Use of surinhibitors for antiviral and antitumor

Abstract

The present application discloses the use of SUR inhibitors for antiviral and antitumor purposes. The SUR inhibitors achieve the antiviral and / or antitumor effects by regulating the Toll-like receptor 4 signaling pathway and activating interferon.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to SUR inhibitors, pharmaceutical compositions thereof, and their uses, particularly SUR inhibitors and their pharmaceutical compositions that exert antiviral and antitumor functions by targeting SUR-activated interferon. Background Technology

[0002] Interferons are among the most important core molecules in the innate immune response, playing a crucial role in antiviral infection and antitumor activity. Interferons can induce the transcription of related genes, inducing an antiviral state in virus-infected cells and uninfected neighboring cells. Interferons can also enhance the activity of natural killer cells (NK cells), macrophages, and T lymphocytes, thereby exerting antitumor and immunomodulatory effects.

[0003] Although several mature interferon drugs have been developed, their widespread application is limited by factors such as limited availability and high cost. Therefore, it is necessary to research and develop natural or synthetic compounds that can induce interferon production.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, this invention, through in-depth research, has discovered that certain compounds or synthesized substances can stimulate cells to induce the production of IFNβ, thereby helping cells resist viral infections. Specifically, this invention includes the following:

[0006] A first aspect of the invention provides the use of SUR inhibitors in the preparation of medicaments for regulating the Toll-like receptor 4 signaling pathway and / or activating interferon.

[0007] A second aspect of the invention provides the use of SUR inhibitors in the preparation of antiviral and / or antitumor drugs.

[0008] In some embodiments, according to the use described in the first or second aspect of the invention, the SUR inhibitor has a structure selected from the following formula:

[0009] Among them, R 1 Selected from the following groups: H, C1-C5 alkyl, halogen, phenyl, R 2 Selected from the following groups: C1-C5 alkyl, phenyl, or

[0010] Among them, R 3 Selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl; or

[0011] Among them, R 4 R 5 and R 6 Each group is independently selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl.

[0012] In some embodiments, according to the use described in the first or second aspect of the invention, the inhibitor has a structure selected from the following formula:

[0013]

[0014]

[0015] In some embodiments, according to the use described in the first or second aspect of the invention, the inhibitor has a structure selected from the following formula:

[0016]

[0017] In some embodiments, according to the use described in the first or second aspect of the invention, the SUR inhibitor comprises siRNA or antibody targeting SUR.

[0018] A third aspect of the present invention provides a pharmaceutical composition comprising a SUR inhibitor and an antitumor drug.

[0019] In some embodiments, according to the pharmaceutical composition of the present invention, the SUR inhibitor has a structure selected from the following formula:

[0020] Among them, R 1 Selected from the following groups: H, C1-C5 alkyl, halogen, phenyl, R 2 Selected from the following groups: C1-C5 alkyl, phenyl, or

[0021] Among them, R 3 Selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl; or

[0022] Among them, R 4 R 5 and R 6Each group is independently selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl.

[0023] In some embodiments, the pharmaceutical composition according to the present invention includes, wherein the inhibitor comprises siRNA or antibody targeting SUR.

[0024] A fourth aspect of the present invention provides a method for in vitro regulation of interferon signaling pathways, comprising the step of contacting a SUR inhibitor with cells, wherein the SUR inhibitor comprises a small molecule compound or siRNA or antibody targeting SUR; preferably, the small molecule compound has a structure selected from the following formula:

[0025] Among them, R 1 Selected from the following groups: H, C1-C5 alkyl, halogen, phenyl, R 2 Selected from the following groups: C1-C5 alkyl, phenyl, or

[0026] Among them, R 3 Selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl; or

[0027] Among them, R 4 R 5 and R 6 Each group is independently selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl.

