Inflammasome inhibitor

Abstract

To provide a novel inflammasome inhibitor.SOLUTION: An inflammasome inhibitor comprising, as an active ingredient, an inhibitor of 3-mercaptopyruvate sulfurtransferase.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to inflammasome inhibitors. [Background technology]

[0002] Inflammasomes are protein complexes composed of signal-recognition proteins (NLRP1, NLRP3, NLRP4, and AIM2), the adaptor protein ASC (apoptosis-associated speck-like protein containing caspase recruitment domain), and caspase-1, and are crucial for regulating inflammatory responses. Inflammasomes, caspase-1 is activated by specific stimuli and induces the maturation of inflammatory cytokines, such as IL-1β and IL-18, leading to inflammation (Figure 1). Among inflammasomes, the NLRP3 inflammasome is known to be activated by a variety of exogenous and endogenous stimuli. Excessive activation of the NLRP3 inflammasome has been implicated in the development of a variety of inflammatory diseases, including hyperuricemia, gout, rheumatoid arthritis, and chronic inflammation in diabetes. Therefore, the NLRP3 inflammasome has recently attracted attention as a potential therapeutic target for these diseases, and research to develop its inhibitors is ongoing worldwide (Non-Patent Document 1).

[0003] The inventors previously discovered that cephalosporin antibiotics, which are considered safe medicines with no side effects and are widely used worldwide, strongly suppress the activation of the NLRP3 inflammasome (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2021 / 177332 [Non-patent literature]

[0005] [Non-Patent Document 1] Nature Medicine (2015) 21, 248-255 Summary of the Invention [Problem to be solved by the invention]

[0006] However, since the cephem antibacterial drug of Patent Document 1 has antibacterial activity, there are concerns that long-term administration may result in the emergence of resistant bacteria and disruption of the intestinal flora.

[0007] The problem to be solved by the present invention is to provide an inflammasome inhibitor that does not have antibacterial activity. [Means for solving the problem]

[0008] The present inventors discovered that inhibitors of 3-mercaptopyruvate sulfotransferase specifically inhibit the activation of NLRP3 inflammasome, and thus completed the present invention.

[0009] The present invention encompasses the embodiments described below. Section 1. An inflammasome inhibitor containing an inhibitor of 3-mercaptopyruvate sulfotransferase as an active ingredient. Section 2. Item 2. The inflammasome inhibitor according to Item 1, wherein the 3-mercaptopyruvate sulfotransferase inhibitor comprises a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka] (In the formula, A is an aromatic ring, B is a moiety having a pyrimidone skeleton, and X is a direct bond or -NH-). Section 3. Item 2. The inflammasome inhibitor according to Item 1, wherein the 3-mercaptopyruvate sulfotransferase inhibitor comprises a compound represented by the following formula (II): [ka] Section 4. Item 2. The inflammasome inhibitor according to Item 1, wherein the inflammasome inhibitor comprises an AIM2 inflammasome inhibitor or an NLRP3 inflammasome inhibitor. Section 5. Item 1. An inflammasome inhibitor for use in the treatment of an inflammatory disease. Section 6. A method for inhibiting ASC speck formation in cells, comprising administering an inhibitor of 3-mercaptopyruvate sulfotransferase to cells derived from a non-human animal or to cells derived from a mammal in vitro. Section 7. A method for inhibiting the formation of inflammasomes in cells, comprising administering an inhibitor of 3-mercaptopyruvate sulfotransferase to cells derived from a non-human animal or to cells derived from a mammal in vitro in an amount effective to suppress the formation of ASC specks. Section 8. A method for inhibiting the release and / or production of inflammatory cytokines from cells, comprising administering to the cells an inhibitor of 3-mercaptopyruvate sulfotransferase in an amount effective to inhibit inflammasome formation. Section 9. Item 9. The method for inhibiting according to any one of Items 6 to 8, wherein the 3-mercaptopyruvate sulfotransferase inhibitor comprises a compound represented by the following formula (II): [ka] Section 10. An inflammatory cytokine inhibitor containing an inhibitor of 3-mercaptopyruvate sulfotransferase as an active ingredient. [Effects of the Invention]

