Broad-spectrum anti-enterovirus peptidomimetic 2CA-1 targeting enterovirus 2C protein and application of broad-spectrum anti-enterovirus peptidomimetic 2CA-1

Abstract

The invention belongs to the field of biological medicine, and discloses a broad-spectrum anti-enterovirus peptidomimetic 2CA-1 targeting enterovirus 2C protein and application of the broad-spectrum anti-enterovirus peptidomimetic 2CA-1. The 2CA-1 can be accurately butted to 2C protein binding pockets of EV71 and CVB3, the helicase activity of the 2CA-1 is effectively inhibited, and the 2CA-1 shows a strong and broad-spectrum antiviral effect on various pathogens such as coxsackie virus, EV-D68 and rhinovirus on the cellular level. More importantly, the 2CA-1 realizes a druggability breakthrough of excellent oral bioavailability in an animal model, and the in-vivo virus load can be remarkably reduced after oral administration. The invention provides an antiviral candidate drug with high-efficiency broad-spectrum activity and ideal oral administration characteristics, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a broad-spectrum anti-enterovirus peptide 2CA-1 targeting enterovirus 2C protein and its applications. Background Technology

[0002] Enteroviruses are a class of positive-sense single-stranded RNA viruses belonging to the genus Enterovirus within the family Picornaviridae. This genus encompasses a variety of common pathogens, such as human enterovirus (EV), Coxsackievirus A and B, echovirus, rhinovirus, and poliovirus. Enterovirus infections are widespread globally, with diverse clinical manifestations ranging from mild to severe: mild cases may present with only low-grade fever, fatigue, or respiratory symptoms, while severe cases can cause herpetic pharyngitis, hand-foot-and-mouth disease, and even serious complications such as aseptic meningitis, myocarditis, acute flaccid paralysis, and encephalitis. Currently, there are no drugs available clinically that can effectively treat or specifically combat these viral infections.

[0003] Herpetic pharyngitis is mainly caused by Coxsackievirus A (CVA2, CVA4, CVA6, etc.) and Coxsackievirus B (CVB1 to CVB5, etc.). The disease often has an acute onset, primarily with low- to moderate fever, occasionally exceeding 40°C, and even triggering febrile seizures. Older children may complain of sore throat, affecting swallowing; infants and young children often present with drooling, refusal to eat, and irritability. Characteristic lesions are concentrated in the pharynx: significant pharyngeal congestion, and within two days of onset, several grayish-white vesicles about 1–2 mm in diameter appear on the oral mucosa, surrounded by a red halo. These lesions rupture after 2–3 days, forming yellow ulcers. The entire course of the disease lasts about 4–6 days, but in a few cases, it can extend to two weeks.

[0004] Hand, foot, and mouth disease is mostly caused by enterovirus 71, CVA6, CVA16, and CVB. Typical clinical manifestations include acute fever, oral pain, loss of appetite, and scattered vesicles or ulcers on the oral mucosa. Simultaneously, maculopapular rashes appear on the hands, feet, and buttocks, rapidly transforming into vesicles surrounded by an inflammatory halo. Some children initially present only with herpetic pharyngitis before developing the typical rash. If the disease progresses rapidly, a small number of children may develop serious complications such as aseptic meningitis and encephalitis.

[0005] Viral myocarditis is mainly caused by Coxsackievirus B1–6 and echovirus, among others. Its clinical manifestations depend on the extent and location of myocardial involvement; mild cases may be asymptomatic, while severe cases can lead to heart failure, cardiogenic shock, or even sudden death. Most patients have a history of upper respiratory or intestinal infection 1–3 weeks prior to onset, followed by cardiac symptoms such as palpitations, chest tightness, chest pain, dyspnea, or edema.

[0006] Respiratory infections are mainly caused by enterovirus D68 (EV-D68), and clinical manifestations can range from common cold-like symptoms to severe respiratory illnesses such as bronchitis and pneumonia, especially triggering acute exacerbations of childhood asthma. Some cases may develop neurological complications, including acute flaccid paralysis (AFM), characterized by sudden weakness and decreased muscle tone in one or more limbs, which can severely impair respiratory muscle function.

[0007] The common cold in humans is primarily caused by rhinoviruses (HRV), typically presenting with upper respiratory tract catarrhal symptoms such as nasal congestion, runny nose, sneezing, and sore throat, usually accompanied by low-grade fever, headache, and general weakness. In infants, the elderly, and immunocompromised individuals, infection can spread to the lower respiratory tract, causing bronchitis, pneumonia, or leading to acute exacerbations of underlying respiratory conditions such as asthma and chronic obstructive pulmonary disease. Summary of the Invention

[0008] The purpose of this invention is to provide a broad-spectrum anti-enterovirus peptide 2CA-1, the chemical formula of which is Cbz-ALFQ-NH2.