[0028] This invention experimentally demonstrates that knocking out SUR1 or using small-molecule inhibitors targeting SUR can effectively activate interferon production. Interferon, as a core molecule mediating antiviral and antitumor activity, is widely recognized in the scientific community. Data from this invention also shows that small-molecule inhibitors, including but not limited to glibenclamide (sometimes referred to as "Gli"), can effectively inhibit viruses and tumors. Therefore, by using small-molecule drugs targeting the SUR protein, interferon can be activated and exert its antiviral and antitumor functions. When combined with immune checkpoint antibody therapy, it can exhibit even better antitumor effects. Attached Figure Description

[0029] Figure 1The images show (A) RT-qPCR detection of IFNβ expression in HT29 cells after treatment with different concentrations of glibenclamide for 24 hours; (B) RT-qPCR detection of IFNβ expression in HT29 cells after treatment with 150 μM glibenclamide for different time periods. RT-qPCR detection of IFNβ expression in (C) HT1080 cells, (D) PANO2 cells, and (E) CT26 cells after treatment with 150 μM glibenclamide is also presented.

[0030] Figure 2 The following images show the expression of IFNβ in (A) peritoneal macrophages and (B) BMDM cells after treatment with Gli at a final concentration of 150 μM for different time periods, as detected by RT-qPCR; (C) heatmap of RNA-seq results for BMDM cells; (D) volcano plot; (E) GO analysis plot; and (F) KEGG analysis plot.

[0031] Figure 3 The following data are shown: (A) RT-qPCR detection of IFNβ expression in HT29 cells after treatment with Gli at a final concentration of 150 μM, followed by STING inhibitor C176, TBK1 inhibitor BX795, TBK1, and IKKε inhibitor IN-1; (B) Gli treatment of STING - / - BMDM cells and (C)TBK1 - / - IFNβ expression was detected by RT-qPCR in HT29 cells; (D)RIG-I - / - Western blot image of HT29 cells; (E) Gli treatment of RIG-I - / - IFNβ expression was detected by RT-qPCR in HT29 cells; (F)RIG-I - / - Western blot image of HT29 cells; (G)Gli treatment of RIG-I - / - IFNβ expression was detected by RT-qPCR in HT29 cells.

[0032] Figure 4 The results show that (A) HT29 cells were treated with Gli at a final concentration of 150 μM, followed by TLR2 inhibitor C29, Myd88 inhibitor, and TLR4 inhibitor Restorvid, and IFNβ expression was detected by RT-qPCR; (B) BMDM cells were pretreated with TLR4 antibody and IgG antibody for 12 h, followed by treatment with Gli and LPS for 3 h, and IFNβ expression was detected by RT-qPCR.

[0033] Figure 5-6 The results of GO pathway enrichment and KEGG pathway enrichment analysis are shown.

[0034] Figure 7The expression of HSV-1 (A) and VSV mRNA (B) was detected by RT-qPCR after HT29 cells were treated with Gli at a final concentration of 150 μM for 3 h with HSV-1 and VSV, respectively.

[0035] Figure 8 The diagram shows (A) a model of subcutaneous tumor creation and drug administration in wild-type Balb / c mice CT26 (n=5); (B) a picture of the tumor; and (C) a tumor growth curve.

[0036] Figure 9 The images show (A) actual images of null / null CT26 subcutaneous tumors in nude mice (n=4); and (B) tumor volume images.

[0037] Figure 10 The images show (A) actual images of subcutaneous tumors of wild-type Balb / c mice CT26 (n=5); and (B) tumor volume diagrams.

[0038] Figure 11 The images show: (A) Subcutaneous pancreatic cancer tumor of C57bl / 6 mouse PANO2 (n=4); (B) Tumor volume; (C) Subcutaneous colorectal cancer tumor of C57bl / 6 mouse MC38 (n=4); (D) Tumor volume; (E) Subcutaneous melanoma tumor of C57bl / 6 mouse B16 (n=3); (F) Tumor volume.

[0039] Figure 12 The results show that (A) IFNβ expression was detected by RT-qPCR after 3 h of treatment with glimepiride, glimepiride, and gliclazide, respectively; and (B) IFNβ expression was detected by RT-qPCR after 3 h of treatment with repaglinide and miglitol calcium tablets, respectively.

