Use of TMED10 as target in treating inflammation

Abstract

Use of an E-TMED10 inhibitor in preparing a product for treating and / or preventing inflammation. The interaction between the E protein of the coronavirus and the TMED10 (E-TMED10) is a potential molecular mechanism for driving excessive inflammation induced by the coronavirus, and is also a potential treatment target for resisting inflammation harmful effects induced by the coronavirus. The interaction between the E protein of the coronavirus and the TMED10 is blocked or reduced, such that the release of pro-inflammatory factors can be reduced, thereby reducing lung inflammation.

Description

Application of TMED10 as a target in the treatment of inflammation Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of TMED10 as a target in treating inflammation, and more particularly to the use of E-TMED10 inhibitors in treating and / or preventing inflammation. Background Art

[0002] The transmembrane EMP24 domain-containing protein (TMED) family is organized into four subfamilies in humans: α (TMED4, 9), β (TMED2), γ (TMED1, 3, 5, 6, 7), and δ (TMED10), comprising nine members. These members are crucial regulators of intracellular protein trafficking, participating in normal embryonic development and the pathogenesis of many human diseases. The prior art publication, "TMED family genes and their roles in human diseases" (Lv Zhou, International Journal of Medical Sciences, 2023), reviews the relationships between TMED family members and human diseases, including cancer, diabetes, neurodegenerative diseases, nonalcoholic fatty liver disease, dilated cardiomyopathy, mucin-1 nephropathy, and Sjögren's syndrome.

[0003] Regarding the relationship between TMED10 and inflammation, the prior art document: A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion (Min Zhang, Cell, 2020) discloses that TMED10 directly mediates the membrane translocation of cytoplasmic proteins lacking signal peptides into liposomes, that is, TMED10 promotes the secretion of inflammatory factors by mediating the non-classical secretion pathway.

[0004] It is well known that excessive and uncontrolled release of proinflammatory cytokines in infections with pathogens such as coronaviruses (such as SARS2, SARS, and MERS) can trigger immunopathogenesis, leading to widespread tissue damage throughout the body, including acute lung injury. This process is associated with the development of multiple organ dysfunction syndrome and an increased risk of mortality. Inflammatory factors circulating in the bloodstream of coronavirus-infected patients, including interleukin-1 (IL1), interleukin-6 (IL6), interleukin-12 (IL12), interferon-γ (IFNγ), and tumor necrosis factor-α (TNF-α), are elevated and strongly correlated with the severity of the disease. However, the precise cellular mechanisms by which coronaviruses modulate host inflammatory responses, particularly in terms of the release of proinflammatory cytokines, remain largely unresolved.

[0005] Summary of the Invention

[0006] To address the deficiencies of the prior art, the present application discloses the molecular mechanism that drives coronavirus-induced excessive inflammation. That is, the envelope protein (E protein) of the coronavirus, rather than other viral proteins, releases and enhances lung inflammation by activating the IL1 family in the UcPS pathway mediated by TMED10. Specifically, the interaction between the coronavirus E protein and TMED10 enhances TMED10 oligomerization and promotes the transport of UcPS cargo to ERGIC. Mechanistically, the E protein of the severe coronavirus has an SS / SD motif that promotes interaction with TMED10, thereby promoting its oligomerization, which is a translocation process necessary for the secretion of various inflammatory factors through UcPS. In addition, the present application further verifies that blocking or reducing the interaction between the E protein and TMED10 can reduce the release of proinflammatory factors and alleviate lung inflammation.

[0007] The present invention provides use of an E-TMED10 inhibitor in preparing a product for treating and / or preventing inflammation.

[0008] E-TMED10 is the interaction between coronavirus E protein (envelope protein) and TMED10, preferably the interaction during the formation of a complex between E protein and TMED10.

[0009] E-TMED10 inhibitors block or reduce the interaction between coronavirus E protein and TMED10.

[0010] Preferably, the E-TMED10 inhibitor treats and / or prevents inflammation by blocking or reducing the oligomerization of TMED10.

[0011] Preferably, the E-TMED10 inhibitor treats and / or prevents inflammation by blocking or reducing the transport of UcPS cargo to ERGIC.

[0012] Preferably, the E-TMED10 inhibitor blocks or reduces the transport of inflammatory factors to vesicles in the UcPS pathway.

[0013] Preferably, the E-TMED10 inhibitor blocks or reduces the translocation of inflammatory factors into the membrane.

[0014] Preferably, the E-TMED10 inhibitor treats and / or prevents inflammation by blocking or reducing the release of inflammatory factors in the UcPS pathway.

[0015] Preferably, the inflammatory factors include inflammatory factors of the IL1 family, such as one or more of IL1β (preferably mIL1β), IL33 (preferably mIL33), IL36α (preferably mIL36α), IL1α (preferably mIL1α) or IL18 (preferably mIL18).

[0016] Preferably, the E-TMED10 inhibitor targets the luminal domain GOLD, the luminal domain CC, the transmembrane domain (TM) or the cytoplasmic C-terminus (CT), and further preferably, targets the cytoplasmic C-terminus of TMED10, preferably targeting a sequence including YLRRFFKAKKLIE (SEQ ID NO: 2), to block or reduce the interaction between E protein and TMED10.

[0017] Preferably, the E-TMED10 inhibitor targets the SS motif or DS motif of the coronavirus E protein to block or reduce the interaction between the E protein and TMED10, wherein S represents serine and D represents aspartic acid.

[0018] Preferably, the SS motif or DS motif is located at positions 60-75, preferably 67-68 or 71-72, of the amino acid sequence of the E protein.

[0019] The inflammation is inflammation caused by coronavirus infection.

[0020] Preferably, the coronavirus comprises an SS motif or a DS motif in its E protein, wherein S represents serine and D represents aspartic acid. Further preferably, the SS motif or the DS motif is located at positions 60-75, preferably 67-68 or 71-72, of the E protein amino acid sequence.

[0021] The coronavirus infects vertebrates, such as humans, mice, pigs, cats, dogs, wolves, cattle or birds, wherein the birds include poultry or birds.

[0022] The coronavirus includes genus α, genus β, genus γ or genus δ, preferably genus β.

[0023] Preferably, the alpha coronavirus includes human coronavirus 229E or a mutant thereof, human coronavirus NL63 or a mutant thereof, human coronavirus HKU1 or a mutant thereof, long-winged bat coronavirus HKU8 or a mutant thereof, horseshoe bat coronavirus HKU2 or a mutant thereof, porcine epidemic diarrhea virus (PEDV) or a mutant thereof, porcine transmissible gastroenteritis virus (TGEV) or a mutant thereof, canine coronavirus (CCoV) or a mutant thereof or feline coronavirus (FCoV) or a mutant thereof One or more of them.

[0024] Preferably, the beta coronavirus includes human coronavirus HKU1 or a mutant thereof, mouse coronavirus or a mutant thereof, house bat coronavirus HKU5 or a mutant thereof, fruit bat coronavirus HKU9 or a mutant thereof, severe acute respiratory syndrome (SARS) or a mutant thereof, MERS or a mutant thereof, SARS-CoV-2 or a mutant thereof, bovine coronavirus (BCoV) or a mutant thereof, human coronavirus OC43 or a mutant thereof, equine coronavirus (ECoV) or a mutant thereof, porcine hemagglutinating encephalomyelitis virus (PHEV) or a mutant thereof, canine respiratory coronavirus (CrCoV) or a mutant thereof, one or more thereof.

[0025] Further preferably, the murine coronavirus comprises one or more of mouse hepatitis virus (MHV), rat coronavirus or avian auklet virus;

[0026] Further preferably, the SARS mutant includes one or more of BJ01, Urbani, Tor2, CUHK or HKV5;

[0027] Further preferably, the SARS-CoV-2 mutant is selected from Alpha, Beta, Gamma, Delta, Lambda or Omiron.

[0028] Preferably, the γ coronavirus includes avian coronavirus such as infectious bronchitis virus (IBV) or its mutant, Beluga whale coronavirus SW1 (BWCoV-SW1) or its mutant;

[0029] Further preferably, avian coronaviruses include coronaviruses that cause infection in poultry such as chickens, turkeys, sparrows, ducks, geese, and pigeons;

[0030] Preferably, the delta coronavirus includes Bulbul coronavirus HKU11 (BuCoV HKU11) or a mutant thereof, Thrush coronavirus HKU12 (ThCoV HKU12) or a mutant thereof, Thrush coronavirus HKU12 (ThCoV HKU12) or a mutant thereof, Asian Leopard Cats Coronavirus (ALCCoV) or a mutant thereof, Chinese ferret-badger Coronavirus (CFBCoV) or a mutant thereof, Porcine Delta Coronavirus (PDCoV) or a mutant thereof, White-eye Coronavirus (WECoV) or a mutant thereof, Sparrow Coronavirus (SPCoV) or a mutant thereof, Magpie robin Coronavirus (MRCoV) or a mutant thereof, Night heron Coronavirus (NHCoV) or a mutant thereof, Wild duck coronavirus (Wigeon Coronavirus (WiCoV) or its mutants, Common Moorhen Coronavirus (CMCoV) or its mutants, one or more of them.