[0009] Another object of the present invention is to provide the application of peptide 2CA-1 in broad-spectrum anti-enterovirus.

[0010] To achieve the above objectives, the present invention adopts the following technical measures:

[0011] The applicant, addressing the shortcomings of the lead peptide 2CL, such as potential non-specific binding due to its cell-penetrating peptide motif and the possibility of its extended structure limiting binding efficiency, analyzed and modified the core sequence of the viral 2C helicase protein. From a large pool of peptides, a peptide-1, 2CA-1, was selected based on optimal safety, broad-spectrum activity, and antibacterial properties. 2CA-1 precisely binds to the 2C protein binding pocket of EV71 and CVB3, effectively inhibiting their helicase activity. At the cellular level, it exhibits potent and broad-spectrum antiviral activity against multiple pathogens, including Coxsackievirus, EV-D68, and rhinovirus. Crucially, 2CA-1 achieved a breakthrough in drug development by demonstrating excellent oral bioavailability in animal models, significantly reducing viral load after oral administration. The chemical formula of the peptide is Cbz-ALFQ-NH2, and its structural formula is:

[0012] .

[0013] The scope of protection of this invention also includes:

[0014] A complex containing peptide 2CA-1.

[0015] The use of peptide 2CA-1 or the above complex in the preparation of drugs for enterovirus infection, wherein the enteroviruses include: enterovirus 71, coxsackievirus A6, A16, B3, echovirus 11, enterovirus D68 and rhinovirus.

[0016] The use of peptide 2CA-1 or the above-mentioned complex in the preparation of drugs for the treatment or prevention of herpetic pharyngitis.

[0017] The use of peptide 2CA-1 or the above-mentioned complex in the preparation of drugs for the treatment or prevention of hand-foot-mouth disease.

[0018] The use of peptide 2CA-1 or the above-mentioned complex in the preparation of drugs for the treatment or prevention of viral myocarditis.

[0019] The use of peptide 2CA-1 or the above-mentioned complex in the preparation of drugs for the treatment or prevention of respiratory tract infections.

[0020] The use of peptide 2CA-1 or the above-mentioned complex in the preparation of drugs for the treatment or prevention of the common cold in humans.

[0021] A broad-spectrum antiviral drug comprising peptide 2CA-1.

[0022] The dosage forms of the drugs described above are pharmaceutically acceptable.

[0023] Preferably, the dosage form is a tablet, capsule, granule, injection, powder, or drop.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] Compared with existing technologies, this invention achieves a significant technological breakthrough: the provided peptide mimic 2CA-1 possesses a novel, compact molecular structure that precisely targets the highly conserved helicase activity pocket of the viral 2C protein, efficiently inhibiting its function. This design strategy yields superior broad-spectrum antiviral properties—experiments have demonstrated potent and consistent inhibitory activity against a variety of pathogens, including Coxsackievirus A6, A16, and B3, Echovirus 11, Enterovirus D68, and Rhinovirus, overcoming the limitations of existing antiviral drugs that typically target only a single or a few viral types. This broad-spectrum inhibitory effect based on key conserved targets enables it to effectively address the public health challenges of rapid mutation of enterovirus populations and the co-circulation of multiple serotypes. This invention is the first to successfully develop a lead compound with both a novel structure and a broad antiviral spectrum, laying an important foundation for establishing a universal therapeutic solution covering multiple enterovirus infections. Attached Figure Description

[0026] Figure 1A-1CResults of assays showing the inhibitory efficiency of peptides 2CA-1, 2CA-2, and 2CA-3 on EV71 in RD cells.

[0027] Figure 2A-2C Results of toxicity assays of peptides 2CA-1, 2CA-2, and 2CA-3 in RD cells.

[0028] Figure 3 The results of assays showing that the peptide 2CA-1 inhibits EV71 cell pathogenesis in RD cells.

[0029] Figure 4 The results of assays showing that the peptide 2CA-1 inhibits EV71 plaque formation in RD cells.

[0030] Figures 5A-5C Results of assays showing the efficiency of peptide 2CA-1 in inhibiting EV71 in various cell types.

[0031] Figures 6A-6C The results of toxicity assays of the peptide 2CA-1 in various cell types were presented.

[0032] Figures 7A-7B A schematic diagram showing the binding pattern of the peptide 2CA-1 to the EV71 2C protein.

[0033] Figure 8 Results showing that peptides 2CA-1, 2CA-2, and 2CA-3 inhibit the helicase activity of EV71 2C protein.

[0034] Figures 9A-9C Results of the detection of antiviral activity of peptides 2CA-1, 2CA-2 and 2CA-3 against EV71 in mice.

