracted genomic DNA was collected in a 1.5 mL EP tube and stored in a -30°C refrigerator. The ASFV genome copy number was detected using the qPCR method recommended by the World Organisation for Animal Health (WOAH) (King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos AD, Drew TW. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fevervirus. J Virol Methods. 2003; 107(1): 53-61).

[0045] 1.6 Virus titer determination (HAD 50 )

[0046] PAMs were plated in 96-well cell culture plates (10 5 After the cells adhered to the wall, the collected virus solution was added to RPMI 1640 medium at 10 -1 ~10 -7The cells were diluted to 8 replicates for each dilution and added to the pre-laid PAMs. 4 days after infection, 10 μL of 1% porcine erythrocyte suspension was added to each well. After 12 hours, the results of the erythrocyte adsorption test in PAMs were observed and recorded. The 50% hemadsorption (HAD) at each time point was calculated using the Reed & Muench method. 50 ), and finally Graphpad Prism 8.0.2 software was used to draw the growth curve (Wang T, Luo R, Zhang J, Lu Z, Li LF, Zheng YH, Pan L, Lan J, Zhai H, Huang S, Sun Y, Qiu HJ. The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKαand IKKβ through the recruitment of TOLLIP. PLoS Pathog. 2023; 19(8): e1011580).

[0047] 1.7 Western blotting

[0048] PAMs were plated in 24-well cell culture plates (2×10 5cells / well), after the cells adhered to the wall, rASFV-Gluc / EGFP (MOI=0.2) was mixed with PCA and infected PAMs. After 2 hours, the supernatant was discarded and replaced with fresh RPMI 1640 medium, and the same concentration of PCA was added to continue the culture. The cells were collected 48 hours after infection, lysed with NP40 lysis buffer (containing protease inhibitor PMSF) at 4°C for 30 minutes, and the supernatant was collected after centrifugation at 12000r / min for 10 minutes. 5× loading buffer was added to it and the protein sample was obtained by boiling water bath for 10 minutes. After SDS-PAGE separation, the samples were transferred to polyvinylidene difluoride membranes and blocked with 5% skim milk at room temperature for 1.5 h; washed three times with Tris-buffered saline with tween (TBST), and the membranes were incubated with mouse anti-p72 (1:200 dilution), rabbit anti-A137R (1:500 dilution) polyclonal antibodies and mouse anti-β-tubulin antibody (1:1000 dilution) as primary antibodies at room temperature for 3 h; washed three times with TBST, and incubated with goat anti-rabbit IgG and goat anti-mouse IgG as secondary antibodies at room temperature for 1 h; washed three times with TBST, and scanned with a near-infrared fluorescence scanning imaging system (Odyssey CLX).

[0049] 1.8 Statistical analysis

[0050] All data in the present invention were analyzed using GraphPad Prism 8.0.1 software, and the significance of the differences between the groups was analyzed by unpaired two-tailed t-test or one-way ANOVA. ns, P ≥ 0.05 indicates no significant difference; *, P < 0.05 indicates a significant difference; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 indicate extremely significant differences.

[0051] 2 Test results

[0052] 2.1 Experimental results of screening compounds that inhibit African swine fever virus from natural small molecule compounds

[0053] Natural small molecule compound library Figure 1 -A, 246 natural small molecule compounds were screened. The inhibitory effect of natural small molecule compounds on ASFV was evaluated by detecting luciferase activity. The results of the luciferase activity detection test are shown in Figure 1 -B shows that at 10 μM, the inhibition rate of PCA and Brefeldin A (BFA) on ASFV reached more than 90%. However, because the cytotoxicity of BFA is too high, the subsequent experiments only studied the inhibitory effect of PCA on ASFV. The results of PCA inhibiting ASFV are shown in Figure 1-C, fluorescence microscopy showed that compared with the DMSO group, 10 μM PCA could significantly inhibit the expression of green fluorescent protein. The above results all indicate that PCA has antiviral activity against ASFV.

[0054] PCA is a type of phenazine compound, and its structure includes a phenazine ring and a carboxyl group. Its chemical structure is as follows: Figure 1 -D as shown.

[0055] In order to confirm that the inhibitory effect of PCA was not due to excessive cytotoxicity, the cell viability of PCA in PAMs after 48 h of incubation was detected by CTG method, and the half-maximal cytotoxic concentrations (CC 50 ); the test results are as follows Figure 1 -E shows the CC of PCA in PAMs 50 It is 470.5μM.

[0056] In this study, rASFV-Gluc / EGFP was used to infect PAMs, the virus titer was determined, and the half-maximal inhibitory concentration (IC) of PCA against ASFV was calculated. 50 ); the test results are as follows Figure 1 -F shows that the IC50 of PCA for ASFV is 1.59 μM and the selectivity index (SI) is 295.9.

[0057] The above experimental results show that PCA has low cytotoxicity at a dose that achieves a significant inhibitory effect on ASFV in PAMs, indicating that PCA has a wide therapeutic window when used to inhibit ASFV in vitro.

[0058] 2.2 Results of inhibition of ASFV replication by different concentrations of PCA

[0059] To further verify the inhibitory effect of PCA on ASFV replication, the dual reporter virus rASFV-Gluc / EGFP was used to evaluate the inhibitory effect of different doses of PCA (0, 5, 10 or 25 μM) on ASFV. Figure 2 -A and Figure 2 -B shows that compared with the DMSO control group, PCA can significantly inhibit the fluorescence expression of ASFV EGFP and luciferase activity in a dose-dependent manner. The results of ASFV genome copy number and virus titer detection are as follows Figure 2 -C and Figure 2-D, as the PCA dose increases, the viral genome level and viral titer decrease significantly. When the PCA concentration reaches 25μM, the inhibitory effect on ASFV can be as high as 100 times or more.

[0060] To confirm the antiviral activity of PCA in wild-type ASFV, this study compared the effects of different doses of PCA (0, 5, 10 or 25 μM) on the expression of p72 and A137R proteins; Western blotting results are shown in Figure 2 As shown in Figure 5-E, PCA can inhibit the expression of p72 and A137R proteins in a dose-dependent manner.

[0061] The above experimental results show that PCA has significant antiviral activity against ASFV in PAMs, further supporting its potential research value as an anti-ASFV drug.

Claims

1. Use of phenazine-1-carboxylic acid or its pharmaceutically acceptable salt in the preparation of drugs for inhibiting animal viruses.

2. The use according to claim 1, characterized in that The animal virus is African swine fever virus.

3. The use according to claim 1, characterized in that The concentration of phenazine-1-carboxylic acid or its pharmaceutically acceptable salt is 25 μM.

4. A pharmaceutical composition for inhibiting animal viruses, comprising: The active ingredient for inhibiting animal viruses and a carrier or auxiliary material acceptable to a pharmaceutical preparation are characterized in that the active ingredient for inhibiting animal viruses is phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof.

5. The use according to claim 4, characterized in that The animal virus is African swine fever virus.

6. The use according to claim 4, characterized in that The concentration of phenazine-1-carboxylic acid or its pharmaceutically acceptable salt is 25 μM.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is prepared into an oral preparation or an injection preparation.

8. The pharmaceutical composition according to claim 7, characterized in that The oral preparation includes powder, granule or oral liquid.

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