[0010] The present invention provides novel inflammasome inhibitors that inhibit inflammasome activation. Because 3-mercaptopyruvate sulfotransferase inhibitors do not affect other immune system signals, they are thought to have fewer side effects than existing anti-inflammatory drugs. Furthermore, 3-mercaptopyruvate sulfotransferase inhibitors are low-molecular-weight compounds with simple structures, offering advantages in terms of ease of production and / or cost. [Brief explanation of the drawings]

[0011] [Figure 1] Schematic diagram of the inflammasome-mediated inflammatory response mechanism. [Figure 2] (A) Experimental schedule for the inhibition of IL-1β release from human monocytes with or without the addition of Alum. (B) Evaluation of IL-1β release by I3MT-3 treatment under Alum stimulation. Sup: culture supernatant, Lys: cell lysate. [Figure 3] Evaluation of IL-1β release by I3MT-3 treatment by ELISA under Alum stimulation. [Figure 4] (A) Experimental schedule for inhibiting IL-1β release from human monocytes with or without the addition of Poly dA:dT. (B) Evaluation of IL-1β release by I3MT-3 treatment with the addition of Poly dA:dT. [Figure 5] Evaluation of IL-1β release by I3MT-3 treatment by ELISA in the presence of Poly dA:dT. [Figure 6] (A) Experimental schedule for the inhibition of IL-1β release from human monocytes with or without gefitinib. (B) Evaluation of NLRP3 inflammasome expression levels by I3MT-3 treatment with gefitinib. [Figure 7] (A) Effect of I3MT-3 on changes in the ASC fraction in the presence of gefitinib. (B) Effect of I3MT-3 on changes in the ASC fraction in the presence of alum. [Figure 8] (A) Experimental schedule for inhibiting ASC speck formation in 293A cells. (B) Effect of I3MT-3 on ASC speck formation. EV: empty vector. [Figure 9]Effect of I3MT-3 on IL-1β release in the presence of pro-caspase-1. [Figure 10] The effect of I3MT-3 on IL-1β release from human monocytes in the presence of Alum. sh Cntl: negative control transfected with non-targeting shRNA control plasmid DNA. sh #1 and sh #2: cells transfected with shRNA targeting different sites of the same gene. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the singular forms "a," "an," and "the" are intended to include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0013] In this specification, the term "comprise" is a concept that encompasses "consist essentially only of" and "consist only of."

[0014] As used herein, "NLRP3 inflammasome" refers to a protein complex consisting of NLRP3, ASC (apoptosis-associated speck-like protein containing caspase recruitment domain), and caspase-1.

[0015] As used herein, the term "AIM2 inflammasome" refers to a protein complex consisting of AIM2, ASC, and caspase-1.

[0016] As used herein, mammals include humans, cows, horses, pigs, monkeys, dogs, cats, mice, rats, rabbits, goats, and sheep, and are preferably humans.

[0017] Hereinafter, embodiments for carrying out the present invention will be described. Note that the embodiment described below shows an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0018] According to a first aspect of the present invention, there is provided an inflammasome inhibitor comprising an inhibitor of 3-mercaptopyruvate sulfotransferase as an active ingredient.

[0019] 3-Mercaptopyruvate sulfurtransferase (3-MST) is one of the active sulfur-producing enzymes, which abstracts a thiol group from 3-mercaptopyruvate to form persulfide (3-MST-SSH). 3-MST inhibitors bind to the reactive S of 3-MST-SSH, preventing the sulfur acceptor from approaching 3-MST and suppressing the generation of active sulfur.

[0020] In some embodiments, the inhibitor of 3-mercaptopyruvate sulfotransferase comprises a compound represented by formula (I): or a pharmaceutically acceptable salt thereof:

[0021] [ka] In the formula, A is an aromatic ring, B is a moiety having a pyrimidone skeleton, and X is a direct bond or —NH—.

[0022] Examples of A include, but are not limited to, an optionally substituted thiophenyl group, an optionally substituted phenyl group, and an optionally substituted naphthyl group. Substituents for the optionally substituted thiophenyl group, the optionally substituted phenyl group, and the optionally substituted naphthyl group include, but are not limited to, an alkyl group having 1 to 6 carbon atoms, a hydroxy group, -C(=O)-NH2, an amino group, etc. The number of substituents is preferably 1, 2, or 3.

