KR101263757B1 - Methods for Screening TLR4(Toll-like receptor 4) Signaling Inhibitors and Uses Thereof - Google Patents

Abstract

The present invention provides a method for screening a Toll-like receptor 4 (TLL4) signaling inhibitor comprising the steps of: (a) contacting a test substance with a Toll-like receptor 4 (TLR4) or TLR4 extracellular domain; And (b) analyzing whether the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4, wherein the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4. TLR4 oligomerization is inhibited to determine it as a TLR4 signaling inhibitor. TLR4 signaling inhibitors of the invention, more preferably thiol-reactive compounds (e.g. SFN), covalently bond with cysteine residues present in the TLR4 or TLR4 extracellular domain to form adducts and ligand-induced TLR4 Both oligomerization and ligand-independent oligomerization of TLR4. Therefore, the composition comprising the TLR4 signaling inhibitor of the present invention or its potency enhancer (for example, SFN) as an active ingredient significantly weakens the inflammatory response and is effective in the treatment of inflammatory diseases, and also improves skin condition and reduces edema. effective.

Description

Methods for Screening TLR4 Signaling Inhibitors and Uses thereof {Methods for Screening Toll-like receptor 4 (TLL4) Signaling Inhibitors and Uses Thereof}

The present invention relates to a method for screening a toll-like receptor 4 (TLR4) signaling inhibitor or an efficacy enhancer thereof.

Natural isothiocyanate derivative 1-isothiocyanate-4- (methylsulfinyl) -butane (sulforaphane or SFN) is abundant in cruciferous vegetables such as broccoli and cauliflower. SFN is well known for its chemopreventive activity, and its activity is phase II involved in the inhibition of phase I enzymes that produce carcinogens and the detoxification of carcinogens. II) mediated through the induction of enzymes (1). In addition, SFN has anti-salt efficacy. SFN reduced the expression of inflammatory proteins such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and TNF-α in LPS-treated macrophages or neurons (2, 3). SFN inhibited LPS-induced excretion of high-mobility group box 1 (HMGB1), which functions as cytokine-like molecules in RAW264.7 cells and is actively excreted by immunostimulation (4). SFN reduced monocyte adhesion to epithelial cells by inhibiting the expression of intercellular adhesion molecule-1, an adhesion molecule, in LPS-treated epithelial cells (5). Dried broccoli diet as a rich source of SFN reduced the infiltration of macrophages into the vascular cell wall, heart and kidney of congenital hypertensive rats (6). Since inflammation is one of the important etiological factors in carcinogenesis, the anti-inflammatory activity of SFN may contribute to anti-cancer efficacy.

Inflammation can be initiated by microbial infection or tissue damage. TLRs are pathogen recognition receptors (PRRs) that detect structural components derived from invading microorganisms or damaged cells / tissues. Activation of TLRs elicits immune and inflammatory responses through activation of cellular signal transduction and expression of immune and inflammatory mediators. TLR4 recognizes LPS, a cell wall component of Gram-negative bacteria that causes systemic inflammatory sepsis. In addition, TLR4 can be activated by endogenous molecules such as heparin sulphate and fibronectin, which are released from damaged tissue sites and cause an aseptic inflammatory response. Activation of TLR4 by LPS results in oligomerization of TLR4 resulting in regression of the adapter molecule. In general, TLR4 signaling is dependent on MyD88 and TRIF (Toll / IL-1R domain containing adapter-inducing IFN-β), which are the major adapter molecules that interact with the cytoplasmic region of TLRs. MyD88 regresses IRAK-4 (IL-1R-associated kinase-4) and IRAK-1, which are further associated with TRAF 6 (TNFR-associated factor 6), leading to IκB kinase (IKK) α / β / γ complexes and MAPKs Results in activation. TRIF is responsible for the activation of the MyD88-independent signaling pathway. TRIF activates receptor interacting protein 1, TANK-binding kinase 1, and IKKε through interaction with TRAF 6 or TRAF 3 (7-11).

Activation of TLR4 significantly promotes the production of pro-inflammatory proteins such as iNOS, COX-2 and TNF-α. Aberrantly regulated TLR activity is known to be closely associated with the development of chronic inflammatory diseases as well as immunological diseases (12). TLRs and downstream signaling components may be effective therapeutic targets for many inflammatory diseases (13, 14). Thus, we investigated whether the well-known anti-inflammatory factor SFN regulates TLRs and downstream signaling pathways and what are the anti-inflammatory targets in the TLR4 signaling pathway regulated by SFN.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to discover toll-like receptor 4 signaling inhibitors or potentiators thereof. As a result, the inventors have found that thiol-reactive compounds (eg, SFN) covalently bond with cysteine residues present in the TLR4 or TLR4 extracellular domain to form adducts, ligand-dependent and ligand-ratios. Inhibits oligomerization of the dependent TLR4 extracellular domain, thereby inhibiting NF-κB activation and interferon regulatory factor 3 (IRF3) activation and inhibiting pro-inflammatory cytokines (eg, TNF-α (tumor necrosis factor-α), The discovery of reduced production of IL (interleukin) -1β or IL-6) led to the completion of the present invention.

It is an object of the present invention to provide a method for screening toll-like receptor 4 (TLR4) signaling inhibitors.

Another object of the present invention is to provide a method for screening an efficacy enhancer of a TLR4 (Toll-like receptor 4) signaling inhibitor.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a Toll-like receptor 4 (TLR4) signaling inhibitor comprising the following steps: (a) Toll-like receptor 4 or TLR4 extracellular domain Contacting the test substance with the test substance; And (b) analyzing whether the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4, wherein the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4. TLR4 oligomerization is inhibited to determine it as a TLR4 signaling inhibitor.

According to another aspect of the present invention, the present invention provides a method for screening a potency enhancer of a toll-like receptor 4 (TLR4) signaling inhibitor comprising the following steps: (a) Toll-like receptor 4 or TLR4 Contacting the TLR4 signaling inhibitor and the test substance with the extracellular domain; (b) contacting a TLR4 signaling inhibitor with a TLR4 or TLR4 extracellular domain as a control; And (c) analyzing the extent to which the TLR4 signaling inhibitor covalently binds to a cysteine residue in the extracellular domain of TLR4 in steps (a) and (b), wherein in (a) the TLR4 signaling inhibitor If the degree of covalent attachment to cysteine residues in the extracellular domain of TLR4 increases, the test substance is considered to be an potentiator of the TLR4 signaling inhibitor.

The present inventors have made extensive efforts to discover toll-like receptor 4 signaling inhibitors or potentiators thereof. As a result, the inventors have found that thiol-reactive compounds (eg, SFN) covalently bond with cysteine residues present in the TLR4 or TLR4 extracellular domain to form adducts, ligand-dependent and ligand-ratios. Inhibits oligomerization of the dependent TLR4 extracellular domain, thereby inhibiting NF-κB activation and interferon regulatory factor 3 (IRF3) activation and inhibiting pro-inflammatory cytokines (eg, TNF-α (tumor necrosis factor-α), It has been found to reduce the production of IL (interleukin) -1β or IL-6).

Innate immune receptors are promising targets that can regulate complex reaction cascades that cause cytokine production, depending on their molecular characteristics, which specifically recognize pathogen ligands and activate various signal cascades to alter gene expression.