[0040] Figure 13 The results show that IFNβ expression was detected by RT-qPCR after HT29 cells were co-treated with (A) glibenclamide, BaCl2, (B) quinine, and (C) TEA for 3 h.

[0041] Figure 14 The results show that HT29 cells were co-treated with Gli and the ATP-sensitive K+ channel agonist minoxidil sulfate (MS) for 3 h, and IFNβ expression was detected by RT-qPCR.

[0042] Figure 15The results show (A) IFNβ expression detected by RT-qPCR after CRISPR-Cas9 knockout of ABCC8 in HT29 cells and treatment with Gli for 3 h; and (B) IFNβ expression detected by RT-qPCR after CRISPR-Cas9 knockout of kir6 in HT29 cells and treatment with Gli for 3 h. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Uses of SUR inhibitors in drug preparation

[0047] In this invention, the terms "sulfonylurea receptor" and "SUR" are used interchangeably.

[0048] A first aspect of this invention provides the use of SUR inhibitors in the preparation of medicaments for regulating the Toll-like receptor 4 signaling pathway and / or activating interferon. "Toll-like receptor 4" is sometimes simply referred to as "TLR4" herein. The inventors have discovered that certain compounds can act as small-molecule inhibitors of SUR, activating the TLR4 signaling pathway, thereby activating interferon production and exerting antiviral and antitumor functions.

[0049] Unless otherwise stated, the interferon mentioned in this article refers to IFNβ.

[0050] Preferably, the SUR inhibitor includes compounds that can target SUR, particularly SUR1.

[0051] Preferably, the SUR inhibitor is a sulfonylurea compound having a structure selected from the following formula:

[0052] Among them, R 1 Selected from the following groups: H, C1-C5 alkyl, halogen, phenyl, R 2 Selected from the following groups: C1-C5 alkyl, phenyl, Alternatively, the SUR inhibitor is a non-sulfonylurea compound having a structure selected from the following formula.

[0053] Among them, R 3 Selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl; or

[0054] Among them, R 4 R 5 and R 6 Each group is independently selected from the following groups: H, C1-C5 alkyl, halogen, substituted or unsubstituted phenyl.

[0055] In some implementations, the SUR inhibitor has the following structure: This compound is also known as gliclazide, with the molecular formula C. 15 H 21 N3O3S.

[0056] In some implementations, the SUR inhibitor has the following structure: This compound is also known as glimepiride, with the molecular formula C. 24 H 34 N4O5S.

[0057] In some implementations, the SUR inhibitor has the following structure: This compound is also known as glibenclamide, with the molecular formula C. 23 H 28 ClN3O5S.

[0058] In some implementations, the SUR inhibitor has the following structure: This compound is also known as chlorpropamide, with the molecular formula C. 10 H 13 ClN2SO3.

[0059] In some implementations, the SUR inhibitor has the following structure: This compound is also known as tolasulfonylurea, with the molecular formula C. 14 H 21 N3O3S.

[0060] In some implementations, the SUR inhibitor has the following structure: This compound is also known as glibenclamide, with the molecular formula C. 27 H 33 N3O6S.

[0061] In addition to the sulfonylurea compounds mentioned above, the inventors have also discovered that non-sulfonylurea compounds can also serve as small molecule inhibitors of SUR.

[0062] In some implementations, the SUR inhibitor has the following structure: This compound is also known as miglitol, with the molecular formula C. 19 H 25 NO3.

[0063] In some implementations, the SUR inhibitor has the following structure: This compound is also known as repaglinide, with the molecular formula C. 27 H 36 N2O4.

