Screening and validation of broad-spectrum anti-influenza virus compounds

Abstract

The present application relates to the field of biotechnology and virology, in particular, the present application relates to screening and verification of a broad-spectrum anti-influenza virus compound, and the present application also relates to the use of a reagent targeting ATP1A1 protein or a composition comprising the reagent targeting ATP1A1 protein in the preparation of a pharmaceutical product.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and virology. Specifically, this application relates to the use of reagents targeting the ATP1A1 protein or compositions containing reagents targeting the ATP1A1 protein in the preparation of pharmaceuticals. Background Technology

[0002] Influenza virus is an acute respiratory infectious disease pathogen, mainly classified into four types: A, B, C, and D. Among them, types A and B have the most significant impact on human health. Influenza A virus (IAV) has an eight-segment single-stranded RNA genome, and its evolutionary plasticity is mainly achieved through two mechanisms: antigenic drift (point mutation) and antigenic shift (gene rearrangement). This rapid evolutionary capacity not only leads to annual seasonal influenza epidemics but has also triggered several catastrophic pandemics. Influenza B virus (IBV) has relatively weak variability and a narrower host range, primarily infecting humans. Although IBV usually does not cause global pandemics, it can still cause local outbreaks or regional epidemics during specific seasons, and its impact on groups such as children and students is often significant.

[0003] The current influenza threat primarily stems from seasonal influenza viruses and zoonotic influenza viruses. Although the H3N8 virus has not yet achieved sustained human-to-human transmission, strains isolated from humans have shown higher pathogenicity compared to avian strains. Furthermore, there are currently no reports of drug screening for the H3N8 virus. On the other hand, seasonal viruses continue to evolve. Surveillance data from Sicily, Italy, in the 2023-2024 season showed that approximately 40% of circulating H1N1 (pdm 2009) strains exhibited partial antigenic drift with the vaccine strains recommended by the World Health Organization (WHO), indicating that the rapid and continuous evolution of the virus is increasingly complicating prevention and control efforts. Meanwhile, novel zoonotic influenza outbreaks also pose a significant concern. Since the first detection of the H5N1 virus in cattle in the United States in March 2024, the outbreak has spread rapidly across multiple states. Most confirmed cases are related to direct contact with infected dairy cows, primarily involving the H5N1 (branch 2.3.4.4b, genotype B3.13) subtype. With the approach of autumn and winter, the dominant circulating strain in 2025 has shifted from H1N1 (pdm 2009) last year to H3N2 (belonging to subgroup J.2.4.1, also recently referred to as "H3N2 subgroup K"). This shift in the dominant circulating strain reveals the pattern of continuous mutation and alternating epidemics of influenza viruses, further increasing the difficulty of influenza prevention and control. Therefore, it is urgent to screen for a promising candidate compound for future development.

[0004] Currently, clinical treatment of influenza virus mainly relies on two classes of direct-acting antiviral drugs: one is neuraminidase inhibitors (such as oseltamivir and zanamivir), which work by blocking viral release; the other is cap-dependent endonuclease inhibitors (such as mabaloxavir), which produce therapeutic effects by inhibiting viral RNA synthesis. However, these drugs have many drawbacks. For example, oseltamivir has a relatively narrow effective therapeutic window, showing the best effect only when used in the early stages of infection (within 48 hours), and drug resistance is becoming increasingly significant with viral strain mutations. Mabaloxavir is only suitable for patients over 12 years of age, and large-scale drug resistance has been observed abroad. Moreover, the existing drug mechanisms are relatively simple, making it difficult to comprehensively address the complex viral dynamics and diverse host responses. Therefore, there is an urgent need to screen for more efficient and reliable broad-spectrum antiviral small molecule compounds. Summary of the Invention

[0005] The applicant of this invention discovered that digoxigenin (Dig) can exert anti-influenza virus activity by targeting the ATP1A1 protein. This invention also verified that Dig has broad-spectrum influenza virus inhibitory activity and can effectively inhibit multiple subtypes of viruses such as H3N8, H1N1, H3N2 and H5N1.

[0006] Therefore, this application provides the use of a reagent targeting the ATP1A1 protein or a composition containing a reagent targeting the ATP1A1 protein in the preparation of a pharmaceutical product, said pharmaceutical product being used for: (i) Prevention and / or treatment of influenza virus infection in the subject; (ii) Inhibit influenza virus replication and / or reproduction in cells; and / or (iii) To prevent and / or treat illnesses caused by influenza virus in the subject.

[0007] In some embodiments, the reagent targeting the ATP1A1 protein is a protein or peptide, a nucleic acid aptamer, or a small molecule compound.