Toll-like receptors (TLRs) belong to a family of structurally related class I single pass transmembrane proteins that function as monitors for pathogen infection. In general, TLRs can be distinguished by target pathogen molecules (eg, highly conserved bacterial proteins, pathogen cell wall components or pathogen-associated nucleic acids) and most mammals are 10 to 15 on the genome. Includes TLRs. TLRs are highly conserved from fruit flies to humans and have structural and functional similarities. TLRs recognize pathogen-associated molecular patterns (PAMPs) expressed in infectious agents, mediate the production of cytokines necessary for effective immunity and exhibit a variety of expression patterns.

TLR4 is a human protein encoded by the TLR4 gene and is very important for the activation of the innate immune system because it recognizes lipopolysaccharides (LPS) of Gram-negative bacteria. TLR4 is also called CD284 (cluster of differentiation 284). TLR4 is most abundantly expressed in the myelomonocytic subpopulation of the placenta and leukocytes. In addition, TLR4 is involved in signal transduction events induced by LPS found in most Gram-negative bacteria.

As mentioned above, activation of LPS-induced TLR4 results in TLR4 oligomerization resulting in recruitment of various adapter molecules (eg, MyD88, TRIF, MD2, etc.).

The present invention provides a method for screening a Toll-like receptor 4 (TLR4) signaling inhibitor comprising the following steps.

(a) contacting a test substance with a TLR4 (Toll-like receptor 4) or TLR4 extracellular domain; And

(b) analyzing whether the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4, and when the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4 Inhibits oligomerization to determine TLR4 signaling inhibitors.

According to the method of the present invention, the test substance to be analyzed is first contacted with the TLR4 or TLR4 extracellular domain of the present invention. Cells comprising the TLR4 or TLR4 extracellular domain of the present invention are not particularly limited, preferably immune cells or skin cells, more preferably epithelial cells, macrophages or fibroblasts. As used to refer to the screening method of the present invention, the term “test material” refers to an unknown material used in screening to test whether it affects oligomerization of the TLR4 or TLR4 extracellular domain of the present invention. The test substance includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

Subsequently, the covalent attachment to the cysteine residue in the TLR4 or TLR4 extracellular domain of the present invention in contact with the test substance is analyzed. This can be done as described below, and as a result of the measurement, when the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4, the test substance can be determined as a TLR4 signaling inhibitor by inhibiting TLR4 oligomerization. .

According to a preferred embodiment of the present invention, the test substance of the present invention reacts with cysteine residues present in the extracellular domain of TLR4 to form adducts. That is, the cysteine residues of the TLR4 extracellular domain are modified by treatment of the test substance of the present invention. More preferably, the cysteine residue of the TLR4 extracellular domain modified by the test substance of the present invention comprises a TLR4 receptor or a cysteine residue in the TLR4 extracellular domain, even more preferably 88, 192, 246, 281, 340, 506, 542 and 609th cysteine residues, or 87, 164, 245, 280, 504, 549 and 606th cysteine residues of mouse TLR4, most preferably 88, 192, 246, of human TLR4 281, 340, 506, 542 and 609 th cysteine residues.

In addition, analysis of the covalent binding to the cysteine residue in the TLR4 or TLR4 extracellular domain of the present invention can be carried out through various immunoassay methods known in the art. For example, changes in the TLR4 or TLR4 extracellular domain may include immunostaining, radioimmunoassay, radioimmunoprecipitation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or competition assay, and Sandwich analysis includes, but is not limited to.

Methods of immunoassay or immunostaining are described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out according to the immunoprecipitation method, a form in which the label is fused to the TLR4 or TLR4 extracellular domain of the present invention can be used for analysis. Immunoprecipitation is a technique of precipitating antigenic proteins in a reaction solution using an antibody that specifically binds to a specific target (eg, a protein or a peptide used as a label). In general, immunoprecipitation techniques include individual protein immunoprecipitation (IP), protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), and the like. Through immunoprecipitation, specific target proteins, preferably the TLR4 or TLR4 extracellular domain of the present invention, can be detected by pulling down from other proteins in a sample. The biggest disadvantage of immunoprecipitation methods is the need for antibodies that can specifically bind to the target. To overcome this disadvantage, a construct is constructed in which a tag for immunoprecipitation is fused to the N-terminus or C-terminus of the target protein. For example, tags that can be fused to a protein terminus include, but are not limited to, various fluorescent proteins (eg, GFP, RFP, CFP, etc.), FLAG, HA, His ₆ or glutathione-S-transferases. It doesn't happen. In the present invention, a construct in which various tags (eg, FLAG, HA, GFP) are fused to a TLR4 or TLR4 extracellular domain, which is a target protein of the present invention, is transfected into cells and various test substances (eg, SFN, Parthenolide, Ethacrynic Acid) and then immunoprecipitation was performed to analyze changes in TLR4 or TLR4 extracellular domains (see Figures 4-6).

On the other hand, when the method of the present invention is carried out according to the method of radioimmunoassay, an antibody labeled with radioisotopes (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) may be used for the TLR4 or TLR4 extracellular domain of the present invention. Can be used to detect.

When the method of the present invention is carried out by ELISA, the present invention comprises the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody to a target (eg, TLR4 or TLR4 extracellular domain) as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable that the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylene diamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) coating an antibody against a target of the invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the cell sample; (Iii) reacting the product of step (ii) with a detecting antibody that has a label generating a signal and which specifically reacts with the TLR4 or TLR4 extracellular domain of the present invention; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

The final signal measurement in the ELISA method and the capture-ELISA method can be performed according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the targets of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to a preferred embodiment of the invention, the test substance of the invention comprises a thiol-reactive compound.

Thiol is a compound comprising a functional group (organic chemically composed of a sulfur-hydrogen bond (-SH), the functional group refers to a thiol group or sulfhydryl groups (sulfhydryl groups). Traditionally, thiol groups, also called mercaptans, are very important in biological terms because they function as functional groups of cysteine residues. When two cysteine residues are in close proximity to each other during protein folding, the thiol group in the cysteine residues forms a cystine with disulfide bonds (-SS-) through oxidation. In addition, sulfhydryl groups in the active site of the enzyme form non-covalent bonds with the enzyme substrate, contributing to catalytic activity. Active site cysteine residues, on the other hand, can react with heavy metal ions (eg, Pb ²⁺ , Hg ²⁺ , Ag ²⁺ ) to inactivate the protein.

According to a preferred embodiment of the present invention, the thiol-reactive compounds of the present invention are sulforaphane, parthenolide, ethacrynic acid, auranofin, cinnamicaldehyde, malein Mead, N-methyl maleimide (NNM), N-ethyl maleimide (NEM), maleic anhydride, diethyl maleate (DEM), 2-AAodoacetamide (IAA), cystine or derivatives thereof, cystamine , Dithioglycolic acid, oxidized glutathione, iodine, hydrogen peroxide, dihydroascorbic acid (DHA), tetrathionate or o-iodosobenzoate (o-iodosobenzoate). More preferably, the thiol-reactive compound of the present invention is SFN, Parthenolide, Ethacrynic Acid, Auranopine or Cinnamicaldehyde, even more preferably SFN, Parthenolide or Ethacrynic Acid, most preferably SFN.

SFN is an organosulfur compound with anticancer, antidiabetic and antibacterial activity. SFN can be obtained from cruciferous vegetables such as broccoli. When a plant is injured, in response, the plant uses a myrosinase to transform the glucosinolate, glucopanin, into sulforaphane (SFN). SFN is broccoli, brussels, cabbage, cauliflower, bok choy, kale, collards, chinese broccoli, broccoli raab, kohlrabi, It is abundant in mustard, turnips, radishes, arugula and watercress.