[0064] The compounds mentioned in this invention belong to the category of known hypoglycemic agents. For example, glibenclamide (Gli) is a second-generation sulfonylurea hypoglycemic agent. It works by inhibiting the ATP-sensitive K+ channel (KATP) of pancreatic β-cells. KATP is an octamer complex composed of four Kir6.x (Kir6.1 or Kir6.2) and four sulfonylurea receptors (SUR; SUR1 or SUR2). The SUR subunit belongs to the ATP-binding cassette (ABC) transporter family, and as a regulatory subunit, it makes Kir6.x sensitive to the inhibition of glibenclamide. Therefore, when glibenclamide binds to the SUR subunit, the KATP channel closes, the cell membrane of pancreatic β-cells depolarizes, calcium ions flow inward, leading to insulin release.

[0065] The inventors have experimentally discovered that the aforementioned small molecule compounds can activate downstream signaling pathways by targeting sulfonylurea receptors (SUR1 and / or SUR2), preferably SUR1, thereby activating interferon. In this document, the terms "activation" or "enhancement" are used interchangeably to refer to promoting IFNβ, preferably increasing the amount of IFNβ or promoting the expression of the IFNβ gene. The amount of IFNβ or the expression of the gene can be determined using any method known in the art, and is not particularly limited thereto. Measurement methods include, but are not limited to, chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR, gene chip technology, or Western blotting.

[0066] In this invention, the downstream signaling pathway preferably includes the TLR4 signaling pathway. Furthermore, the inventors have discovered that the activation of IFNβ by the small molecule compounds mentioned herein does not depend on the cGAS-STING and RIG-I-MAVS pathways.

[0067] It is understood that the compounds mentioned above in this invention also include their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" means a salt that, within reasonable medical judgment, is suitable for contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and bases.

[0068] In addition to the small molecule compounds mentioned above, the SUR inhibitors of the present invention can also be activated by genetic engineering techniques, such as targeted knockout or knockdown of SUR1 (which can be used interchangeably with ABCC8), or by using SUR1 siRNA, thereby activating downstream signaling pathways (preferably the TLR4 signaling pathway), upregulating the expression of interferon genes or interferon-induced genes, thereby activating interferon and promoting antiviral and antitumor functions.

[0069] In some embodiments, gene knockout agents include, but are not limited to, CRISPR / Cas9 systems, interfering RNA, or antisense oligonucleotides. While gene knockout agents are used in some embodiments, this does not imply any limitation on the SUR inhibitors of the present invention. Therefore, the SUR inhibitors of the present invention further include antibodies, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, or fragments derived therefrom.

[0070] Pharmaceutical Composition

[0071] In another aspect, the present invention provides a pharmaceutical composition comprising the SUR inhibitor and antitumor drug described herein, and optionally a pharmaceutically acceptable excipient, carrier, or diluent.

[0072] The term "pharmaceutically acceptable excipient, carrier, or diluent" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in the transport or delivery of a drug from one organ or site of the body to another organ or site of the body. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation and does not harm the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants, such as sodium dodecyl sulfonate, magnesium stearate, and polyoxyethylene-polypropylene copolymers, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0073] Another aspect of the invention provides "an antiviral and / or antitumor method" comprising the step of administering a therapeutically effective amount of the SUR inhibitor or pharmaceutical composition described herein to a subject. The term "subject" as used herein refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, and the like who will receive specific treatment. Generally, "subject" and "patient" are used interchangeably in this invention, both referring to a test subject.

[0074] As used in this invention, the terms “give,” “apply,” or “administer” mean the introduction of a SUR inhibitor or a pharmaceutical composition containing it through implantation, absorption, ingestion, injection, inhalation, or other means.

[0075] As used herein, the term "effective amount" refers to the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeutic effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function. Generally, the effective amount as used herein varies depending on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or symptom, subject being treated, etc., but can still be routinely determined by those skilled in the art.

[0076] The term "antiviral and / or antitumor" as used in this invention refers to the improvement of a condition before or after the onset of a disease or dysfunction. This degree of remission or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. In this invention, the term "treatment" refers to therapeutic treatments and preventative or therapeutic measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the progression of viral and / or neoplastic diseases. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). "Treatment" also refers to an extension of survival compared to the expected survival without treatment. Those who need treatment include those who already have the condition or disorder, those who are prone to the condition or disorder, or those who need to prevent the condition or disorder.

