Application of salinomycin and its derivatives in the preparation of anti-FIPV drugs

Abstract

The present invention discloses the application of salinomycin and its derivatives in the preparation of anti-feline infectious peritonitis virus (FIPV) drugs, which belongs to the field of biomedicine technology. Using computer-aided drug design, salinomycin and its derivatives were screened to bind to fAPN receptors and have potential anti-FIPV ability. Further antiviral test results show that salinomycin and its derivatives have a significant inhibitory effect on FIPV, and the derivative M1 has a higher inhibition rate and lower cytotoxicity than salinomycin. The derivative M1 is a compound formed by chemical coupling reaction between the carboxyl group on the C1 position of salinomycin and the amino group on 2-thiophenecarboxaldehydehydrazone. The present invention has the advantages of good efficacy and high safety for the treatment of feline infectious peritonitis (FIP).

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of salinomycin and its derivatives in the preparation of anti-FIPV (Feline Infectious Peritonitis Virus) drugs. Background Art

[0002] Feline infectious peritonitis (FIP) is a highly contagious, complex, and severe disease of cats, primarily caused by the feline infectious peritonitis virus (FIPV). Its overall incidence rate is approximately 10%, and it is characterized by fibrinous and granulomatous serositis and protein-rich serous effusions within the body cavity. The disease has a high mortality rate. Currently, FIP is treated primarily with immunosuppressive and anti-inflammatory drugs, such as high-dose steroids, or with broad-spectrum antibiotics and antivirals to prevent secondary infection. These drugs not only have significant side effects but also provide only symptomatic treatment, temporarily delaying the disease progression and failing to reduce the high mortality rate associated with FIP. Developing new, broad-spectrum, and effective drugs requires identifying drug targets. It is reported that APN (cell surface aminopeptidase N) is an important receptor of coronavirus. It plays an important role in the invasion and infection of many coronaviruses such as porcine transmissible gastroenteritis virus (TGEV), human coronavirus 229E (HCoV-229E), feline infectious peritonitis virus (FIPV), canine coronavirus (CCV) and porcine deltacoronavirus (PDCoV). It is regarded as one of the key targets of coronavirus drugs. Therefore, screening inhibitors against feline aminopeptidase N (fAPN) receptor is a promising approach to develop FIPV drugs.

[0003] Salinomycin is a polyether monocarboxylic acid antibiotic produced by Streptomyces albus. + 、Na + and Rb + It binds tightly, disrupting the normal ion balance inside and outside the membrane, and has a strong inhibitory effect on harmful Gram-positive bacteria, molds, and coccidia in the intestine. This substance is a widely used poultry anticoccidial drug worldwide, characterized by high efficacy, broad spectrum, low resistance, and low residue. In recent years, salinomycin has also been used against coronaviruses, but salinomycin and its derivatives have not been used against FIPV.

[0004] Salinomycin itself has a high toxicity problem, which limits its application in humans and animals. Chemical modification is an important way to alter drug activity, and structurally diverse derivatives can be obtained through techniques such as chemical synthesis. Currently, chemical modifications of salinomycin mainly focus on dehydroxylation, acylation, or hydrogenation reduction of C18-C19 at the C20 position. There are also reports on carboxyl substitution at the C1 position. For example, Huczyński and Antoszczak et al. combined floxuridine and silybin with the C1 carboxyl group to form complexes, which showed enhanced antibacterial efficacy. Other literature reports that the C1 carboxyl group was linked to an L-amino acid methyl ester, which has a strong antibacterial effect on leukemia cell proliferation and doxorubicin-resistant colon cancer cells. However, compounds derived from monosubstituted carboxyl groups, such as halogenation, nitration, or N-benzylamine amidation, have weaker antitumor and antibacterial activities than the original salinomycin.

[0005]

[0006] This project used computer-aided drug design and compound modification technologies to synthesize salinomycin derivatives, and obtained a new compound M1 with a replaced carboxyl group at the C1 position. This not only reduced the toxicity of salinomycin, but also enhanced the anti-FIPV effect targeting fAPN. After searching, no relevant literature has been reported. Summary of the Invention

[0007] The purpose of the present invention is to provide the use of salinomycin and its derivatives in the preparation of anti-FIPV drugs. The present invention also provides a salinomycin derivative and a synthesis method thereof.

