CN112870359A - TRIM41 inhibitor, anti-inflammatory effect and application thereof - Google Patents

Abstract

The present disclosure relates to tri-motif E3 ubiquitin ligase 41(TRIM41) inhibitors, their anti-inflammatory effects and uses. In particular, the present disclosure provides for the use of a TRIM41 inhibitor (e.g., siRNA) in the preparation of a product for preventing and/or treating an inflammatory disease and/or condition in a subject, particularly inflammation associated therewith in combating viral and bacterial infections. TRIM41 inhibitors are effective in reducing proinflammatory cytokine levels (especially high proinflammatory cytokine levels caused by infection), and provide a useful strategy for the prevention and/or treatment of inflammation.

Description

TRIM41 inhibitor, anti-inflammatory effect and application thereof

Technical Field

The present application belongs to the field of biotechnology and medicine. In particular, the present application relates to the effects, mechanisms of action, methods of implementation and uses of the tri-motif E3 ubiquitin ligase 41(TRIM41, tripartite motif E3 ubiquitin ligand 41) and inhibitors thereof (e.g., small interfering RNAs) in inflammatory diseases and/or conditions (e.g., viral or bacterial infectious diseases).

Background

Pathogen infection (including viral infection and bacterial infection) is a common clinical disease, and can cause local and systemic infectious manifestations in the body, and can even ultimately lead to severe consequences such as septic shock, endotoxic shock, and Multiple Organ Dysfunction Syndrome (MODS). Once severe infection leads to shock or multiple organ failure, high mortality (-50-80%) manifestations are caused.

Common clinical viral or bacterial infections, such as influenza virus, herpes simplex virus, listeria, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis and the like, can cause local and systemic inflammatory reactions of the body, and cause the occurrence of infectious diseases of different degrees.

For virus infection patients, the current antiviral drugs mainly aim at multiple links of virus replication, but have limited effect on completely eliminating viruses, and the elimination of the viruses also depends on the immune system of the body. When the virus persists, it may induce a cytokine storm in the body, leading to systemic inflammatory responses, shock and MODS.

In patients with bacterial infections, physicians generally use high doses of antibiotics to achieve a rapid and complete kill of the pathogens, but often neglect the other aspect that large amounts of toxins are released after the death of the pathogens, and these toxins cause severe systemic symptoms. There are two main classes of toxins that induce systemic inflammation: endotoxins (the lipopolysaccharide component of the cell wall of gram-negative bacteria) and superantigens (including the peptidoglycan/lipoteichoic complex of gram-positive bacteria, exotoxins, and the like).

Shock and multiple organ failure caused by pathogen infection remains a significant challenge for clinical prevention and treatment.

The human body can detect pathogen invasion and initiate host anti-pathogenic microbial responses, such as the production of type I interferons and proinflammatory cytokines, through Pattern Recognition Receptors (PRRs) in the innate immune system. Nucleic acids (including DNA and RNA) are important genetic information carriers for all organisms (including viruses, bacteria) and are the major structures recognized by the innate immune system. Intracellular nucleic acid-recognizing PRRs include several endosomal members of the Toll-like receptor (TLR) family as well as cytoplasmic-recognized DNA and RNA receptors.

TLR is one of the most studied PRR families [ Kawai t. et al, immunity.2011; 34: 637-50 ]. TLRs are single-pass transmembrane proteins with an extracellular domain that contain leucine-rich repeats that are used to recognize constitutively expressed molecules common to most pathogenic microorganisms, i.e., Pathogen-associated molecular patterns (PAMPs). It also contains a transmembrane domain and a cytoplasmic Toll/IL-1receptor (TIR) domain responsible for transducing signals to downstream adaptor proteins, including TRIF and MyD 88.

10 TLRs were identified separately in humans, 4 of which were involved in nucleic acid recognition, TLR3, TLR7, TLR8 and TLR9, respectively. TLR3 recognizes double-stranded RNA (dsRNA) in viral molecules [ Takeuchi, o. et al, immunity.1999; 11: 443-451; ozinsky, a. et al, Proc Natl Acad Sci usa.2000; 97: 13766-; alexoulou, l. et al, nature.2001; 413: 732-; human TLR7 and TLR8 genes map to chromosome Xp22, recognize single-stranded RNAs (ssRNAs) in viral molecules [ Heil, f. et al, science.2004; 303: 1526-; human TLR9 maps to chromosome 3p21.3 and recognizes CpG motifs in bacterial and viral DNA or synthetic CpG oligonucleic acids (CpG ODNs) [ Hemmi, h. et al, nature.2000; 408: 740-.

These receptors recognize pathogenic nucleic acids in the endosome and act through two signaling pathways: a MyD88 (myoid differentiation factor 88) dependent pathway and a TRIF (Toll/IL-1receptor (TIR) -domain containing adapter protein inducing IFN- β) dependent pathway. MyD88 activates downstream IRAKs, TRAF6, TAK1, IKK and MAPK signaling molecules, and finally leads to the early activation of NF-kB and AP1 and mediates the production of proinflammatory cytokines such as TNF-alpha, IL-6, IL-1 beta and nitric oxide; activation of TRIF activates TBK1, TRAF3 and IRF3, ultimately leading to the production of type I interferons and the activation of interferon inducible genes, such as CXCL10 and CCL5 et al [ Akira, S. et al, nat. Rev. Immunol.4, 499-511 (2004); liew, f.y. et al, nat. rev. immunol.5, 446-458 (2005).