[0077] The therapeutic dosage of this invention can vary widely. Generally, the dosage of the compounds, pharmaceutical compositions, or adjuvants used in this invention is well known to those skilled in the art. The dosage can be administered as a single dose or in several doses, such as two, three, or four doses. The administered dosage is within the range that clinicians or laboratory personnel can anticipate, and the dosage can be appropriately adjusted, for example, through efficacy and safety testing, to obtain the optimal dosage.

[0078] The total dose required for each treatment can be divided into multiple administrations or administered as a single dose. The compounds, pharmaceutical compositions, or adjuvants of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs, with dosage adjustments.

[0079] There are no particular limitations on the administration method of the SUR inhibitor or pharmaceutical composition of the present invention. Exemplary administration methods include, but are not limited to, oral administration, intramuscular injection, intravenous injection, intravenous drip, enema, spray, external application, or intraperitoneal injection.

[0080] Solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules. In these solid dosage forms, the inhibitor or pharmaceutical composition is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) a filler or compatibilizer, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) a binder, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) a humectant, such as glycerin; (d) a disintegrant, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) a slowing agent, such as paraffin; (f) an absorption accelerator, such as a quaternary ammonium compound; (g) a wetting agent, such as cetyl alcohol and glyceryl monostearate; (h) an adsorbent, such as kaolin; and (i) a lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0081] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the inhibitor or pharmaceutical composition can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the SUR inhibitor or pharmaceutical composition of the present invention may also be formed into microcapsules with one or more of the excipients described above.

[0082] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the SUR inhibitors or pharmaceutical compositions of the present invention, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof.

[0083] In addition to these inert diluents, the SUR inhibitors or pharmaceutical compositions of the present invention may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0084] In addition to the SUR inhibitors or pharmaceutical compositions of the present invention, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0085] SUR inhibitors or pharmaceutical compositions intended for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0086] Dosage forms of the SUR inhibitors or pharmaceutical compositions of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The SUR inhibitors or pharmaceutical compositions of the present invention are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0087] The SUR inhibitors or pharmaceutical compositions of the present invention can be administered alone or in combination with other therapeutic agents. Examples of other therapeutic agents include, but are not limited to, drugs that can be used for antiviral and / or antitumor purposes, such as antibody therapeutics, chemical drugs, miscellaneous drugs, alkylating agents, antimetabolites, antitumor antibiotics, hormonal drugs, immunomodulators, antimicrobial agents, etc.

[0088] In some implementations, other therapeutic agents, such as anti-tumor drugs, include, but are not limited to, anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, and anti-CD antigen antibodies.

[0089] In this invention, tumors include hematologic malignancies, solid tumors, or combinations thereof. In some embodiments, the hematologic malignancies include, but are not limited to, acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0090] In some embodiments, the solid tumors include, but are not limited to, gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, cervical cancer, endometrial cancer, mesothelioma, pancreatic cancer, or combinations thereof.

[0091] In this invention, "virus" includes, but is not limited to, general DNA and RNA viruses, as well as retroviruses. Examples of DNA viruses include the Parvoviridae, Papillaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Hepatoviridae families. Examples of RNA viruses include the Picornaviridae, Clonorviridae, Rhabdoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Reoviridae, RNA Oncologicviridae, and Filaviridae families. In particular, it includes DNA viruses such as Herpesviridae and Adenoviridae, and RNA viruses such as Paramyxoviridae, Orthomyxoviridae, Picornaviridae, Coronaviridae, Clonorviridae, and Rhabdoviridae. This includes varicella-zoster virus (VZV) of the Varicellavirus genus (Herpesviridae family), herpes simplex virus types I and II (HSV-I, II) of the Herpes simplex virus genus (Herpesvirus family); adenovirus types 3 and 7 (AdV3, 7) of the Adenovirus genus (Adenoviridae family); measles virus (MV) of the Measlesvirus genus (Paraxviridae family), mouse pneumonia virus, respiratory syncytial virus (RSV) and parainfluenza virus-I of the Pneumovirus genus (Pneumovirus family); influenza virus (A3V) of the Influenza Virus genus (Orthomyxoviridae family), and the mouse lung-adapted strain FM1 of influenza virus subtype A; small RNA Enteroviruses (Viridaceae family) include poliovirus type III (PVIII), echoviruses 6 and 11 (ECH06, 11), Coxsackieviruses B (CVB3, CVB4, CVB5, CVB6), Coxsackievirus A16 (CVA16), and novel enterovirus 71 (EV71); SARS virus (Coronaviruses) (Coronaviridae family); rubella virus (RV) (Russiaviruses) (Blephaviridae family); and vesicular stomatitis virus (VSV) (Varicaviridae family).