[0008] In some embodiments, the agent targeting the ATP1A1 protein is digoxigenin (Dig) or its prodrug, solvate, or pharmaceutically acceptable salt.

[0009] In some embodiments, the isohydroxydigitoxin prodrug includes a carrier prodrug of isohydroxydigitoxin (e.g., a glycoside prodrug, a fatty acid ester prodrug, an amino acid ester prodrug, a phosphate ester / sulfate ester prodrug) and an antibody-drug conjugate (ADC) prodrug.

[0010] In some embodiments, the glycoside prodrug of the isohydroxydigitoxinin includes digoxin, lanatoside C, deslanoside, and metildigoxin.

[0011] In some embodiments, the solvates of the isohydroxydigitoxinogen include polar solvates of the isohydroxydigitoxinogen (e.g., hydrates, methanolic compounds, or ethanolic compounds).

[0012] In some embodiments, pharmaceutically acceptable salts of the isohydroxydigitoxin include inorganic salts, organic acid salts, inorganic base salts, and organic base salts of the isohydroxydigitoxin (e.g., sodium salts, potassium salts, calcium salts, meglumine salts, hydrochloride salts, nitrates, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, acetates, propionates, butyrates, oxalates, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, or benzenesulfonate).

[0013] In some implementations, the influenza virus includes both influenza A and influenza B viruses.

[0014] In some embodiments, the influenza A virus includes influenza A subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18.

[0015] In some implementations, the influenza B virus includes influenza B viruses of the Victoria and Yamagata lineages.

[0016] In some embodiments, the influenza A virus includes influenza A viruses of subtypes H3N8, H1N1, H3N2, and H5N1.

[0017] In some implementations, the influenza B virus includes the Victoria lineage of influenza B virus.

[0018] This invention verifies that reagents targeting the ATP1A1 protein (e.g., Dig) have broad-spectrum anti-influenza virus effects. Among these, reagents targeting the ATP1A1 protein (e.g., Dig) are particularly effective against the H5N1 subtype of influenza A virus. Therefore, in some embodiments, the influenza A virus is the H5N1 subtype of influenza A virus.

[0019] In some implementations, the subject is a mammal.

[0020] In some implementations, the subject is a bovine, equine, suidae, canine, feline, rodent, or primate.

[0021] In some implementations, the subject is a human, rat, monkey, pig, or dog.

[0022] In some implementations, the cell is a mammalian cell.

[0023] In some embodiments, the cells are cells of bovines, equines, suidaes, canines, felines, rodents, or primates.

[0024] In some embodiments, the cells are human cells, mouse cells, monkey cells, pig cells, or dog cells.

[0025] In some implementations, the diseases include influenza, uncomplicated infections, pneumonia (e.g., severe pneumonia), acute or severe acute respiratory infections, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, and septic shock.

[0026] In some implementations, the uncomplicated infection includes fever, cough, and sore throat.

[0027] In some embodiments, the pharmaceutical product also contains a pharmaceutically acceptable carrier and / or excipient.

[0028] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0029] In some embodiments, the drug also contains additional active ingredients.

[0030] In some embodiments, the additional active ingredient includes an antiviral agent.

[0031] In some embodiments, the additional active ingredients include amantadine, rimantadine, emfuvirdi, maraviro, acyclovir, ganciclovir, valacyclovir, famciclovir, sodium phosphonoformate, lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, efavirenz, nevirapine, saquinavir, oseltamivir, zanamivir, ribavirin, and interferon.

[0032] It is understood that the pharmaceutical products of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. The specific dosage form used depends on the intended route of administration and therapeutic purpose.

[0033] In some embodiments, the pharmaceutical product of the present invention comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0034] In some embodiments, the medicine is an injection, infusion, tablet, capsule, spray, aerosol, or rinse.

[0035] In some implementations, the drug is present in unit dose form.

[0036] In some embodiments, the drug contains 1-1000 mg (e.g., 1-800 mg, 1-500 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-20 mg, or 1-10 mg) of isohydroxydigitoxin or its prodrug, solvate or pharmaceutically acceptable salt.

[0037] It is understood that the medicaments of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. In some embodiments, the route / method of administration is parenteral (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose.

[0038] In some embodiments, the drug is a drug that is targeted for release into the gastrointestinal tract, or a drug that is released in a controlled manner into the gastrointestinal tract.

[0039] In some embodiments, the medicine is formulated for oral administration.

[0040] In some implementations, the drug is a parenteral drug.

[0041] In some embodiments, the parenteral administration is selected from intravenous, intramuscular, subcutaneous, intranasal, transmucosal, sublingual, rectal, transdermal, inhalation, or any combination thereof.