When pathogenic microorganisms invade the cytoplasm and replicate within the cell, their nucleic acids are recognized by nucleic acid receptors within the cytoplasm and trigger downstream immune responses. The two most prominent receptors are RIG-I, which recognizes RNA with triphosphate at the 5' end, and cGAS, which recognizes double-stranded DNA.

RIG-I like receptors are DExD/H-box domain containing proteins required for cytoplasmic recognition of double stranded RNA and induction of pro-inflammatory cytokines and interferon production [ Yoneyama m, et al, nat. immunol.2004; 5: 730-37 ]. RIG-I is an important member of the RIG-I like receptor (RLR) family, which recognizes viruses in the Paramyxoviridae family, such as Sendai virus and Newcastle disease virus; viruses in the flaviviridae family, such as encephalitis b virus, dengue virus, west nile virus and hepatitis c virus; viruses in the rhabdoviridae family, such as vesicular stomatitis virus; reovirus in the reoviridae family, et al [ Gitlin l. et al, Proc Natl Acad Sci usa.2006; 103: 8459-64; kato h. et al, nature.2006; 441: 101-5; fredericksen BL. et al, j.virol.2008; 609: 609-16; lo Y-m. et al, j.virol.2008; 82: 335-45 ]. When RIG-I recognizes viral nucleic acid and is activated, it can recruit the adaptor protein mavs (mitogenic anti viral signaling) and mediate the production of pro-inflammatory cytokines such as TNF- α, IL-6, IL-1 β and interferons by activating IKK and TBK1, ultimately leading to the activation of NF- κ B and IRF3 [ Seth RB. et al, cell.2005; 669-82 ].

The natural immune nucleic acid receptor signal pathway is used as the first barrier of the body's innate immunity, and can eliminate pathogens by inducing the production of proinflammatory cytokines and interferons, thereby maintaining the stability of the body's internal environment. However, this signal is activated by a twolip sword, and excessive pro-inflammatory cytokine production while eliminating pathogens can lead to various inflammatory and autoimmune diseases.

Abnormal activation of nucleic acid receptors of the Toll-like receptor (TLR) family (e.g., TLR3, TLR7, TLR8 and TLR9), cytoplasmic RNA receptors (e.g., RIG-I and MDA5) and cytoplasmic DNA receptors (e.g., cGAS and several other STING-related DNA receptors) is closely associated with inflammatory and autoimmune diseases and has been extensively studied [ Yin, q. et al, annu. rev immunol. 2015; 33,393-416; cao, X, nat.rev.immunol.2016; 16, 35-50; chen q. et al, nat. immunol.2016; 17: 1142-49; crowl, j, et al, annu.rev.immunol.2017; 35,313-. Activation of the innate immune nucleic acid recognition signaling pathway must be tightly regulated to prevent its over-activation, particularly leading to the overproduction of inflammatory factors. Therefore, the search for inflammation-modulating molecules and the discovery of new anti-inflammation targets have been the focus of natural immunity research.

Ubiquitin (Ub) modification plays a crucial role in the regulation of innate and adaptive immunity. TRIM family proteins are defined as E3 ubiquitin ligases, characterized by an N-terminal RING-finger domain, one or two zinc finger domains (called B1 and/or B2 BOX), one coiled coil structure and a different C-terminal domain. TRIM family proteins regulate important cell life activities such as intracellular signaling, natural immune regulation, transcriptional regulation, autophagy, and tumors [ Ozato, k. et al, nat. rev. immunol.2008; 8,849-; hatakeyama, S et al, Trends biochem. sci.2017; 42,297- & ltSUB & gt 311 ].

TRIM41 is also a TRIM protein containing the PRY-SPRY domain. However, the biological and immunological functions of TRIM41 are poorly understood. TRIM41 was reported to potentially interact with PKC kinases and NOD2, and may inhibit transcription of the hepatitis b virus gene [ Chen, d, et al, j.biol.chem.2007; 282,33776-33787; zhang, S, PLoS one.2013; 8, e 70001; thiebaut, R, et al, PLoS one.2016; 11, e0165420 ]. Recently, TRIM41 was found to act as an E3 ubiquitin ligase for influenza a virus nucleoprotein, leading to influenza a virus nucleoprotein degradation [ Patil, g. et al, j.virol.2018; 492, e00905-e00918 ].

In view of the foregoing, there is a strong need in the art to develop an immunologically active substance that is effective against inflammatory diseases and/or conditions (e.g., over-production of pro-inflammatory cytokines during viral or bacterial infection).

Disclosure of Invention

The present application provides novel uses of small interfering RNA of TRIM41 protein for effective inhibition of inflammatory diseases and/or conditions (e.g., pro-inflammatory cytokine production following viral or bacterial infection).

In a first aspect of the present application, there is provided the use of an inhibitor of TRIM41 in the manufacture of a product for the prevention and/or treatment of an inflammatory disease and/or condition in a subject.

In some embodiments, TRIM41 is a material selected from the group consisting of: trim41 gene, mRNA, cDNA of Trim41, TRIM41 precursor, and TRIM41 protein.