[0023] Examples of B include, but are not limited to, groups represented by the following formulae (III-1) and (III-2).

[0024] [ka]

[0025] In formula (III-1), R 1 and R 2 are each independently an alkyl group having 1 to 6 carbon atoms, a hydroxy group, -C(=O)-NH2, or an amino group.

[0026] In formula (III-2), ring D is an optionally substituted thiophenyl group, an optionally substituted phenyl group, or an optionally substituted naphthyl group. Substituents for the optionally substituted thiophenyl group, the optionally substituted phenyl group, and the optionally substituted naphthyl group of ring D include an alkyl group having 1 to 6 carbon atoms, a hydroxy group, -C(=O)-NH2, an amino group, etc. The number of substituents is preferably 1, 2, or 3.

[0027] In some embodiments, the inhibitor of 3-mercaptopyruvate sulfotransferase is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein in formula (I), A is an optionally substituted naphthyl group, X is a direct bond, B is a group represented by formula (III-1), and R 1 and R 2 are each independently an alkyl group having 1 to 6 carbon atoms, a hydroxy group, -C(=O)-NH2, or an amino group.

[0028] In some embodiments, the inhibitor of 3-mercaptopyruvate sulfotransferase comprises a compound represented by formula (II):

[0029] [ka]

[0030] The inflammasome inhibitor according to the first aspect of the present invention may include an AIM2 inflammasome inhibitor or an NLRP3 inflammasome inhibitor.

[0031] A pharmaceutically acceptable salt of a compound represented by formula (I) is a salt having the desired pharmacological activity of a compound represented by formula (I), and refers to a salt prepared from a pharmaceutically acceptable base or acid, including an inorganic base or organic base, and an inorganic acid or organic acid.

[0032] Examples of inorganic bases include alkali metals (e.g., Na, K) and alkaline earth metals (e.g., Ca, Mg). Examples of organic bases include triethylamine, pyridine, etc. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid, etc. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid, etc.

[0033] Pharmaceutically acceptable salts of the compounds of formula (I) may be solvates with organic solvents and / or water.

[0034] The compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be produced by a known method, or can be obtained as a commercially available product.

[0035] The inflammasome inhibitor of the first aspect of the present invention can be used in subjects that are mammals including humans (e.g., humans, cows, horses, pigs, monkeys, dogs, cats, mice, rats, rabbits, goats, sheep, etc.), and is preferably used in humans.

[0036] The dosage form of the inflammasome inhibitor may be, for example, an oral agent, an injection, a suppository, or the like, with an oral agent or an injection being preferred. These dosage forms can be prepared by formulation methods known and commonly used by those skilled in the art. The dosage form can be selected depending on the drug.

[0037] The amount of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to be incorporated into each dosage unit varies depending on the symptoms of the subject to which it is to be administered or on the dosage form, but is generally about 250 to 2000 mg per dosage unit for oral preparations, about 1000 to 6000 mg for injections, and about 250 mg for suppositories. The daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the dosage forms described above varies depending on the symptoms, body weight, age, sex, etc. of the subject and cannot be determined in general, but is usually about 1000 to 6000 mg, preferably 1000 to 2000 mg, per day for an adult (body weight 50 kg), and is preferably administered once or in divided doses about two to three times a day.

[0038] The inflammasome inhibitor of the first aspect of the present invention inhibits inflammasome formation and therefore suppresses the production of inflammatory cytokines in immune cells and / or their release from immune cells. Suppression of the production and / or release of inflammatory cytokines in a subject's immune cells is thought to suppress the inflammatory response and be effective in preventing or treating inflammatory diseases in a subject. Examples of inflammatory cytokines include at least one selected from the group consisting of IL-1α, IL-1β, IL-6, IL-18, IL-33, TNF-α, and HMGB1. In a preferred embodiment, the inflammatory cytokine is at least one selected from the group consisting of IL-1β and IL-18.