[0035] Figures 10A-10C Results of assays on the antiviral activity of peptide 2CA-1 against EV71 in mouse muscle, lung and brain.

[0036] Figure 11 The results of toxicity testing of the peptide 2CA-1 in mice were shown.

[0037] Figures 12A-12C The peptide 2CA-1 exhibits oral activity with the aid of adjuvants.

[0038] Figures 13A-13F Results of assays on the efficiency of peptide 2CA-1 in inhibiting multiple enteroviruses in RD cells. Detailed Implementation

[0039] This invention relates to novel antiviral peptide mimics (named 2CA-1, 2CA-2, and 2CA-3) targeting the 2C protein of enteroviruses (EVs) and their applications. The specific embodiments of this invention will be described in detail below. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all derived from commercial channels or are publicly available materials. The peptide mimics 2CA-1, 2CA-2, and 2CA-3 were all synthesized by Nanjing Genscript Biotech Co., Ltd., as detailed below:

[0040] The chemical formula of 2CA-1 is Cbz-ALFQ-NH2, and its structural formula is: ;

[0041] The chemical formula of 2CA-2 is {5-F-benzoicacid}-ALFQ-NH2, and its structural formula is: ;

[0042] The chemical formula of 2CA-3 is: Benzoyl-ALFQ-NH2, and its structural formula is: .

[0043] Example 1:

[0044] Determination of the inhibitory efficiency of peptides 2CA-1, 2CA-2 and 2CA-3 on EV71 in RD cells

[0045] 1. Experimental Materials

[0046] RD cells, DMEM medium (Thermo), and serum (Gibco) were purchased from Invitrogen. The Total RNA Extraction Kit (Omega) and One-Step qRT-PCR Kit (Takara) were purchased from Yuming. DEPC water was used for RNA extraction and qRT-PCR. The entire experiment was conducted in an RNase-free environment.

[0047] 2. Experimental Procedure

[0048] (1) Use RD cells to form 24-well plates.

[0049] (2) When the culture reaches 70%-80% confluence, replace the DMEM medium containing 10% serum with DMEM medium containing 2% serum (0.5 mL of 2% serum DMEM medium is added to each well), and add 5 μL of 1×10 6 PFU / mL EV71 virus.

[0050] (3) After 1 h, the supernatant was discarded and different peptides were added to the supernatant at final concentrations of 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM and 20 μM respectively, with the group without peptides as the control.

[0051] (4) EV71 virus (VR-1432), see Fang, Y., et al. Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviralinfection in vivo. Immunity. 2021 Oct 12;54(10):2231-2244.e6. Samples were collected 24 h after infection and RNA was extracted using the Total RNA Extraction Kit.

[0052] (5) Discard the supernatant, add 350 μL of TRK lysis buffer to the well, and place it on a shaker for 5 min.

[0053] (6) Add 350 μL of 70% ethanol (DEPC) to the well and place it on a shaker for 5 min.

[0054] (7) Remove the solution from the well and transfer it to the RNA extraction column. Centrifuge at 12000 g for 1 min.

[0055] (8) Load the solution in the recovery tube back onto the column and centrifuge at 12000 g for 1 min.

[0056] (9) Add RNA washing buffer 1 and centrifuge at 12000 g for 30 s.

[0057] (10) Add RNA washing buffer 2 and centrifuge at 12000 g for 1 min.

[0058] (11) Repeat step (10).

[0059] (12) Empty column 12000 g, 2 min, to completely remove residual RNA washing buffer.

[0060] (13) Add 50 μL of DEPC water and centrifuge at 12000 g for 2 min.

[0061] (14) Take 2 μL of RNA sample and perform a fluorescence quantitative experiment using a one-step qRT-PCR kit.

[0062] The results are as follows Figures 1A-1C Table 1-3 shows the results of the anti-EV71 effects of peptides 2CA-1, 2CA-2, and 2CA-3 in RD cells: Peptide 2CA-1 showed an IC50 value of 100% in RD cells. 50 1.609 μM ( Figure 1A ), peptide 2CA-2 in RD cells IC 50 1.734 μM ( Figure 1B ), peptide 2CA-3 IC50 in RD cells 50 1.126 μM ( Figure 1C ).

[0063] Table 1

[0064] .

[0065] Table 2

[0066] .

[0067] Table 3

[0068] .

[0069] Example 2:

[0070] Assay for the toxicity of peptides 2CA-1, 2CA-2, and 2CA-3 in RD cells

[0071] 1. Experimental Materials

[0072] RD cells; DMEM medium (Thermo) and serum (Gibco) were purchased from Invitrogen; CCK-8 reagent (MCE) was purchased from Startup subsidiary.

[0073] 2. Experimental Procedure

[0074] In the process of antiviral activity, peptides must not only inhibit viruses but also ensure that they are non-toxic to cells. Therefore, this indicator is detected by a cytotoxicity test, with untreated cells serving as the control group.