According to a preferred embodiment of the invention, the test substance of the invention (eg, SFN) inhibits ligand-dependent and ligand-independent TLR4 oligomerization.

According to a preferred embodiment of the invention, the test substance of the invention (eg SFN) inhibits LPS-, TLR4-, MyD88- and IKKβ (IκB kinase β) -induced NF-κB activation.

According to a preferred embodiment of the invention, the test substance of the invention (eg SFN) inhibits LPS- and TLR4-induced interferon regulatory factor 3 (IRF3) activation.

More preferably, the test substance of the present invention, even more preferably SFN, reduces the production of pro-inflammatory cytokines. Pro-inflammatory cytokines include tumor necrosis factor (TNF), interleukin-1α, 1L-1β, IL-6, IL-8, IL-18, interferon γ, HMG-1, platelet- Activating factor (platelet-activating factor (PAF) or macrophage migration inhibitory factor (MIF)), more preferably TNF-α, IL-1β or IL-6.

According to a preferred embodiment of the present invention, the test substance of the present invention (eg, SFN) can treat an inflammatory disease.

According to a preferred embodiment of the present invention, the inflammatory disease which can be treated by the composition comprising the above-described test substance as an active ingredient is an inflammatory skin disease [eg, asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis arthritis, contact dermatitis, atopic dermatitis, psoriasis, acne, atopic rhinitis (hay fever), allergic dermatitis (eczema), chronic sinusitis or seborrheic dermatitis], bone disease, gastritis, gout Gout arthritis, ulcers, chronic bronchitis, acute lung injury, pulmonary inflammation, airway hypersensitivity, delayed-type hypersensitivity, food sensitivity, wound-healing, inflammatory bowel disease (e.g. Crohn's disease, ulcerative colitis) ), Acute or chronic inflammatory diseases such as ankylosing spondylitis, gluten-sensitive enteropathy, pneumonia, pulmonary shock, vasculitis and bursitis; Autoimmune diseases such as lupus, rheumatic polymyalgia, scleroderma, Wegener's granulomatosis, temporal arteritis, cryoglobulinemia and multiple sclerosis; Rejection of transplants; Cancers including solid cancers such as lungs, CNS, intestines, kidneys and pancreas; Alzheimer's disease; Atherosclerosis, insulin resistance, obesity; Viral infections (eg HIV or influenza); Chronic viral infections (eg, Epstein-Barr virus, cytomegalovirus, herpes simplex virus) or capillary dilatation ataxia, more preferably pneumonia, atherosclerosis, insulin resistance, obesity or cancer .

According to a preferred embodiment of the present invention, the cancer which can be treated by the composition containing the above-described test substance as an active ingredient is breast cancer, kidney cancer, neuroendocrine cancer, gastric cancer, lung cancer, ovarian cancer, liver cancer, bronchial cancer, non-phosphorus Head cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer, prostate cancer, bone cancer, brain cancer, skin cancer, thyroid cancer, parathyroid cancer or ureter cancer.

The present invention also provides a method for screening for potency enhancers of Toll-like receptor 4 (TLR4) signaling inhibitors comprising the following steps:

(a) contacting a TLR4 signaling inhibitor and a test substance with a TLR4 (Toll-like receptor 4) or TLR4 extracellular domain;

(b) contacting a TLR4 signaling inhibitor with a TLR4 or TLR4 extracellular domain as a control; And

(c) analyzing the extent to which the TLR4 signaling inhibitor covalently binds to a cysteine residue in the extracellular domain of TLR4 in steps (a) and (b), wherein in step (a) the TLR4 signaling inhibitor is If the degree of covalent binding to cysteine residues in the extracellular domain of TLR4 increases, the test substance is considered to be an potentiator of the TLR4 signaling inhibitor.

Since the method of the present invention includes the above-described method for screening the TLR4 signaling inhibitor of the present invention, the contents in common between the two are omitted in order to avoid excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-described TLR4 signaling inhibitor (eg thiol-reactive compound) or potency enhancer thereof; And (b) a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described TLR4 signaling inhibitor or potency enhancer thereof.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, or the like. Can be.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the preferred dosage of the pharmaceutical composition of the present invention is 0.0001-1000 μg per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a composition for improving skin conditions comprising the above-described TLR4 signaling inhibitor of the present invention or an efficacy enhancer thereof as an active ingredient.

Since the method of the present invention includes the above-described TLR4 signaling inhibitor of the present invention or an efficacy enhancer thereof as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

As demonstrated in the Examples below, the TLR4 oligomerization inhibitors of the invention, preferably thiol-reactive compounds, more preferably SFN, parthenolide or ethacrynic acid, most preferably SFN are ligand-dependent and Ligand-independent TLR4 oligomerization was inhibited. That is, the SFN of the present invention inhibited LPS-, TLR4-, MyD88- and IKBβ (IκB kinase β) -induced NF-κB activation, and inhibited LPS- and TLR4-induced interferon regulatory factor 3 (IRF3) activation. Weakened. In addition, the SFN of the present invention attenuated the inflammatory response by reducing the production of pro-inflammatory cytokines (eg, TNF-α, IL-1β or IL-6). In an inflammatory animal model based on these results, it was found that the TLR4 oligomerization inhibitor of the present invention significantly attenuates LPS-induced inflammation in vivo in mice (see FIG. 9). This effect was as strong as indomethacin, a well-known anti-inflammatory drug. Therefore, the composition of the present invention is very effective for improving the skin condition.

According to a preferred embodiment of the invention, the improvement of the skin condition by the TLR4 oligomerization inhibitor of the invention is an improvement of dermatitis or edema.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for screening a Toll-like receptor 4 (TLR4) signaling inhibitor or an efficacy enhancer thereof.

(b) A thiol-reactive compound, preferably SFN, which is a TLR4 signaling inhibitor of the invention, covalently bonds with cysteine residues present in the TLR4 or TLR4 extracellular domain to form adducts.

(c) Thiol-reactive compounds, preferably SFNs, which are TLR4 signaling inhibitors of the invention, inhibit both ligand-induced oligomerization of TLR4 and ligand-independent oligomerization of TLR4.

(d) SFNs of the present invention inhibit LPS-, TLR4-, MyD88- and IKKβ (IκB kinase β) -induced NF-κB activation and inhibit LPS- and TLR4-induced interferon regulatory factor 3 (IRF3) activation Weakens.

(e) SFN of the present invention reduces the production of pro-inflammatory cytokines (eg, TNF-α, IL-1β or IL-6), thereby attenuating the inflammatory response.

(f) Therefore, the composition comprising the TLR4 signaling inhibitor of the present invention or its potency enhancer (e.g. SFN) as an active ingredient significantly weakens the inflammatory response to be effective in the treatment of inflammatory diseases and improves and swells the skin condition. It is also effective in reducing.