[0039] The inflammatory disease may preferably be an inflammatory disease accompanied by increased release of inflammatory cytokines from immune cells due to the formation of the NLRP3 inflammasome, and the immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0040] The inflammatory disease includes at least one selected from the group consisting of gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, pneumoconiosis, cryopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS). These inflammatory diseases are autoinflammatory diseases, and the onset of these diseases is thought to be related to the NLRP3 inflammasome.

[0041] According to a second aspect of the present invention, there is provided a method for inhibiting ASC speck formation in a cell, which comprises administering to the cell an inhibitor of 3-mercaptopyruvate sulfotransferase.

[0042] The 3-mercaptopyruvate sulfotransferase inhibitor may include a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is as described for the inflammasome inhibitor of the first aspect.

[0043] In some embodiments, administering to a cell comprises administering to a cell from a non-human animal.

[0044] In some embodiments, administering to a cell comprises administering to a cell of mammalian origin in vitro.

[0045] In some embodiments, administering to a cell comprises administering to a cell from a mammal in vivo.

[0046] The cells derived from a non-human animal may be cells present in the non-human animal, cells isolated from the non-human animal, or cultured cells. The cells derived from a non-human animal are preferably immune cells derived from the non-human animal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0047] The mammal-derived cells may be cells present in a mammal, cells isolated from a mammal, or cultured cells. The mammal-derived cells are preferably immune cells derived from a mammal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0048] According to a third aspect of the present invention, there is provided a method for inhibiting inflammasome formation in a cell, comprising administering to the cell an inhibitor of 3-mercaptopyruvate sulfotransferase in an amount effective to suppress the formation of ASC specks.

[0049] The 3-mercaptopyruvate sulfotransferase inhibitor may include a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is as described for the inflammasome inhibitor of the first aspect.

[0050] In some embodiments, administering to a cell comprises administering to a cell from a non-human animal.

[0051] In some embodiments, administering to a cell comprises administering to a cell of mammalian origin in vitro.

[0052] In some embodiments, administering to a cell comprises administering to a cell from a mammal in vivo.

[0053] The cells derived from a non-human animal may be cells present in the non-human animal, cells isolated from the non-human animal, or cultured cells. The cells derived from a non-human animal are preferably immune cells derived from the non-human animal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0054] The mammal-derived cells may be cells present in a mammal, cells isolated from a mammal, or cultured cells. The mammal-derived cells are preferably immune cells derived from a mammal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0055] According to a fourth aspect of the present invention, there is provided a method for inhibiting the release and / or production of inflammatory cytokines from cells, comprising administering to the cells an inhibitor of 3-mercaptopyruvate sulfotransferase in an amount effective to inhibit inflammasome formation.

[0056] The 3-mercaptopyruvate sulfotransferase inhibitor may include a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is as described for the inflammasome inhibitor of the first aspect.

[0057] The inflammatory cytokine includes at least one selected from the group consisting of IL-1α, IL-1β, IL-6, IL-18, IL-33, TNF-α, and HMGB 1. In a preferred embodiment, the inflammatory cytokine is at least one selected from the group consisting of IL-1β and IL-18.

[0058] In some embodiments, administering to a cell comprises administering to a cell from a non-human animal.

[0059] In some embodiments, administering to a cell comprises administering to a cell of mammalian origin in vitro.

[0060] In some embodiments, administering to a cell comprises administering to a cell from a mammal in vivo.

[0061] The cells derived from a non-human animal may be cells present in the non-human animal, cells isolated from the non-human animal, or cultured cells. The cells derived from a non-human animal are preferably immune cells derived from the non-human animal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0062] The mammal-derived cells may be cells present in a mammal, cells isolated from a mammal, or cultured cells. The mammal-derived cells are preferably immune cells derived from a mammal. The immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0063] According to a fifth aspect of the present invention, there is provided an inflammatory cytokine inhibitor comprising, as an active ingredient, an inhibitor of 3-mercaptopyruvate sulfotransferase or the inflammasome inhibitor of the first aspect.

[0064] Inhibitors of 3-mercaptopyruvate sulfotransferase may include compounds represented by formula (I) above or pharmaceutically acceptable salts thereof.