[0092] Methods for modulating interferon signaling pathways

[0093] This invention provides a method for in vitro modulation of interferon signaling pathways. In some embodiments, the interferon signaling pathway includes the SUR1-TLR4-IFNβ axis.

[0094] In some implementations, regulation includes promoting, activating, upregulating, and / or enhancing interferon signaling-related pathways, preferably the TLR4 signaling pathway, thereby enhancing, increasing, and / or upregulating the amount or activity of IFNβ, or increasing the amount of interferon genes and / or interferon-induced genes, or promoting the expression of interferon genes and / or interferon-induced genes.

[0095] In some implementation schemes, regulation includes at least one of the following:

[0096] (a) Reduce viral load and / or activity;

[0097] (b) Enhance or improve the interferon response;

[0098] (c) Reduce the expression of virus-derived mRNA.

[0099] The method of the present invention for in vitro modulation of interferon signaling pathways includes the step of contacting a SUR inhibitor with cells. Cells include cancer cell lines or immune cells, examples of which include, but are not limited to, HT29 cells, HT1080 cells, Pan02 cells, and CT26 cells. Examples of immune cells include, but are not limited to, natural killer cells, macrophages, and T lymphocytes.

[0100] Example

[0101] This embodiment uses glibenclamide as an exemplary small molecule inhibitor.

[0102] 1. Glibenclamide can activate IFNβ in tumor cells.

[0103] As an FDA-approved drug, glibenclamide's safety is guaranteed. This example demonstrates that treatment of cells with appropriate concentrations of glibenclamide leads to upregulation of interferon and interferon-stimulated genes (ISGs) expression, exhibiting antiviral and antitumor effects.

[0104] RT-qPCR analysis after treating HT29 cells with different concentrations of Gli for 24 hours revealed that Gli induced interferon IFNβ expression in a concentration-dependent manner, with the strongest activation observed at a final concentration of 150 μM. Therefore, this concentration was used in subsequent experiments. RT-qPCR analysis of IFNβ expression in HT29 cells treated with a final concentration of 150 μM Gli for different time periods showed that the strongest activation occurred after 3 hours of treatment. Therefore, subsequent experiments were conducted with a 3-hour treatment period. Gli also induced IFNβ expression in HT1080 cells, Pan02 cells, and CT26 cells (e.g., ...). Figure 1 (As shown).

[0105] 2. Glibenclamide activates IFNβ in primary cells.

[0106] Treatment of mouse peritoneal macrophages (bone marrow-derived macrophages, BMDM) with Gli significantly activated IFNβ production in both Gli-derived peritoneal macrophages and BMDM cells. Furthermore, RNA-seq analysis of BMDM cells revealed that Gli activated a series of positively immunomodulatory cytokines (such as...). Figure 2 (As shown).

[0107] 3. Glibenclamide activates IFNβ independently of the cGAS-STING and RIG-I-MAVS pathways.