[0031] Preferably, the coronavirus comprises an SS motif or a DS motif in its E protein, wherein S represents serine and D represents aspartic acid;

[0032] Preferably, the SS motif or DS motif is located at positions 60-75, preferably 67-68 or 71-72, of the amino acid sequence of the E protein.

[0033] Preferably, the inflammation includes pneumonia, nephritis, hepatitis or splenitis.

[0034] Preferably, the inflammation includes lung damage caused by immune cell infiltration.

[0035] The E-TMED10 inhibitors include small molecules, traditional Chinese medicine, traditional Chinese medicine extracts, antibodies, RNAi (interfering RNA), reagents for knocking out TMED10, or cell therapy drugs.

[0036] Preferably, the E-TMED10 inhibitor includes an agent that expresses or overexpresses the cytoplasmic domain of the coronavirus E protein, such as a vector that expresses or overexpresses the cytoplasmic domain of the coronavirus E protein.

[0037] Preferably, the vector can be a viral vector or a non-viral vector.

[0038] Preferably, the viral vector comprises: one or more of a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector or a herpesvirus vector.

[0039] Preferably, the non-viral vector comprises any one or more of liposomes, lipid nanoparticles, polymers, polypeptides, antibodies, aptamers or N-acetylgalactosamine (GalNAc).

[0040] Preferably, the cytoplasmic domain of the coronavirus E protein comprises SEQ ID NO: 1 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more homology to SEQ ID NO: 1, or having a substitution, deletion, or insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0041] Preferably, the vector comprises a nucleotide sequence encoding the cytoplasmic domain of the coronavirus E protein, preferably comprising a single copy or two or more copies of the nucleotide sequence, for example, 1, 2, 3, 4, 5, 6, 7 or 8 copies.

[0042] Preferably, the cytoplasmic domain of the coronavirus E protein is expressed or overexpressed in the immune cells.

[0043] Preferably, the immune cells include one or more of lymphocytes, monocytes, macrophages, NK cells, eosinophils, neutrophils or natural killer cells.

[0044] Preferably, the E-TMED10 inhibitor includes an agent that knocks out or silences TMED10 protein or gene.

[0045] Preferably, knockout or silencing can be achieved by methods such as shRNA, siRNA, CRISPR / Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology, or homing endonuclease.

[0046] Preferably, the E-TMED10 inhibitor is selected from progesterone or its analogs, dichlorophenol or its analogs, nitazoxanide or its analogs.

[0047] The progesterone analogue has the general formula (I) or a pharmaceutically acceptable salt, solvate or metabolite thereof:

[0048] in:

[0049] X is O or NOCH3.

[0050] R1 and R2 are independently selected from H, -C1-C6 alkyl, halogen, -OH, -C1-C6 alkoxy, or R1 and R2 do not exist and a double bond is formed between the carbon atoms to which R1 and R2 are connected; preferably, R1 is -CH3; preferably, R2 is H or halogen; preferably, R1 and R2 do not exist and a double bond is formed between the carbon atoms to which R1 and R2 are connected.

[0051] R3 is selected from H, -C1-C6 alkyl, phenyl or substituted phenyl; preferably, R3 is H or where R 13 Selected from H, -C1-C6 alkyl, -C1-C6 alkoxy, -NR 14 R 15 、-COR 14 、-SR 14 、-SOR 14 or-SO2R 14 , where R 14 and R 15 Independently selected from H or C1-C6 alkyl; further preferably, R3 is H or More preferably, R 13 It is -N(CH3)2.

[0052] R4 and R5 are independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy; preferably, R4 is H; preferably, R5 is -CH3.

[0053] R6 is selected from H, -C1-C6 alkyl, -(CH2) m -halogen, -(CH2) m OR 16 , R 16 Selected from H, -C(=CH2)R 17 Or-COR 17, wherein m is an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6); preferably, m is 1, 2 or 3; preferably, R 17 Selected from -C1-C 12 Alkyl; further preferably, R 17 is H, -CH3, -CH2-CH3 or n is an integer of 1-6 (eg, 1, 2, 3, 4, 5 or 6).

[0054] Preferably, R6 is selected from -CH3, -CH2-halogen, -CH2-OR 16 .

[0055] R7 is selected from H, -OH, -OCOC1-C 12 Alkyl, or R7, R8 and adjacent carbon atoms form an oxygen-containing heterocycle; preferably, R7 is selected from H, -OH, -OCOC1-C6 alkyl (for example, -OCOCH3, -OCO(CH2)2CH3).

[0056] Preferably, R7, R8 and adjacent carbon atoms form a five-membered heterocyclic ring containing 1 or 2 oxygen atoms, more preferably, R7, R8 and adjacent carbon atoms form

[0057] R8 and R9 are independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy, or R8 and R9 and the connected carbon atom form Preferably, R8 and R9 are H or R8 and R9 and the carbon atom to which they are attached form

[0058] R 10、 R 11 and R 12 Independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy.

[0059] Preferably, R 10 It is H or -CH3.

[0060] Preferably, R 11 H or F.

[0061] Preferably, R 12 It is H or -CH3.

[0062] In one embodiment of the present invention, the progesterone analogue is selected from:

[0063] Preferably, the treatment and / or prevention of inflammation includes any one or more of the following:

[0064] A) Inhibit the secretion of inflammatory factors;

[0065] B) reduce E protein-promoted TMED10 oligomerization;

[0066] C) reduce the translocation of inflammatory factors into the membrane;

[0067] D) reduce the transfer of UcPS cargo to ERGIC;

[0068] E) Reduce the transport of inflammatory factors into vesicles via the UcPS pathway.

[0069] The present invention also provides a method for treating and / or preventing inflammation, comprising administering an effective amount of an E-TMED10 inhibitor to a subject in need thereof.

[0070] The E-TMED10 inhibitor can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0071] The E-TMED10 inhibitor can be in any suitable dosage form, such as a gastrointestinal dosage form or a parenteral dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, and the like.

[0072] Various dosage forms of the E-TMED10 inhibitor can be prepared according to conventional production methods in the pharmaceutical field.

[0073] The weight ratio of the active ingredient (E-TMED10 inhibitor) in the various dosage forms is 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).

[0074] The amount of E-TMED10 inhibitor administered to the subject is 0.0001-1000 mg, for example 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 800, 1000 mg.

[0075] The present invention further provides a use of progesterone or an analog thereof in treating and / or preventing inflammation by blocking or reducing the interaction between coronavirus E protein and TMED10.

[0076] The abbreviation "TMED10" of the present invention is the full name of "Transmembrane emp24 domain-containing protein 10", which is a protein containing a transmembrane emp24 domain.

[0077] The abbreviation "ERGIC" in the present invention is ER-Golgi intermediate compartment, which stands for endoplasmic reticulum-Golgi intermediate region.

[0078] The abbreviation "UcPS" in the present invention stands for unconventional protein secretion, which stands for non-classical secretion.

[0079] The abbreviation "THU" in the present invention stands for TMED10-channeled UcPS, which represents the TMED10-mediated non-classical secretion pathway.

[0080] The "halogen" described in the present invention includes F, Cl, Br or I.

[0081] The "alkyl" mentioned in the present invention includes straight-chain alkyl, branched-chain alkyl, cycloalkyl, or a combination thereof, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc.

[0082] The "C1-C6 alkoxy" mentioned in the present invention includes alkoxy groups containing 1, 2, 3, 4, 5 or 6 carbon atoms, such as methoxy, ethoxy and the like.

[0083] The "substituted phenyl" described in the present invention includes phenyl groups having 1, 2, 3, 4 or 5 substituents on the phenyl ring, and the substituents include alkyl, halogen, -alkoxy, -OH, -CN, -amide, -COOH, -amino or substituted amino groups.

[0084] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0085] The "pharmaceutically acceptable salt" of the present invention refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base, wherein the acid or base includes an inorganic acid or base or an organic acid or base. The inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, hydroiodic acid, or sulfuric acid. The inorganic base is selected from calcium, magnesium, lithium, sodium, zinc, aluminum, or potassium. The organic acid is selected from formic acid, glycolic acid, propionic acid, acetic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, glutamic acid, benzoic acid, stearic acid, alginic acid, benzenesulfonic acid, glucuronic acid, pamoic acid, or galacturonic acid. The organic base is selected from diethanolamine, choline, procaine, lysine, or 1,2-ethylenediamine.

[0086] As used herein, a "solvate" refers to a physical association of a compound of the invention with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, a solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solution phases and isolatable solvates. Representative solvates include ethanolates, methanolates, and the like.

[0087] The "metabolites" mentioned in the present invention refer to products obtained by chemical degradation of the compounds of the present invention under physiological conditions in vivo.

[0088] The term "overexpression" as used in the present invention refers to the expression level of the target gene being higher than that in the original cell, and also includes the expression of the target gene that was not expressed in the original cell line.