[0075] The steps are as follows:

[0076] (1) Use RD cells to seed 96-well cell plates, 100 μL per well.

[0077] (2) When the peptide reaches 70%-80% confluence, replace the DMEM medium containing 10% serum with DMEM medium containing 2% serum, and add peptides 2CA-1, 2CA-2 or 2CA-3 at a certain concentration gradient so that the final peptide concentrations in the wells are 0.977 μM, 1.953 μM, 3.906 μM, 7.813 μM, 15.625 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM and 500 μM respectively.

[0078] (3) After adding the peptide for 24 hours, collect the sample and add 10 μL of live cell detection reagent CCK-8 to each well and mix well.

[0079] (4) Place at 37℃ for 2 hours.

[0080] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of OD450.

[0081] The results are as follows Figure 2A-2C As shown in Table 4-6, with the cell viability of untreated cells as 100%, the CC of each peptide was calculated. 50 2CA-1's CC 50 >500 μM ( Figure 2A ), 2CA-2 of CC 50 >500 μM ( Figure 2B ), 2CA-3 of CC 50 515.7 μM ( Figure 2C ).

[0082] Based on the results of Examples 1 and 2, 2CA-1 showed higher inhibition efficiency against EV71 while ensuring safety. Therefore, this peptide was used for further experiments.

[0083] Table 4

[0084] .

[0085] Table 5

[0086] .

[0087] Table 6

[0088] .

[0089] Example 3:

[0090] Assay for 2CA-1 inhibiting EV71 cytopathic effect in RD cells

[0091] 1. Experimental Materials

[0092] RD cells; DMEM medium (Thermo) and serum (Gibco) were purchased from Invitrogen.

[0093] 2. Experimental Procedure

[0094] (1) Use RD cells to form 24-well plates.

[0095] (2) When the culture reaches 70%-80% confluence, replace the DMEM medium containing 10% serum with DMEM medium containing 2% serum (0.5 mL of 2% serum DMEM medium is added to each well), and add 5 μL of 1×10 6 PFU / mL EV71 virus.

[0096] (3) After 1 h, the supernatant was discarded and peptide 2CA-1 was added to a final concentration of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM and 10 μM respectively, with the group without peptide added as the control.

[0097] (4) Samples were collected 24 h after EV71 virus infection, and the cytopathic effects were observed and images were taken under a microscope that could capture images.

[0098] The results of the detection of anti-EV71 cytopathic effect of peptide 2CA-1 in RD cells are as follows: Figure 3 As shown, 2CA-1 can attenuate cytopathic effects induced by EV71 infection, and the degree of attenuation of the cytopathic effect increases in a concentration-dependent manner with increasing 2CA-1 concentration.

[0099] Example 4:

[0100] 2CA-1 inhibits EV71 plaque assay in RD cells

[0101] 1. Experimental Materials

[0102] RD cells; DMEM medium (Thermo), serum (Gibco), 2×MEM medium (Gibco) were purchased from Ingenic Semiconductor, low melting point agarose (Biofrox), paraformaldehyde, and crystal violet were purchased from Startup subsidiary.

[0103] 2. Experimental Procedure

[0104] (1) Use RD cells to form 12-well plates.

[0105] (2) When the culture reaches 70%-80% confluence, replace the DMEM medium containing 10% serum with DMEM medium containing 2% serum (the amount of 2% serum DMEM medium added to each well is 1 mL), adding 10 μL of 1×10⁻⁶ DMEM medium to each well. 6 PFU / mL EV71 virus.

[0106] (3) After 1 h, the supernatant was discarded and peptide 2CA-1 was added to a final concentration of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM and 10 μM respectively, with the group without peptide added as the control.

[0107] (4) After adding the peptide for 1 h, discard the supernatant, add 1.5% low melting point agarose mixed in a 1:1 ratio and a solution containing 6% serum and 2% 2×MEM, wait for it to solidify in the well, and invert it in a 37℃ incubator for culture.

[0108] (5) After 2 days, when the cells showed obvious lesions, staining was performed. A staining solution containing 4% paraformaldehyde and 1% crystal violet was added to the wells and stained at 4 °C for 2 h. The gel in the wells was rinsed off with tap water, and the number of empty spots in each well was observed and recorded.

[0109] (6) Samples were collected 24 h after EV71 virus infection, and the cytopathic effects were observed and images were taken under a microscope with image acquisition capability.

[0110] The results of plaque detection of peptide-2CA-1 against EV71 virus in RD cells are as follows: Figure 4 As shown, 2CA-1 significantly inhibited plaque formation induced by EV71 infection, and this inhibitory effect increased with increasing concentration in a dose-dependent manner.