[0108] To investigate the mechanism by which Gli activates IFNβ, HT29 cells were co-treated with the STING inhibitor C176, the TBK1 inhibitor BX795, and the TBK1 and IKKε inhibitor IN-1, respectively. The results showed that Gli activation of IFNβ was not affected by the STING inhibitor BX795, while STING… - / - BMDM results showed that Gli activation of IFNβ was unaffected, indicating that Gli activation of IFNβ is independent of the STING-CGAS signaling pathway. The inhibitors BX795 (TBK1), TBK1, and IN-1 (IKKε) all significantly inhibited the production of Gli activation of IFNβ. Knockout of TBK1 significantly inhibited the production of Gli activation of IFNβ, indicating that Gli activation of IFNβ is dependent on TBK1.

[0109] Next, RIG-I was knocked out in HT29 cells. The results showed that knocking out RIG-I did not affect Gli activation of IFNβ, indicating that Gli activation of IFNβ is independent of the RIG-I-MAVS signaling pathway. IRF3 is downstream of TBK1, and IFNβ expression depends on the transcription factor IRF3. Does Gli activation of IFNβ depend on IRF3? In this example, IRF3 was knocked out in HT29 cells, and then WT HT29 cells and IRF3-knockout cells were treated with Gli, respectively. The results showed that knocking out IRF3 almost completely inhibited Gli activation of IFNβ, indicating that Gli activation of IFNβ depends on IRF3 (e.g., Figure 3 (As shown).

[0110] 4. Glibenclamide activates IFNβ through the TLR4 signaling pathway.

[0111] Toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and induce the body to produce a variety of pro-inflammatory factors, chemokines, and type I interferons, playing an indispensable role in the innate immune response. So, does Gli exert its effect through TLRs? In this study, HT29 cells were co-treated with TLR2 inhibitors, TLR4 inhibitors, Myd88 inhibitors, and TRIF inhibitors, respectively. The results showed that TLR2 inhibitors did not inhibit Gli activation of IFNβ, while TLR4 inhibitors significantly inhibited Gli activation of IFNβ; Myd88 inhibitors inhibited Gli activation of IFNβ in a concentration-dependent manner; and TRIF inhibitors significantly inhibited Gli activation of IFNβ.

[0112] Next, BMDM cells were pretreated with a TLR4 antibody for 12 hours, followed by treatment with Gli and LPS for 3 hours. The results showed that the TLR4 antibody significantly inhibited the activation of IFNβ by Gli and LPS, while the IgG antibody did not affect the activation of IFNβ by Gli and LPS. This indicates that Gli and LPS, similarly, activate IFNβ by transmitting signals downwards through TLR4. Then, RNA-Seq was performed on samples of BMDM cells treated with Gli and Gli plus a TLR4 inhibitor for 3 hours. The results showed that the TLR4 inhibitor significantly inhibited the activation of Gli, including Il1a, Il1b, Tnf, Cxcl10, Ptgs2, IFNβ, and Il6. GO pathway enrichment analysis mainly focused on signaling pathways such as IL6, IL8, NF-κB, and TLR4. KEGG pathway enrichment analysis, in addition to focusing on the TLR signaling pathway, also enriched in Th1 and Th2 signaling pathways (e.g., IL6, IL8, NF-κB, and TLR4). Figure 4-6 (As shown).

[0113] 5. Glibenclamide has antiviral effects.

[0114] To investigate the antiviral effect of Gli, HT29 cells were co-treated with Gli at a final concentration of 100 μM and HSV-1 and VSV at an MOI of 0.1 for 3 hours, and the viral genes were then analyzed. The results showed that the Gli group had significant antiviral activity (e.g., Figure 7 (As shown).

[0115] 6. Glibenclamide has anti-tumor effects.

[0116] To investigate the antitumor effect of Glibenzyl ether (Glibenzyl ether) in mice, CT26 colorectal cancer was induced by subcutaneous inoculation with Balb / c mice, followed by intraperitoneal injection of Glibenzyl ether. PBS was used as a control. Tumor volume and mouse weight were monitored periodically. The results showed that Glibenzyl ether inhibited tumor growth, indicating that intraperitoneal injection of Glibenzyl ether has an antitumor effect (e.g., Figure 8 (As shown).

[0117] 7. Intraperitoneal injection of glibenclamide in nude mice showed no antitumor effect.