[0065] As is clear from the description of the present specification, an inhibitor of 3-mercaptopyruvate sulfotransferase or an inflammasome inhibitor of the first aspect can be used as an active ingredient of an inflammatory cytokine inhibitor that inhibits the release and / or production of inflammatory cytokines.

[0066] The administration target, dosage, and administration form of the inflammatory cytokine inhibitor of the fifth aspect of the present invention can be those described for the inflammasome inhibitor of the first aspect.

[0067] In the second to fourth aspects of the inhibition method and the fifth aspect of the proinflammatory cytokine inhibitor, the 3-mercaptopyruvate sulfotransferase inhibitor preferably comprises a compound represented by the following formula (II): The compound represented by formula (II) is referred to as I3MT-3.

[0068] [ka]

[0069] According to a sixth aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of an inflammatory disease, comprising an effective amount of an inflammasome inhibitor of the first aspect, or a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0070] The inflammatory disease may preferably be an inflammatory disease accompanied by increased release of IL-1β from immune cells due to the formation of NLRP3 inflammasome, and the immune cells are preferably monocytes, macrophages, dendritic cells, or a combination thereof.

[0071] The inflammatory disease may include, for example, at least one selected from the group consisting of gout, pseudogout, rheumatoid arthritis, hyperuricemia, type II diabetes, pneumoconiosis, cryopyrinopathy, and cryopyrin-associated periodic fever syndrome (CAPS).

[0072] The inflammasome inhibitor of the first aspect, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof are as described for the inflammasome inhibitor of the first aspect.

[0073] When the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is contained in a pharmaceutical composition, it is mixed with a pharmaceutical carrier as necessary, and various administration forms can be adopted depending on the purpose of prevention or treatment.

[0074] Pharmaceutical carriers are various organic or inorganic carrier substances commonly used as formulation materials, and are formulated as excipients, binders, disintegrants, lubricants, colorants in solid formulations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, sweeteners, stabilizers, etc. can also be used as needed.

[0075] The dosage form of the pharmaceutical composition may be, for example, an oral agent, an injection agent, a suppository, etc., and the oral agent or injection agent is preferred. These dosage forms can be prepared by formulation methods known and commonly used by those skilled in the art.

[0076] The pharmaceutical composition of the sixth aspect of the present invention can be used in mammalian subjects including humans, and is preferably used in humans.

[0077] The dosage form of the pharmaceutical composition may be, for example, an oral agent, an injection agent, a suppository, etc., and the oral agent or injection agent is preferred. These dosage forms can be prepared by formulation methods known and commonly used by those skilled in the art.

[0078] The amount of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to be incorporated into each dosage unit varies depending on the symptoms of the subject to which it is to be administered or on the dosage form, but is generally about 250 to 2000 mg per dosage unit for oral preparations, about 1000 to 6000 mg for injections, and about 250 mg for suppositories. The daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the dosage forms described above varies depending on the symptoms, body weight, age, sex, etc. of the subject and cannot be determined in general, but is usually about 1000 to 6000 mg, preferably 1000 to 2000 mg, per day for an adult (body weight 50 kg), and is preferably administered once or in divided doses about two to three times a day.

[0079] According to a seventh aspect of the present invention, there is provided use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for producing an inflammasome inhibitor, which may be an AIM2 inflammasome inhibitor or an NLRP3 inflammasome inhibitor.

[0080] In the sixth and seventh aspects above, the details of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the subjects to be administered, the dosage, and the administration form are as described for the inflammasome inhibitor of the first aspect of the present invention.

[0081] According to an eighth aspect of the present invention, there is provided use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a proinflammatory cytokine inhibitor.

[0082] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0083] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or literature procedures in the art. [Example]

[0084] Example 1: Suppression of NLRP3-activating stimulus-dependent inflammatory cytokine release using I3MT-3 (method) THP-1 cells were cultured in RPMI medium supplemented with 10% FBS and differentiated for 3 hours with 12-0-tetradecanoylphorbol 13-acetate (PMA) for this experiment. Purified human monocytes were plated and cultured overnight (O / N) to allow differentiated cells to adhere to the plate. Subsequently, 200 μg / mL of Alum (Imject Alum, Thermo Scientific, an aqueous solution containing aluminum hydroxide and magnesium hydroxide) and I3MT-3 were administered at the specified concentrations. Cells and culture supernatants were collected 4 hours later. The schedule is shown in Figure 2 (A).