[0089] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0090] The "effective amount" of the present invention refers to the amount or dosage of the drug of the present invention that provides the desired treatment or prevention after single or multiple doses are administered to a subject or organ.

[0091] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0092] The "subject" described in the present invention can be a human or non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, zoo animal, commercial animal, pet, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0093] The inflammation described in the present invention can be inflammation occurring in any tissue, including but not limited to the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxillary bone, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicle, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, and leukocytes. Further preferably, the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (e.g., allergic rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity reactions, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis, or interstitial cystitis.

[0094] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0095] The term "homology" as used in the present invention refers to that, in terms of using an amino acid sequence or a nucleotide sequence, those skilled in the art can adjust the sequence according to actual work needs, while ensuring that the structure or function is similar to that of a known sequence, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0097] Figure 1: Coronavirus E protein promotes UcPS. (A-B) mIL1β secretion in HEK293T cells transfected with mIL1β-HA alone or co-transfected with FLAG-tagged SARS2 protein. (C) Secretion of mature forms of IL1s and IL6 was examined in HEK293T cells expressing or not expressing Myc-E-SARS2. (D) IL1β secretion in WT or GSDMD-KO THP-1 cells expressing or not expressing Myc-E-SARS2. THP-1 cells were induced to differentiate and treated with 50 ng / ml LPS overnight and 2 mM ATP for 30 min. (E) mIL1β secretion in HEK293T cells transfected with mIL1β-HA alone or co-transfected with coronavirus Myc-E. (F) mIL1β secretion in THP-1 cells expressing or not expressing coronavirus Myc-E. Differentiated cells were treated with LPS and ATP. (G) mIL33 secretion in HEK293T cells transfected with mIL33-FLAG alone or with Myc-E from the coronavirus. (H) IL33 secretion in BEAS-2B cells expressing or not expressing the indicated Myc-E from the coronavirus. (IL) C57BL / 6WT mice were injected with AAV-GFP, AAV-E-SARS2, or AAV-E-229E, stimulated with LPS, and euthanized. Lung expression of E-SARS2, E-229E, or GFP was confirmed by immunofluorescence (K, left panel). (I) Serum IL1β levels were measured by ELISA (mean ± SD). (J) Quantitative PCR analysis of IL6 mRNA expression in tissues (mean ± SD). (L) H&E staining (K, right panel) and quantitative analysis of inflammatory cell infiltration were performed. p values ​​(one-way ANOVA, n = 5, *: p value < 0.05; **: p value < 0.01; ***: p value < 0.001). Scale bar, 50 μm. (MO) Secretion of mIL1β-HA in HEK293T cells transfected with WT, SARS2 (MN), or SARS (O) mutants, either alone or together with Myc-tagged E. Lysate represents cell lysate, and medium represents culture medium.

[0098] Figure 2: Coronavirus E protein promotes UcPS. (A) Secretion of mIL1β from HEK293T cells expressing mIL1β-HA alone or with varying amounts of Myc-E-SARS2. Bar graph represents LDH release (mean ± SD). (B) Secretion of mIL1β from HEK293T cells expressing mIL1β-HA alone or with untagged, Myc-, or FLAG-tagged E-SARS2. E protein expression was detected using an anti-E antibody. (C) HEK293T cells expressing Myc-E WT or SARS2 T9I were treated with 50 μg / mL CHX in the absence or presence of 10 μM MG-132 and 0.5 μg / mL BafA1. E and RPN1 levels were measured. (D) Quantification of normalized protein levels of Myc-E-SARS2 WT (blue) or T9I (red) in (C) (mean ± SD). p value (two-way ANOVA, n=3, *: p value < 0.05).

[0099] Figure 3: E-regulated UcPS is THU-dependent. (A) Immunofluorescence in U2OS cells expressing GFP-ERGIC53 and the coronavirus Myc-E. Scale bar, 10 μm. (B) Immunofluorescence in U2OS cells expressing GFP-ERGIC53, TMED10-V5, and Myc-E-SARS2. Scale bar, 10 μm. (C) mIL1β-HA secretion in WT and TMED10-KO HEK 293T cells expressing or not Myc-E-SARS2. (D) IL1β secretion in WT and TMED10-KO THP-1 cells in the presence or absence of Myc-E-SARS2, TMED10-V5, or both. Differentiated cells were treated with LPS and ATP. (E) mIL33 secretion in WT or TMED10-KO BEAS-2B cells in the presence or absence of Myc-E-SARS2 or TMED10-V5 expression. (FI) WT and TMED10-KO mice injected with AAV-GFP or AAV-E-SARS2 were stimulated with LPS and euthanized. Lung expression of E-SARS2 or GFP was verified by IF (H, left panel). (F) Serum IL1β levels were measured by ELISA (mean ± SD). In (G), IL6 mRNA expression levels in the corresponding tissues were analyzed (mean ± SD). (I) Inflammatory cell infiltration was analyzed and quantified by H&E staining (H, right panel). p values ​​(one-way ANOVA, n = 5, ***: p value < 0.001). Scale bar, 50 μm. (J) Co-IP was performed using HEK293T cells expressing Myc-E-SARS2 and TMED10-V5 or TMEDΔ10CT-V5. (K) GST pull-down analysis of MBP-Myc-SARS2-E protein using GST and GST-TMED10. (L) Pull-down analysis of MBP-Myc-SARS2-E using Ctrl, TMED6-CT, or TMED10-CT short peptides. (M) Changes in mIL1β secretion in TMED10-KO-THP-1 cells transfected with or without Myc-E-SARS2 in the presence of TMED10-WT or the TMED10ΔCT mutant. (N) Co-immunoprecipitation (Co-IP) of TMED10-V5 and Myc-E proteins from different coronaviruses in HEK293T cells. (O) Cross-linking assay using HEK293T cells expressing TMED10-V5 in the absence or presence of Myc-E proteins from different coronaviruses.

[0100] Figure 4: E-mediated UcPS via THU. (A-B) Secretion of FLAG-tagged mIL33 (A) and mIL36A (B) was detected in WT or TMED10-KO HEK293T cells in the presence or absence of Myc-E-SARS2 expression. (C) Cross-linking assay using HEK293T cells expressing TMED10-V5 in the absence or presence of mIL1β, Myc-E-SARS2, or both. (D) Cross-linking assay using HEK293T cells expressing TMED10ΔCT-V5 in the absence or presence of Myc-E.

[0101] Figure 5: E protein promotes membrane translocation of THU. (A) Schematic diagram of the in vitro translocation assay. Briefly, proteoliposomes containing either GST-tagged E or TMED10, alone or with GST-tagged E or TMED10, were incubated with purified mIL1β-FLAG. Proteinase K digestion was performed to determine the amount of intramembrane cargo. (B) In vitro membrane translocation of mIL1β-FLAG from control or GST-TMED10-containing proteoliposomes in the presence or absence of SARS2-containing GST-Myc-E. (C) Schematic diagram of the cell-free membrane translocation assay. Briefly, membrane fractions collected at 100,000 × g from HEK293T cells expressing control (ctrl), Myc-E-SARS2, TMED10-V5, or both after differential centrifugation were incubated with HEK293T cell cytoplasm, recombinant mIL1β-FLAG, and a GTP and ATP regeneration system (ATPR) at 30°C for 1.5 h. OptiPrep gradient ultracentrifugation was then performed to remove free protein. The top-floating membranes were collected and translocated IL1β was determined using a proteinase K protection assay. (D) Cell-free translocation of mIL1β-FLAG in the presence or absence of TMED10, Myc-E-SARS2, or both. Membrane fractions were collected from TMED10KO HEK293T cells. (E) Cell-free membrane translocation of mIL1β-FLAG in the absence or presence of Myc-tagged E2677 from different coronaviruses. (FG) GFP complementation analysis (F). Cells expressing GFP(1-10)-TMED10 were transfected with a negative control (NC) or mIL1β-GFP11, while either left untransfected (ctrl) or transfected with Myc-tagged E2677 from the indicated coronaviruses. (G) Complementation GFP signals were analyzed by FACS and quantified (mean ± SD). p values ​​(one-way ANOVA, n = 4, *: p value < 0.05; **: p value < 0.01; ***: p value < 0.001).

[0102] Figure 6: Purified proteins and SARS E-facilitated cargo transport. (A) Coomassie blue staining of proteins used in the in vitro translocation assay, showing two different protein concentrations. (B) In vitro membrane translocation of mIL1β-FLAG in control or GST-TMED10-containing liposomes in the absence or presence of GST-Myc-E-SARS. (C) Immunoblot showing expression of GFP(1-10)-TMED10, mIL1β-GFP11, and Myc-tagged E of the coronavirus from Figure 5G.

