Use of phenoxazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting African swine fever virus

Abstract

The application discloses a use of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof in preparation of a medicine for inhibiting African swine fever virus. The application uses a double reporter virus rASFV-Gluc / EGFP co-expressing green fluorescent protein and Gaussia luciferase to screen 246 natural small molecule compounds, and finds that phenazine-1-carboxylic acid has a significant inhibiting effect on replication of the ASFV; further evaluation of the inhibiting effect finds that the half maximal cytotoxicity concentration of PCA on target cells of primary porcine alveolar macrophages is 470.5 muM, and the half maximal inhibitory concentration of PCA on the ASFV in PAMs is 1.59 muM. According to a dose-dependent inhibiting experiment result, when the concentration of PCA is 25 muM, the inhibiting effect on the ASFV can reach more than 100 times. The application has application prospects in preparation of medicines or preparations against the ASFV.

Description

TECHNICAL FIELD

[0001] The present application 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 a medicine for inhibiting African swine fever virus, belonging to the field of antiviral application of phenazine small molecule compounds. BACKGROUND

[0002] African swine fever (ASF) is an acute, severe, and highly contagious infectious disease caused by African swine fever virus (ASFV) infection in 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 full-length genome of ASFV is 170-194 kb, which 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 the attenuated genotype I strain, and the 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 a broad antiviral compound. Virol Sin. 2023; 38(3): 459-469). The large genome and complex virion structure of ASFV, combined with the evolving genotype, have seriously hindered the development of ASF vaccines. Currently, there is no safe and effective commercial vaccine or antiviral drug to prevent and control ASF. Therefore, the development of anti-ASFV drugs as an alternative vaccine prevention strategy is particularly important.Previous studies have shown that certain compounds have good inhibitory effect on ASFV in vitro, and further exploration of new antiviral drugs can help to 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] Currently, it has been found that some drugs or compounds can inhibit the replication of ASFV through different mechanisms, for example, Ailanthone (AIL) exerts 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, Zheng X, Tan X. ASFV transcription reporter screening system identifies ailanthone as a broad antiviral compound. Virol Sin. 2023; 38(3): 459-469). Recent studies have found that Bis-benzylisoquinoline alkaloids (BBAs) inhibit the internalization and replication of ASFV by disrupting the function of late endosomes / lysosomes, while other drugs or compounds such as Triapine, Cytarabine hydrochloride, Dihydromyricetin, Tetrandrine, Berbamine, Pentagastrin, Cangrelor, Fostamatinib and Polygalic acid, etc. have also been shown to significantly inhibit the replication of ASFV in vitro. However, whether the above drugs or compounds can exert anti-ASFV efficacy in pigs needs further study. Therefore, it is of great significance to continue to explore more candidate anti-ASFV drugs and compounds and to analyze their mechanisms of action for formulating 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, and 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 Y L, Sun S, Shen Y, et al. Development and application of microbial pesticide Shenzimycin [J]. Chinese Journal of Pesticides, 2014, 16(4): 387-393). So far, there has been no research showing that PCA has inhibitory effect on ASFV or other viruses. Summary of the Invention

[0005] The main objective of this invention is to apply phenazine-1-carboxylic acid or its pharmaceutically acceptable salt to the preparation of drugs that inhibit ASFV.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] One aspect of the present invention is the application of phenazine-1-carboxylic acid or its pharmaceutically acceptable salt in the preparation of 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 the phenazine-1-carboxylic acid or its pharmaceutically acceptable salt is 25 μM.

[0010] Salts, solvates, or prodrugs of phenazine-1-carboxylic acid formed by base addition are also included in this invention.

[0011] Basic addition salts can be prepared in situ during the final separation and purification of the compounds of this 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, salts based on alkali metal or alkaline earth metal cations, such as lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary ammonium salts and amine cations, including ammonium, tetramethylammonium, tetraethylammonium methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium. Other representative organic amines that can be used to form basic addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, etc.

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

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

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

[0015] A preferred embodiment of the present invention provides a pharmaceutical composition for inhibiting animal viruses, comprising: an active ingredient for inhibiting animal viruses and a pharmaceutically acceptable carrier or excipient; 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 the phenazine-1-carboxylic acid or its pharmaceutically acceptable salt is 25 μM.