[0008] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0009] Based on the fAPN receptor, the applicant used computer-aided drug design and found that the molecular docking score of M1 (Total Score = 12.22) was higher than that of salinomycin (Total Score = 11.01), and the hydrogen bond binding sites of the two were different, indicating that salinomycin and its derivative M1 both have the potential to bind to fAPN, and the derivative M1 has better binding ability than salinomycin.

[0010] On this basis, the applicant discovered through further experiments that: 5 μM salinomycin has a 60.01% inhibitory effect on FIPV, but is toxic to cat kidney cells (CRFK), with a cell viability of 51.04%; 5 μM M1 can significantly inhibit the copy number of FIPV in infected CRFK, and 10 μM M1 has a 105.01% inhibitory effect on FIPV, and has no toxic effect on cat kidney cells at this concentration, with a cell viability of 147.26%.

[0011] The above results show that salinomycin and its derivative M1 have significant inhibitory effects on FIPV and can become new choices for the preparation of anti-FIPV drugs. The inhibitory effect and cytotoxicity of the derivative M1 are better than those of salinomycin.

[0012] The chemical structure of salinomycin is as follows:

[0013]

[0014] The derivatives include but are not limited to pharmaceutically acceptable prodrugs, isomers, salts, crystal forms of salinomycin, or any other derivatives that can be directly or indirectly administered according to the needs of an animal.

[0015] Among them, the salinomycin derivative M1 is a compound formed by a chemical coupling reaction between the carboxyl group at the C1 position of salinomycin and the amino group on 2-thiophenecarboxaldehydehydrazone. The chemical structure of the salinomycin derivative M1 is as follows:

[0016]

[0017] The present invention further provides a method for synthesizing the salinomycin derivative M1: using DMF as a reaction solvent and NHS and EDC as coupling agents, salinomycin is reacted with 2-thiophenecarboxaldehyde hydrazone, so that the hydroxyl group at the C1 position of salinomycin and the amino group on the 2-thiophenecarboxaldehyde hydrazone undergo a chemical coupling reaction.

[0018] Wherein, the molar ratio of salinomycin to 2-thiophenecarboxaldehydehydrazone is 1:1-5.

[0019] Wherein, the reaction temperature is 20-50°C.

[0020] The present invention also provides an anti-FIPV drug, the active ingredient of which is salinomycin or its derivative M1.

[0021] The beneficial effects of the present invention are:

[0022] The present invention is based on the fAPN receptor and uses computer-aided drug design and molecular docking to screen out compounds that effectively inhibit FIPV, meeting the clinical treatment needs of feline infectious peritonitis and overcoming the problem of insufficient efficacy of existing drugs against FIPV.

[0023] The present invention modifies the C1 position of salinomycin, which not only improves the inhibition rate of FIPV but also reduces cytotoxicity. It has the advantages of good efficacy and high safety in treating feline infectious peritonitis.

[0024] The salinomycin derivative provided by the invention has the advantages of simple synthesis process, low cost, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The binding sites and spatial distribution of salinomycin and its derivative M1 to fAPN were simulated by PyMOL 2.4;

[0026] Figure 2 Mass spectrometric identification of M1.

[0027] Figure 3 M1 NMR 1 H spectrum identification.

[0028] Figure 4 M1 NMR 13 C spectrum identification.

[0029] Figure 5 The inhibitory effects of salinomycin and M1 on CRFK cells infected with FIPV. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the technical solution of the present invention, the present invention will be described in detail below with reference to specific embodiments. The specific embodiments described are only part of the embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these specific embodiments, and these improved technical solutions are also within the scope of protection of the present invention.

[0031] Unless otherwise specified, the reagents and materials used in the following examples can be obtained through commercial channels.

[0032] Example 1 Computer-aided drug design screening of small molecule inhibitors targeting fAPN

[0033] 1. SWISS-MODEL modeling

[0034] There is no crystal structure of fAPN in the PDB, so the CDS sequence of fAPN in the NCBI database (NCBI Reference Sequence: NM_001009252) was used for online virtual modeling on the SWISS-MODEL website (https: / / swissmodel.expasy.org / ), and the template was human APN (PDB ID: 6U7F).

[0035] The modeling results showed GMQE=0.84, QMEANDisCo Global=0.87±0.05, and Seq Identity=79.25%. The results met the standards and could be used for subsequent drug screening based on molecular docking.

[0036] 2. According to the Basics Manual SYBYL-X, the modeled fAPN crystal structure and the small molecule compound library were prepared for docking. Then, docking screening was performed in Screen and GeomX modes respectively to screen out the top-ranked small molecule compounds.