[0103] Figure 7: The SS / DS motif of Es-SSC regulates THU. (A) Amino acid sequence alignment of the C-terminal portion of the coronavirus E protein (SARS-CoV E protein sequence: MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV (SEQ ID NO: 3)). The SS / DS residues of Es-SSC are marked in red. The corresponding residues of Es-MSC are marked in green. (B) Secretion of mIL1β-HA in HEK293T cells transfected with either the WT or AA mutant of Myc-E from a severe coronavirus. (C) Co-IP using HEK293T cells expressing TMED10-V5 and either the WT or AA mutant of Myc-E from a severe coronavirus. (D) Absence of cell membrane translocation of mIL1β-FLAG in the presence or absence of the WT or AA mutant of Myc-E from SARS-CoV. (E) IL1β secretion in THP-1 cells expressing the WT or AA mutant of Myc-E, a severe coronavirus. Cells were differentiated and treated with LPS and ATP. (F) mIL1βHA secretion in HEK293T cells transfected with Myc-SARS2-E and the WT or SS mutant of Myc-E, a mild coronavirus. (G) Co-IP using HEK293T cells expressing TMED10-V5 and Myc-SARS2-E and the WT or SS mutant of Myc-E, a mild coronavirus. (H) IL1β secretion in THP-1 cells expressing Myc-SARS2-E and the WT or SS mutant of Myc-E, a mild coronavirus. (IK) No cell membrane translocation of mIL1β-FLAG in the absence or presence of Myc-SARS2-E and the WT or SS mutant of Myc-E, a mild coronavirus.

[0104] Figure 8: Localization and secretion regulation of Es-SSC SS / DS motif mutants and the effect of TMED10-CT on E-regulated secretion. (A) Immunofluorescence of U2OS cells expressing GFP-ERGIC53 and Myc-tagged severe coronavirus E AA mutants. (B) mIL1β-HA secretion in HEK293T cells expressing Myc-E-SARS2 in the presence of increasing amounts of GFP-TMED10-CT.

[0105] Figure 9: E-TMED10 interaction is required for E-induced inflammation. (A) Amino acid sequence and domain structure of the SARS-2-E protein. The C-terminal region of SARS-2-E (ECT) that blocks the E-TMED10 interaction is highlighted in orange. (B) Co-IP was performed using HEK293T cells expressing TMED10-V5 and Myc-tagged E-SARS-2 in the absence or presence of GFP or GFP-tagged E-SARS-2 CT (GFP-ECT). (C) mIL1β-HA secretion in HEK293T cells transfected with or without Myc-tagged E-SARS-2, or in the absence or presence of increasing amounts of GFP or GFP-ECT. (D) THP-1 cells expressing GFP or GFP-ECT were used, followed by differentiation and mIL1β secretion assays. Differentiated cells were treated with LPS and ATP. (E) Cross-linking experiments were performed using HEK293T cells expressing TMED10-V5 in the absence or presence of Myc-E-SARS-2, GFP, or GFP-ECT. (F) In the absence or presence of Myc-E-SARS2, GFP, or GFP-ECT, mIL1β-FLAG lacks cell membrane translocation. (GJ) C57BL / 6 mice co-injected with AAV-E-SARS2 and AAV-GFP or AAV-GFP-ECT were stimulated with LPS and euthanized. Lung expression of E-SARS2, GFP, and GFP-ECT was verified by IF (I, left and middle panels). (G) Serum IL1β levels were measured by ELISA (mean ± SD). IL6 mRNA expression levels in the corresponding tissues were analyzed in (H) (mean ± SD). Inflammatory cell infiltration was analyzed by H&E staining (I, right panel) and quantified (J). p values ​​(two-tailed t-test, n = 5, **: p value < 0.01; ***: p value < 0.001). Scale bar, 50 μm.

[0106] Figure 10: UPA and progesterone are inhibitors of E-induced secretion and inflammation. (A) Schematic diagram of the high-throughput compound screening system based on the complementary NanoLuc luciferase cargo secretion assay. (B) Heatmap shows the mean mIL1β-HiBiT secretion levels in the presence of each compound from two independent experiments, normalized to the secretion levels in the presence of DMSO. The numbers to the right of the heatmap indicate the compound number within each row. The heatmap displays log2 values. (C) Relative quantification of mIL1β secretion levels in HEK293T cells in the presence of the indicated compounds (mean ± SD). p-values ​​(one-way ANOVA, n = 3, *: p-value < 0.05; **: p-value < 0.01; ****: p-value < 0.0001). (D-E) mIL1β secretion in THP-1 cells expressing coronavirus Myc-E after 4 hours of treatment with 10 μM UPA (D) or progesterone (E). Cells were differentiated, treated with LPS, and then treated with ATP.

[0107] Figure 11: UPA and progesterone are inhibitors of E-induced secretion and inflammation. (A) Co-IP analysis of HEK293T cells expressing TMED10-V5 and Myc-E-SARS2 in the absence or presence of 10 μM UPA or progesterone. (B) Cross-linking experiments were performed using HEK293T cells expressing TMED10-V5 (without or with Myc-E-SARS2) in the absence or presence of 10 μM UPA or progesterone. (C) No cell membrane translocation of mIL1β-FLAG in the absence or presence of Myc-E-SARS2, UPA, or progesterone (P4).

[0108] Figure 12: C57BL / 6 mice infected with AAV-E-SARS2 were injected with UPA, then stimulated with LPS and euthanized. Immunofluorescence confirmed the expression of E-SARS2 in the lung (C, upper panel). (A) Serum IL1β levels were measured by ELISA (mean ± SD). (B) Analysis of IL6 mRNA expression levels in specific tissues (mean ± SD). Inflammatory cell infiltration was analyzed by H&E staining (C, lower panel) and quantification (D). p value (two-tailed t-test, n = 5, **: p value < 0.01). Scale bar, 50 μm.

[0109] Figure 13: Quantification of relative secretion levels of mIL1β in HEK293T cells in the presence or absence of compounds (mean ± SD). p value (one-way ANOVA, n = 3, *, p value < 0.05; **: p value < 0.01; ****: p value < 0.0001).

[0110] Figure 14: Chemical structures and names of progesterone and its analogs with (Y) or without (N) inhibitory effect on mIL1β secretion.

[0111] Figure 15: E-regulated UcPS and inflammation in MHV-infected mice. (A) mIL1β-FLAG secretion and Caspase-3 cleavage in HEK293T-mCC1a cells after 36 hours of infection with or without MHV-A59 (MOI of 0.005, 0.05, and 0.5). (BC) IL1β secretion in BMDMs from WT, TMED10-KO (B), or GSDMD-KO (C) mice after infection with MHV-A59 (MOI 0.1, 36 hours). (D) IL1β secretion in BMDMs infected with MHV-A59 in the presence of GFP or GFP-ECT. (E) IL1β secretion in BMDMs infected with MHV-A59 in the absence or presence of 10 μM UPA or progesterone. (FI) Ifnar injected with AAV-GFP or AAV-GFP-ECT - / - Mice were infected with MHV-A59 (2×10 4 PFU) and euthanasia were performed 4 days later. The expression of GFP or GFP-ECT in the lung was verified by IF (H, upper panel). (F) Serum IL1β levels were determined by ELISA (mean ± SD). The expression levels of IL6 mRNA in the corresponding tissues were analyzed in (G) (mean ± SD). Inflammatory cell infiltration was analyzed by H&E staining (H, lower panel) and quantified in (I). p value (two-tailed t-test, n=5, *: p value <0.05; ***: p value <0.001). Scale bar, 50 μm. (JM) Infected with MHV-A59 (2×10 4 Ifnar of PFU) - / - Mice were intraperitoneally injected with DMSO, UPA (1 mg / kg), or progesterone (1 mg / kg) for 3 consecutive days and euthanized. (J) Serum IL1β levels were measured by ELISA (mean ± SD). IL6 mRNA expression levels in the corresponding tissues were analyzed in (K) (mean ± SD). Inflammatory cell infiltration was analyzed (L) and quantified (M) by H&E staining. p values ​​(one-way ANOVA, n = 5, *: p value < 0.05; ***: p value < 0.001). Scale bar, 50 μm.

[0112] Figure 16: E-mediated UcPS model. In brief, the E protein of severe coronaviruses (SARS, SARS2, and MERS) strongly interacts with TMED10, activating THU-mediated release of inflammatory factors. DETAILED DESCRIPTION

[0113] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0114] Materials and methods used in the examples

[0115] 1. Plasmids and cells

[0116] Mature forms of IL1 family proteins (IL1α, IL1β, IL18, IL33, IL36α), IL6, TMED10-V5 and TMED 10ΔCT-V5 mutant plasmids are as described in the literature A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652.e615 (2020)). FLAG-tagged expression plasmids of SARS2 proteins (E, M, S, N, orf3a, orf6, orf7b and orf8) are as described in the literature A systematic and molecular study of subcellular localization of SARS-CoV-2 proteins (J. Zhang et al., Signal Transduct Target Ther 5, 269 (2020).). E-SARS2 expression plasmids with or without N-terminal Myc tags were amplified from template PCR and inserted into the FUGW vector. Expression plasmids for the E proteins of SARS, MERS, 229E3, HKU1, and OC43 were generated by DNA synthesis and subsequently inserted into a FUGW vector with a Myc tag at the N-terminus. E protein mutants were constructed using the QuickChange method. GFP-ECT was generated by PCR amplification from 38-71aa of the C-terminal triplet of E-SARS2 (wherein the amino acid sequence at positions 38-71 is RLCAYCCNIVNVSLVKPSFYVYSRYKNLNSSRVP (SEQ ID NO: 1)) and inserted into a FUGW vector with a GFP tag at the N-terminus. E-SARS2 and E-SARS were also inserted into pGEX4T1 or pET28a vectors with GST or MBP and Myc tags for protein purification. The plasmids used for purification of mIL1β and TMED10 proteins were described in the literature A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652.e615 (2020)).

