Use of human cathelicidin derivatives for the preparation of a medicament against enteroviruses

Abstract

The application provides application of a human-derived CATHELICIDIN derivative in preparation of an antienterovirus drug, and the human-derived CATHELICIDIN derivative is a peptide shown in the following amino acid sequence or a pharmaceutically acceptable salt or a pharmaceutically acceptable derivative thereof; FRKSK EKIGK XFKRI VQRIX DFLRN LV, wherein X is a hydrophobic amino acid. The derivative is highly efficient against enterovirus, and can treat or prevent diseases caused by enterovirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and applied technology, specifically relating to the application of human CATHELICIDIN derivatives in the preparation of anti-enterovirus drugs. Background Technology

[0002] Enteroviruses are a large family of viruses, encompassing many that cause human diseases. Enterovirus 71 (EV71) is a common pathogen causing hand, foot, and mouth disease (HFMD). HFMD typically affects children under 6 years old and is a serious public health threat, particularly in the Asia-Pacific region. China reports approximately 2 million cases of HFMD annually, with nearly 1 million of these being EV71 infections. Infection usually causes rashes and oral ulcers; some patients infected with EV71 experience severe neurological symptoms, leading to acute flaccid paralysis and death. To date, there are no effective vaccines or drugs against EV71 for clinical treatment, making the development of anti-EV71 drugs an urgent priority.

[0003] Polypeptides possess advantages such as high specificity, strong tolerability, few side effects (the final metabolite is amino acids), and low likelihood of inducing drug resistance, making them ideal candidates for the treatment of viral infections. For example, Chinese invention CN201810508832.2 "A class of antiviral polypeptides and their preparation methods and applications" discloses a class of polypeptides with antiviral activity against various viruses such as cucumber mosaic virus and barley stripe mosaic virus; Chinese invention CN200910191485.6 "Human α-defensin 5 antiviral mutant polypeptides and their preparation methods and applications" discloses the role of human α-defensin 5 (HD5) derivatives in preventing and treating viral infections such as HSV, HPV, and HIV; and Chinese invention CN201010257748.1 "A scorpion-derived antiviral active polypeptide and its uses" discloses the application of a scorpion-derived active polypeptide AVP-W3 in the preparation of treatments or prevention of diseases caused by HCV virus.

[0004] The human body mainly produces two major classes of antimicrobial peptides: the Defensin family peptides and the Cathelicidin family peptides. The Defensin family encompasses a variety of antimicrobial peptides, while the Cathelicidin family contains only one polypeptide in human cells: LL-37 (hCAP18). LL-37 contains 37 amino acids, with its primary amino acid sequence being LLGDF FRKSK EKIGK EFKRI VQRIKDFLRN LVPRT ES. It is mainly expressed in bone marrow cells and epithelial cells of many organs, and possesses antibacterial, antiviral, wound-healing, and immunomodulatory functions.

[0005] Human cathelicidin derivatives are derived from a core polypeptide of 27 amino acids (DL-37, amino acid sequence: FRKSK EKIGK EFKRI VQRIK DFLRN LV). Through the substitution of hydrophobic and cationic amino acid residues, peptides with stronger antiviral activity, namely LL-18 and FF-18, were obtained. Isogai et al. (2003) demonstrated that LL-18 and FF-18 peptides can attenuate the activity of Porphyromonas spp. LPS and induce membrane disruption, exhibiting antibacterial effects against Porphyromonas and Prevotella species. Kuroda et al. (2012) found that FF-18 has an antiproliferative effect on the colon cancer cell line HCT116. Qin et al. (2019) found that FF-18 can alleviate the progression of sepsis-induced acute lung injury. Chinese invention patent document CN200480003209.8 discloses the use of LL-37 and its derivatives in the preparation of wound healing drugs; Chinese invention patent document CN201810742563.6 discloses an LL-37 derivative and its application in antibacterial and antifungal effects; US patent document US10546739, "Anti-viral activity of cathelicidin peptides," discloses the antiviral activity of LL-37, including viruses such as poxvirus, herpesvirus, vaccinia virus, and papillomavirus. However, currently published documents or patents do not describe the antiviral activity of DL-37-derived peptides against enteroviruses. Summary of the Invention

[0006] To address the problems of the prior art, this invention provides the application of human cathelicidin derivatives in the preparation of anti-enterovirus drugs. These derivatives possess highly efficient anti-enterovirus activity and can treat or prevent diseases caused by enteroviruses.