[0037] 3. PyMOL 2.4 Simulation of Binding

[0038] The compound ranked high in the above scores was subjected to binding simulation, and it was found that there were many hydrogen bonds and strong binding potential. Based on the molecular structure of salinomycin, SBDD optimization was performed, and the derivative of salinomycin, M1, was designed and docked. The amino acid sites where fAPN binds to salinomycin and M1 were found. The binding hydrogen bonds are marked with dotted lines, and salinomycin, M1 and the binding residue sites are presented in stick form. Regions are represented as surfaces.

[0039] The results are shown in Table 1. The docking score of M1 (Total Score = 12.22) is higher than that of salinomycin (Total Score = 11.01), and their Crash and Polar values ​​are comparable, indicating that M1 may have better binding potential to fAPN than salinomycin.

[0040] Table 1 Scoring table for the binding of salinomycin and its derivative M1 to fAPN after SYBYL-X 2.1.1 simulation screening

[0041] Total Score Crash Polar M1 12.2208 -3.822 5.2338 Salinomycin 11.0091 -3.994 5.5992

[0042] like Figure 1 As shown, the results showed that salinomycin can bind to ASN-349, ALA-350, GLU-379, ARG-380, ASP-438, THR-861 and SER-898 sites through hydrogen bonding forces; M1 can bind to GLU-379, ARG-380, GLU-417, ARG-830 and SER-862 sites of fAPN through hydrogen bonding forces, and the binding sites of the two are different.

[0043] Example 2 Chemical synthesis and structure confirmation of M1

[0044] 1. Extraction of Salinomycin

[0045] Salinomycin raw material (Qilu, China) with a purity of 24% was used as the starting material, which was dissolved in dichloromethane (DCM), then filtered and extracted three times with acidified water (pH = 1.5). The organic phase was dried over anhydrous Na2SO4 for at least 2 h, and the solvent was removed under reduced pressure to obtain a relatively pure salinomycin sample.

[0046] 2. Chemical synthesis of 2-thiophenecarboxaldehyde hydrazone

[0047] To 70 mL of N,N-dimethylformamide (DMF), add 10-20 mL of hydrazine hydrate (CAS: 10217-52-4), followed by the slow dropwise addition of 30-40 mL of 2-thiophenecarboxaldehyde (CAS: 98-03-3). The reaction mixture is stirred at 25°C for 1-4 hours. The solvent is removed under reduced pressure to yield the crude product. Yield: 87%.

[0048] 3. Chemical Synthesis of M1

[0049] Salinomycin (100 mg), N-hydroxysuccinimide (NHS, 245 mg) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 408 mg) were added to N,N-dimethylformamide (DMF, 12 mL) in sequence, and the reaction mixture was stirred for 2-6 h, and 2-thiophenecarboxaldehyde hydrazone (200-300 mg) was added. The reaction mixture was stirred for 12-16 h. The reaction product was monitored by thin layer chromatography (TLC) (n-hexane / ethyl acetate = 1 / 1, relative migration value Rf = 0.25) and observed after carbonization with 5% vanillin sulfuric acid / ethanol solution. The product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2 / 1). The yield was 46%. The product was analyzed by MS, 1 H-NMR and 13 The salinomycin derivative M1 was characterized by various techniques including C-NMR.

[0050] like Figure 2 As shown, the molecular mass of M1 is 858.5, and the mass spectrometer in hydrogenation mode shows the correct molecular mass (859.5014), proving that the product contains the target product.

[0051] like Figure 3 As shown, the NMR of M1 1 H spectrum confirmed that this species was the target product.

[0052] like Figure 4 As shown, the NMR of M1 13 The C spectrum confirmed that the species was the target product.

[0053] Example 3 Inhibitory Effects of Salinomycin and Its Derivatives on FIPV

[0054] 1. Cell Culture and Starvation Treatment

[0055] 2.5×10 5 and 5×10 4Gently tap the perimeter of the culture plate to evenly distribute the cells. Once the cells have settled to the bottom of the plate, place the plate in a 37°C, 5% CO2 incubator. When the cells have grown to 70%-80%, remove the culture medium and wash the cells with sterile PBS. Add serum-free DMEM medium and, after 12 hours, allow the cell cycle to synchronize and remove the culture medium.