1 shows the result of inhibiting LPS-induced activation of TLR4 signaling and expression of target genes. A, RAW264.7 cells were pretreated with SFN (10 and 20 μM) for 1 hour and then further treated with LPS (5 ng / ml) for 30 minutes. The levels of IRAK-1 and actin protein were analyzed in cell lysates using immunoblotting. Luciferase reporter plasmids comprising IFN-β promoter domains (IFN-β PRDIII-I) (C) at B and C, NF-κB binding sites (2x) (B) or IRF3 binding sites, were transferred to RAW264.7 cells. Transfection. Transfected cells were pretreated with SFN (10 and 20 μM) for 1 hour and then further treated with LPS (5 ng / ml) for 6 hours. Luciferase and β-galactosidase enzyme activities were analyzed in cell lysates. Relative luciferase activity (RLA) was calculated by normalizing to β-galactosidase activity. The value means the mean value ± standard error (n = 3). * p <0.05; ** p <0.01. D, RAW264.7 cells were pretreated with SFN (10 and 20 μM) for 1 hour and then further treated with LPS (5 ng / ml) for 6 hours. The levels of COX-2 and actin protein were analyzed in cell lysates using immunoblotting. Veh, control.
2 shows that SFN inhibits ligand-independent activation of TLR4. As indicated, a luciferase reporter plasmid comprising an NF-κB binding site (2 ×) or an IRF3 binding site (IFN-β PRDIII-I) (C) and a continuously activated TLR4 (CD4-TLR4) or WT TLR4 Expression plasmid was transfected into 293T cells (A and B) or RAW264.7 cells (C). After 24 hours, SFN (10 and 20 μM) was treated with cells for an additional 8 hours and RLA was measured. The value means the mean value ± standard error (n = 3). Veh, control; * p <0.05; ** p <0.01.
FIG. 3 shows that SFN inhibits NF-κB activation induced by MyD88 and IKKβ but does not inhibit IRF3 activation induced by TRIF and TBK1. As indicated, transfect the 293T cells with a luciferase reporter plasmid comprising an NF-κB binding site (2 ×) or an IRF3 binding site (IFN-β PRDIII-I) and an expression plasmid of MyD88, IKKβ, TRIF or TBK1. Sean. SFN (10 and 20 μM) were treated with cells for an additional 8 hours and RLA was measured. The value means the mean value ± standard error (n = 3). Veh, control; * p <0.05; ** p <0.01.
4 shows that SFN inhibits oligomerization of TLR4. Ba / F3 cells expressing A, Flag-TLR4, GFP-TLR4, MD-2, CD14, and NF-κB reporter genes were pretreated with SFN (10 and 20 μM) for 1 hour and then LPS (50 ng / ml). Was further treated for 20 minutes. Cells were then immunoprecipitated with anti-Flag antibody and immunoblotted with anti-GFP (top panel) and anti-Flag (bottom panel). B, another batch of Ba / F3 cells were pretreated with SFN (10 and 20 μM) for 1 hour and then further treated with LPS (1 ng / ml) for 8 hours. Luciferase enzyme activity was measured in cell lysates. 293T cells transfected with C, HA- and Flag-ΔTLR4 were further treated with SFN (10 and 20 μM) for 8 hours. Cells were then immunoprecipitated with anti-HA antibody and immunoblotted with anti-Flag (top panel) and anti-HA (bottom panel). 293T cells transfected with D, Flag-ΔTLR4 and NF-κB reporter gene were further treated with SFN (10 and 20 μM) for 8 hours. The value means the mean value ± standard error (n = 3). Veh, control; * p <0.05; ** p <0.01.
5 shows that thiol donors block the inhibitory effect of SFN on TLR4 oligomerization and NF-κB activation. In Ba / F3 cells expressing the A, Flag-TLR4, GFP-TLR4, MD-2, CD14, and NF-κB reporter genes, SFN (20 μM) is absent or present in DTT (300 μM) or NAC (2 mM). After treatment for 1 hour under LPS (50 ng / ml) was further treated for 20 minutes. Cells were then immunoprecipitated with anti-Flag antibody and immunoblotted with anti-GFP or anti-Flag. B, another batch of Ba / F3 cells were treated as described in FIG. 5A followed by additional treatment with LPS (1 ng / ml) for 8 hours. Luciferase enzyme activity was measured in cell lysates. The value means the mean value ± standard error (n = 3). Veh, control; ** p <0.01.
6 is a result showing the effect of parthenolide and ethacrynic acid on LPS-induced oligomerization of TLR4. Parthenolide (Par; 5 and 10 μM) to Ba / F3 cells expressing luciferase reporter genes comprising A and C, Flag-TLR4, GFP-TLR4, MD-2, CD14 and NF-κB binding sites and Ethacrynic acid (EA; 50 and 100 μM) was pretreated for 1 hour followed by additional treatment with LPS (50 ng / ml) for 20 minutes. Cells were then immunoprecipitated with anti-Flag antibody and immunoblotted with anti-GFP (top panel) and anti-Flag (bottom panel). B and D, different batches of Ba / F3 cells were pretreated with Par (5 and 10 μM) and EA (50 and 100 μM) and then further treated with LPS (1 ng / ml) for 8 hours. Luciferase enzyme activity was measured in cell lysates. The value means the mean value ± standard error (n = 3). Veh, control; * p <0.05; ** p <0.01.
7 is a result showing that the inhibitory effect of SFN on TLR4-dependent signaling is observed in Nrf2-KO cells as well as WT cells. MEFs isolated from A, WT or Nrf2-KO mice were transfected with the luciferase reporter gene containing the NF-κB binding site. Cells were pretreated with SFN (20 μM) for 1 hour and then further treated with LPS (100 ng / ml) for 8 hours. Luciferase enzyme activity was measured in cell lysates. The value means the mean value ± standard error (n = 3). MEFs isolated from B, WT or Nrf2-KO mice were pretreated with SFN (10 and 20 μM) for 1 hour and then further treated with LPS (1 μg / ml) for 15 minutes. The levels of phospho-Ser ⁴⁷³ -Akt ( p -Akt) and Akt protein were analyzed in cell lysates using immunoblotting. Expression plasmids of the luciferase reporter gene, including TLR4 (ΔTLR4) and NF-κB binding sites, which were constantly activated in C, WT or Nrf2-KO MEFs, were transfected. Cells were further treated with SFN (20 μM) for 9 hours and then luciferase enzyme activity was measured in cell lysates. The value means the mean value ± standard error (n = 3). Veh, control; *** p <0.001.
8 shows that SFN blocks LPS-induced production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 in mice. Mice were orally administered SFN (25 mg / kg) for 24 hours and 1 hour before challenged with LPS (7.5 mg / kg, intraperitoneal injection). Two hours after LPS treatment, serum samples were obtained. Serum concentrations of TNF-α, IL-1β and IL-6 were measured using specific ELISA. The value means the mean value ± standard error (n = 3-4). PBS was used as a vehicle (vehicle). * p <0.05; *** p <0.001.
9 shows results that SFN reduces skin inflammation induced by LPS infusion in mice. SFN (10 and 30 mg / kg) or indomethacin (INDO; 3 mg / kg) LPS (50 μg / 25 μl in PBS) in the middle of the right ear of the mouse 24 and 1 hour before and 6 hours after intradermal injection ) Was administered orally. A, LPS was injected 24 hours after weighing both ears. The untreated left ear was used for standardization of individual variations and the ratio of right ear to left ear was presented as ear swelling values. The value means the mean value ± standard error (n = 3-5). * p <0.05; ** p <0.01. B, after weighing, the biopsy tissue was fixed and subjected to H & E staining for histological examination. Representative pictures are presented. Veh, control.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

animal

Animal care and research protocols were conducted in accordance with the guidelines of the AMOREPACIFIC R & D Center or the Institutional Animal Care and Use Committee of Gwangju Institute of Technology. Mice were purchased from Orient Bio (Seoul, South Korea) and acclimated under specific pathogen-free conditions in animal facilities for at least 1 week prior to experiment. Mice were housed at 23 ± 3 ° C. in a breeding room adjusted to a relative humidity of 40-60%. The light was automatically adjusted to 12 hours of light and 12 hours of dark cycle.

reagent

SFN was purchased from Sigma-Aldrich (St. Louis, Mo.). Purified LPS was purchased from List Biological Laboratory (Campbell, CA) and dissolved in endotoxin-free water. Antibodies to IRAK-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to COX-2 were purchased from Cayman Chemical (Ann Arbor, MI). Unless otherwise noted, all other reagents were purchased from Sigma-Aldrich.