In some embodiments, the inhibitor is selected from the group consisting of for TRIM 41: small interfering rna (sirna), shRNA, miRNA, antisense oligonucleotide, zinc finger protein, CRISPR/Cas9 gene editing product, antibody, chemical inhibitor.

In some embodiments, the inhibitor is selected from the group consisting of a naturally purified substance, a modified naturally purified substance, a semi-synthetic substance, and a chemically synthetic substance.

In some embodiments, the inhibitor is derived from a mammal.

In some embodiments, the inhibitor is derived from: humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits).

In some embodiments, the inhibitor is an siRNA of TRIM41 selected from the group consisting of:

(a) has the sequence shown in SEQ ID NO: 1-12, or a pharmaceutically acceptable salt thereof;

(b) an RNA molecule which is homologous to the RNA sequence in (a) and has the function of inhibiting TRIM 41; and/or

(c) And (b) an RNA molecule derived from (a) or (b) by substituting, deleting or adding one or more bases in the RNA sequence of (a) or (b) and having an effect of inhibiting TRIM 41.

In some embodiments, the inhibitor is an siRNA of TRIM41 selected from the group consisting of: SEQ ID NO: 1-12 in any pair of the sequences of RNA molecules.

In some embodiments, the inhibitor is an siRNA of TRIM41, the sequence of which is selected from the group consisting of: 1 and 2,3 and 4, 5 and 6, 7 and 8, 9 and 10, and 11 and 12.

In some embodiments, the inflammatory disease and/or condition is: pathogen infection, such as viral (e.g., DNA virus or RNA virus), bacterial, rickettsial, mycoplasma, fungal, spirochete, and/or parasitic; physical factors such as high temperature, low temperature, radioactive material damage, mechanical damage; chemical factors, such as exogenous toxins, endogenous toxins; allergic reactions, such as hypersensitivity, autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, ulcerative colitis, etc.).

In some embodiments, the inflammatory disease and/or condition comprises one or more selected from the group consisting of: overproduction of proinflammatory cytokines (e.g., IL-6, TNF α, type I interferons (e.g., IFN β), IL-1), such as inflammatory factor storm; viral or endotoxic shock or death; inflammatory injury or multiple organ failure of an organ, for example the organ is selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine; chronic inflammatory diseases caused by viral infection.

In some embodiments, the pathogen may be selected from, but is not limited to: viruses, bacteria, rickettsia, mycoplasma, fungi, spirochetes, and parasites. For example, the pathogen may be one or more selected from the group consisting of: influenza virus, vesicular stomatitis virus, herpes simplex virus, Listeria, coronavirus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, enterococcus faecalis.

In some embodiments, the subject to be administered is a mammal, e.g., a human, a non-human primate (e.g., orangutan, ape), a rodent (e.g., rat, mouse, guinea pig), a pet (e.g., cat, dog), a farm animal (e.g., horse, cow, sheep, pig, rabbit).

In some embodiments, the subject is a subject with a natural immunodeficiency/impairment and/or treatment of an interferon antiviral infection that is ineffective or prone to side effects, such as a subject with or susceptible to an autoimmune disease, such as an IFN insufficiency or an overactive subject (e.g., a subject with inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, tuberculosis, chronic gastrointestinal disease).

In some embodiments, the product is a pharmaceutical composition or kit, e.g., in a form suitable for administration by a formulation selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, and transdermal administration.

In another aspect of this document, there is provided the use of an inhibitor of TRIM41 in the manufacture of a product for the treatment or co-treatment of a pathogen infectious disease and/or disorder. Wherein the inhibitor of TRIM41 is as described hereinbefore.

In some embodiments, the use of the product is as described above. The pathogen is one or more selected from the group consisting of: viruses, bacteria, rickettsia, mycoplasma, fungi, spirochetes and/or parasites, e.g. selected from influenza virus, vesicular stomatitis virus, herpes simplex virus, listeria, coronavirus (e.g. COVID-19), escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis.

In another aspect, there is provided a pharmaceutical composition or kit comprising:

(A) a therapeutically and/or prophylactically effective amount of an inhibitor of TRIM 41;

(B) a pharmaceutically or immunologically acceptable carrier or excipient;

(C) optionally, one or more other active substances for the prevention and/or treatment of inflammatory diseases and/or symptoms.

In some embodiments, the inhibitor, pharmaceutical composition or kit of TRIM41 and uses thereof are as described herein.

In some embodiments, the additional active substance may be selected from, but is not limited to, the group consisting of: clinical commonly used antiviral drugs, such as tricyclic amines, pyrophosphates, protease inhibitors, nucleosides, antisense oligonucleotides, interferon, ribavirin, amantadine, oseltamivir, etc.; antibiotics, such as β -lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenics, macrolides, antifungal antibiotics, antitubercular antibiotics; clinically, common immunosuppressive agents, such as glucocorticoid, cyclophosphamide, chloroquine, cyclosporine A, tripterygium wilfordii, traditional Chinese medicine preparations and anti-TNF monoclonal antibodies.

In other aspects, also provided herein is a method for preventing and/or treating an inflammatory disease and/or symptom, comprising administering to a subject in need thereof an effective amount of a TRIM41 inhibitor or pharmaceutical composition or kit described herein.