[0007] This invention is achieved through the following technical solution:

[0008] The application of human CATHELICIDIN derivatives in the preparation of anti-enterovirus drugs, wherein the human CATHELICIDIN derivative is a peptide or a pharmaceutically acceptable salt or a pharmaceutically acceptable derivative thereof, as shown in the following amino acid sequence;

[0009] FRKSK EKIGK XFKRI VQRIX DFLRN LV

[0010] Wherein, X is a hydrophobic amino acid.

[0011] The use of human CATHELICIDIN derivatives in the preparation of medicaments for the treatment or prevention of diseases caused by enteroviruses, wherein the human CATHELICIDIN derivative is a peptide with the following amino acid sequence, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable derivative thereof;

[0012] FRKSK EKIGK XFKRI VQRIX DFLRN LV

[0013] Wherein, X is a hydrophobic amino acid.

[0014] Preferably, X is leucine or phenylalanine.

[0015] Preferably, the amino acid sequence of the peptide is FRKSK EKIGK LFKRI VQRIL DFLRN LV, or FRKSKEKIGK FFKRI VQRIF DFLRN LV.

[0016] Preferably, the pharmaceutically acceptable salt is an acetate, carbonate, phosphate, sulfate, trifluoroacetate, or chloride.

[0017] Preferably, the pharmaceutically acceptable derivative is an ester or an amide.

[0018] Preferably, the enterovirus is poliovirus, Coxsackievirus, or echovirus, or enterovirus type 68, 69, 70, or 71.

[0019] Preferably, the enterovirus is enterovirus 71.

[0020] Preferably, the disease is hand-foot-mouth disease, viral pharyngitis, viral myocarditis, pulmonary edema, or encephalitis.

[0021] Preferably, the dosage form of the drug is tablets, granules, pills, capsules, or oral liquid.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention is the first to apply cathelicidin derivatives to the research of anti-enterovirus drugs. Results show that it possesses good anti-enterovirus activity and can treat or prevent diseases caused by enteroviruses. Experiments were conducted to determine its optimal concentration for effective antiviral treatment while minimizing toxic side effects, thus alleviating the current shortage of anti-enterovirus drugs. Furthermore, this invention explores the mechanism of action of the peptide, which exerts its antiviral effect by directly binding to the virus and inhibiting its entry into host cells. This invention provides an important reference for the development of antiviral drugs and has promising applications in treating diseases caused by enterovirus infections. Attached Figure Description

[0024] Figure 1 In the table, (a) shows the effect of LL-18 and FF-18 on the viability of enterovirus EV71-infected cells: viral infection caused a decrease in cell viability, while LL-18 and FF-18 were able to save cell viability; (b) shows the protein levels of the virus in the cells, and treatment with LL-18 and FF-18 reduced the expression level of viral proteins.

[0025] Figure 2 In the figures, (a) shows the viral load in brain and muscle tissues, and LL-18 treatment reduced the viral load; (b) shows the mortality rate of infected mice, and LL-18 treatment reduced the mortality rate of mice.

[0026] Figure 3 In the image, (a) is a schematic diagram of CABS-dock simulating the binding of LL-18 to the virus; (b) is the result of immunoprecipitation of LL-18 peptide with viral capsid protein, showing that LL-18 can directly interact with viral capsid proteins VP1-VP3.

[0027] Figure 4 LL-18 and FF-18 inhibited viral invasion of host cells. (a) shows the effect of LL-18 on viral adsorption; (b) shows the effect of FF-18 on viral adsorption; (c) shows the effect of LL-18 on viral internalization; and (d) shows the effect of FF-18 on viral internalization. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0029] The human-derived CATHELICIDIN derivatives of this invention are the peptides shown below and their pharmaceutically acceptable salts or derivatives.