[0117] WT and TMED10-KO HEK293T, U2OS, WT, TMED10-KO, and GSDMD-KO THP-1 cells were grown and maintained as described in A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652.e615 (2020)). BEAS-2B cells were provided by Dr. Rao Lun (Tsinghua University). Bone marrow-derived macrophages (BMDMs) were isolated from 6-week-old male C57BL / 6 mice and differentiated using standard protocols.

[0118] 2. Reagents and Antibodies

[0119] DSS was obtained from Thermo, GTP, ATP, proteinase K, protease inhibitor cocktail, anti-V5 agarose beads, anti-Myc agarose beads were obtained from Sigma, and phenylmethylsulfonyl fluoride (PMSF) was obtained from Amresco. Mouse anti-FLAG, anti-tubulin, anti-GST, goat anti-IL1β, rabbit anti-IL1β, anti-V5, anti-HA, anti-GFP, anti-TMED10 and anti-ERGIC53 antibodies, HRP and Alexa Fluor conjugated secondary antibodies were as described in the literature A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652.e615 (2020)). Mouse anti-Myc and rabbit anti-Caspase3 were purchased from CST, and rabbit anti-IL33 was purchased from Proteintech. Rabbit anti-SARS2-E antibodies were obtained by immunizing rabbits with a synthetic peptide of the last 25 residues of the E-SARS2 protein by Abclonal.

[0120] 3. Transfection, Lentiviral Transduction, and Secretion Assay

[0121] DNA constructs were transfected into cells using PEI (Polysciences, Inc.) for HEK293T and X-tremeGENE HP (Roche) for U2OS according to product instructions. Lentiviral infection was used to express TMED10, E and mutants, GFP-ECT in THP-1 or BEAS-2B cells. The pLX304 plasmid containing TMED10-V5 or the FUGW plasmid containing the indicated coronavirus E was transfected into HEK293T cells together with VSVG and psPAX2 for 72 hours to produce lentivirus. The supernatant was collected to infect the indicated cells. Secretion assays were performed as described in the literature A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652. e615 (2020)). Briefly, cells were replaced with DMEM for 1 hour or induced overnight with 50 ng / ml LPS in RPMI-1640 plus 10% FBS, followed by treatment with 2 mM ATP in saline (147 mM NaCl, 10 mM HEPES pH 7.4, 13 mM glucose, 2 mM CaCl2, 1 mM MgCl2, 2 mM KCl) for 30 minutes. The culture medium was concentrated using an Amicon filter (Millipore), and cell lysates were collected. Immunoblotting was performed to determine the amount of cargo in the culture medium and cells. LDH assays (Thermo) were performed according to the manufacturer's instructions.

[0122] 4. Immunofluorescence and fluorescence complementation

[0123] For immunofluorescence staining of lung tissue, samples were fixed in 4% PFA, dehydrated in 30% sucrose solution for 24 h, and embedded in Tissue-Tek OCT compound. Frozen blocks were cut into 10-μm-thick sections.

[0124] For immunofluorescence, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Cells were permeabilized with 0.1% Triton X-100 diluted in PBS for 3 minutes at room temperature. Samples were blocked with 10% FBS diluted in PBS for 1 hour and incubated with primary antibodies for 1 hour. After multiple washes, samples were incubated with secondary antibodies for 40 minutes at room temperature. Fluorescence images were acquired using an Olympus FV3000 confocal microscope. Quantification was performed using ImageJ.

[0125] For fluorescence complementation, cells expressing GFP(1-10)-TMED10-V5 and IL1β-FLAG-GFP11 were transfected with coronavirus E plasmid. The GFP signal in the cells was collected by CytoFlex LX (Beckman) and analyzed by FlowJo software.

[0126] 5. Co-immunoprecipitation and in vitro peptide / GST pull-down assay

[0127] For immunoprecipitation, cells were lysed on ice for 30 minutes in IP buffer (50 mM Tris / HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 10% glycerol) containing a protease inhibitor cocktail, and the lysate was cleared by centrifugation. The resulting supernatant was incubated with the indicated agarose and rotated at 4°C for 3 hours. The agarose was then washed five times with IP buffer before immunoblotting.

[0128] For peptide pull-down analysis, synthetic peptides were conjugated to agarose beads using AminoLink Plus coupling resin (Thermo) according to the product instructions. 2 mg of purified SARS-2 MBP-Myc-E protein was incubated with 15 μL of peptide-coupled beads in IP buffer and rotated at 4°C for 3 hours. The agarose beads were then washed three times with IP buffer and immunoblotted.

[0129] For GST pull-down analysis, proteins were purified and GST or GST-TMED10 was incubated with GSH-agarose (GE) (blocked with 10% FBS) in IP buffer for Co-IP and rotated at 4°C for 1 hour. The GST- or GST-TMED10-loaded beads were then collected and incubated with MBP-Myc-E of SARS-2 for 2 hours at 4°C. The beads were washed three times and then subjected to immunoblotting.

[0130] 6. Cross-linking analysis

[0131] For DSS cross-linking assays, cells were suspended in PBS containing 0.25 mM DSS for 30 minutes at room temperature. The reaction was quenched with 20 mM Tris, and samples for immunoblotting were prepared as described in the literature A translocation pathway for vesicle-mediated unconventional protein secretion (M. Zhang et al., Cell 181, 637-652. e615 (2020)).

[0132] 7. In vitro translocation and cell-free translocation experiments

[0133] For in vitro translocation assays, GST-Myc-E, GST-TMED10, and His-mIL1β-FLAG proteins were expressed in Escherichia coli and purified. Total lipids extracted from HEK293T cells were incubated with TMED10 and / or E protein to generate proteoliposomes. mIL1β protein was further incubated with the proteoliposomes for in vitro translocation. After translocation, membrane fractions were collected by flotation, followed by proteinase K digestion and immunoblotting.

[0134] For cell-free translocation analysis, cell homogenates of WT or TMED10-KO HEK293T cells expressing Myc-E or TMED10-V5 were subjected to differential centrifugation (3 k×g, 100 k×g). Membrane fractions were obtained from the 100 k×g pellet and resuspended in 100 μL B88 (20 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate, protease and phosphatase inhibitors, 0.3 mM DTT). HEK293T cytosol (3 mg / mL final concentration), ATP regeneration system (40 mM creatine phosphate, 0.2 mg / mL creatine phosphate kinase, and 1 mM ATP), GTP (0.15 mM), 10 μg mIL1β-FLAG protein, and different membrane fractions (0.2 mg / mL PC content final concentration) were incubated at 30°C for 1.5 hours. After translocation, membrane fractions were collected by flotation using an Opti-prep gradient, digested with proteinase K, and detected by immunoblotting.

[0135] 8. AAV infection and lipopolysaccharide (LPS) stimulation

[0136] Experiments with mice were approved by the Institutional Animal Care and Use Committee of Tsinghua University. Mice were housed in a SPF facility with ventilated cages under controlled temperature and light conditions and had ad libitum access to food and water. C57BL / 6J mice were purchased from the Laboratory Animal Resource Center of Tsinghua University. TMED10fl / fl mice (C57BL / 6) were constructed by GemPharmatech Co., Ltd., China. TMED10-inducible whole-body knockout mice were generated by hybridization of TMED10fl / fl with Cre-ERT (provided by Dr. Xiaoyu Hu from Tsinghua University). Eight-week-old male mice were intraperitoneally injected with tamoxifen (80 mg / kg) or corn oil for 5 consecutive days. Seven days after the last tamoxifen injection, mice were further injected with pneumophilic AAVs (GFP, GFP-ECT for SARS-CoV, or Myc-E for coronavirus) using endotracheal intubation. Four weeks later, mice were challenged with LPS (15 mg / kg). Fifteen hours after LPS stimulation, mice were euthanized, and serum, lungs, spleen, liver, and kidneys were collected for ELISA, immunofluorescence, RT-qPCR, and H&E staining.

[0137] 9. MHV infection

[0138] MHV-A59 was propagated in 17-Cl-1 cells, and the titer of the virus was determined by endpoint dilution assay. For cell infection assays, 293T-mCC1a or BMDM cells were infected with MHV-A59 for 2 h in serum-free medium and then cultured in complete medium (10% FBS and 1% Pan-Strep) for 36 h. Cells were treated with different drugs for 4 h in serum-free medium, and the culture medium was collected to detect mIL1β secretion. For mouse infection assays, 8-12 week old Ifnar mice were used. - / - Mice (a gift from Dr. You Fuping of Peking University) were anesthetized with isoflurane and inoculated intranasally with MHV-A59 (2×10 4 PFU). The body weight and health status of the mice were observed and recorded every day. The mice were euthanized on day 4 to collect serum and different tissues for further testing.