In other aspects, also provided herein is a method for treating or aiding in the treatment of a pathogen infectious disease and/or disorder, the method comprising administering to a subject in need thereof an effective amount of a TRIM41 inhibitor or a pharmaceutical composition or kit as described herein.

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.

FIG. 1: after the small interfering RNA is used for interfering TRIM41 of mouse abdominal cavity macrophages, the generation of proinflammatory cytokines after virus stimulation can be obviously reduced. Wherein:

FIGS. 1a and 1b show the change in the expression level of TRIM41 after viral stimulation;

FIG. 1c is the efficiency test of TRIM41 small interfering RNA;

FIGS. 1d and 1e show the fluorescent quantitative PCR detection of the mRNA level of proinflammatory cytokines;

FIGS. 1f and 1g show the levels of proinflammatory cytokine protein measured by ELISA.

P < 0.01; p < 0.001; p < 0.0001. Ctrl represents a control group, Trim41KD represents a Trim41 interference group, and Mock is the same DMEM culture solution group used for virus resuspension.

FIG. 2: the production of proinflammatory cytokines was significantly reduced in bone marrow-derived macrophages from Trim 41-deficient mice after treatment with pathogen-derived nucleic acids or pathogens.

FIG. 2a shows the detection of inflammatory factors by ELISA after treatment of cells with pathogen-derived nucleic acids; wherein Liposome is Liposome for transfecting nucleic acid, and is used as a control group;

FIG. 2b shows the detection of pro-inflammatory cytokines by ELISA after direct treatment of cells with pathogens; wherein Mock is a DMEM culture solution for resuspending the pathogen, and is used as a control group.

***，P<0.001；****，P<0.0001。Trim41^+/+Representing the wild type control Trim41^-/-Represents the TRIM41 expression defect group.

FIG. 3: trim 41-deficient mice are less inflammatory after viral infection.

FIG. 3a is a peripheral blood serum ELISA assay;

figure 3b is a mouse lung HE staining analysis.

****，P<0.0001。Trim41^+/+Representing the wild type control Trim41^-/-Represents the TRIM41 expression-deficient group, and the mock represents the control group.

FIG. 4: western Blot analysis of bone marrow-derived macrophages from Trim 41-deficient mice, the activation of inflammation-associated signaling pathways was significantly reduced after treatment with virus.

FIG. 5: ELISA analysis shows that after small interfering RNA is used for interfering TRIM41 of human-derived macrophage line THP1, the production of proinflammatory cytokines after virus stimulation can be obviously reduced.

P < 0.001; p < 0.0001. Ctrl represents a control group, Trim41KD represents a Trim41 interference group; mock is DMEM medium for resuspension of the pathogen, here used as a control.

Detailed Description

Through a large number of researches and animal model experiments, the applicant finds that the interference or knockout of the expression of TRIM41 can effectively inhibit the production of proinflammatory cytokines and improve the organ functional state in inflammatory diseases or symptoms (such as bacterial or viral infection). The present applicant has completed the present application on this basis.

Specifically, the research on the application of the genes related to inflammation is a hotspot of the research on molecular biology and cell biology of inflammation, the expression of the genes promoting inflammation is knocked down by using the small interfering RNA, and the application of the genes in the prevention and treatment of inflammation is an effective technology for manual intervention of bacterial infection, so that the genes have wide application prospects in functional genome research and gene treatment related to inflammation.

The inventor finds out through research that: trim41 deficiency or knockdown significantly inhibited the inflammatory response. In VSV and HSV-1 infection models, it is observed that the Trim41 defect can obviously inhibit the production of proinflammatory cytokines and I-type interferon, and Trim41 defective mice have slight lung injury induced by VSV and HSV-1, which suggests that interference in knocking down Trim41 may have application prospects in treatment of infection-related inflammatory diseases. Thus, the present application provides methods and strategies for the use of small interfering RNA of Trim41 in the prevention and treatment of inflammation associated with infectious diseases, particularly lung injury caused by available infections.

The application aims at novel Trim41 small interfering RNA with anti-inflammatory effect, researches the generation of proinflammatory cytokines of macrophages in immune cells under the action of microbial components, and verifies the therapeutic effect of the small interfering RNA on infection-related inflammation. Experiments prove that: 1) the interference of knocking down Trim41 in macrophage can inhibit the generation of virus-induced proinflammatory cytokine and type I interferon; 2) trim 41-deficient macrophages produce less pro-inflammatory cytokines and type I interferons upon pathogen infection; 3) in VSV and HSV-1 infection models, the Trim 41-deficient mice have fewer peripheral blood proinflammatory cytokines and type I interferons and less lung inflammation damage; 4) interfering with knockdown Trim41 inhibiting macrophage inflammation-related signaling; 5) interference with knock-down of Trim41 in human macrophage lines inhibited the production of proinflammatory cytokines and type I interferons following viral stimulation.

The application proves that the inhibitor of Trim41 can inhibit the expression of proinflammatory cytokines and reduce inflammation and organ injury. These findings confirm that inhibitors of Trim41 (e.g., small interfering RNAs) are expected to be effective means for the treatment and prevention of inflammatory diseases (e.g., infection-related inflammation).

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

TRIM41 and inhibitor thereof

As used herein, the term "TRIM 41" has its broadest meaning and includes the TRIM41 gene, mRNA of TRIM41, cDNA, TRIM41 precursor, TRIM41 protein, modified or cleaved products, or active fragments thereof.