[0042] In some implementations, the parenteral administration is performed via intravenous infusion.

[0043] On the other hand, this application provides a high-throughput screening method for broad-spectrum anti-influenza virus compounds, the method comprising: Step (1): Provide recombinant influenza virus expressing reporter protein and the compound to be screened; Step (2): The recombinant virus is brought into contact with the cells while, before, or after the cells are treated with the compound; Step (3): Under the condition that the reporter protein is able to generate a signal, measure the signal level (e.g., fluorescence intensity) of the cell. The recombinant influenza virus, relative to the wild-type influenza virus, contains a nucleotide sequence encoding the reporter protein at a position between nucleotides 500 and 501 of the NS gene corresponding to SEQ ID NO: 1.

[0044] In some implementations, the wild-type influenza virus is an influenza A virus and an influenza B virus (e.g., an influenza A virus of the H3N8 subtype).

[0045] In some embodiments, the reporter protein includes green fluorescent protein (GFP) and luciferase.

[0046] In some embodiments, in step (2), after treating the cells with the compound, the recombinant virus is brought into contact with the cells.

[0047] In some embodiments, the cells include eukaryotic cells and prokaryotic cells, such as A549 cells.

[0048] In some embodiments, the NS gene of the recombinant influenza virus is shown in SEQ ID NO: 2.

[0049] In some implementations, the signal level is the fluorescence intensity of GFP.

[0050] In some embodiments, the method further includes: Step (4): Compare the measured value in step (3) with the signal level measured without treatment with the compound, and obtain the following ratio: (measured value without treatment with the compound - measured value in step (3)) / measured value without treatment with the compound; In some embodiments, the method further includes: Step (5): Based on the ratio obtained in step (4), generate the dose-response curve of the compound and thereby obtain the EC. 50 According to the EC 50 The anti-influenza virus activity of the compound was evaluated.

[0051] The method of the present invention can achieve high-throughput detection. In some embodiments, the number of cells mentioned in steps (1)-(3) is multiple. The anti-influenza virus activity of multiple different compounds can be measured simultaneously in multiple cells. For example, the number of cells measured in step (3) is not less than 100, such as not less than 500, not less than 800, or not less than 1000.

[0052] In some implementations, step (3) is determined by fluorescence microscopy or a high-content imaging system.

[0053] The method of this invention enables broad-spectrum detection of compounds. In some embodiments, the recombinant influenza virus described in steps (1)-(3) is of multiple subtypes. Cells can be treated simultaneously with multiple recombinant influenza viruses to determine the inhibitory activity of the compound against different influenza viruses. For example, the recombinant influenza virus used in step (2) includes influenza A and influenza B viruses, such as influenza A subtypes H3N8, H1N1, H3N2, and H5N1, and influenza B viruses of the Victoria and Yamagata lineages.

[0054] In some embodiments, the broad-spectrum anti-influenza virus compound includes a reagent targeting the ATP1A1 protein or a composition containing a reagent targeting the ATP1A1 protein.

[0055] In some embodiments, the broad-spectrum anti-influenza virus compound includes isohydroxydigitoxinogen or its prodrug, solvate, or pharmaceutically acceptable salt.

[0056] Terminology Definition

[0057] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0058] As used herein, the term "ATP1A1 protein" is a membrane protein, the α-1 subunit of the sodium-potassium ATPase, responsible for establishing and maintaining the electrochemical gradient of Na and K ions across the plasma membrane. ATP1A1 proteins are widely distributed in various organisms, such as mammals, birds, and amphibians. ATP1A1 proteins exhibit high conservation across different organisms, particularly among mammals, with at least 95% sequence similarity among different mammals.

[0059] As used in this article, digoxigenin is a cardiac glycoside-based therapeutic agent widely used in the treatment and management of congestive heart failure and other heart diseases. Its molecular formula is C0. 23 H 34 O5, with a molecular weight of 390.51 and CAS number 1672-46-4, has the following chemical structural formula: .

[0060] It should be understood that the isohydroxydigitoxinogen of this application may exist in its free form for therapeutic purposes, or, where appropriate, in its pharmaceutically acceptable derivative form. In this application, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compound of this application or its metabolites or residues. Therefore, when "isohydroxydigitoxinogen" is mentioned herein, it is intended to encompass all of the aforementioned derivative forms.

[0061] As used herein, the term "pharmaceuticalally acceptable salt" includes inorganic or organic acid salts of isohydroxydigitoxin, as well as inorganic or organic base salts, such as sodium, potassium, calcium, lithium, meglumine, hydrochloride, hydrobromide, nitrate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or dihydroxynaphthate, etc. A review of suitable salts can be found, for example, in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of this application are known to those skilled in the art.