Cell culture

RAW264.7 cells (mouse monocyte cell line, ATCC TIB-71) and 293T cells [human embryonic kidney cell; Dr. Daniel Hwang (provided by the Western Human Nutrition Research Center, USDA) received 10% (v / v) heat-inactivated FBS (HyClone, Logan, UT), 100 U / ml penicillin and 100 mg / ml streptoto Cultured in DMEM containing mycin. As previously described (15), TLR4 (Flag-tagged or GFP-tagged), cluster of differentiation (CD) 14, leukocyte Ag 96 (MD-2; Flag-tagged) and NF-κB luciferase reporter genes were Ba / F3 cells, which express IL-3-dependent mouse pro-B cell lines (provided by Kensuke Miyake, Ph.D., provided by Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan), were cultured. Embryonic fibroblasts were maintained in IMDM (Invitrogen, Carlsbad, Calif.) Containing 10% FBS and antibiotics from 13.5 day mouse embryos of wild type and nrf2- disrupted genotype 16 (17). Cells were maintained at 37 ° C., 5% CO ₂ / air environment.

Plasmid

Frank Mercurio (Signal Pharmaceuticals, San Diego, Calif.) Provided an NF-κB (23) -luciferase reporter construct. Kindly, K. Fitzgerald (University of Massachusetts Medical School, Worcester, Mass.) Provided IFN-β-positive regulatory domain III-I (PRDIII-I) -luciferase reporter plasmid and WT TBK1 expression plasmid. The heat shock protein 70-β-galactosidase reporter plasmid was obtained from R. Modlin (University of California, Los Angeles, CA). Constitutively active chimeric CD4-TLR4 was obtained from CA Janeway, Jr. (Yale University, New Haven, CT). WT TLR4 expression plasmids were obtained from A. Hajjar (University of Washington, Seattle, WA). Wild type MyD88 was obtained from J. Tschopp (University of Lausanne, Lausanne, Switzerland). TRIF expression plasmids were obtained from S. Akira (Osaka University, Osaka, Japan). WT IKKβ was obtained from M. Karin (University of California, San Diego, Calif.). For transfection, all DNA constructs were made on a large scale using EndoFree Plasmid Maxi kit (Qiagen, Chatsworth, Calif.).

Transfection and Luciferase Assays

Luciferase plasmids and expression plasmids of various signaling components were transfected into RAW264.7 or 293T cells using SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. The heat shock protein 70-β-galactosidase reporter plasmid was co-transfected as an internal control. The total amount of transfected plasmids was identified by complementation with the corresponding empty vector. Luciferase and β-galactosidase enzyme activities were determined using a luciferase assay system and a β-galactosidase enzyme system (Promega, Madison, Wis.) According to the manufacturer's instructions. To determine relative luciferase activity, luciferase activity was normalized by β-galactosidase activity.

Immunoblotting

Equal amounts of cell extracts were separated on SDS-PAGE and transferred electrically to polyvinylidene difluoride membranes. Membranes were blotted with primary antibodies and then reacted with HRP-conjugated secondary antibodies (Amersham Biosciences, Arlington Heights, IL). The detected bands were visualized using an increased chemiluminescence system (Amersham Biosciences).

Immune precipitation

Ba / F3 cells stably expressing Flag-TLR4, GFP-TLR4, CD14 and MD-2 and hemagglutinin (HA) -ΔTLR4 and Flag-ΔTLR4 with persistent activity in truncated forms of TLR4 ( Protein extracts were prepared from 293T cells transfected with 18). The supernatant extracted from Ba / F3 lysate and 293T lysate was reacted with monoclonal anti-Flag antibody (Sigma-Aldrich) and anti-HA (Roche, Mannheim, Germany) for 4 hours, and then 70 μl of 50% protein A-Agarose (Amersham Biosciences) was reacted with shaking at 4 ° C. overnight. After washing four times with lysis buffer, immune complexes were solubilized in Laemmli sample solution. Receptive immune complexes were separated by SDS-PAGE. For Ba / F3 cell lysates, membranes were immunoblotted with anti-Flag antibodies and rabbit anti-GFP antibodies, and for 293T cell lysates, membranes were immunoblotted with anti-Flag antibodies and anti-HA antibodies.

TLR4 in-solution cleavage and micro-liquid chromatography-tandem mass spectrometry analysis

The extracellular domain of purified human TLR4 (27-631 amino acids) or mouse TLR4 (26-629 amino acids) was determined using J.-O. Obtained from Lee (Korea Advanced Institute of Science and Technology, Daejeon, Korea; 19, 20), In-solution digestion and micro-liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis Previously, 2 μg of each protein was reacted with SFN (100 μM) at 37 ° C. for one hour. In-solution cleavage was performed with chymotrypsin and trypsin to produce peptides with various cleavage sites. The extracellular domain of SFN-modified TLR4 was digested with cleavage buffer containing chymotrypsin [100 mM Tris-HCl (pH 7.8) and 10 mM CaCl ₂ ; Enzyme to substrate ratio of 1:50] at 37 ° C. for 12 hours. After thermodenatured chymotrypsin, aliquots of chymotrypsin-treated samples were further cleaved with trypsin at 37 ° C. for 12 hours (enzyme to substrate ratio of 1:50). Protein cleavage was finally stopped by adding 90% formic acid (final concentration of 5%). Micro-LC-MS / MS analysis was performed with chymotrypsin-treated samples or chymotrypsin / trypsin-treated samples. MS / MS spectra were investigated using TurboSequest against a homemade protein database containing human and mouse TLR4 sequences. Bioworks version 3.1 was used to filter the findings, and the following Xcorr values and filtering criteria were applied for peptides of different charge states: 1.8 for single charged peptides; 2.5 for a bivalent peptide; For trivalent peptides, 3.5; And 0.08 for ΔCn peptide in all charge states.

Analysis of Serum Cytokines

Female C57BL / 6 mice were administered orally with a control vehicle (PBS) or SFN (25 mg / kg) 24 and 1 hour prior to intraperitoneal injection of LPS (7.5 mg / kg). Two hours after LPS injection, blood was obtained through retro-orbital bleeding and coagulated at room temperature. Serum was separated by centrifugation and stored at −80 ° C. until analysis. The concentrations of cytokines TNF-α, IL-1β and IL-6 were measured using a Quantikine ELISA kit (R & D Systems, Minneapolis, MN) specific for each cytokine according to the manufacturer's instructions.

In vivo skin inflammation experiment

Controls (PBS with 3% DMSO) or SFN (10 and 30 mg) 24 hours and 1 hour before and 6 hours after intradermal injection of LPS (50 μg / 25 μl in PBS) in the center of the right ear of female BALB / c mice / kg) or indomethacin (3 mg / kg) was administered orally. To assess LPS-induced inflammation and edema, 24 hours after injection of LPS, 6-mm raw samples of both ears were obtained and weighed. The untreated left ear was used for standardization of individual variations and the ratio of right ear to left ear was presented as ear swelling values. After weighing, biopsy tissues were fixed and H & E stained for histological examination by histologists unknown to the treatment group.

Statistical analysis

Immunoblot data is presented along with representative figures derived from three or more independent experiments. Other data are expressed as mean ± standard error. Differences between groups were analyzed by the Student t test (significantly assessed when p <0.05).