TRIM41 can be derived from humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits), and the like.

The sequence of Trim41 may be, for example: in humans [ Homo sapiens (human) ] corresponding to NCBI Gene ID: 90933, respectively; in mice [ Mus musculus (house mouse) ] corresponding to NCBI Gene ID: 211007; corresponding to NCBI Gene ID: 303088. the protein sequence of TRIM41 can be, for example: GI: 42516572/116242826, GI: 81889232/124358936, GI:197927145, etc.

As used herein, the term "inhibitor of TRIM 41" or "inhibitor" has its broadest meaning and refers to a substance that is capable of reducing or attenuating or eliminating the level or function of TRIM 41. Inhibitors may include, but are not limited to: substances which reduce the transcriptional and translational expression level of Trim41 (such as substances which knock down or knock out the expression of Trim41), substances which inhibit the function of Trim41 and the like.

In some embodiments, inhibitors of TRIM41 include, but are not limited to: substances inhibiting TRIM41 (such as compounds, e.g., small molecule compounds), antibodies against upstream metabolic enzymes produced by TRIM41 or precursors thereof (e.g., antibodies against IRG1 or OGDH), siRNAs (e.g., siRNAs against TRIM41 as set forth in any of SEQ ID NOS: 1-12), miRNAs, antisense oligonucleotides, CRISPR/Cas9 gene editing products, zinc finger proteins, compounds inhibiting the function and/or activity of TRIM41 or precursors thereof, promoter elements and/or expression vectors that down-regulate the expression level of genes or mRNAs or proteins of TRIM 41.

One of ordinary skill in the art can design or obtain a corresponding inhibitor for TRIM41 to produce an effect of preventing and/or treating an inflammatory disease and/or disorder by blocking or inhibiting the expression or activity of the enzyme.

Product and application thereof

The disclosure also provides a product, which may be a pharmaceutical composition or kit, comprising an effective amount of a TRIM41 inhibitor of the disclosure, and a pharmaceutically or immunologically acceptable carrier. As used herein, the terms "active" or "active ingredient" are used interchangeably to refer to TRIM41 inhibitor.

The inhibitors and products of the present disclosure are useful for preventing and/or treating inflammation or diseases and/or conditions associated therewith. For example, inflammation caused by infection, which may be a DNA virus and/or RNA virus infection, such as an infection caused by one or more viruses selected from the group consisting of: herpes simplex virus, vesicular stomatitis virus, influenza virus, encephalomyocarditis virus, Sendai virus, hepatitis B virus, adenovirus, poxvirus, small DNA virus, adeno-associated virus.

Inflammatory diseases and/or conditions may include, but are not limited to, one or more selected from the group consisting of: overproduction of proinflammatory cytokines (e.g., IL-6, TNF α, type I interferons (e.g., IFN β), IL-1), such as inflammatory factor storm; viral or endotoxic shock or death; inflammatory injury or multiple organ failure of an organ, for example the organ is selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine; chronic inflammatory diseases caused by viral infection. As used herein, the terms "pro-inflammatory cytokine", "pro-inflammatory factor" and "inflammatory factor" are used interchangeably.

The inhibitors or products of the present disclosure may be used in different subjects, preferably mammalian subjects, e.g., humans, pets, livestock. Furthermore, the inhibitors or products of the present disclosure have advantages in preventing or treating subjects with natural immunodeficiency, e.g., subjects with natural immunodeficiency/impairment and/or treatment of interferon antiviral infection that is ineffective or prone to side effects, such as subjects with or susceptible to autoimmune diseases, such as subjects with IFN insufficiency or overexcitation (e.g., subjects with inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, tuberculosis, chronic gastrointestinal disorders).

As used herein, the term "pharmaceutically/immunologically acceptable" ingredient is one that is suitable for use in humans and/or animals without undue adverse effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a prophylactic and/or therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art and a full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub.Co., N.J.1991.

Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

An immunologically acceptable adjuvant may also be included in the products of the present disclosure, for example, the adjuvant is selected from: aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21.

The active substance in the product of the present disclosure accounts for 0.001-99.9 wt% of the total weight of the composition; preferably 1 to 95 wt%, more preferably 5 to 90 wt%, and even more preferably 10 to 80 wt% of the total weight of the composition. The balance can be pharmaceutically acceptable carriers and other additives.

As used herein, the term "unit dosage form" refers to a dosage form that is desirable for a single administration of a product of the present disclosure for ease of administration, including, but not limited to, various solid agents (e.g., tablets), liquids, capsules, sustained release agents.

In other embodiments of the present disclosure, the product is in unit dosage form or multiple dosage form. In other embodiments of the present disclosure, 1 to 6 doses of the composition of the present disclosure, such as 1 to 3 doses, or 1 dose, are applied daily.

It will be appreciated that the effective dose of inhibitor or drug employed may vary with the severity of the subject to be administered or treated. The specific condition is determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, desired effect), and is within the judgment of a skilled physician.

The products of the present disclosure may be in solid form (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (e.g., oral liquid) or other suitable form. The route of administration may be, but is not limited to, depending on the type of inhibitor: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus, or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, etc.