[0062] As used herein, the term "isohydroxydigitoxin prodrug" refers to a derivative with novel physical, chemical, and biological properties obtained by modifying the chemical structure of isohydroxydigitoxin. It does not exhibit the pharmacological effects of the parent drug (i.e., isohydroxydigitoxin) but is converted into the parent drug in vivo to exert its effect. In this paper, the term "carrier prodrugs" refers to prodrugs formed by covalently linking isohydroxydigitoxin to a small molecule carrier motif (e.g., sugar, fatty acid, amino acid, phosphate). Based on the different covalent bonds and carriers, carrier prodrugs can be classified into glycoside prodrugs, fatty acid ester prodrugs, amino acid ester prodrugs, and phosphate / sulfate ester prodrugs. In this paper, the term "antibody-drug conjugate (ADC) prodrug" refers to an ADC formed by conjugating isohydroxydigitoxin to an antibody, which delivers hydroxydigitoxin through the activity of the antibody.

[0063] Isohydroxydigitoxinogen can exist as a solvate (preferably a hydrate) comprising a polar solvent, particularly water, methanol, or ethanol, which constitutes the structural elements of the isohydroxydigitoxinogen crystal. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric. It should be understood that any solvate of isohydroxydigitoxinogen used in the treatment of the diseases or infections described in this application, although potentially providing different properties (including pharmacokinetic properties), will yield isohydroxydigitoxinogen upon absorption into the subject, thus the use of isohydroxydigitoxinogen specifically encompasses the use of any solvate of isohydroxydigitoxinogen.

[0064] As used herein, the term "targeting" refers to the process by which a drug, carrier, probe, or therapeutic agent selectively accumulates or acts on a specific target site at the molecular, cellular, or tissue level through a specific recognition mechanism, while minimizing the impact on non-target regions. In some embodiments, the reagent targeting the ATP1A1 protein is a reagent that specifically recognizes and binds to the ATP1A1 protein.

[0065] As used in this article, the term "aptamer" refers to a class of short single-stranded DNA or RNA oligonucleotides. They bind to target molecules with high specificity through a specific three-dimensional conformation and are characterized by high stability, low synthesis cost, and non-immunogenicity.

[0066] As used herein, the term "corresponding position" or "corresponding to... position" refers to the nucleotide position at an equivalent position in the two sequences being compared when performing an optimal alignment, i.e., when aligning the two sequences to obtain the highest percentage identity. For example, the expression "corresponding to the position between nucleotides 500 and 501 of the NS gene shown in SEQ ID NO:1" means, when performing an optimal alignment of a sequence with SEQ ID NO:1, i.e., when aligning a sequence with SEQ ID NO:1 to obtain the highest percentage identity, the nucleotide position at an equivalent position in the compared sequence between nucleotides 500 and 501 of SEQ ID NO:1.

[0067] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). th (ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, delayed absorption agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Delayed absorption agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of an active ingredient in a drug, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0068] As used herein, the term "treatment" aims to alleviate, reduce, improve, or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, when a subject receives a therapeutic amount of the compound or composition as described herein, one or more indications and symptoms show an observable and / or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0069] As used herein, the term "prevention" aims to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms before the onset of the relevant medication. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after the administration of the relevant medication, or lessening the severity of subsequently occurring related symptoms, can be considered as "prevention" of the onset or development of the disease.

[0070] Beneficial effects

[0071] The applicant of this invention established a platform for screening anti-influenza A virus drugs through extensive experiments. Based on this platform, high-throughput screening was performed to identify digoxigenin (Dig) from a library of 3104 FDA-approved drugs. The anti-influenza virus activity of Diig was verified through cell experiments and mouse experiments. Furthermore, this invention also verified that Diig has broad-spectrum influenza virus inhibitory activity, effectively inhibiting influenza A viruses (e.g., H3N8, H1N1, H3N2, and H5N1 subtypes) and influenza B viruses (e.g., the Victoria lineage). Further, this invention verified the mechanism of Diig's activity, which involves targeting the host protein ATP1A1, thereby inducing the degradation of NP proteins via proteasome and lysosome pathways.

[0072] Therefore, this application provides a target for an anti-influenza virus drug and a drug that can effectively combat influenza viruses.

[0073] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0074] Figure 1This diagram shows the screening flowchart for anti-H3N8-GFP drugs and the composition of the FDA-approved drug library. Among them, Figure 1 A is a flowchart of the screening process for anti-H3N8-GFP drugs; Figure 1 B is a diagram of the FDA-approved drug library.