[0118] To rule out the influence of the immune system on Gli, a CT26 colorectal cancer model was established in null / null mice. Gli was administered via intraperitoneal injection and oral administration, respectively. The results showed that Gli did not inhibit tumor growth, indicating that Gli has no antitumor effect in null mice (e.g., Figure 9 (As shown).

[0119] 8. Glibenclamide can enhance the therapeutic effect of anti-PD-1 therapy.

[0120] To investigate whether Gli can synergize with anti-PD-1 immunotherapy, CT26 subcutaneous tumor results showed that, compared with other groups, the Gli plus anti-PD-1 group had the smallest tumor volume, indicating that Gli can synergize with anti-PD-1 immunotherapy (such as...). Figure 10 (As shown).

[0121] 9. Oral administration of glibenclamide has an antitumor effect.

[0122] Gli is a first-line oral drug. To study its oral efficacy, pancreatic cancer cells were subcutaneously inoculated with PANO2 in C57bl / 6 mice. Compared with intraperitoneal injection, oral administration significantly reduced tumor volume in MC38 colorectal cancer and B16 melanoma. In summary, this indicates that Gli has an antitumor effect (e.g., Figure 11 (As shown).

[0123] 10. Studies on the activation of IFNβ by other sulfonylurea drugs

[0124] This embodiment further demonstrates, through experiments, that other sulfonylurea drugs, such as glimepiride, glibenclamide, and gliclazide, can also activate IFNβ; while other non-sulfonylurea drugs that can inhibit SUR1 / 2, such as repaglinide and miglitol calcium tablets, have also been shown to activate IFNβ (e.g. Figure 12 (As shown).

[0125] Commonly used pharmacological K+ channel inhibitors include Ba... 2+ To determine whether K+ channels affect Gli activation of IFNβ, HT29 cells were co-treated with BaCl2, TEA, and quinine (inhibitors of inward rectified K+ channels, including those with inward rectified K+ channels, tetraethylamine (TEA, voltage-gated K+ channel inhibitor), and quinine (two-pore domain K+ channel inhibitor, K2P). The results showed that BaCl2 and quinine inhibited Gli activation, while TEA at high concentrations had a slight inhibitory effect (e.g., BaCl2 and quinine). Figure 13 (As shown).

[0126] 11. ATP-sensitive K+ channel agonists antagonize glibenclamide-activated interferon.

[0127] As one of the representative ATP-sensitive K+ channel inhibitors, is the activation of IFNβ by Gli affected by agonists? In this example, HT29 cells were co-treated with Gli for 3 h using the ATP-sensitive K+ channel agonist minoxidil sulfate (MS). The results showed that different concentrations of MS could inhibit the activation of Gli (e.g., ...). Figure 14 (As shown).

[0128] 12. Knocking out ABCC8 (SUR1) activates IFNβ, while knocking out kir6 does not affect glibenclamide's activation of IFNβ.

[0129] To investigate the effects of sulfonylurea drugs on the receptor ABCC8, ABCC8 was knocked out in HT29 cells using CRISPR-Cas9 (or ABCC8 siRNA). The results showed that HT29 cells with ABCC8 knockout expressed high levels of IFNβ without any further treatment. Subsequently, knocking out the ATP-sensitive K+ channel regulatory subunit kir6 showed that knocking out kir6 did not affect Gli activation of IFNβ (e.g., Figure 15 (As shown).

[0130] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. The use of SUR inhibitors in the preparation of antiviral and / or antitumor drugs, characterized in that, The virus is HSV-1 or VSV, the tumor is colorectal cancer or pancreatic cancer, and the SUR inhibitor is glibenclamide.

2. The use according to claim 1, characterized in that, The SUR inhibitor regulates the Toll-like receptor 4 signaling pathway and activates interferon.

3. The application of glibenclamide and anti-PD-1 antibody in the preparation of antitumor drugs, characterized in that, The tumor is either colorectal cancer or pancreatic cancer.

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