In addition, the products of the present disclosure may also contain other active agents useful for ameliorating and treating inflammatory diseases or conditions, including but not limited to: clinical commonly used antiviral drugs, such as tricyclic amines, pyrophosphates, protease inhibitors, nucleosides, antisense oligonucleotides, interferon, ribavirin, amantadine, oseltamivir, etc.; antibiotics, such as β -lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenics, macrolides, antifungal antibiotics, antitubercular antibiotics; clinically, common immunosuppressive agents, such as glucocorticoid, cyclophosphamide, chloroquine, cyclosporine A, tripterygium wilfordii, traditional Chinese medicine preparations and anti-TNF monoclonal antibodies.

Also, the inhibitors or products of the present disclosure may be used in combination with each other, and may also be used in combination with other drugs and therapeutic means for the treatment of inflammatory diseases or conditions.

Examples

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.

Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred embodiments and materials described herein are intended to be exemplary only.

Small interfering RNA

The sequence of the small interfering RNA used in the examples is shown below:

example 1: interference with knock-down of Trim41 inhibits proinflammatory cytokine production after viral infection of macrophages

Materials and methods

C57BL/6J mice, male, week-old 6-8 weeks, purchased from Shanghai Biky laboratory animals, Inc. Intraperitoneal injection of mice was performed with 3% sodium thioglycolate broth (from merck), four days after injection, mice were sacrificed by cervical dislocation, epidermis was cut to expose peritoneum, peritoneal cavity was repeatedly washed with serum-free DMEM medium to obtain mouse peritoneal macrophages, and the ratio of mouse peritoneal macrophages was 4 × 10⁵The concentration of individual/ml was plated into cell culture plates for subsequent experiments.

Vesicular stomatitis virus (VSV, ATCC VR-1238) and herpes simplex virus (HSV-1, F-strain, ATCC VR-260) were both purchased from ATCC. Different viruses were stimulated by adding different calculated MOI values (see below) to the cell culture.

Mouse TRIM41 small interfering RNA from Shanghai Jima gene, according to the instruction steps with Lipofectamine RNAiMAX liposome (from life detection company), adding cell culture solution to perform RNA interference experiment, the small interfering RNA final concentration is 20nM, and the interference time is 48 hours.

Western blot was performed according to a method commonly used in laboratories, cytokine mRNA levels were detected by fluorescent quantitative PCR, and cytokine protein levels in culture supernatants were detected using ELISA assay kits (purchased from Biolegend) for the respective factors according to the instructions.

Detailed experimental procedure

First, mouse abdominal macrophages were treated with VSV virus (MOI ═ 1) and HSV-1 virus (MOI ═ 5) for various periods of time (as shown in the figure), RNA and protein were collected, and changes in expression level of TRIM41 were detected by fluorescence quantitative PCR and Western blot assay. Then, after the knocking-down efficiency of Trim41 small interfering RNA is verified, the small interfering RNA is used for subsequent experiments, and the proinflammatory cytokine mRNA and protein level when the virus infects macrophages after interfering Trim41 is detected.

Results and discussion

The results of analysis of the expression level of Trim41 are shown in FIGS. 1a and 1b, respectively. The results show that: the expression level of TRIM41 was significantly reduced after viral infection.

The results of the efficiency validation of small interfering RNA of Trim41 are shown in FIG. 1 c. The results show that: each siRNA has an interference effect on Trim41, wherein the interference efficiency of siRNA1 is strongest, and the siRNA is used for subsequent experiments.

Proinflammatory cytokine mRNA levels after viral stimulation are shown in figures 1d, 1 e: the interfering Trim41 inhibits the production of mrnas for proinflammatory cytokines and type I interferons.

The ELISA results are shown in FIGS. 1f and 1 g: interfering Trim41 inhibits the production of pro-inflammatory cytokines and type I interferon proteins. Mock in the figure is DMEM medium for resuspending the pathogen, here used as a control.

The results of this example show that: the small interfering RNA of Trim41 can inhibit the production of proinflammatory cytokines and type I interferon after virus infection macrophages.

Example 2: trim41 deficiency renders mouse bone marrow-derived macrophages under viral nucleic acid analogs and viral processing Thereafter, the ability to produce proinflammatory cytokines and type I interferons is reduced

Materials and methods

Trim 41-deficient mice Committee the Shanghai Square model BiologyTechnology development company Limited constructs, targets based on S129 embryonic stem cells, and implants the target into C57 pseudopregnant mice to obtain F1 generation. Raising under SPF environment, hybridizing F1 mouse with C57BL/6J for over 10 generations, genotyping, and identifying Trim41^+/+And Trim41^-/-Mice, for subsequent experiments.

Taking the bone marrow cells of the mice at 6-8 weeks, and Tris-NH₄Breaking erythrocytes with Cl, neutralizing with DMEM (10% FBS), and resuspending the cells after centrifugation in BMDM induction medium (DMEM, 10% FBS, 20ng/ml M-CSF) at 2X 10⁶The amount of each well is paved on a 6-well plate for culture, and bone marrow-derived macrophages (BMDM) can be obtained after 5-6 days. Cells were collected and replated as needed for subsequent experiments.

Vesicular stomatitis virus (VSV, ATCC VR-1238), Sendai virus (SeV, ATCC VR-105), herpes simplex virus (HSV-1, F-strain, ATCC VR-260), vaccinia virus (VACV, ATCC VR-1536) and Listeria (LM, ATCC 19115) were all purchased from ATCC. Different viruses calculated different MOI values (see below) were added to the cell culture medium for stimulation.