[0018] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinylpyrrolidone, low molecular weight dextran, sodium chloride, calcium gluconate, or calcium phosphate; the excipients may be antioxidant complexing agents, fillers, matrix materials, etc.

[0019] In a preferred embodiment of the present invention, the pharmaceutical composition is prepared into an oral formulation or an injectable formulation; preferably, the oral formulation includes a powder, granules, or an oral liquid.

[0020] The compositions of the present invention are generally intended for oral, parenteral, intravenous, intramuscular, subcutaneous, or other injectable routes of administration. Where appropriate, the composition may be presented in discrete dose units, by any method well known in the pharmaceutical field. Pharmaceutically suitable compositions for oral administration are listed as individually packaged units, such as powders, granules, or oral solutions, each containing a predetermined amount of the 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, for example by injection (push or continuous infusion), and may be administered in unit dosage form in small-dose ampoules or in multi-dose containers with added preservatives. The compositions may be in the form of suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0021] This invention utilizes a dual reporter virus, rASFV-Gluc / EGFP, co-expressing green fluorescent protein and Gaussian luciferase. Screening of 246 natural small molecule compounds revealed that PCA significantly inhibits ASFV replication. After evaluating its cytotoxicity, it was found that PCA exhibits low cytotoxicity against PAMs (polyacrylamide) and CC (cytotoxicity). 50 The concentration reached 470.5 μM. Within the safe concentration range, the anti-ASFV activity of PCA in PAMs was further evaluated. The results showed that PCA had a significant inhibitory effect on ASFV, with an IC50 concentration of 470.5 μM. 50The value was 1.59 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 was more than 100 times higher. These data indicate that PCA has high practical value and application potential. Therefore, this invention reveals that PCA has a significant inhibitory effect on ASFV in PAMs, providing a new target for developing strategies to control ASFV, and holds promise for developing highly efficient and low-cost anti-ASFV drugs based on PCA through strategies such as combination therapy or organic synthesis.

[0022] The following examples are provided to illustrate that the invention is not limited thereto and should not be considered as limiting the invention. The abbreviations have the following meanings:

[0023] As used in this invention, "salt" refers to those salts that retain the biological potency and properties of the parent compound and are biologically harmless or otherwise harmless at the administered dose. Salts of the compounds of this invention can be prepared from inorganic or organic bases.

[0024] The compounds of this invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic bases. The term "pharmaceutically acceptable salt" means a salt that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and is comparable to salts in terms of a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. described pharmaceutically acceptable salts in detail in *Pharmaceutical Science* in 1977.

[0025] The term "pharmaceutically acceptable" describes materials that are not biologically or otherwise undesirable, that is, materials that do not cause undesirable biological effects at unacceptable levels or interact in a harmful manner.

[0026] As used in this invention, the term "composition" is intended to cover products containing specified amounts of specified ingredients, and any products produced directly or indirectly from combinations of specified amounts of specified ingredients. Attached Figure Description

[0027] Figure 1 The image shows the results of screening PCA from 246 natural small molecule compounds; among them, Figure 1 -A is a schematic diagram of the screening of small molecule compounds against ASFV; Figure 1 -B is a graph showing the inhibition rate of small molecule compounds against ASFV 36 h after rASFV-Gluc / EGFP infection of PAMs, based on luciferase activity detection.

[0028] Figure 1-C represents the inhibitory effect of PCA on green fluorescence expression after 36 hours of rASFV-Gluc / EGFP infection of PAMs; Figure 1 -D is the chemical structural formula of PCA; Figure 1 -E represents the half-maximal toxicity concentration (IC50) of PCA for PAMs; Figure 1 -F is the half-maximal inhibitory concentration (ICP-C) plot of PCA against ASFV in PAMs;

[0029] Figure 2 Figure 1 shows the results of ASFV replication inhibition by different doses of PCA; where Figure 2 -A is a graph showing the inhibitory effect of PCA on green fluorescence expression 48 h after infection with rASFV-Gluc / EGFP and treatment with different doses (5, 10 or 25 μM) of PCA or DMSO (0 μM PCA treatment). The scale bar is 400 μm. Figure 2 -B is a graph showing the effect of PCA (0, 5, 10 or 25 μM) on luciferase produced by rASFV-Gluc / EGFP infected with PAMs. Figure 2 -C represents the inhibition effect of PCA (0, 5, 10 or 25 μM) on ASFV genome copy; Figure 2 -D represents the titer of PCA (0, 5, 10, or 25 μM) against ASFV virus (HAD). 50 The inhibition effect diagram; Figure 2 -E represents the inhibitory effect of PCA (0, 5, 10 or 25 μM) on ASFV p72 and A137R proteins 48 h after ASFV-WT (ASFV HLJ / 18) infection of PAMs. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0031] 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 different concentrations of compounds.