[0030] FRKSK EKIGK XFKRI VQRIX DFLRN LV

[0031] Wherein, X is any hydrophobic amino acid, preferably leucine (L) and phenylalanine (F), referred to in this invention as LL-18 and FF-18, respectively. The amino acid sequence of LL-18 is FRKSK EKIGK LFKRI VQRIL DFLRN LV, and the amino acid sequence of FF-18 is FRKSK EKIGK FFKRI VQRIF DFLRN LV.

[0032] The pharmaceutically acceptable salts include, for example, antagonistic acetates, carbonates, phosphates, sulfates, trifluoroacetates, and chlorides, with phosphates being a preferred salt. Examples of the pharmaceutically acceptable derivatives are esters or amides.

[0033] The application of the human cathelicidin derivative in the preparation of anti-enterovirus drugs.

[0034] The use of the human cathelicidin derivative in the preparation of drugs for the treatment or prevention of diseases caused by enteroviruses.

[0035] The enteroviruses include: poliovirus serotypes 1, 2, and 3; Coxsackievirus A1-24 and B1-6; echovirus 1-34; and enteroviruses 68, 69, 70, and 71, with enterovirus 71 (EV71) being the preferred type.

[0036] The diseases mentioned include hand-foot-mouth disease, viral pharyngitis, viral myocarditis, pulmonary edema, encephalitis, etc.

[0037] The dosage forms of the drug include tablets, granules, pills, capsules, or oral liquids.

[0038] This invention also discloses the dosage of the human cathelicidin derivative in the preparation of a medicament for the prevention or treatment of EV71 infection. The medicament effectively inhibits EV71 infection at a final concentration of 0.3–20 μM. In a preferred embodiment, the peptide exhibits highly efficient inhibitory activity against EV71 infection. In in vitro experiments, the peptide exerts a cell-protective effect at a final concentration of 0.3 μM, and achieves approximately 100% protection at 1.5 μM. In animal experiments, administration of the peptide significantly reduces viral load in mice, effectively reducing mortality after viral infection.

[0039] This invention discloses the mechanism of action of the peptide against enteroviruses. The peptide can directly bind to enteroviruses and inhibit their entry into cells, effectively inhibiting viral infection in the early stages of the viral life cycle.

[0040] Example 1: Determination of the in vitro antiviral activity of the peptide by CTG (Cell Titer-Glo) luminescence assay and Western blotting

[0041] Principle and Purpose: ATP (adenosine triphosphate) participates in various enzymatic reactions in organisms and is an indicator of cellular metabolism; its content directly reflects the number and state of cells. Western blotting involves staining gel electrophoresis-treated cell or tissue samples with specific antibodies. By analyzing the location and depth of staining, information about the expression of specific proteins in the analyzed cells or tissues can be obtained, thus reflecting the number of virus-infected cells.

[0042] Methods: EV71 virus was incubated with different concentrations of peptides (DL-37, LL-18, FF-18) or PBS for 1 hour at room temperature. The mixture was then added to human rhabdomyosarcoma cells (RD) with approximately 90% confluence. After culturing at 37°C in a 5% CO2 incubator for 24 hours, the culture medium was aspirated, and 100 μl of CTG (CELL TITER-GLO) solution was added. Cells were lysed by shaking for 5 minutes and then transferred to 384-well plates. The luminescence value was detected using a BioTek NEO2 microplate reader, and the relative cell viability was calculated. The same procedure was performed on the cells, and after 24 hours, cells were collected for SDS-PAGE and Western blot analysis. Cell viability without peptides and with an equal amount of virus served as negative and positive controls, respectively.