[0111] Example 5:

[0112] Determination of the inhibitory efficiency of 2CA-1 on EV71 in various cell types

[0113] 1. Experimental Materials

[0114] Vero cells, 293T cells, and Huh7 cells; DMEM medium (Thermo) and serum (Gibco) were purchased from Invitrogen; Total RNA Extraction Kit (Omega) and One-Step qRT-PCR Kit (Takara) were purchased from Yuming Corporation; DEPC water was used in RNA extraction and qRT-PCR; and the entire experiment was conducted in an RNase-free environment.

[0115] 2. The experimental procedure is the same as in Example 1.

[0116] The results are as follows Figures 5A-5C Table 7-9 shows the results of the anti-EV71 effect of peptide-2CA-1 in different cell types: peptide-2CA-1 showed an IC50 value of 1 in Vero cells. 50 2.929 μM ( Figure 5A IC50 in 293T cells 50 2.769 μM ( Figure 5BIn Huh7 cells, IC 50 4.014 μM ( Figure 5C ).

[0117] Table 7

[0118] .

[0119] Table 8

[0120] .

[0121] Table 9

[0122] .

[0123] Example 6:

[0124] 2CA-1 toxicity assay in various cell types

[0125] 1. Experimental Materials

[0126] Vero cells, 293T cells, and Huh7 cells; DMEM medium (Thermo) and serum (Gibco) were purchased from Invitrogen; CCK-8 reagent (MCE) was purchased from Startup subsidiary.

[0127] 2. The experimental procedure is the same as in Example 2.

[0128] The results are as follows Figures 6A-6C As shown in Table 10-12, with the cell viability of untreated cells as 100%, the CC of each peptide was calculated. 50 In Vero cells, CC 50 >500 μM ( Figure 6A CC in 293T cells 50 >500 μM ( Figure 6B CC in Huh7 cells 50 >500 μM ( Figure 6C ).

[0129] Table 10

[0130] .

[0131] Table 11

[0132] .

[0133] Table 12

[0134] .

[0135] Example 7:

[0136] Schematic diagram of the binding pattern of peptide 2CA-1 to EV71 2C protein.

[0137] To investigate whether 2CA-1 can act as a keyhole disruptor, helicase activity was inhibited by blocking the pocket of the 2C ATPase domain. Molecular docking simulations showed that 2CA-1 can bind tightly to the keyhole structure of the EV71 2C ATPase domain. Figure 7A Their binding strength may even exceed the interactions observed at the interface between natural 2C monomers. Similar docking studies have been conducted on CVB3 2C proteins with a more pronounced keyhole structure. Figure 7B The results showed that 2CA-1 could also bind tightly to the cavity, suggesting that this peptide mimic may be equally effective against group B enteroviruses.

[0138] Example 8:

[0139] Peptides 2CA-1, 2CA-2, and 2CA-3 inhibit the helicase activity of EV71 2C protein.

[0140] 1. Experimental Materials

[0141] Baculovirus expressing MBP-EV71 2C protein was used. Fall armyworm cells (Sf9) were obtained from the China Center for Type Culture Collection (CCTCC). SF-HM culture medium was purchased from WhiteJ Technology Co., Ltd., maltose-binding protein (MBP) packing material was purchased from NEB, and Amicon Ultra-30 kDa ultrafiltration tubes were purchased from Millipore. Binding buffer (pH 7.4): 20 mM Tris-HCl (pH 7.4), 0.5 M EDTA, 200 mM NaCl, 10 mM β-mercaptoethanol, 5% anhydrous ethanol (v / v), and 10% glycerol (v / v). Elution buffer: 10 mM maltose solution and 50 mM HEPES solution at pH 7.5.

[0142] A 42nt long single-stranded RNA labeled with HEX fluorescently and a 54nt long single-stranded RNA complementary to the HEX-labeled RNA strand.

[0143] 2. Experimental Procedure

[0144] 2.1 In vitro expression and purification of EV71 2C protein. The experimental procedure was based on the following: Xia H, Wang P, Wang GC, Yang J, Sun X, Wu W, Qiu Y, Shu T, Zhao X, Yin L, Qin CF, Hu Y, Zhou X. Human Entererovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone. PLoS Pathog. 2015 Jul 28;11(7):e1005067.doi: 10.1371 / journal.ppat.1005067. PMID: 26218680; PMCID: PMC4517893.

[0145] 2.2 Peptide 2CT-NG, peptide-2CA-1, 2CA-2, and 2CA-3 inhibit the helicase activity of EV71 2C protein in vitro.

[0146] (1) Using HEX-labeled strand at a concentration of 0.2 pmol / μL, add complementary strand RNA of the same concentration and anneal to prepare HEX-labeled double-stranded dsRNA substrate.