Experiment result

SFN Inhibits Ligand-Dependent and -Independent Activation of TLR4

To investigate whether SFN regulates TLR4 activation, macrophages (RAW264.7) cells are stimulated with LPS, a representative TLR4 agonist in the presence of SFN, followed by activation of downstream signaling molecules and expression of inflammatory genes. Investigate.

Activation of TLR4 by the ligand causes regression of MyD88, which MyD88 complexes with IRAK-4 and IRAK-1 resulting in phosphorylation and degradation of IRAK-1. Thus, IRAK-1 degradation is one of the readouts for early events of TLR4 activation. SFNs in macrophages blocked IRAK-1 degradation induced by LPS (FIG. 1A). In addition, SFN is LPS-induced, as confirmed by the reporter assay (FIG. 1B-1C) using a luciferase reporter gene comprising an NF-κB binding site or an IRF3 binding site (IFN-β PRDIII-I). The activation of transcription factors (eg, NF-κB and IFN regulatory factor 3 (IRF3)) was inhibited. In addition, LPS-induced COX-2 expression was reduced by SFN (FIG. 1D). The above results showed that SFN inhibits the activation of ligand-induced TLR4.

CD4 replacement of the extracellular domain of TLR4 continued to activate NF-κB without ligand (21) and eliminated the need for ligands to activate intracellular signaling pathways through overexpression of wild type TLR4. Thus, we investigated the activation of TLR4 signaling stimulated with CD4-TLR4 or TLR4 (WT) in the presence of SFN to determine whether SFN can inhibit ligand-independent activation of TLR4. SFN inhibited NF-κB activation induced by overexpression of persistently activated TLR4 (CD4-TLR4) or TLR4 (WT) in 293T cells (FIGS. 2A-2B). In addition, IRF3 activation induced by CD4-TLR4 was reduced by SFN in RAW264.7 cells (FIG. 2C).

From the above results, it was found that SFN can inhibit ligand-dependent and ligand-independent activation of TLR4. Furthermore, the results described above mean that the inhibitory target of SFN may not be the site of interaction between the ligand and the receptor.

SFN inhibits MyD88- and IKKβ-induced activation of downstream signaling but does not inhibit TRIF- and TBK1-induced activation

To identify anti-inflammatory targets of SFN in the TLR4 signaling pathway, we examined downstream signaling molecules affected by SFN. In general, the TLR4 signaling pathway consists of a MyD88-dependent pathway and a TRIF-dependent pathway. The MyD88 pathway of TLR4 leads to the activation of NF-κB through the activation of the IKKβ complex, while the TRIF pathway of TLR4 leads to the activation of IRF3 through the activation of TBK1 / IKKε. To determine whether SFN affects MyD88-dependent or TRIF-dependent pathways, 293T cells were transfected with signaling components fused with the NF-κB reporter gene of the MyD88 pathway and the IRF3 reporter gene of the TRIF pathway, respectively. Sean. SFN blocked the activation of NF-κB induced by overexpression of MyD88 or IKKβ in 293T cells (Figures 3A-3B), indicating that the target of SFN is in the MyD88 pathway. It has been reported that SFN directly inhibits the kinase activity of IKKβ and the DNA binding capacity of NF-κB (2). Therefore, the above results may refer to the inhibitory activity of SFN on the activation of MyD88- or IKKβ-induced NF-κB.

In contrast, SFN did not inhibit the activation of IRF3 induced by overexpression of TRIF (FIG. 3C). Furthermore, SFN did not inhibit the activation of IRF3 induced by TBK1, a kinase present downstream of TRIF (FIG. 3D). The above results mean that the target of the SFN is not TRIF or a downstream signaling component of TRIF. Although SFN did not inhibit TRIF- and TBK1-induced IRF3 activation, SFN could inhibit the activation of TLR4 ligands (eg LPS)-and TLR4 (CD4-TLR4) -induced IFR3 (FIG. 1C and 2C). Thus, the above results indicate that SFN may have other inhibitory targets present upstream of TRIF in addition to IKKβ and NF-κB, for example another inhibitory target of SFN may be a target at the receptor level.

SFN inhibits thiol-dependent oligomerization of TLRs

Receptor oligomerization is one of the initiating steps leading to activation of TLR4 and intracellular signaling pathways (15, 22). Oligomerization of TLR4 provides a cytoplasmic platform for the association of downstream signaling molecules that deliver extracellular stimuli into intracellular signaling events. Therefore, we conducted an immunoprecipitation study using BaF3 cells stably expressing both Flag-TLR4 and GFP-TLR4 to examine how SFN affects oligomerization of TLR4. After LPS stimulation, GFP-TLR4 co-immunoprecipitated with Flag-TLR4, resulting in oligomer formation through association of Flag-TLR4 with GFP-TLR4. SFN attenuated the binding of LPS-induced Flag-TLR4 and GFP-TLR4, meaning that SFN inhibited ligand-induced oligomerization of TLR4 (FIG. 4A). In addition, the inhibitory effect of SFN on NF-κB activation was also observed in the same Ba / F3 cells (FIG. 4B).

In order to further investigate whether SFN inhibits ligand-independent oligomerization of TLR4, we have HA-tagged or flag-tagged truncated TLR4 (ΔTLR4) that can form oligomers with sustained activity and without ligand stimulation. (18) was used for immunoprecipitation assay. SFN inhibited TLR4 oligomerization and NF-κB activation induced by ΔTLR4 (FIGS. 4C-4D). The above results showed that SFN inhibits both ligand-dependent and ligand-independent oligomerization of TLR4. Furthermore, the above results indicate that attenuation of NF-κB activation by SFN results in an inhibitory effect on receptor oligomerization in addition to direct inhibition of NF-κB.

Since it is well known that SFN interacts with the sulfhydryl moiety in the protein (23), we investigated whether SFN interacts with cysteines in the TLR4 protein. SFN is reacted with purified extracellular domain (27-631 amino acids) of mouse TLR4 or extracellular domain of mouse TLR4 (26-629 amino acids) to form adducts using micro-LC-MS / MS analysis Was investigated. SFN is the 88, 192, 246, 281, 340, 506, 542 and 609th cysteine of human TLR4 out of the 16 cysteines present in each extracellular domain and 87, 164, 245, 280, 504, 549 and mouse TLR4; Adducts were formed with the 606th cysteine (Table 1). Of the cysteines described above, the 88, 192, 246, 340, 506 and 542 th cysteine of human TLR4 and the 164, 245, 504 and 549 th cysteine of mouse TLR4 are free cysteines and other cysteines form disulfide bonds. The above results showed that SFN can bind cysteine residues on the extracellular domain of TLR4. We investigated whether inhibition of TLR4 oligomerization by SFN is dependent on reactivity with sulfhydryl groups. Treatment with thiol donors DTT or N-acetyl-L-cysteine (NAC) blocked the inhibitory effect of SFN on TLR4 oligomerization and NF-κB activation (FIG. 5). The above results indicate that the inhibitory effect of SFN on TLR4 activation is due to its reactivity to sulfhydryl moieties. In agreement, another thiol-reactive compound, parthenolide (5 and 10 μM), also significantly reduced LPS-induced TLR4 oligomerization and NF-κB activation (FIGS. 6A-6B). Ethacrynic acid (EA) significantly reduced NF-κB activation at the same concentrations, while weakly attenuating LPS-induced TLR4 oligomerization at relatively high concentrations of 50 and 100 μM (FIGS. 6C-6D). ).