[0043] Results Analysis: The results are as follows Figure 1 As shown in (a), equal amounts of DL-37 did not exhibit significant antiviral activity. LL-18 and FF-18 enhanced the viability of virus-infected cells, indicating that the substitution of hydrophobic amino acids significantly enhanced the antiviral activity of DL-37. At 0.3 μM, LL-18 and FF-18 could exert antiviral activity against enterovirus 71. At 1.5 μM, the cell viability of the LL-18 and FF-18 treated groups was almost similar to that of the negative control group, indicating that LL-18 and FF-18 have good antiviral activity in vitro. Figure 1 (b) shows that the application of 1.5 μM LL-18 and FF-18 can significantly reduce the protein level of the virus in cells, indicating that the viral infection is suppressed.

[0044] Example 2: The peptide can effectively protect mice infected with the virus.

[0045] Principle and Purpose: Mice are the primary animal model for evaluating drug efficacy, and intraperitoneal injection is a common route of administration. MP4 is a mouse-adapted EV71 strain; a certain dose of MP4 induces typical clinical symptoms in mice, such as hind limb paralysis and death. This model can be used to explore the in vivo antiviral activity of the described peptide. TCID 50 The viral titer refers to the amount of virus required to induce cytopathic effect (CPE) in culture plate wells or test tubes. It is used to characterize the viral titer and is often used to identify the in vivo antiviral effect of drugs by detecting viral titers in mice.

[0046] Methods: An appropriate amount of MP4 virus was incubated with an equal volume of PBS or LL-18 for one hour. Six 3-day-old ICR newborn mice were intraperitoneally infected with the virus at a dose of 50 μl per mouse. After 48 hours, the mice were euthanized by dislocation, and equal masses of muscle and brain tissue were collected, homogenized in PBS, centrifuged, and filtered to release the virus from the cells. The viral TCID was then measured. 50 Meanwhile, an appropriate amount of MP4 virus was incubated with an equal volume of PBS or LL-18 for one hour, and 50 μl / mouse was injected into 3-day-old ICR newborn mice for intraperitoneal infection. Eight mice were in each group, and the mortality rate of the mice was observed.

[0047] Results Analysis: The results are as follows Figure 2 As shown in (a)-(b), the viral titers in the brain and muscle tissues of mice treated with the peptide LL-18 were significantly reduced; and compared to the viral injection group, Figure 2 (c) shows that the survival rate of mice in the LL-18 administration group was significantly increased, the mortality rate of infected mice was reduced, and the mice were effectively protected, indicating that the LL-18 peptide can also exert antiviral effects in vivo.

[0048] Example 3: CABS-dock simulates the binding of the peptide to the virus.

[0049] Principle and Purpose: CABS-dock does not require pre-estimation of binding sites. Instead, based on a given protein receptor structure and peptide sequence, it simulates and searches for binding sites starting from the peptide's random conformation and position. This allows for flexibility in peptide structure and small fluctuations in receptors. The simulated model is filtered and clustered (grouped by similar binding modes and similar peptide conformations) to calculate the rational binding patterns between proteins and peptides.

[0050] Method: Use CABS-dock web server( CABS-dock:server forProtein-peptide docking was used to analyze the binding of the virus to the peptide LL-18. The viral protein receptor structure was derived from PDB. The EV71 structure of PDBID 4AED and the amino acid sequence of the LL-18 peptide were input for docking analysis, with other parameters left at default.

[0051] Results analysis: such as Figure 3 As shown in (a), the peptide LL-18 binds to the viral capsid protein and has binding sites for viral capsid proteins VP1, VP2, and VP3. Theoretically, the peptide LL-18 binds directly to the virus, suggesting that the peptide may exert its antiviral effect by directly binding to the virus.

[0052] Example 4: Detection of the interaction between the peptide and viral capsid protein using immunoprecipitation technique.

[0053] Principle and Purpose: Co-immunoprecipitation (Co-IP) is based on the specific binding between antibodies and antigens and is used to detect and determine protein-protein interactions under physiological conditions. Its basic principle involves lysing cells under non-denaturing conditions, adding specific antibodies to the cell lysate, allowing the antibodies to form antigen-antibody complexes with known antigens. If a protein interacting with a known antigen is present in the system, that protein will also precipitate as a complex. After elution, the protein interacting with the known antigen can be obtained.