[0147] (2) Annealing process: The reaction system is at 75℃ for 3 min, and the temperature of the reaction system is reduced to 25℃ at a rate of 1℃ per minute, and then at 25℃ for 2 min.

[0148] (3) Prepare the target protein and double-stranded substrate according to the standard unwinding reaction system. Add 5 μg of peptides 2CA-1, 2CA-2 and 2CA-3 respectively, and set up single-stranded, double-stranded and MBP controls. Single-stranded samples need to be boiled at 75℃ for 3 min, then on ice for 2 min.

[0149] (4) After mixing the prepared system, place it at 37°C and react for 50 min.

[0150] (5) Perform electrophoresis on the mixture after the reaction.

[0151] (6) Finally, the HEX signal was obtained by direct scanning using Typhoon 9500.

[0152] During electrophoresis, single-stranded molecules migrate faster than double-stranded molecules. Therefore, if the MBP-2C protein possesses helicase activity, it can open the double-stranded dsRNA substrate, releasing single-stranded RNA, resulting in two bands in the lane. Lane 1 (without protein reaction) serves as the positive control, lane 2 (single-stranded RNA prepared by boiling at 75°C) serves as the negative control, and lane 3 (MBP protein) serves as the positive control. EV71 Lane 4 served as the negative control, while 2CT-NG (a truncated polypeptide of 2CL, with the amino acid sequence TIEALFQ) (lane 5) served as the positive control. Lanes 2CA-1, 2CA-2, and 2CA-3 (lanes 6-8) were the experimental groups. Figure 8 As shown in lane 4, EV71 2C exhibits helicase activity, capable of unwinding double-stranded dsRNA substrates; the helicase activity of 2C can be inhibited; peptides 2CA-1, 2CA-2, and 2CA-3 can all inhibit the helicase activity of 2C (lanes 6-8), while the MBP control does not affect the helicase activity of 2C (lane 3). These results indicate that peptides 2CA-1, 2CA-2, and 2CA-3 can indeed inhibit the helicase function of EV71 2C.

[0153] Example 9:

[0154] Antiviral activity of peptides 2CA-1, 2CA-2 and 2CA-3 against EV71 in mice

[0155] (1) Forty-eight 8-day-old ICR suckling mice were randomly divided into five groups: one group of nine suckling mice was challenged with the virus and injected with an equal volume of PBS (vehicle) as a positive control; three groups of ten suckling mice were challenged with the virus and injected with peptides 2CA-1, 2CA-2, and 2CA-3, respectively; and one group of nine suckling mice was neither challenged nor given any medication as a negative control. These 39 suckling mice were administered the drug via intraperitoneal injection at a dose of 10... 5 Attack on PFUEV71.

[0156] (2) At the same time as the attack, the three groups were given intraperitoneal injections of 10 mg / kg of 2CA-1, 2CA-2 and 2CA-3 peptides as the treatment group, and one group was injected with an equal amount of PBS as the control group.

[0157] (3) Both peptides and PBS were injected twice a day until the 7th day after challenge.

[0158] (4) Observe the clinical symptoms and mortality of suckling mice, and record the daily weight changes until day 21.

[0159] (5) Clinical symptoms are judged by the clinical scoring system: 0 points for healthy, 1 point for slow and hunched movements, 2 points for weakness of one limb, 3 points for paralysis of one limb, 4 points for paralysis of both limbs, and 5 points for death.

[0160] The results are as follows Figure 9A As shown, all the negative control group (Mock) mice survived, while all 9 mice in the challenge-only group (without drug administration) died on day 11, with a mortality rate of 100%. In contrast, 8 mice in the 2CA-1 administration group, 7 mice in the 2CA-2 administration group, and 9 mice in the 2CA-3 administration group survived. Figure 9B As shown, the average body weight of the group that was only challenged with the virus but not given any medication was slightly lower than that of the group that was challenged with the virus and given medication at the time of survival. Figure 9C As shown, the clinical scores of the group that was only challenged with the virus but did not receive any medication were significantly higher than those of the group that received medication after being challenged with the virus.

[0161] The above results indicate that 2CA-1, 2CA-2, and 2CA-3 can all effectively treat lethal doses of EV71 in suckling mice and prevent them from dying. Figures 9A-9C Based on the results and drug safety, peptide 2CA-1 is the most suitable for drug use.

[0162] Example 10:

[0163] Assay of antiviral activity of peptide-2CA-1 against EV71 in mouse muscle, lung and brain

[0164] (1) Nine 8-day-old ICR suckling mice were randomly divided into three groups: one group of three suckling mice was challenged with the virus and injected with an equal volume of PBS (vehicle) as a positive control; another group of three suckling mice was challenged with the virus and injected with peptide 2CA-1; and the third group of three suckling mice was not challenged with the virus and did not receive any medication as a negative control. Six of these suckling mice were administered the drug via intraperitoneal injection at a dose of 10... 5 Virus attack using PFU EV71.