[0075] Figure 2 This shows the effect of Dig on EC infected with H3N8-GFP and H1N1-GFP. 50 .in, Figure 2 A shows the effect of Dig on EC infected with H3N8-GFP. 50 ; Figure 2 B shows the effect of Dig on EC infected with H1N1-GFP. 50 .

[0076] Figure 3 The inhibitory effect of Dig on IBV was shown in A549 cells.

[0077] Figure 4 The inhibitory effect of Dig on different subtypes of H1N1 influenza virus was shown in different cell lines. Figure 4 A, 4B, and 4C show the inhibitory effects of Dig on different subtypes of H1N8 influenza virus (H3N8, H1N1, H3N2, and H5N1) in A549, AT, and hAOs cell lines, respectively.

[0078] Figure 5 Dig showed a protective effect in H3N8-infected mice. Figure 5 A shows the experimental flowchart; Figure 5 B shows the changes in mouse body weight and survival rate; Figure 5 C shows the H&E staining of mouse lung tissue sections.

[0079] Figure 6 Dig showed a protective effect in H1N1-infected mice. Figure 6 A shows the experimental flowchart; Figure 6 B shows the changes in mouse body weight and survival rate; Figure 6 C shows the H&E staining of mouse lung tissue sections.

[0080] Figure 7 The results show that Dig targets the host protein ATP1A1, thereby inducing the degradation of NP protein via the proteasome and lysosome pathways. Figure 7 A shows the predicted interaction between Dig and ATP1A1; Figure 7 B shows the knockdown of ATP1A1 by siRNA; Figure 7 C shows the effect of Dig application and ATP1A1 knockdown on NP protein levels; Figure 7D shows the effect of MG132 and CQ application on NP protein levels.

[0081] Sequence information

[0082] Information on a portion of the sequence involved in this invention is provided below.

[0083] Detailed Implementation

[0084] The invention will now be described with reference to the following examples, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995.

[0085] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0086] Example 1. Establishing a high-throughput screening platform for compounds against human H3N8-GFP virus.

[0087] This embodiment constructs an anti-H3N8-GFP screening platform. First, H3N8-GFP, a human H3N8 recombinant virus, was constructed. It was obtained by inserting the GFP gene sequence downstream of the NS1 gene sequence in the NS segment of the A / Henan / 4-10 / 2022 (H3N8) virus (rescued in the laboratory). The nucleotide sequence of the NS gene of the A / Henan / 4-10 / 2022 (H3N8) virus is shown in SEQ ID NO: 1. The sequence encoding GFP is inserted between nucleotides 500 and 501 corresponding to SEQ ID NO: 1. The nucleotide sequence of the modified NS gene of H3N8-GFP is shown in SEQ ID NO: 2.

[0088] Therefore, in H3N8-GFP, GFP expression is closely related to viral replication. After the virus infects the host cell, GFP mRNA is expressed along with viral replication and translated into protein. The intensity of the resulting fluorescence signal is positively correlated with the level of viral replication. If the applied compound has anti-influenza A virus activity, it will block the viral life cycle, leading to reduced NS segment replication, decreased GFP mRNA transcription, and reduced fluorescent protein synthesis, ultimately resulting in a decrease in fluorescence intensity.

[0089] The drug library used in this embodiment contains 3104 compounds. Figure 1 (B) encompasses multiple categories, including drugs related to nerve signal transduction, metabolism regulation, endocrinology and hormones, and angiogenesis. The positive control drug used is baloxavir (Tao Shu, T14495), a viral RNA polymerase inhibitor that blocks viral mRNA synthesis by inhibiting viral endonuclease activity. The negative control used is DMSO. The fluorescence intensity detection method can be any commonly used method in the field, such as reading CPE or using immunofluorescence detection. In this embodiment, the detection method uses a PE high-content imaging system to quantify the fluorescence signal. The specific experimental steps are as follows.

[0090] Use Echo ® A 650 sonic liquid processor was used to dispense 3104 small molecule compounds (final concentration 10 µM) into 384-well plates using sonic dispensing. 0.1% DMSO was added to the negative control wells, and 20 nM baloxavir was added to the positive control wells. A549 cells (5 × 10³ cells per well) were added to the plates containing the compounds. After 6 hours of incubation, H3N8-GFP virus (MOI = 0.2) was diluted with DMEM without fetal bovine serum (FBS) for inoculation. One hour after infection, the medium was replaced with virus-free medium for further culture. Twenty-four hours post-infection, the plates were washed three times with PBS. Cell nuclei were counterstained with DAPI (1 µg / ml, 10 min). Fluorescence signals were quantitatively analyzed using a PE high-content imaging system, and Dig (Digoxigenin) (Tao Shu, T3212) was selected as a candidate compound against H3N8-GFP virus (e.g., ...). Figure 1 (As shown in A).