[0054] Methods: VP1-HA, VP2-HA, VP3-HA, LL18-eGFP, and eGFP expression vectors were constructed. These vectors were co-transfected into 293T cells in pairs to co-express the target proteins. Cells were harvested 48 h after transfection and the proteins were released in cell lysis buffer containing 1% protease inhibitor. HA-antibody and beads-protein A / G (magnetic beads) were added to co-precipitate the proteins. The beads were collected and added to LDS loading buffer. After denaturation, SDS-PAGE and Western blot analysis were performed.

[0055] Results analysis: The results of the co-precipitation immunoprecipitation are as follows: Figure 3 As shown in (b), after the HA antibody binds to the beads-protein A / G magnetic beads, it specifically recognizes and binds to VP1-HA, VP2-HA, and VP3-HA proteins. LL18-eGFP protein was detected in the proteins adsorbed by the magnetic beads, but eGFP protein was not detected, indicating that LL-18 has bound to the viral capsid protein.

[0056] Example 5: qPCR detection of the effect of the peptide on viral adsorption and internalization

[0057] Principle and Purpose: At 4℃, the virus only adsorbs onto cells, and its internalization is inhibited. Therefore, by quantifying the virus adsorbed onto cells with or without the addition of the peptide using qPCR, the effect of the peptide on virus adsorption can be detected. At 4℃, after ensuring that the amount of virus adsorbed by the control group and the experimental group is the same, the peptide is added and treated for 1 hour. Then, the cells are transferred to 37℃ to allow the virus to internalize. The effect of the peptide on virus internalization can be detected by quantifying the internalized virus using qPCR.

[0058] Methods: 1.5 μM LL-18 / FF-18 or an equal volume of PBS was incubated with an appropriate amount of viral particles at room temperature for 1 h. RD cells with a confluence of approximately 90% were added, and the mixture was aspirated after adsorption at 4 °C for 1 h. The cells were washed three times with cold PBS, the cells were collected, RNA was extracted, reverse transcribed, and qPCR was used to detect the viral VP1 mRNA level to detect the effect of the peptide on viral adsorption.

[0059] An appropriate amount of viral particles were added to RD cells with a confluence of approximately 90%. After adsorption at 4°C for 1 hour, the particles were aspirated. The cells were washed three times with cold PBS, and 1.5 μM LL-18 / FF-18 or an equal volume of PBS was added. After incubation for 1 hour, the particles were aspirated. After adding culture medium, the cells were transferred to 37°C to allow the adsorbed virus to internalize and enter the cells. After 1 hour, the cells were treated with trypsin for 1 minute, washed three times with cold PBS to remove uninternalized virus, and the cells were harvested. RNA was extracted, reverse transcribed, and qPCR was used to detect the viral VP1 mRNA level to detect the effect of the peptide on viral internalization.

[0060] Results analysis: such as Figure 4 As shown, the qPCR quantitative results indicate that the amount of virus adsorbed on the cell surface or internalized is significantly reduced under the action of the peptides LL-18 and FF-18, indicating that the peptides LL-18 and FF-18 inhibit viral adsorption and internalization, and also confirming that the peptides LL-18 and FF-18 play an antiviral role in the early stage of viral life.

Claims

1. The application of human cathelicidin derivatives in the preparation of anti-enterovirus drugs, characterized in that, The human-derived cathelicidin derivative is a peptide or a pharmaceutically acceptable salt thereof, as shown in the following amino acid sequence. The amino acid sequence is FRKSK EKIGK LFKRI VQRIL DFLRN LV, or FRKSK EKIGK FFKRIVQRIF DFLRN LV; The enterovirus in question is enterovirus 71.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salts are acetates, carbonates, phosphates, sulfates, trifluoroacetates, or chlorides.

3. The application according to claim 1, characterized in that, The dosage form of the drug is tablets, granules, pills, capsules, or oral liquid.

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