SFN forms an adduct with cysteines present in the TLR4 extracellular domain. sample Combined position Xcorr ΔCn Charge state Human TLR4 Cys88 4.087 0.67 +2 Cys192 3.1873 0.7725 +2 Cys246 4.5803 0.7752 +3 Cys281 3.9585 0.8489 +2 Cys340 3.6012 0.6699 +2 Cys506 3.2717 0.8529 +2 Cys542 2.6685 0.564 +2 Cys609 2.9741 0.6263 +2 Mouse TLR4 Cys87 4.5986 0.704 +3 Cys164 2.9526 0.8235 +2 Cys245 3.0517 0.7491 +2 Cys280 4.8984 0.736 +3 Cys504 3.654 0.6314 +2 Cys549 3.2568 0.7103 +2 Cys606 3.717 0.6236 +2

SFN is well known to activate the keap-1 / Nrf2 pathway leading to antioxidant and phase II detoxifying enzymes. To investigate whether Nrf2 is involved in the inhibitory potency of SFN on TLR4-dependent signaling, we determined TLR4-dependent in mouse embryonic fibroblasts (MEFs) derived from wild-type or Nrf2-knockout (KO) mice. The effect of SFN on signaling was analyzed. Inhibitory effects of SFN on LPS-induced NF-κB activation and Akt phosphorylation were observed to a similar extent in Nrf2-KO MEFs as well as wild-type MEFs (FIGS. 7A-7B). In addition, SFN inhibited to a similar extent TLR4-induced NF-κB activation, which is always active in both wild-type MEFs and Nrf2-KO MEFs (FIG. 7C). The above results indicate that KO of Nrf2 does not block the inhibitory effect of SFN on TLR4-dependent signaling.

Taken together, our results demonstrate that SFN inhibits oligomerization of TLR4 thiol-dependently, leading to a decrease in activation of downstream signaling pathways and target gene expression.

SFN attenuates in vivo the LPS-induced inflammation in an inflammatory animal model

To investigate whether SFN exhibited similar inhibitory effects on in vivo LPS signaling, serum levels of cytokines were measured after LPS injection into mice treated with or without SFN. When mice were treated with LPS, serum levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were markedly increased, whereas increased oral cytokine production was observed with oral administration of SFN to mice. It was greatly reduced (FIG. 8). To once again validate the in vivo inhibitory effect of SFN on LPS signaling, we used an LPS-derived skin inflammation animal model. Skin inflammation was induced by intradermal injection of LPS in the middle of the right ear of the mouse and examined 24 hours after the ear was obtained for evaluation of LPS-induced inflammation and edema. As can be seen from the increased ear weight, LPS injection induced ear edema. However, oral administration of SFN reduced ear edema and weight (FIG. 9A). Histological analysis showed that the ears injected with LPS showed moderate skin irritation and severe edema, while the SFN treated ears showed at least to mild skin irritation and minimal edema (FIG. 9B). The protective effect of SFN against LPS-induced ear skin inflammation was just as strong as that of indomethacin (positive control), a well-known non-steroidal anti-inflammatory drug. Taken together, the blockade of TLR4 signaling by SFN resulted in reduced production of inflammatory cytokines and in vivo reduced skin inflammation and edema in experimental inflammatory animal models.

Additional discussion

SFN has been known to have various intracellular targets to exhibit anticancer or anti-inflammatory activity. The anti-inflammatory activity of SFN has been partly explained by its inhibitory effect on NF-κB activation. SFN inhibited the transcriptional activity of NF-κB, nuclear translocation of p65, degradation of IκBα and IKKβ kinase activity in prostate cancer cell lines (24). Reduced nuclear transfer of NF-κB has been reported in the ventricular and renal medulla of broccoli fed animals (6). SFN inhibited both NF-κB metastasis and IκBα degradation in LPS-stimulated right aortic endothelial cells (ECs) (5), whereas SFN inhibited nuclear transfer of NF-κB and degradation of IκBα in LPS-stimulated macrophages. DNA binding of NF-κB was inhibited without interfering with (2). Although the mechanism of NF-κB inhibition varies by cell / tissue and stimulus, the above results indicate that activation of NF-κB was inhibited by SFN. Nevertheless, our results consistently showed that NF-κB activation induced by LPS, TLR4, MyD88 or IKKβ was attenuated by SFN. Regardless of the various stimuli used in this study, downregulation of NF-κB by SFN was at least partially due to the direct inhibition of IKKβ and NF-κB by the inhibitory effect of SFN on NF-κB activation in TLR4 signaling. That means you can. Interestingly, our results showed that SFN did not inhibit IRF3 activation induced by downstream molecules such as TRIF and TBK1, whereas it inhibited LPS- and TLR4-induced IRF3 activation. The above results indicate that the target of SFN may be present upstream of TRIF in addition to direct inhibition of IKKβ and NF-κB. Because of this, we quickly investigated whether SFN influences receptor oligomerization. Our results demonstrated that SFN inhibits both ligand-induced oligomerization of TLR4 and ligand-independent oligomerization of TLR4, which means that TLR4 oligomerization is a novel anti-inflammatory target of SFN. Receptor oligomerization is one of the initiation steps of TLR activation, which leads to activation of downstream molecules (15, 19, 25). TLR4 interacts with TLR4 to form homodimers (15, 22). TLR2 binds to TLR6 or TLR1 depending on the nature of the ligand to form a heterodimer (26, 27). TLR3 oligomerization requires recognition of dsRNA and subsequent activation of downstream signaling (28, 29). Although TLR monomers may induce NF-κB activation to some extent, oligomerization maximizes the activation of TLRs (22). Forced oligomerization of chimeric TLRs with extracellular domains replaced with CD4 or integrin chains resulted in ligand-independent activation of downstream transcription factors (21, 22). Thus, receptor oligomerization is an important event that causes maximal activation of TLRs. Our results demonstrate that SFN inhibits TLR4 oligomerization to reduce activation of downstream transcription factors and expression of inflammatory proteins. Furthermore, this means that receptor oligomerization may be an effective target to modulate TLR activity and may be an efficient target to reduce the risk of TLR-related inflammatory diseases. Although the regulation of TLR4 oligomerization and its importance require further confirmation in vivo in future studies, our in vivo studies have shown that oral administration of SFN has shown a protective effect against LPS-induced inflammation, This means that the inhibitory effect of SFN on TLR4 oligomerization can disrupt the anti-inflammatory response in vivo .