[0091] Dig, or isohydroxydigitoxinogen, has the chemical formula C0. 23 H 34 O5, see structural diagram. Figure 1 A. Dig is the aglycone (ligand) moiety of a naturally occurring cardiac glycoside. Chemically, it belongs to the class of cardiac steroid lactones and is an important structural component of drugs such as digoxin. Dig retains some of the cardiac stimulating activity of digoxin by inhibiting the activity of Na+ on cardiomyocytes. + / K + -ATPase enhances myocardial contractility. In the field of biotechnology, it is often used as a marker for antibody preparation or as a detection standard.

[0092] Example 2. Validation of Dig's in vitro anti-influenza virus activity

[0093] Furthermore, this embodiment verifies the anti-IAV (influenza A virus) and anti-IBV (influenza B virus) activity of the screened compound Dig through in vitro experiments.

[0094] This embodiment detected the EC of the screened compounds. 50 The models used were A549 cells (ATCC), primary mouse lung epithelial cells (ATs) (isolated and cultured in the laboratory), and human alveolar organoids (hAOs) induced by ipsc (cultured in the laboratory). The virus strains used were H3N8 / H3N8-GFP (prepared in Example 1), H1N1 (A / Puerto Rico / 8 / 1934(H1N1)) (preserved in the laboratory) / H1N1-GFP (Shandong Medical University), H3N2 (A / CHINA / LZP / 2017 (H3N2)) / H3N2-GFP (rescued in the laboratory), H5N1 (A / Duck / JIANGXI / PY380 / 2023 (H5N1)) (preserved in the laboratory), and IBV (B / Lee / 1940) (preserved in the laboratory). The construction methods for strains H1N1-GFP and H3N2-GFP are the same as those for H3N8-GFP in Example 1. Both strains are obtained by inserting the GFP gene sequence downstream of the NS1 gene sequence in the NS segment of the wild-type strain. The insertion position corresponds to the insertion position of GFP in H3N8-GFP. The specific experimental steps are as follows.

[0095] Cells were seeded at an appropriate density in 12-well plates before infection. For viral infection, A549 cells were co-incubated for 1 hour with H3N8-GFP (MOI = 0.2), H1N1-GFP (MOI = 0.05), H3N2-GFP (MOI = 0.1), H5N1 (MOI = 0.1), or IBV (MOI = 0.2), respectively. ATs were infected with a 10-fold diluted viral titer for 1 hour, while hAOs were infected with a 20-fold diluted viral titer for the same duration. After infection, the culture medium was aspirated, cells were washed once with phosphate-buffered saline (PFS), and then cultured for another 24 hours in maintenance medium containing different concentrations of Dig.

[0096] First, we measured the effect of Dig on EC infected with H3N8-GFP and H1N1-GFP viruses.50 The results showed that Dig was effective against H3N8-GFP-infected EC 50 The wavelength was 176.9 nm, which was observed in EC1N1-GFP infected with H1N1. 50 206.7 nm (e.g.) Figure 2 (As shown). Furthermore, we examined the inhibitory effects of Dig on H3N8, H1N1, H3N2, H5N1, and IBV. The results indicate that Dig can significantly inhibit IBV replication in A549 cells (as shown). Figure 3 As shown in the figure), it exhibited good broad-spectrum anti-influenza virus effects at all levels (A549 cell line, Ats, hAOs), demonstrating significant and efficient inhibitory capabilities against H3N8, H1N1, H3N2, and H5N1 subtypes of influenza A virus and influenza B virus. Among these, the inhibitory effect against the H5N1 subtype was the best (e.g., Figure 4 (As shown).

[0097] Example 3. In vivo verification of the anti-IAV activity of the screened compounds

[0098] Furthermore, this embodiment verifies the anti-IAV activity of the screened compound Dig through in vivo experiments; the model used is BALB / c mice (Zhiyuan Biomedical Technology Co., Ltd.); the virus strain used is H3N8 A / Henan / 4-10 / 2022 (2×10 6 PFU) and H1N1 A / Puerto Rico / 8 / 1934 (150 PFU).