[0139] 10. Compound Screening

[0140] A rapid secretion assay using complementary NanoLuc leuciferace was used to screen inhibitors of E-SARS2-induced mIL1β secretion. HEK293T cells expressing mIL1β-HiBiT and Myc-E-SARS2 were treated with different compounds from an FDA-approved drug library (Selleck, L3000) for 8 hours. The culture medium was collected and then incubated with LgBiT protein and substrate for 10 minutes, followed by fluorescence intensity analysis. The results of two independent experiments were normalized. Compounds whose fluorescence intensity decreased by more than 0.75 times relative to DMSO treatment in both experiments were selected as candidates.

[0141] Example 1: Coronavirus E protein promotes UcPS

[0142] Among inflammatory factors, IL1 family members, especially IL1β and IL33, play a role as key mediators in the upstream of the cascade release of inflammatory factors. Previous studies involving protein-protein interactions have shown that viral proteins in coronaviruses (such as SARS2) may induce the release of IL1 family factors such as IL1β and IL33, and both proteins are secreted through the non-classical secretion (UcPS) pathway. In order to clarify the mechanism by which coronavirus proteins induce the inflammatory factor UcPS, the effect of a single SARS2 protein on the secretion of mature IL1β (mIL1β) was studied. The secretion assay used the secretion system established in the literature Translocation of interleukin-1βinto a vesicle intermediate in autophagy-mediated secretion (M. Zhang, SJ Kenny, L. Ge, K. Xu, R. Schekman,. elife 4, e11205 (2015).

[0143] Notably, expression of the E protein enhanced IL1β secretion, while the effects of other SARS2 factors (N, S, M, ORF3a, ORF6, ORF7b, and ORF8) were relatively minor (Figures 1A and 1B). Further experiments revealed that the effect of the E protein on IL1β secretion was dose-dependent (Figure 2A), and this effect was independent of the specific tag used, as both untagged and Myc- or FLAG-tagged E enhanced IL1β secretion (Figure 2B). In addition, the E protein also promoted UcPS of other IL1 family inflammatory factors, including mIL33, mIL36A, mIL1α, and mIL18, but had no effect on the secretion of the classic secreted protein IL6 (Figure 1C).

[0144] Previous studies have shown that gasdermin D (GSDMD) regulates IL-1β release in inflammatory cells. Interestingly, when E protein was expressed in GSDMD-knockout THP-1 cells, it was found to enhance the secretion of mature IL-1β, indicating that E-regulated UcPS is independent of GSDMD (Figure 1D). Furthermore, E protein expression had minimal effects on caspase-3 cleavage or LDH release, indicating that E-regulated UcPS is not due to cell death (Figure 2A).

[0145] To investigate whether the E proteins of various coronaviruses affect UcPS, we examined the secretion of mIL1β in non-inflammatory and inflammatory cells. Surprisingly, we observed that the E proteins of severe coronaviruses (Es-SSC), including SARS-2, SARS, and MERS, significantly enhanced mIL1β secretion, whereas mild coronaviruses (Es-MSC), such as 229E, HKU1, and OC43, had only a weak effect (Figure 1E and Figure 1F). Similar trends were observed when mIL33 secretion was examined in non-inflammatory cells and BEAS-2B lung bronchial epithelial cells (Figure 1G and Figure 1H).

[0146] To elucidate the role of E protein in promoting inflammation under physiological conditions, adeno-associated virus (AAV) was used to express the E proteins of SARS2 (E-SARS2, a representative of Es-SSC) and 229E (E-229E, a representative of Es-MSC) in the lungs of C57BL / 6 mice (Figure 1K, left). After stimulation with low-dose lipopolysaccharide (LPS), it was found that the expression of E-SARS2, but not E-229E, led to increased inflammation, as evidenced by increased serum IL1β levels (Figure 1I). This was accompanied by a corresponding increase in IL6 mRNA production in the lungs, kidneys, liver, and spleen (Figure 1J). In addition, compared to E-229E, the expression of E-SARS2 enhanced the infiltration of immune cells into the lungs (Figure 1K, right and Figure 1L). These findings indicate that the coronavirus E protein promotes the UcPS of inflammatory factors, and the effects of different E proteins are associated with the severity of symptoms after infection.

[0147] Previous evidence has established that coronavirus E proteins function as viroporins, forming ion channels that activate inflammasomes and potentially promote IL1β maturation and secretion. Contrary to expectations, mutants of E-SARS2 and E-SARS with inactivated ion channel activity promoted the secretion of mIL1β-HA with the same efficiency as wild-type (WT) E protein in non-inflammatory cells (Figure 1M-O). These results suggest that ion channel activity is unlikely to directly regulate the secretion of mIL1β itself. Notably, the T9I mutation in the E protein, present in omicron strains of SARS2, did not affect E's action on the UcPS (Figure 1M). However, this mutation significantly reduced the protein's stability (Figures 2C and 2D), potentially impairing sustained UcPS triggering by E. E protein instability and impaired channel activity may contribute to the attenuation of inflammation during omicron infection.

[0148] Example 2: E-SARS2-regulated UcPS is dependent on TMED10

[0149] Previous studies have determined that coronavirus E protein is primarily localized to the endoplasmic reticulum-Golgi intermediate zone (ERGIC), an organelle that is a hub for regulating the secretion of IL1 family members through the TMED10-channelled UcPS (THU) pathway. Importantly, the E protein of the severe symptom-causing coronavirus (Es-SSC) is primarily localized to the ERGIC (Figure 3A). Notably, E-SARS2 was observed to colocalize with TMED10 on the ERGIC (Figure 3B), suggesting that E-mediated UcPS regulation may be related to the THU pathway. Interestingly, E-SARS2 failed to enhance the secretion of mature IL1β (mIL1β) in TMED10 knockout (KO) cells (Figure 3C), and similar situations were observed for the secretion of mIL33 and mIL36α (Figures 4A and 4B).

[0150] Further analysis of endogenous mIL1β and mIL33 secretion in THP-1 or BEAS-2B cells showed that in the absence of TMED10, E-regulated UcPS was significantly impaired, and this effect could be restored when TMED10 was re-expressed (Figure 3D and Figure 3E). This finding further confirmed that E-SARS2 promotes UcPS through the THU pathway. In TMED10 knockout mice, compared with wild-type mice, the expression of E-SARS2 failed to increase serum IL1β levels, IL6 production in different tissues, and lung damage caused by immune cell infiltration (Figure 3F-I). In summary, these results provide sufficient evidence that E-SARS2 promotes UcPS of mIL1β and other inflammatory factors through the THU pathway.

[0151] To understand how coronavirus E protein regulates UcPS through THU. Co-immunoprecipitation (Co-IP) and GST pull-down assays revealed that E-SARS2 protein interacts with TMED10 (Figure 3J and Figure 3K). Notably, E-SARS2 interacted weakly with TMED10 lacking the C-terminal domain (TMED10ΔCT mutant) (Figure 3J). Furthermore, in a pull-down assay, E-SARS2 was found to specifically interact with the C-terminal (CT) peptide of TMED10, but not with the CT of TMED6 (Figure 3L). These findings strongly suggest a direct binding interaction between TMED10-CT and E-SARS2. In TMED10 knockout (TMED10KO) THP-1 cells, E-SARS2 effectively restored IL1β secretion when full-length TMED10 was reintroduced, but this restoration was not achieved when the TMED10ΔCT variant was used (Figure 3M). These findings highlight the indispensable role of the association between the C-terminal domain of TMED10 (TMED10-CT) and E-SARS2 in E-SARS2-mediated UcPS regulation.

[0152] Previous studies have shown that TMED10 has the ability to form oligomers and may serve as a protein channel responsible for regulating protein translocation. Notably, expression of E-SARS-CoV-2 was observed to enhance the formation of TMED10 oligomers in the presence and absence of the secretory cargo IL1β (Figure 4C). This effect depended on the interaction between TMED10 and E-SARS-CoV-2, as the oligomerization of the TMED10ΔCT mutant was not affected by expression of E-SARS-CoV-2 (Figure 4D).

[0153] Crucially, Es-SSCs interacted more strongly with TMED10 than Es-MSCs (Figure 3N), thereby more effectively increasing TMED10 oligomerization (Figure 3O). These results suggest that TMED10 may be a host target of Es-SSCs to promote the inflammatory cytokine UcPS.