[0165] (2) At the same time as the virus challenge, one group was given an intraperitoneal injection of 10 mg / kg of 2CA-1 peptide as the treatment group, and another group was given an equal amount of PBS as the control group.

[0166] (3) Both peptides and PBS were injected twice a day until the 7th day after challenge.

[0167] (4) Observe the clinical symptoms of suckling mice. After the positive control group showed symptoms, all 9 mice were euthanized and muscle, lung and brain tissues were collected in 1 mL of Trizol solution.

[0168] (5) After grinding the mouse tissue, add 200 μL of chloroform, shake vigorously to mix, and let stand for 5 min.

[0169] (6) After centrifuging at 12000 g for 15 min at 4℃, take the upper aqueous phase of the solution into a new EP tube.

[0170] (7) Add an equal volume of isopropanol, invert and mix well, and let stand for 20 min.

[0171] (8) Centrifuge at 12000 g for 15 min at 4℃, discard the supernatant and add 1 mL of 75% ethanol, then gently invert to mix.

[0172] (9) Centrifuge at 12000 g for 15 min at 4℃, discard the supernatant and dry at room temperature.

[0173] (10) Dissolve in 50 μL of DEPC water.

[0174] (11) Take 2 μL of RNA sample and perform a fluorescence quantitative experiment using a one-step qRT-PCR kit.

[0175] The results of the assay for the antiviral activity of peptide 2CA-1 against EV71 in mouse muscle, lung and brain are as follows: Figures 10A-10C As shown in Table 13, it has a significant inhibitory effect on the virus.

[0176] Table 13

[0177] .

[0178] Example 11:

[0179] Toxicity assay of peptide-2CA-1 in mice

[0180] Twenty 8-day-old ICR suckling mice were randomly divided into four groups: one group of five suckling mice was injected with 25 mg / kg of 2CA-1 peptide, another group of five suckling mice was injected with 50 mg / kg of 2CA-1 peptide, another group of five suckling mice was injected with 100 mg / kg of 2CA-1 peptide, and a group of five suckling mice was injected with an equal volume of PBS (vehicle) as a negative control.

[0181] The peptide and PBS were injected only once, and the mortality of the suckling mice was observed and the daily weight changes were recorded until day 14.

[0182] The results are as follows Figure 11 As shown, all suckling mice in the negative control group (Mock) survived, as did all suckling mice in the three groups treated with different doses of the peptide 2CA-1. These results indicate that 2CA-1 is non-toxic at a dose of 100 mg / kg and does not cause death in suckling mice.

[0183] Example 12:

[0184] Detection of antiviral activity of peptide-2CA-1 against EV71 in mice

[0185] (1) Twenty-four 8-day-old ICR suckling mice were randomly divided into two groups: one group of 12 suckling mice was challenged with the virus and administered an equal volume of PBS (vehicle) via gavage as a positive control; the other group of 12 suckling mice was challenged with the virus and administered the peptide 2CA-1 at a dose of 20 mg / kg via gavage. The 24 suckling mice were administered the drug via intraperitoneal injection at a dose of 10 mg / kg. 5 Virus attack using PFU EV71.

[0186] (2) Both the peptide and PBS were administered by gavage twice a day until the 7th day after the challenge.

[0187] (3) Observe the clinical symptoms and mortality of suckling mice, and record the daily weight changes until the 14th day.

[0188] (4) Clinical symptoms are judged by the clinical scoring system: 0 points for healthy, 1 point for slow and hunched movements, 2 points for weakness of one limb, 3 points for paralysis of one limb, 4 points for paralysis of both limbs, and 5 points for death.

[0189] The results are as follows Figure 12A As shown, in the group of suckling mice that were only challenged with the virus but not treated, 9 mice died and 3 survived on day 14. In the group of suckling mice treated with 2CA-1, all 12 mice survived, with a rescue rate of 66.7%. Figure 12B As shown, the average body weight of the group that was only attacked with the virus but not given any medication was lower than that of the group that was attacked with the virus and then given medication. Figure 12C As shown, the clinical scores of the group challenged but not treated were significantly higher than those of the group treated after challenge. These results indicate that 2CA-1 can effectively treat lethal doses of EV71 in suckling mice and prevent them from dying.

[0190] Example 13:

[0191] Determination of the inhibitory efficiency of peptide-2CA-1 on multiple enteroviruses in RD cells

[0192] 1. Experimental Materials

[0193] RD cells; DMEM medium (Thermo) and serum (Gibco) were purchased from Invitrogen. Total RNA extraction kit (Omega) and One-step qRT-PCR kit (Takara) were purchased from Yuming Corporation. DEPC water was used for RNA extraction and qRT-PCR. The entire experiment was conducted in an RNase-free environment.