[0085] Proteins were extracted from cell culture supernatants by methanol / chloroform precipitation. Briefly, cell-free supernatants were mixed with methanol:chloroform at a ratio of 5:5:1 (cell culture supernatant / methanol / chloroform), the mixture was vortexed, and centrifuged at 15,000 rpm for 15 minutes. The clear upper layer was discarded, and 1000 μl of methanol was added to the middle layer. The mixture was centrifuged at 15,000 rpm for 10 minutes, and the liquid layer was removed. The protein pellet was dried and resuspended in 8 M urea. Cells were lysed in ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, and 1% protease and phosphatase inhibitor mix (Nacalai Tesque). Protein samples extracted from cell culture supernatants and cells were separated by SDS-PAGE and analyzed by Western blotting (WB).

[0086] (result) As shown in Figure 2(B), IL-1β was not released without the addition of Alum (leftmost lane). Addition of Alum resulted in extracellular release of IL-1β, but the addition of I3MT-3 reduced the amount of IL-1β released, indicating that I3MT-3 inhibited IL-1β release.

[0087] As shown in Figure 3, the amount of IL-1β produced by adding Alum was evaluated by ELISA. IL-1β production was confirmed with the addition of Alum, but the amount of IL-1β production decreased in a concentration-dependent manner with the addition of I3MT-3, indicating that Alum inhibited IL-1β production (Student's t-test, NS not significant, *<0.05, **<0.01, ***<0.001).

[0088] Example 2: Suppression of AIM2 activating stimulus-dependent inflammatory cytokine release using I3MT-3 (method) THP-1 cells were cultured in RPMI medium supplemented with 10% FBS and differentiated for 3 hours with 12-0-tetradecanoylphorbol 13-acetate (PMA) for this experiment. Purified human monocytes were plated and cultured overnight (O / N) to allow differentiated cells to adhere to the plate. Subsequently, AIM2 inflammasome stimulators, Poly dA:dT 3 μg / mL and I3MT-3, were administered at the specified concentrations. Cells and culture supernatants were collected 4 hours later. The schedule is shown in Figure 4(A).

[0089] (result) As shown in Figure 4(B), IL-1β was not released without the addition of Poly dA:dT. The addition of Poly dA:dT resulted in extracellular release of IL-1β, but the addition of I3MT-3 reduced the amount of IL-1β released, demonstrating its suppression.

[0090] As shown in Figure 5, the amount of IL-1β produced by adding Poly dA:dT was evaluated by ELISA. IL-1β production was confirmed with the addition of Poly dA:dT, but the amount of IL-1β production decreased in a concentration-dependent manner with the addition of I3MT-3, indicating that IL-1β production was suppressed (Student's t-test, NS not significant, *<0.05, **<0.01, ***<0.001).

[0091] Example 3: Inhibition of inflammasome formation using I3MT-3 (method) The ASC oligomerization (inflammasome formation) assay was performed according to the method described in BioProtoc. 7(10):. doi:10.21769 / BioProtoc.2292 with some minor modifications. Briefly, cells were washed with PBS and harvested in buffer A (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose, 1% protease inhibitor cocktail). Cells were lysed by shearing 10 times through a 27-gauge needle, and the cell lysate was centrifuged at 600 g to remove bulk nuclei and unbroken cells. The resulting supernatant was centrifuged at 17,700 g to pellet ASC oligomers. The pellet was resuspended in CHAPS buffer (20 mM HEPES-KOH (pH 7.5), 5 mM MgCl2, 0.5 mM EGTA, 0.1% CHAPS, 1% protease inhibitor cocktail), reacted with 1.5 mM disuccinimidyl phosphate (DSS) for 30 minutes, and quenched with SDS sample buffer.