[0154] Example 3: E protein promotes the membrane translocation process of THU

[0155] In THU, TMED10 plays a crucial role in facilitating cargo transport into the ERGIC. This process involves the introduction of signal-peptide-less UcPS cargo into the vesicular transport system to initiate the intracellular UcPS pathway. To assess whether E proteins promote the entry of secretory cargo into vesicles, an established in vitro membrane translocation assay was performed (Figure 5A). As previously described, the presence of TMED10 on liposomes effectively promoted the translocation of mIL1β into liposomes, as evidenced by its protective effect against proteinase K digestion. Notably, E-SARS2 alone in liposomes had no cargo transport capacity, but when combined with TMED10, it significantly enhanced TMED10-promoted mIL1β translocation (Figures 5B and 6A). A similar enhancement of TMED10-mediated translocation was also observed with E-SARS (Figures 6A and 6B). These findings confirm that both E-SARS2 and E-SARS directly promote the transport of UcPS cargo into vesicles through TMED10.

[0156] Given the difficulty in purifying all other coronavirus E proteins, a cell-free UcPS cargo translocation assay was developed using membrane fractions from cells expressing various E proteins (Figure 5C). Similar to the liposome assay, E-SARS-CoV-2 promoted mIL1β translocation in the presence of TMED10 (Figure 5D). Furthermore, Es-SSC promoted mIL1β translocation, while Es-MSC had a minimal effect (Figure 5E). To further confirm the differential effects of E proteins on cargo entry into vesicles, a GFP complementation assay was performed to assess UcPS cargo translocation into the ERGIC (Figure 5F). In this experiment, the GFP1-10 fragment was fused to the luminal end of TMED10 to assess the translocation of GFP11-tagged mIL1β into the ERGIC lumen, where GFP complementation occurs. As shown in Figures 5G and 6C, Es-SSC, but not Es-MSC, again enhanced mIL1β entry into the ERGIC. These results collectively suggest that Es-SSCs increase UcPS of inflammatory factors by promoting cargo transport in the THU pathway.

[0157] Example 4: The SS / DS motif in Es-SSCs determines their effects on THU

[0158] Sequence alignment revealed the presence of the SS / DS motif in Es-SSC but not in Es-MSC (Figure 7A). Alteration of the SS / SD motif did not affect the ERGIC localization of Es-SSC (Figure 8A). However, this mutation significantly impaired the ability of Es-SSC to enhance mIL1β secretion in non-inflammatory and inflammatory cells (Figures 7B and 7E), hindered its interaction with TMED10, and its ability to promote cargo transport (E-SARS2) (Figures 7C and 7D). This suggests that the SS / SD motif is essential for Es-SSC to upregulate the THU pathway.

[0159] In contrast, introduction of the SS motif into Es-MSCs increased their interaction with TMED10, modulated secretion, and enhanced cargo transport (Figure 7F-K). Thus, the SS / DS motif of the severe coronavirus E protein plays a key role in the UcPS that promotes inflammatory cytokines.

[0160] Example 5: E-TMED10 interaction is required for E-induced inflammation

[0161] The data show that the interaction between E and TMED10 promotes TMED10 oligomerization and cargo translocation, thereby releasing a variety of inflammatory factors through the THU pathway. A further purpose of this example is to investigate whether blocking the E-TMED10 complex can reduce inflammation induced by E expression. E is a small protein composed of a luminal N-terminal domain, a transmembrane domain, and a C-terminal (CT) cytoplasmic domain (Figure 9A). The association of E-SARS2 with TMED10-CT suggests a potential interaction between the cytoplasmic portion of E-SARS2 and the CT of TMED10. In order to disrupt the E-TMED10 interaction, TMED10-CT or E-SARS2-CT was overexpressed. Notably, the expression of green fluorescent protein-tagged E-SARS2-CT (GFP-ECT), but not TMED10-CT expression, effectively blocked the E-TMED10 interaction (Figures 8B and 9B). Similarly, in HEK293T cells and THP-1 cells, expression of GFP-ECT showed a dose-dependent inhibition of E-SARS2-induced mIL1β secretion (Figure 9C and Figure 9D). In cross-linking and translocation experiments, GFP-ECT reduced E-promoted TMED10 oligomerization and membrane translocation of mIL1β (Figure 9E and Figure 9F). In a mouse model, expression of GFP-ECT led to reduced inflammation, as manifested by reduced levels of IL1β in serum and corresponding reductions in IL6 production in the lungs, kidneys, liver, and spleen (Figure 9G-J). These findings indicate that the E-TMED10 interaction plays an important role in promoting the release of inflammatory factors induced by E-SARS2 through the THU pathway.

[0162] Example 6: Progesterone and its analogs inhibit E-induced inflammatory cytokine release

[0163] The above examples demonstrate that E protein induces the release of inflammatory factors through the THU pathway, which may cause SARS2 and other coronaviruses to induce severe inflammation, leading to severe symptoms. Subsequently, a study was conducted on the development of E-induced UcPS inhibitors, which may help alleviate the inflammation caused by severe coronavirus infection. To facilitate this study, a rapid secretion analysis method using complementary NanoLuc luciferase was established. The analysis was then combined with high-throughput compound screening to identify chemical regulators of E-mediated UcPS (Figure 10A).

[0164] Among approximately 2,500 FDA-approved drugs, progesterone and some of its analogs were found to exhibit a strong inhibitory effect on E-stimulated UcPS (Figures 10B and 10C). Progesterone and its analog, ulipristal acetate (UPA), both effectively inhibited E-SSC-induced IL1β release in THP-1 cells (Figures 10D and 10E). Similar to the peptides of ECT, progesterone and UPA blocked the interaction between E and TMED10, inhibited E-induced TMED10 oligomer formation, and prevented E-enhanced mIL1β translocation (Figure 11). These findings suggest that these two drugs act through a mechanism similar to ECT. In mouse experiments, UPA alleviated lung inflammation caused by E-SARS2 expression (Figure 12). It is therefore concluded that progesterone and its analogs inhibit E-induced UcPS by disrupting the interaction between E and TMED10.

[0165] To further elucidate the structural features necessary for the inhibitory function of progesterone and its analogs in regulating TMED10-mediated UcPS, 23 progesterone analogs were evaluated for their effects on E-enhanced mIL1β secretion (Figure 14). Interestingly, it was observed that modifications at position 11 on the steroid core of the progesterone analogs, such as the introduction of a carbonyl or hydroxyl group, largely abolished their inhibitory effects (Figure 13). However, the presence of an N,N-dimethylaniline modification at position 11, as seen in UPA, did not diminish the inhibitory effect (Figure 13). In addition, various modifications at position 17 were observed in progesterone analogs, but these modifications did not significantly affect the activity of these compounds in regulating E-enhanced UcPS in the secretion assay (Figure 13).

[0166] Example 7: TMED10 regulates UcPS, an inflammatory factor induced by MHV infection

[0167] To investigate the role of TMED10-regulated UcPS in the release of inflammatory factors during coronavirus infection, an infection model was established in HEK293T cells and bone marrow-derived macrophages (BMDMs) using mouse hepatitis virus (MHV). Similar to the effect observed on E protein expression, MHV infection triggered a dose-dependent release of mIL1β in HEK293T cells expressing the MHV receptor mCC1a (Figure 15A). Importantly, during MHV infection, cell death-promoting Caspase-3 cleavage was not increased, suggesting that mIL1β release is more likely attributable to UcPS rather than cell death (Figure 15A).

[0168] In BMDMs, MHV infection-induced mIL1β release was found to be dependent on TMED10 rather than GSDMD (Figures 15B and 15C), highlighting the involvement of the THU pathway. Furthermore, expression of ECT or administration of progesterone (or UPA) effectively blocked MHV infection-induced mIL1β secretion (Figures 15D and 15E), highlighting the significance of the E-TMED10 interaction in this process.

[0169] Using Ifnar - / - In a mouse lung infection model of E-deficient mice, MHV infection resulted in severe lung infection, with mIL1β release, lung immune cell infiltration, and upregulated IL6 expression in multiple organs, effects similar to those caused by expression of E protein in the presence of mild LPS stimulation. Notably, expression of ECT or progesterone (or UPA) treatment attenuated inflammation following MHV infection, further confirming the role of these interventions in modulating the inflammatory response (Figure 15F-M).

[0170] Based on the above experiments, this application reveals a key molecular interaction, namely the E-TMED10 interaction, which triggers the release of THU-mediated inflammatory factors, leading to severe inflammation during coronavirus infection, and proposes a model for this (see Figure 16). The findings obtained from cell, molecular and mouse experiments strongly emphasize the potential of targeting the E-TMED10 interaction as a therapeutic strategy to combat the lethal inflammatory factor storm induced by SARS-CoV and other highly toxic coronaviruses.

[0171] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0172] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Use of an E-TMED10 inhibitor in the preparation of a product for treating and / or preventing inflammation, characterized in that: E-TMED10 is the interaction between coronavirus E protein and TMED10. E-TMED10 inhibitors block or reduce the interaction between coronavirus E protein and TMED10. The inflammation is inflammation caused by coronavirus infection.

2. The use according to claim 1, characterized in that The E-TMED10 inhibitor treats and / or prevents inflammation by blocking or reducing the oligomerization of TMED10.

3. The use according to claim 1 or 2, characterized in that The E-TMED10 inhibitors treat and / or prevent inflammation by blocking or reducing the transport of UcPS (unclassical secretion) cargo to ERGIC.