[0032] 1. Experimental Methods

[0033] 1.1 Detection of Gaussian luciferase (Gluc) activity

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

[0035] 1.2 EGFP detection

[0036] After mixing rASFV-Gluc / EGFP (MOI = 0.2) with PCA (0, 5, 10 or 25 μM), PAMs (10 5 Cells / wells were used to observe the expression of green fluorescence using an inverted fluorescence microscope 48 hours after infection.

[0037] 1.3 Screening of anti-ASFV compounds

[0038] rASFV-Gluc / EGFP (MOI = 0.2) was mixed with the selected natural small molecule compound (10 μM) or dimethyl sulfoxide (DMSO) and then used to infect PAMs. After 36 h of infection, the cell culture supernatant was aspirated, inactivated at 70 °C, and then added to opaque 96-well plates (20 μL / well). 30 μL of Gluc substrate was added to each well, and the reaction was allowed to proceed for 10 min. Once the fluorescence signal stabilized, Gluc activity was detected using a focused multi-functional fluorescence analyzer. DMSO was used as a negative control, and the culture medium served as a blank control.

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

[0040] 1.4 Cytotoxicity Detection

[0041] PAMs were pre-seeded into 96-well cell culture plates (10) 5(Cells / well). After cell adhesion, different concentrations of PCA were added and incubated at 37°C and 5% CO2. After 48 hours, CTG reagent was added to lyse the cells. The firefly luciferase (Fluc) in CTG reagent reacts with the ATP produced by cell lysis in a redox reaction, causing bioluminescence. DMSO was used as a negative control, and the culture medium was used as a blank control. Each concentration of the compound was tested in triplicate. Cell viability was calculated by detecting the relative light units (RLUs) produced by bioluminescence using a focusing multifunction fluorescence analyzer.

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

[0043] 1.5 qPCR detection

[0044] PAMs were seeded in 24-well cell culture plates. After cell attachment, rASFV-Gluc / EGFP (MOI = 0.2) was mixed with PCA (5, 10, or 25 μM) and used to infect the PAMs. After 2 hours, the supernatant was discarded, and the culture medium was replaced with fresh PCA-only medium for further incubation. After 48 hours of infection, cells and culture supernatant were collected. The samples were added to a nucleic acid extraction or purification kit, and ASFV genomic DNA was extracted using an automated nucleic acid extractor. The extracted genomic DNA was collected in 1.5 mL EP tubes and stored at -30°C. 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 fever virus. J Virol Methods. 2003; 107(1):53-61).

[0045] 1.6 Virus titer determination (HAD) 50 )

[0046] PAMs were seeded in 96-well cell culture plates (10) 5(cells / well), after the cells adhered, the collected virus solution was cultured in RPMI 1640 medium at 10... -1 ~10 -7 The solution was diluted to the desired concentration, with eight replicates for each dilution, and added to pre-spread PAMs. Four days after infection, 10 μL of 1% porcine erythrocyte suspension was added to each well, and the erythrocyte adsorption assay results in the PAMs were observed and recorded after 12 hours. The half-maximal hematoxylin and eosinophil (HAD) at each time point was calculated using the Reed & Muench method. 50 Finally, the growth curve was plotted using Graphpad Prism 8.0.2 software (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 IKKα and IKKβ through the recruitment of TOLLIP. PLoS Pathog. 2023; 19(8):e1011580).