[0194] Related viruses include

[0195] 1. CVA6 (17ES4 / QD / CHN / 2017, Reference: Su Z, Shi X, Zhang F, Chai Q, Gong J, Wang Z. Genome Sequence of a Human Coxsackievirus A6 Strain Isolated from a Severe Hand, Foot, and Mouth Disease Case in Qingdao, China, in 2017. Microbiol Resour Announc. 2020 Apr 23;9(17):e01449-19. doi: 10.1128 / MRA.01449-19. PMID: 32327512; PMCID: PMC7180285.)、

[0196] 2. CVA16 (strain CA16 / GD09 / 24, Reference: Han JF, Yu N, Pan YX, He SJ, Xu LJ, Cao RY, Li YX, Zhu SY, Zhang Y, Qin ED, Che XY, Qin CF. Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice. Virus Res. 2014 Jan 22;179:212-9. doi: 10.1016 / j.virusres.2013.10.020. Epub 2013 Nov 6. PMID: 24211607.)、

[0197] 3.CVB3 (Woodruff variant, Editor:Knowlton KU, Jeon ES, Berkley N,Wessely R, Huber S. A mutation in the puff region of VP2 attenuates themmyocarditic phenotype of an infectious cDNA of the Woodruff variant ofcoxsackievirus B3. J Virol Nov;70(11):7811-8 doi:10.1128 / JVI.70.11.7811-7818.1996;

[0198] 4.Echo11 (strain GWCMC01 / GZ / CHN / 2019, Editor:Wang C, Yang R, YangF, Han Y, Ren Y, Xiong X, Wang X, Bi Y, Li L, Qiu Y, Xu Y, Zhou X. Echovirus11 infection induces pyroptotic cell death by facilitating NLRP3 inflammasome activation 2022 Aug 26;18(8):e1010787 PMID: 36026486;

[0199] 5.EVD68 (US / MO / 14-18947, Reference: Liu 12;33(2):267-278.e4. doi: 10.1016 / j.chom.2024.12.015.Epub 2025 Jan 10. PMID: 39798568.)

[0200] 6.HRV A1B (USA / 2021 / WAM55M, Reference: Goya S, Wendm ST, Xie H, NguyenTV, Barnes S, Shankar RR, Sereewit J, Cruz K, Pérez-Osorio AC, Mills MG,Greninger AL. Genomic Epidemiology and Evolution of Rhinovirus in Western Washington State, 2021-2022. J Infect Dis. 2025 Feb 4;231(1):e154-e164. doi:10.1093 / infdis / jiae347. PMID: 38963827; PMCID: PMC11793040.).

[0201] 2. The experimental procedure is the same as in Example 1.

[0202] The results are shown in Figure 13 and Table 14-19. The efficacy of peptide 2CA-1 in inhibiting various enteroviruses in RD cells was as follows: 2CA-1 had an IC50 of [missing information] against CVA6 virus. 50 1.547 μM ( Figure 13A ), for the IC of the CVA16 virus 50 1.656 μM ( Figure 13B ), for the IC of the CVB3 virus 50 4.433 μM ( Figure 13C ), for the IC of the Echo11 virus 50 4.074 μM ( Figure 13D ), for the IC of the EVD68 virus 50 3.218 μM ( Figure 13E ), for HRV A1B virus IC 50 4.173 μM ( Figure 13F ).

[0203] Table 14

[0204] .

[0205] Table 15

[0206] .

[0207] Table 16

[0208] .

[0209] Table 17

[0210] .

[0211] Table 18

[0212] .

[0213] Table 19

[0214] .

[0215] The foregoing has provided a detailed description of the peptides and their applications provided by this invention. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. An artificially synthesized peptidomimetic 2CA-1, the chemical formula of which is: Cbz-ALFQ-NH2, and the structural formula of which is: 。 2. A complex containing the peptidomimetic 2CA-1 of claim 1.

3. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment of enterovirus infection, the enterovirus being enterovirus 71, coxsackievirus A6, A16, B3, echovirus 11, enterovirus D68 or / and rhinovirus.

4. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment or prevention of herpangina.

5. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment or prevention of hand, foot and mouth disease.

6. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment or prevention of viral myocarditis.

7. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment or prevention of respiratory tract infection.

8. Use of the peptidomimetic 2CA-1 of claim 1 or the complex of claim 2 in the preparation of a medicament for the treatment or prevention of common cold in human.

9. A broad-spectrum anti-enterovirus medicament, the medicament comprising the peptidomimetic 2CA-1 of claim 1.

10. The medicament of any of claims 3-8 or claim 9, the medicament being in a pharmaceutically acceptable dosage form.

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