[0099] Six-week-old specific pathogen-free male BALB / c mice were randomly assigned to experimental groups, with five mice in each group. Two hours before intranasal viral infection, the mice were administered either PBS (solvent control group) or Dig (5 mg / kg) via gavage. Subsequently, the animals were infected with H3N8 virus via intranasal instillation (dose 2 × 10⁻⁶). 6 (PFU) or H1N1 virus (dose of 150 PFU). Specific timelines for compound administration, viral infection, euthanasia, and sample collection are detailed below. Figure 5 A and Figure 6 A. Throughout the study, the mice's weight changes and survival curves were monitored daily.

[0100] Figure 5 B and 5C show the results of Dig treatment in mice against H3N8 virus. Figure 6B and 6C show the results of Dig treatment in mice against H1N1 virus. In vivo experiments showed that Dig could increase the survival rate of mice infected with H3N8 and H1N1, maintain stable body weight, and H&E staining of lung tissue sections showed that Dig could reduce lung damage caused by viral infection and reduce viral load in the lungs, indicating that Dig also has broad-spectrum anti-H1N1 virus activity in vivo.

[0101] Example 4. Dig targets the host protein ATP1A1, thereby inducing NP protein to travel via the proteasome and lysosome pathway. degradation

[0102] This embodiment utilizes the CB-DOCK2 website to perform molecular docking analysis on the target sites of Dig; and uses Dig to treat overexpressed NPs to study the mechanism of action.

[0103] Specifically, molecular docking analysis using CB-DOCK2 predicted a high-affinity interaction between Dig and ATP1A1. To functionally validate ATP1A1 as its target, we knocked down the ATP1A1 gene using siRNA, resulting in a significant reduction in the inhibitory effect of Dig on H3N8 infection. The amino acid sequence of the ATP1A1 protein is shown in SEQ ID NO: 3; the siRNAs used were siRNA-1 (SEQ ID NO: 4 and 5), siRNA-2 (SEQ ID NO: 6 and 7), and siRNA-3 (SEQ ID NO: 8 and 9), and the nucleotide sequence of the negative control siNC is shown in SEQ ID NO: 10 and 11.

[0104] Subsequently, we investigated the downstream effects of Dig binding to ATP1A1. Given that Dig treatment reduces the level of viral NP protein after infection, we assessed whether Dig directly induces NP protein degradation. In cells overexpressing NP protein, Dig significantly reduced NP protein abundance, an effect that was reversed by the proteasome inhibitor MG132 (Selleck, S2619) and the autophagy inhibitor chloroquine (CQ) (Selleck, S6999). In summary, these findings reveal a novel antiviral mechanism: Dig triggers the degradation of influenza virus NP protein (e.g., NP protein degradation) via the ubiquitin-proteasome and autophagy pathways by binding to host ATP1A1. Figure 7 (As shown).

[0105] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use in the preparation of a pharmaceutical product containing a reagent targeting the ATP1A1 protein or a composition containing a reagent targeting the ATP1A1 protein, said pharmaceutical product being used for: (i) Prevention and / or treatment of influenza virus infection in the subject; (ii) Inhibit influenza virus replication and / or reproduction in cells; and / or (iii) To prevent and / or treat illnesses caused by influenza virus in the subject.

2. The use according to claim 1, wherein, The reagent targeting the ATP1A1 protein is a protein or peptide, a nucleic acid aptamer, or a small molecule compound. Preferably, the reagent targeting the ATP1A1 protein is digoxigenin (Dig) or its prodrug, solvate or pharmaceutically acceptable salt; Preferably, the prodrug of isohydroxydigitoxin includes a carrier prodrug of isohydroxydigitoxin (e.g., a glycoside prodrug, a fatty acid ester prodrug, an amino acid ester prodrug, a phosphate ester / sulfate ester prodrug) and an antibody-drug conjugate (ADC) prodrug. Preferably, the glycoside prodrug of the isohydroxydigitoxinin includes digoxin, lanatoside C, deslanoside, and metildigoxin. Preferably, the solvates of the isohydroxydigitoxinogen include polar solvates of the isohydroxydigitoxinogenogen (e.g., hydrates, methanolic compounds, or ethanolic compounds). Preferably, the pharmaceutically acceptable salts of the isohydroxydigitoxin include inorganic salts, organic acid salts, inorganic base salts, and organic base salts of the isohydroxydigitoxin (e.g., sodium salts, potassium salts, calcium salts, hydrochloride salts, nitrates, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, acetates, propionates, butyrates, oxalates, trimethylacetate, adipates, alginates, lactates, citrates, tartrates, succinates, maleates, fumarates, aspartate, gluconate, benzoates, methanesulfonates, ethanesulfonates, or benzenesulfonates).