[0047] 1.7 Western blotting

[0048] PAMs were seeded onto 24-well cell culture plates (2×10⁻⁶). 5Cells were cultured in wells (cells / well). After cell adhesion, rASFV-Gluc / EGFP (MOI = 0.2) was mixed with PCA and used to infect PAMs. Two hours later, the supernatant was discarded, and the medium was replaced with fresh RPMI 1640 medium, with the same concentration of PCA added for further culture. Cells were collected 48 hours post-infection and lysed in NP40 lysis buffer (containing the protease inhibitor PMSF) at 4°C for 30 min. The supernatant was then collected after centrifugation at 12000 rpm for 10 min. 5× loading buffer was added, and the mixture was boiled for 10 min to obtain protein samples. After separation by SDS-PAGE, the samples were transferred to a polyvinylidene fluoride membrane and blocked with 5% skim milk at room temperature for 1.5 h. The membrane was then washed three times with Tris-buffered saline with Tween (TBST). The membrane was incubated with mouse anti-p72 (1:200 dilution), rabbit anti-A137R (1:500 dilution) polyclonal antibody, and mouse anti-β-tubulin antibody (1:1000 dilution) as primary antibodies for 3 h at room temperature. The membrane was then washed three times with TBST and incubated with goat anti-rabbit IgG and goat anti-mouse IgG as secondary antibodies for 1 h at room temperature. The membrane was then washed three times with TBST and scanned using a near-infrared fluorescence scanning imaging system (Odyssey CLX).

[0049] 1.8 Statistical Analysis

[0050] All data in this invention were analyzed using GraphPad Prism 8.0.1 software. The significance of differences between groups was determined using unpaired two-tailed t-tests or one-way ANOVA. P ≥ 0.05 indicates no significant difference; * indicates significant difference; ** indicates significant difference; P < 0.01; *** indicates highly significant difference; and **** indicates extremely significant difference.

[0051] 2. Experimental Results

[0052] 2.1 Results of screening compounds from natural small molecule compounds to inhibit African swine fever virus

[0053] Natural small molecule compound library such as Figure 1 As shown in -A, 246 natural small molecule compounds were screened. The inhibitory effect of these natural small molecule compounds on ASFV was evaluated by detecting luciferase activity. The results of the luciferase activity assay are shown below. Figure 1 As shown in Figure -B, at 10 μM, both PCA and brefeldtin A (BFA) achieved inhibition rates of over 90% against ASFV. However, due to the high cytotoxicity of BFA, subsequent experiments only investigated the inhibitory effect of PCA on ASFV. The results of PCA's inhibition of ASFV are shown in Figure -B. Figure 1As shown in Figure C, fluorescence microscopy revealed that 10 μM PCA significantly inhibited the expression of green fluorescent protein compared to the DMSO group. These results all indicate that PCA possesses antiviral activity against ASFV.

[0054] PCA is a type of phenazine compound whose structure includes a phenazine ring and a carboxyl group. Its chemical structural formula is shown below. Figure 1 -D is shown.

[0055] To confirm that the inhibitory effect of PCA was not due to excessive cytotoxicity, cell viability after 48 hours of incubation with PCA in PAMs was first assessed using the CTG method, and the half-maximal cytotoxic concentrations (CC) were calculated. 50 ); test results are as follows Figure 1 As shown in -E, PCA in PAMs CC 50 The value is 470.5 μM.

[0056] This experiment used rASFV-Gluc / EGFP to infect PAMs, measured viral titers, and calculated the half-maximal inhibitory concentration (IC50) of PCA against ASFV. 50 ); test results are as follows Figure 1 As shown in -F, the IC50 of PCA for ASFV is 1.59 μM, and the Selectivity Index (SI) is 295.9.

[0057] The above experimental results indicate that PCA has low cytotoxicity at the dose that achieves significant inhibition of ASFV in PAMs, suggesting that PCA has a wide therapeutic window when used in vitro to inhibit ASFV.

[0058] 2.2 Results of PCA Inhibition of ASFV Replication at Different Concentrations

[0059] To further verify the inhibitory effect of PCA on ASFV replication, the inhibitory effect of different doses of PCA (0, 5, 10 or 25 μM) on ASFV was evaluated using the dual reporter virus rASFV-Gluc / EGFP. Figure 2 -A and Figure 2 -B indicates that, compared with the DMSO control group, PCA significantly inhibited ASFV EGFP fluorescence expression and luciferase activity in a dose-dependent manner. ASFV genome copy number and viral titer results are as follows... Figure 2 -C and Figure 2As shown in Figure -D, the viral genome level and viral titer decreased significantly with increasing PCA dosage. When the PCA concentration reached 25 μM, the inhibitory effect on ASFV could be more than 100-fold.

[0060] To confirm the antiviral activity of PCA against 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 below. Figure 2 As shown in Figure -E, PCA can inhibit the expression of p72 and A137R proteins in a dose-dependent manner.

[0061] The above experimental results indicate 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; wherein the animal virus is African swine fever virus.

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

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