The viral nucleic acid analogue poly (I: C) (tlrl-picwlv), 5' ppp-dsRNA (tlrl-3prna), HSV-60(tlrl-HSV60n), ISD (tlrl-isdn) were purchased from InvivoGen, co-incubated with the nucleic acid analogue according to the instructions for liposome interferon (purchased from polyplus), and stimulated by the addition of macrophage culture supernatant.

The ELISA assay for cytokines in cell culture was the same as in example 1.

Detailed experimental procedure

The nucleic acid analogues and liposome mixtures were added to macrophage culture supernatants (FIG. 2a) or different groups of bone marrow-derived macrophages were treated with RNA viruses VSV, SeV and Listeria (MOI ═ 1), DNA viruses HSV-1 and VACV (MOI ═ 5) as described above and the cell culture supernatants were analyzed by ELISA for production of proinflammatory cytokines TNF α, IL-6 and IFN- β. Liposome in FIG. 2a represents the control group of liposomes alone, and Mock in FIG. 2b is DMEM medium for resuspending the pathogen, here used as a control group.

Results and discussion

The results are shown in FIGS. 2a and 2 b.

The results show that: after the nucleic acid analogue stimulates or a pathogen infects bone marrow-derived macrophages, the defect of Trim41 can obviously inhibit the production of proinflammatory cytokines and type I interferon.

The results of this example show that: trim 41-deficient cells produce less pro-inflammatory cytokines and type I interferons upon infection.

Example 3: trim 41-deficient mice have a weaker inflammatory response after infection

Materials and methods

Acquisition of defective mice, pathogen source and cytokine level detection as in example 2, HE staining of mouse tissue organs was performed by google bio.

Detailed experimental procedure

Trim 41-deficient mice were injected intraperitoneally with 1X 10⁷(PFU) VSV or 1X 10⁸(PFU) HSV-1, after 12 hours serum was isolated from peripheral blood of mice and ELISA was used to detect IL-6 and IFN- β levels. After 24 hours, the lung of the mouse was taken and subjected to HE staining to observe the level of inflammatory lesions.

Results and discussion

The effect of the Trim41 deficiency on peripheral proinflammatory cytokine production after mouse infection is shown in FIG. 3a, and the effect on lung inflammation is shown in FIG. 3 b.

The results show that: trim 41-deficient mice produced significantly lower pro-inflammatory cytokines upon pathogen infection; meanwhile, the lung inflammation is less damaged. In the wild type mouse lung, more infiltration of nuclear-profused inflammatory cells and tissue damage was observed. In FIG. 3b the simulant is a balanced salt PBS solution for dilution of the pathogen as a no-infection control.

The results show that: the deficiency of Trim41 inhibits pro-inflammatory cytokines and inflammatory injury after pathogen infection in mice.

Example 4: trim41 deficiency inhibits inflammatory signals after virus infection of mouse macrophages

Materials and methods

The obtaining of the defective mouse, the culture of macrophage derived from bone marrow and the source of pathogen are the same as the example 2, and the Western blot detection is still carried out according to the conventional flow in the laboratory.

Detailed experimental procedure

Trim41 was treated with RNA virus VSV (MOI ═ 1) and DNA virus HSV-1(MOI ═ 5)^+/+And Trim41^-/-The bone marrow-derived macrophages are collected, and Western blot is used for detecting activation of inflammation-related signal channels.

Results and discussion

The Western blot analysis results are shown in FIG. 4.

The results show that: after the virus infects bone marrow-derived macrophages, the defect of Trim41 can obviously inhibit the activation of inflammation-related signal pathways NF-kappa B, TBK1-IRF3, JNK and P38, and has no obvious influence on ERK signals. And Trim41 deficiency had no significant effect on the unstimulated group.

Example 5: trim41 interfering with human macrophage THP1 can obviously inhibit proinflammatory cells after virus infection Generation of factors

Materials and methods

The THP1 cell line (ATCC TIB-202) was purchased from ATCC. The human TRIM41 small interfering RNA is designed and synthesized by the Shanghai Jima gene. The virus was obtained and treated in the same manner as in example 1.

Detailed experimental procedure

THP1 cells at 3X 10⁵The mixture is spread into a cell culture plate at a concentration of one/ml, and simultaneously 100ng/ml of PMA is added to induce the mixture to differentiate and adhere to the wall for 24 hours, and after the mixture adheres to the wall, an interference experiment and cytokine detection are carried out according to the method in the embodiment 1.

The efficiency of human small interfering RNA of Trim41 was verified. H-siRNA1, which has the strongest interference efficiency, was selected for subsequent experiments.

The production of proinflammatory cytokines TNF α, IL-6 and IFN- β in cell culture supernatants were analyzed by ELISA by treating THP1 cells with small interfering RNA of Trim41, treating different groups of THP1 cells with RNA virus VSV (MOI ═ 1) and DNA virus HSV-1(MOI ═ 5).

Results and discussion

The ELISA results are shown in FIG. 5.

The results show that: the interference of Trim41 in THP1 can effectively inhibit the production of proinflammatory cytokines and I-type interferon protein.