SFN has sulfhydryl-modifying activity to form thionoacyl adducts through a reaction between thiol in the protein and the isothiocyanate moiety of SFN (23). SFN has been known to temporarily deplete intracellular glutathione levels (5). SFN modulates several cysteine residues in the Keap1 protein to regulate Nrf2 activation (23). According to the LC-MS / MS analysis of the present invention, SFN reacted with cysteine residues in the extracellular domain of TLR4 form an adduct and the thiol donor reverses impairment of SFN-induced receptor oligomerization and NF-κB activation. As such, the inhibitory effect of SFN on TLR4 oligomerization is dependent on its reactivity with thiol groups. This means that the reactivity of SFN to the sulfhydryl moiety is one of the factors contributing to the inhibition of receptor oligomerization and can modulate the activity of TLR4 through modification of the sulfhydryl residues of TLR4. Consistently, other thiol-reactive compounds such as patenolide and ethacrynic acid could also inhibit LPS-induced TLR4 oligomerization and NF-κB activation. In addition, according to the results of previous studies by the inventors, auranofin and acinamaldehyde reacting with thiol groups were also able to inhibit TLR4 oligomerization (30, 31). The efficacy of inhibiting TLR4 oligomerization appears to vary depending on the nature of the compound. In this study, we newly identified that thiol-reactive compounds react with cysteine residues of the TLR4 extracellular domain to form adducts. This means that thiol modification may be a novel strategy for developing anti-inflammatory therapies by modulating TLR4 oligomerization. It is well known that the use of TLR ligands initiates oligomerization of the extracellular domain to initiate regression of the adapter and downstream signal transduction into the intracellular domain (15, 19, 25, 29). Although complete loss of the TLR4 extracellular domain results in a lack of activity (32), truncated TLR4 with some extracellular domains always activated and spontaneously formed oligomers when overexpressed (18, 33). This means that the extracellular domain has important functions in the formation of receptor oligomers and activation of downstream signaling. In addition, when cells are exposed to a chemical, the extracellular domain of the membrane receptor may be the first target to contact the chemical before the chemical encounters the intracellular cytoplasmic compartment. Therefore, the present inventors have concentrated on whether SFN can modify the extracellular domain of TLR4. The results of the inventors revealed that SFN reacts with cysteine residues in the TLR4 extracellular domain to form an adduct. Further studies are needed to determine what the function of cysteine is in the oligomer formation process and whether the modification by SFN is directly linked to the inhibition of TLR4 oligomerization. In addition, it is not possible to exclude the possibility that SFN may modify other domains of TLR4 or other molecules in the receptor complex, and these events contribute to the inhibitory effect of SFN on TLR4 signaling. In experiments with DTT and NAC, we used concentrations where these reducing agents did not affect TLR4 oligomerization and signaling on their own (results not shown). Thus, these reducing agents appear to block the inhibitory effect of SFN by reacting with SFN rather than TLR4. Park et al. (19) found that oligomerization of TLR4 is highly dependent on noncovalent interactions such as hydrogen bonding, hydrophobic and hydrophilic interactions rather than disulfide bonds. LPS binds to large hydrophobic pockets in MD-2 and directly mediates the dimerization of the two TLR4-MD-2 complexes. The hydrophobic R2 lipid chain interacts with two phenylalanines (F440 and F463) and one leucine (L444) of TLR4. Hydrophobic residues of MD-2 (V82, M85, L87, I124 and F126) support the hydrophobic interface. The 3-hydroxy groups of the G123, K125 and R90 of MD-2, and the LPS R2 chain form hydrogen bonds with S416, N417, E439 and Q436 of TLR4, respectively. In addition, two phosphate groups in the LPS also ionic interact with clusters of positively charged residues in TLR4 and MD2, contributing to receptor multimerization. In addition, TLR4 forms direct dimerization by contacting other TLR4 at the central site of the C-terminal domain in the extracellular domain through hydrogen bonding and hydrophobic interactions. As confirmed by LC-MS / MS analysis, modification of TLR4 by SFN causes significant interference on the interaction between LPS, MD-2 and TLR4 dimers, as SFN binds to many cysteine residues in TLR4. This can lead to blocking of oligomerization.

Anti-inflammatory activity of SFN is often explained by activation of the keap-1 / Nrf2 pathway. Lin et al. (34) reported that SFN inhibits LPS-induced expression of inflammatory genes Nrf2-dependently. According to this report, the inhibitory effects of SFN on the mRNAs of TNF-α, IL-1β, COX-2 and iNOS, and proteins of COX-2 and iNOS were blocked in Nrf2-KO mice. However, although the extent of the inhibitory effect of SFN in Nrf2-KO macrophages was very small compared to wild type cells, the inhibitory effect of SFN was still observed on the production of IL-1β, PGE ₂ and nitrite. According to our results, the inhibitory effect of SFN on TLR4 signaling was still observed in wild type MEFs as well as Nrf2-KO MEFs. This means that in addition to Nrf2 activation, other mechanisms function on the anti-inflammatory activity of SFN on TLR4-mediated signaling. Taken together, the results of the present inventors suggest that TLR4 oligomerization is a novel molecular target for the anti-inflammatory activity of SFN. TLR4 is closely associated with various inflammatory conditions, including pneumonia, atherosclerosis, insulin resistance, obesity and cancer. Thus, regulation of TLR4 activity by SFN may exert beneficial effects on the onset of chronic diseases.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (15)

A method for screening a Toll-like receptor 4 (TLR4) signaling inhibitor comprising the following steps:
(a) contacting a test substance with a cell comprising a TLR4 (Toll-like receptor 4) or a TLR4 extracellular domain; And
(b) analyzing whether the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4, and when the test substance covalently binds to a cysteine residue in the extracellular domain of TLR4 Inhibits oligomerization to determine TLR4 signaling inhibitors.
The method of claim 1, wherein the test substance reacts with cysteine residues present in the extracellular domain of TLR4 to form adducts.
The method of claim 1, wherein the cysteine residues of the TLR4 extracellular domain modified by the test substance are 88, 192, 246, 281, 340, 506, 542, and 609th cysteine residues of human TLR4.
The method of claim 1 wherein the test substance is a thiol-reactive compound.
The method of claim 4, wherein the thiol-reactive compound is SFN.
The method of claim 1, wherein the test substance inhibits ligand-dependent and ligand-independent TLR4 oligomerization.
The method of claim 1, wherein the test substance inhibits lipopolysaccharide (LPS)-, TLR4-, MyD88-, and IκB kinase β-induced NF-κB activation.
The method of claim 1, wherein the test substance inhibits LPS- and TLR4-induced interferon regulatory factor 3 (IRF3) activation.
The method of claim 1, wherein the test substance reduces the production of pro-inflammatory cytokines.
10. The method of claim 9, wherein the pro-inflammatory cytokine is tumor necrosis factor-α, interleukin-1β, or IL-6.
The method of claim 1, wherein the test substance is capable of treating an inflammatory disease.
The method of claim 11, wherein the inflammatory disease is asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, allergic dermatitis, Chronic sinusitis, seborrheic dermatitis, gastritis, gout, gout arthritis, ulcers, chronic bronchitis, pneumonia, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid Muscle pain, temporal arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity or viral infection.
A method for screening a potentiator of a toll-like receptor 4 (TLR4) signaling inhibitor comprising the following steps:
(a) contacting a TLR4 signaling inhibitor and a test substance with a cell comprising a Toll-like receptor 4 (TLL4) or TLR4 extracellular domain;
(b) contacting a TLR4 signaling inhibitor with a cell comprising a TLR4 or TLR4 extracellular domain as a control; And
(c) analyzing the extent to which the TLR4 signaling inhibitor covalently binds to a cysteine residue in the extracellular domain of TLR4 in steps (a) and (b), wherein in step (a) the TLR4 signaling inhibitor is If the degree of covalent binding to cysteine residues in the extracellular domain of TLR4 increases, the test substance is considered to be an potentiator of the TLR4 signaling inhibitor.
14. The method of claim 13, wherein said potentiator enhances the efficacy of treating a inflammatory disease of a TLR4 signaling inhibitor.
The method of claim 14, wherein the inflammatory disease is asthma, eczema, psoriasis, allergy, rheumatoid arthritis, psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, allergic dermatitis, Chronic sinusitis, seborrheic dermatitis, gastritis, gout, gout arthritis, ulcers, chronic bronchitis, pneumonia, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, rheumatoid Muscle pain, temporal arteritis, multiple sclerosis, solid cancer, Alzheimer's disease, arteriosclerosis, obesity or viral infection.

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