3. The use according to claim 1 or 2, wherein, The influenza viruses include influenza A and influenza B viruses; Preferably, the influenza A virus includes influenza A subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18; Preferably, the influenza B virus includes influenza B viruses of the Victoria and Yamagata lineages; Preferably, the influenza A virus includes influenza A viruses of subtypes H3N8, H1N1, H3N2, and H5N1; Preferably, the influenza B virus includes the Victoria lineage of influenza B virus.

4. The use according to any one of claims 1-3, wherein, The subjects were mammals; Preferably, the subject is a bovine, equine, suidae, canine, feline, rodent, or primate. Preferably, the subject is a human, rat, monkey, pig, or dog.

5. The use according to any one of claims 1-3, wherein, The cells in question are mammalian cells; Preferably, the cells are cells of bovine animals, equine animals, suidae animals, canine animals, feline animals, rodents, or primates. Preferably, the cells are human cells, mouse cells, monkey cells, pig cells, or dog cells.

6. The use according to any one of claims 1-5, wherein, The diseases mentioned include influenza, uncomplicated infections, pneumonia (such as severe pneumonia), acute or severe acute respiratory infections, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, and septic shock. Preferably, the uncomplicated infection includes fever, cough, and sore throat.

7. The use according to any one of claims 1-6, wherein, The drug also contains pharmaceutically acceptable carriers and / or excipients; Preferably, the pharmaceutically acceptable carrier and / or excipients include pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. Preferably, the medicine further contains other active ingredients (e.g., antiviral agents). Preferably, the additional active ingredients include amantadine, rimantadine, emfuvirdi, maraviro, acyclovir, ganciclovir, valacyclovir, famciclovir, sodium phosphonoformate, lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, efavirenz, nevirapine, saquinavir, oseltamivir, zanamivir, ribavirin, and interferon.

8. The use according to any one of claims 1-7, wherein, The medicine is an injection, infusion, tablet, capsule, spray, aerosol, or rinsing solution; Preferably, the drug is present in unit dose form; Preferably, the drug contains 1-1000 mg (e.g., 1-800 mg, 1-500 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-20 mg, or 1-10 mg) of isohydroxydigitoxin or its prodrug, solvate or pharmaceutically acceptable salt.

9. The use according to any one of claims 1-8, wherein, The drug is a drug that is targeted for release into the gastrointestinal tract, or a drug that is released in a controlled manner into the gastrointestinal tract; Preferably, the medicine is formulated for oral administration.

10. The use according to any one of claims 1-8, wherein, The drug is a parenteral drug; Preferably, the parenteral administration is selected from intravenous, intramuscular, subcutaneous, intranasal, transmucosal, sublingual, rectal, transdermal, inhalation, or any combination thereof; Preferably, the parenteral administration is performed via intravenous infusion.

11. A high-throughput screening method for broad-spectrum anti-influenza virus compounds, the method comprising: Step (1): Provide recombinant influenza virus expressing reporter protein and the compound to be screened; Step (2): The recombinant virus is brought into contact with the cells while, before, or after the cells are treated with the compound; Step (3): Under the condition that the reporter protein is able to generate a signal, measure the signal level (e.g., fluorescence intensity) of the cell. The recombinant influenza virus, relative to the wild-type influenza virus, contains a nucleotide sequence encoding the reporter protein at a position between nucleotides 500 and 501 of the NS gene corresponding to SEQ ID NO:

1.

12. The method of claim 11, wherein, The method has one or more technical features selected from the following: (i) The wild-type influenza viruses include influenza A viruses and influenza B viruses (e.g., influenza A virus of the H3N8 subtype). (ii) The reporter proteins include green fluorescent protein (GFP) and luciferase; (iii) In step (2), after treating the cells with the compound, the recombinant virus is brought into contact with the cells; (iv) The cells include eukaryotic cells and prokaryotic cells, such as A549 cells; Preferably, the NS gene of the recombinant influenza virus is as shown in SEQ ID NO:

2.

13. The method of claim 11 or 12, wherein, The method further includes: Step (4): Compare the measured value in step (3) with the signal level measured without treatment with the compound, and obtain the following ratio: (measured value without treatment with the compound - measured value in step (3)) / measured value without treatment with the compound; Preferably, the method further includes step (5): generating a dose-response curve of the compound based on the ratio obtained in step (4) and thereby obtaining EC. 50 According to the EC 50 The anti-influenza virus activity of the compound was evaluated.

14. The method according to any one of claims 11-13, wherein, The broad-spectrum anti-influenza virus compounds include reagents that target the ATP1A1 protein or compositions containing reagents that target the ATP1A1 protein. Preferably, the broad-spectrum anti-influenza virus compound comprises isohydroxydigitoxinogen or its prodrug, solvate or pharmaceutically acceptable salt.

Images