4. The use according to any one of claims 1 to 3, characterized in that: The E-TMED10 inhibitor blocks or reduces the transport of inflammatory factors to vesicles in the UcPS pathway.

5. The use according to any one of claims 1 to 3, characterized in that: The E-TMED10 inhibitor blocks or reduces the translocation of inflammatory factors into the membrane.

6. The use according to any one of claims 1 to 3, characterized in that: The E-TMED10 inhibitor treats and / or prevents inflammation by blocking or reducing the release of inflammatory factors in the UcPS pathway.

7. The use according to claim 4 or 5, characterized in that The inflammatory factors include inflammatory factors of the IL1 family, such as one or more of IL1β, IL33, IL36α, IL1α or IL18.

8. The use according to claim 1, characterized in that The E-TMED10 inhibitor targets the cytoplasmic C-terminus of TMED10, preferably targeting a sequence including YLRRFFKAKKLIE (SEQ ID NO: 2).

9. The use according to claim 1, characterized in that The E-TMED10 inhibitor targets the SS motif or DS motif of the coronavirus E protein, wherein S represents serine and D represents aspartic acid.

10. The use according to claim 1, characterized in that The inflammation includes pneumonia, nephritis, hepatitis or splenitis.

11. The use according to claim 1, characterized in that The inflammation includes lung damage caused by immune cell infiltration.

12. The use according to claim 1, characterized in that The coronavirus infects vertebrates, such as humans, mice, pigs, cats, dogs, wolves, cattle or birds, wherein the birds include poultry or birds.

13. The use according to claim 1, characterized in that The coronavirus includes genus α, genus β, genus γ or genus δ, preferably genus β.

14. The use according to claim 13, characterized in that The alpha coronavirus includes human coronavirus 229E or a mutant thereof, human coronavirus NL63 or a mutant thereof, human coronavirus HKU1 or a mutant thereof, long-winged bat coronavirus HKU8 or a mutant thereof, horseshoe bat coronavirus HKU2 or a mutant thereof, porcine epidemic diarrhea virus (PEDV) or a mutant thereof, porcine transmissible gastroenteritis virus (TGEV) or a mutant thereof, canine coronavirus (CCoV) or a mutant thereof or feline coronavirus (FCoV) or a mutant thereof One or more of them.

15. The use according to claim 13, characterized in that The beta coronavirus includes human coronavirus HKU1 or a mutant thereof, mouse coronavirus or a mutant thereof, house bat coronavirus HKU5 or a mutant thereof, fruit bat coronavirus HKU9 or a mutant thereof, severe acute respiratory syndrome (SARS) or a mutant thereof, MERS or a mutant thereof, SARS-CoV-2 or a mutant thereof, bovine coronavirus (BCoV) or a mutant thereof, human coronavirus OC43 or a mutant thereof, equine coronavirus (ECoV) or a mutant thereof, porcine hemagglutinating encephalomyelitis virus (PHEV) or a mutant thereof, canine respiratory coronavirus (CrCoV) or a mutant thereof, one or more thereof; Preferably, the murine coronavirus comprises one or more of mouse hepatitis virus (MHV), rat coronavirus or avian auklet virus; Preferably, the SARS mutant includes one or more of BJ01, Urbani, Tor2, CUHK or HKV5; Preferably, the SARS-CoV-2 mutant is selected from Alpha, Beta, Gamma, Delta, Lambda or Omiron.

16. The use according to claim 13, characterized in that The gamma coronavirus includes avian coronaviruses such as infectious bronchitis virus (IBV) or its mutants, Beluga whale coronavirus SW1 (BWCoV-SW1) or its mutants; Preferably, the avian coronavirus includes coronaviruses that cause infection in poultry such as chickens, turkeys, sparrows, ducks, geese, and pigeons.

17. The use according to claim 13, characterized in that The delta coronavirus includes Bulbul coronavirus HKU11 (BuCoV HKU11) or its mutant, Thrush coronavirus HKU12 (ThCoV HKU12) or its mutant, Thrush coronavirus HKU12 (ThCoV HKU12) or its mutant, Asian Leopard Cats Coronavirus (ALCCoV) or its mutant, Chinese ferret-badger Coronavirus (CFBCoV) or its mutant, Porcine Delta Coronavirus (PDCoV) or its mutant, White-eye Coronavirus (WECoV) or its mutant, Sparrow Coronavirus (SPCoV) or its mutant, Magpie robin Coronavirus (MRCoV) or its mutant, Night heron Coronavirus (NHCoV) or its mutant, Wild duck coronavirus (Wigeon Coronavirus (WiCoV) or its mutants, Common Moorhen Coronavirus (CMCoV) or its mutants, one or more of them.

18. The use according to any one of claims 1 to 17, characterized in that: The coronavirus comprises an SS motif or a DS motif in its E protein, wherein S represents serine and D represents aspartic acid.

19. The use according to claim 1, characterized in that The E-TMED10 inhibitors include small molecules, traditional Chinese medicines, traditional Chinese medicine extracts, antibodies, RNAi, reagents for knocking out TMED10, or cell therapy drugs.

20. The use according to claim 19, characterized in that The E-TMED10 inhibitors include agents that express or overexpress the cytoplasmic domain of the coronavirus E protein, such as vectors that express or overexpress the cytoplasmic domain of the coronavirus E protein; Preferably, the vector can be a viral vector or a non-viral vector.

21. The use according to claim 20, characterized in that The cytoplasmic domain of the coronavirus E protein includes SEQ ID NO:

1.

22. The use according to claim 20, characterized in that The vector comprises a nucleotide sequence encoding the cytoplasmic domain of the coronavirus E protein; It is preferred to include a single copy or two or more copies of the nucleotide sequence.

23. The use according to claim 20, characterized in that Express or overexpress the cytoplasmic domain of coronavirus E protein in immune cells.

24. The use according to claim 23, characterized in that The immune cells include one or more of lymphocytes, monocytes, macrophages, NK cells, eosinophils, neutrophils or natural killer cells.

25. The use according to claim 19, characterized in that The E-TMED10 inhibitor is selected from progesterone or its analogues, dichlorophenol or its analogues, nitazoxanide or its analogues.

26. The use according to claim 25, characterized in that The progesterone analogue has the general formula (I) or a pharmaceutically acceptable salt, solvate or metabolite thereof: wherein X is selected from O or NOCH3; R1 and R2 are independently selected from H, -C1-C6 alkyl, halogen, -OH, -C1-C6 alkoxy, or R1 and R2 are absent and a double bond is formed between the carbon atoms to which R1 and R2 are attached; R3 is selected from H, -C1-C6 alkyl, phenyl or substituted phenyl; R4 and R5 are independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy; R6 is selected from H, -C1-C6 alkyl, -(CH2) m -halogen, -(CH2) m OR 16 , R 16 Selected from H, -C(=CH2)R 17 Or-COR 17 , wherein m is an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6), R 17 Selected from -C1-C 12 alkyl; R7 is selected from H, -OH, -OCOC1-C 12 Alkyl, or R7, R8 and adjacent carbon atoms form an oxygen-containing heterocyclic ring; R8 and R9 are independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy, or R8 and R9 and the connected carbon atom form R 10 、R 11 and R 12 Independently selected from H, -C1-C6 alkyl, halogen, -OH or -C1-C6 alkoxy.

27. The use according to claim 26, characterized in that in: R1 is -CH3; R2 is H or halogen; Preferably, R1 and R2 are absent and a double bond is formed between the carbon atoms to which R1 and R2 are attached; R3 is H or where R 13 Selected from H, -C1-C6 alkyl, -C1-C6 alkoxy, -NR 14 R 15 、-COR 14 、-SR 14 、-SOR 14 or-SO2R 14 , where R 14 and R 15 Independently selected from H or C1-C6 alkyl; Preferably, R3 is H or Preferably, R 13 is -N(CH3)2; R4 is H; R5 is -CH3; m is 1, 2, or 3; R6 is selected from -CH3, -CH2-halogen, -CH2-OR 16 ; R 17 is H, -CH3, -CH2-CH3 or n is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6); R7 is selected from H, -OH, -OCOC1-C6 alkyl (e.g., -OCOCH3, -OCO(CH2)2CH3); Preferably, R7, R8 and adjacent carbon atoms form a five-membered heterocyclic ring containing 1 or 2 oxygen atoms, more preferably, R7, R8 and adjacent carbon atoms form R8 and R9 are H or R8 and R9 and the carbon atom to which they are connected form R 10 is H or -CH3; R 11 H or F; R 12 It is H or -CH3.

28. The use according to claim 26, characterized in that The progesterone analogue is selected from:

29. The use according to claim 1, characterized in that The treatment and / or prevention of inflammation includes any one or more of the following: A) Inhibit the secretion of inflammatory factors; B) reduce E protein-promoted TMED10 oligomerization; C) reduce the translocation of inflammatory factors into the membrane; D) reduce the transfer of UcPS cargo to ERGIC; E) Reduce the transport of inflammatory factors into vesicles via the UcPS pathway.

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