All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Further, it should be understood that various changes or modifications can be made to the present application by those skilled in the art after reading the above teachings of the present application, and these equivalents also fall within the scope of the present application as defined by the appended claims.

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Claims (10)

1. Use of an inhibitor of Trimotif E3 ubiquitin ligase 41(TRIM41) for the preparation of a product for the prevention and/or treatment of an inflammatory disease and/or condition in a subject.

2. The use of claim 1, wherein TRIM41 is a substance selected from the group consisting of: trim41 gene, mRNA, cDNA of Trim41, TRIM41 precursor, and TRIM41 protein.

3. The use of claim 1, wherein the inhibitor is selected from the group consisting of the inhibitors against TRIM 41: small interfering rna (sirna), shRNA, miRNA, antisense oligonucleotide, zinc finger protein, CRISPR/Cas9 gene editing product, antibody, chemical inhibitor;

for example, the inhibitor is: natural purified substances, modified natural purified substances, semisynthetic substances, chemically synthesized substances;

for example, the inhibitor is derived from a mammal;

for example, the inhibitor is derived from: humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits).

4. The use of claim 1, wherein said inhibitor is an siRNA of TRIM41 selected from the group consisting of:

(a) has the sequence shown in SEQ ID NO: 1-12, or a pharmaceutically acceptable salt thereof;

(b) an RNA molecule which is homologous to the RNA sequence in (a) and has the function of inhibiting TRIM 41; and/or

(c) And (b) an RNA molecule derived from (a) or (b) by substituting, deleting or adding one or more bases in the RNA sequence of (a) or (b) and having an effect of inhibiting TRIM 41.

5. The use of claim 1, wherein the inflammatory disease and/or condition is: pathogen infection, such as viral (e.g., DNA virus or RNA virus), bacterial, rickettsial, mycoplasma, fungal, spirochete, and/or parasitic; physical factors such as high temperature, low temperature, radioactive material damage, mechanical damage; chemical factors, such as exogenous toxins, endogenous toxins; allergic reactions, such as hypersensitivity, autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, ulcerative colitis, etc.);

for example, the inflammatory disease and/or condition comprises one or more selected from the group consisting of: overproduction of proinflammatory cytokines (e.g., IL-6, TNF α, type I interferons (e.g., IFN β), IL-1), such as inflammatory factor storm; viral or endotoxic shock or death; inflammatory injury or multiple organ failure of an organ, for example the organ is selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine; chronic inflammatory diseases caused by viral infections;

for example, the pathogen is selected from: viruses, bacteria, rickettsia, mycoplasma, fungi, spirochetes, and parasites;

for example, the pathogen is one or more selected from the group consisting of: influenza virus, vesicular stomatitis virus, herpes simplex virus, Listeria, coronavirus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, enterococcus faecalis.

6. The use according to claim 1, wherein the subject is a mammal, such as a human, a non-human primate (e.g. orangutan, ape), a rodent (e.g. rat, mouse, guinea pig), a pet (e.g. cat, dog), a domestic animal (e.g. horse, cow, sheep, pig, rabbit);

for example, the subject is a subject with a natural immunodeficiency/impairment and/or treatment of an interferon antiviral infection that is ineffective or poorly effective or prone to side effects, such as a subject with or susceptible to an autoimmune disease, such as an IFN insufficiency or an overactive subject (e.g., a subject with inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, tuberculosis, chronic gastrointestinal disease).

7. Use according to claim 1, wherein the product is a pharmaceutical composition or kit, for example in a form suitable for administration by a formulation selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, and transdermal administration.

8. Use of an inhibitor of Trimotif E3 ubiquitin ligase 41(TRIM41) for the preparation of a product for the treatment or co-treatment of a pathogen infectious disease and/or disorder,

for example, the inhibitor of TRIM41 is as mentioned in any one of claims 2 to 4;

the use of the product is as mentioned in any of claims 6 to 7; and/or

The pathogen is one or more selected from the group consisting of: viruses, bacteria, rickettsia, mycoplasma, fungi, spirochetes and/or parasites, e.g. selected from influenza virus, vesicular stomatitis virus, herpes simplex virus, listeria, coronavirus (e.g. COVID-19), escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, enterococcus faecalis.

9. A pharmaceutical composition or kit comprising:

(A) a therapeutically and/or prophylactically effective amount of an inhibitor of TRIM 41;

(B) a pharmaceutically or immunologically acceptable carrier or excipient;

(C) optionally, one or more other active substances for the prevention and/or treatment of inflammatory diseases and/or symptoms.

10. The pharmaceutical composition or kit of claim 9, wherein,

an inhibitor of TRIM41 as mentioned in any one of claims 2 to 4;

the use of the pharmaceutical composition or kit is as mentioned in any one of claims 5 to 7; and/or

The other active substance is selected from the group consisting of: clinical commonly used antiviral drugs, such as tricyclic amines, pyrophosphates, protease inhibitors, nucleosides, antisense oligonucleotides, interferon, ribavirin, amantadine, oseltamivir, etc.; antibiotics, such as β -lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenics, macrolides, antifungal antibiotics, antitubercular antibiotics; clinically, common immunosuppressive agents, such as glucocorticoid, cyclophosphamide, chloroquine, cyclosporine A, tripterygium wilfordii, traditional Chinese medicine preparations and anti-TNF monoclonal antibodies.

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