[0056] 2. Immunofluorescence assay (IFA) to determine the anti-FIPV effect of the drug

[0057] Basal medium containing FIPV (MOI = 0.1) was added to each well and incubated at 37°C and 5% CO2. The plates were incubated for 1 hour to ensure complete infection, after which the inoculum was removed. Salinomycin and its derivative M1 were dissolved in DMSO to prepare the corresponding stock concentrations. The cells were then overlaid with DMEM medium containing 2% serum containing the corresponding drug. Blank and control groups were also included in the experiment. Cytopathic effects were observed 48 hours after infection, and cells were subjected to indirect immunofluorescence assays and photographed.

[0058] The results are as follows Figure 5 As shown, 5 μM salinomycin and M1 at the same concentration can significantly inhibit CRFK-infected FIPV, and the effect of 5 μM M1 is better than that of salinomycin at the same concentration.

[0059] 3.TCID 50 Methods for measuring FIPV titers

[0060] Dilute the virus solution and dilute the cell culture virus solution 10 times in increments. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 . Take the virus solution of appropriate dilution and mix it with the culture medium at a ratio of 1:1, and inoculate it into a 96-well cell culture plate. Inoculate 6 wells for each dilution, 100 μL per well, and set up virus control and cell control groups at the same time. Culture in a 37°C incubator with 5% CO2 for 48 hours. Observe and record the pathological changes (if more than 70% of the cells show pathological changes, it is considered infected). Use Spearman-Karber TCID 50 Methods Virus titer was calculated.

[0061] The results are shown in Table 2. 5 μM salinomycin had a 60.01±0.83% inhibitory effect on FIPV, but was toxic to CRFK cells, with cell viability of only 51.04±2.10%. 5 μM M1 significantly inhibited the copy number of FIPV in CRFK cells infected with 5 μM M1, while 10 μM M1 had a 105.01±5.30% inhibitory effect on FIPV and was non-toxic to CRFK cells at this concentration, with cell viability of 147.26±1.51%. Orlistat was used as a negative control in this experiment and had no significant inhibitory effect on FIPV at 80 μM.

[0062] Table 2 Anti-FIPV activity of salinomycin and its derivative M1 in CRFK cells

[0063]

[0064]

[0065] In summary, the present invention found that salinomycin and its derivative M1 can significantly inhibit FIPV in CRFK cells, and a low dose (5 μM) in the cells can achieve an ideal inhibitory effect. However, 5 μM salinomycin has obvious toxic side effects on CRFK cells, and its application is limited. M1 at 5-10 μM has a good inhibitory effect on FIPV in CRFK cells. At 10 μM, the inhibition rate is even as high as 105.01 ± 5.30%, and there is no obvious toxic effect at this concentration, which provides a new method for the drug treatment of FIP in clinical production.

[0066] Studies have shown that salinomycin and M1 have a significant inhibitory effect on FIPV. The mechanism may be that they competitively bind to feline aminopeptidase N (fAPN), blocking the virus from binding to and invading host cells, thereby achieving a therapeutic or preventive effect. The present invention provides a new drug option for the prevention and control of coronaviruses, including FIPV. The salinomycin is an animal-specific antibiotic that is currently widely used in animal husbandry clinical treatment. The drug has a strong inhibitory and killing effect on most Gram-positive bacteria, fungi and various coccidia, is not easy to produce drug resistance and cross-resistance, is excreted rapidly, and has the characteristics of high efficiency, broad spectrum, low drug resistance and low residue. Salinomycin and M1 can be quickly used in clinical prevention and control of FIPV, which can greatly save development time and cost and maximize the use of resources.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a salinomycin derivative in the preparation of a medicament for inhibiting feline infectious peritonitis virus, characterized in that: The salinomycin derivative is a compound formed by a chemical coupling reaction between the carboxyl group at the C1 position of salinomycin and the amino group on 2-thiophenecarboxaldehydehydrazone, and its chemical structure is as follows: 。 2. The use according to claim 1, characterized in that The synthesis method of the derivative comprises the following steps: using DMF as a reaction solvent, NHS and EDC as coupling agents, reacting salinomycin with 2-thiophenecarboxaldehyde hydrazone, and causing a chemical coupling reaction between the carboxyl group at the C1 position of salinomycin and the amino group on the 2-thiophenecarboxaldehyde hydrazone.

3. The use according to claim 2, characterized in that: The molar ratio of salinomycin to 2-thiophenecarboxaldehydehydrazone is 1:1-5.

4. The use according to claim 2, wherein: The reaction temperature is 20-50°C.

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