[0092] THP-1 cells were cultured in RPMI medium supplemented with 10% FBS and differentiated for 3 hours with 12-0-tetradecanoylphorbol 13-acetate (PMA) for this experiment. Purified human monocytes were plated and cultured overnight (O / N) to allow differentiated cells to adhere to the plate. Subsequently, 20 μM gefitinib and I3MT-3 were administered at the specified concentrations, and the cells and culture supernatants were collected 6 hours later. The schedule is shown in Figure 6(A).

[0093] The effect of I3MT-3 on ASC migration to the insoluble fraction was examined using the same schedule as described above and in Example 1. Cells were lysed in ice-cold lysis buffer containing DISC lysis buffer Tx-1.0 and 1% protease and phosphatase inhibitor mixture (Nacalai Tesque). The cell lysate was centrifuged at 15,000 rpm, and the resulting supernatant was used as the soluble fraction sample. Undisrupted cells were added to DISC lysis buffer Tx-1.0, shaken for 5 minutes, and then centrifuged at 15,000 rpm for 15 minutes. The undisrupted pellet was then lysed in ice-cold lysis buffer containing RIPA 10 and 1% protease and phosphatase inhibitor mixture (Nacalai Tesque). The insoluble fraction sample was then separated by SDS-PAGE and analyzed by Western blotting (WB).

[0094] (result) As shown in Figure 6(B), the NLRP3 inflammasome complex was not formed without the addition of gefitinib. The addition of gefitinib induced the formation of the NLRP3 inflammasome complex, but the addition of I3MT-3 reduced the expression level of the NLRP3 inflammasome.

[0095] As shown in Figures 7(A) and (B), ASC did not transfer to the insoluble fraction without the addition of gefitinib or Alum. The addition of gefitinib and Alum resulted in the transfer of ASC to the insoluble fraction, but the addition of I3MT-3 reduced the amount of ASC in the insoluble fraction.

[0096] Example 4 Inhibition of ASC speck formation using I3MT-3 (method) HEK293A cells were cultured in DMEM medium supplemented with 5% FBS, plated, and cultured overnight (O / N) until the cells adhered to the plate. Then, empty vector (EV) and ASC plasmid were transfected into the cells using pei-Max. 5.5 hours later, I3MT-3 was added at the specified concentration. The overnight cultured cells were then harvested. The schedule is shown in Figure 8(A).

[0097] An ASC oligomerization assay was carried out in the same manner as in Example 3.

[0098] (result) As shown in Figure 8(B), overexpression of ASC in cells resulted in ASC oligomerization. Addition of I3MT-3 attenuated the band indicating ASC oligomerization, indicating that I3MT-3 inhibited the formation of ASC specks.

[0099] Example 5 Inhibition of Caspase-1 Activation with I3MT-3 (method) HEK293A cells were cultured in DMEM medium supplemented with 5% FBS, plated, and cultured overnight (O / N) until the cells adhered to the plate. Then, empty vector, IL-1β precursor (Pro-IL-1β), and caspase-1 precursor (Pro-Caspase-1) plasmids were transfected into the cells using pei-Max. 5.5 hours later, I3MT-3 was added at the specified concentration. The cells were then harvested. The same schedule as in Figure 8(A) was used.

[0100] (result) As shown in Figure 9, expression of the IL-1β precursor alone did not result in IL-1β release. Overexpression of the caspase-1 precursor resulted in IL-1β release, but the amount of IL-1β released was reduced by the addition of I3MT-3.

[0101] Example 6 Suppression of inflammatory cytokine release in 3-MST KD cells using I3MT-3 (method) THP-1 cells were transfected with shRNA to generate 3-MST knockdown (KD) cells. The resulting cells were cultured in RPMI medium supplemented with 10% FBS and differentiated for 3 hours with 12-0-tetradecanoylphorbol 13-acetate (PMA) for this experiment. Purified human monocytes were plated and cultured overnight (O / N) to allow the differentiated cells to adhere to the plate. Subsequently, Alum 200 μg / mL and I3MT-3 were administered at the specified concentrations, and the cells and culture supernatant were collected 4 hours later. The same schedule as in Figure 2(A) was used.

[0102] (result) As shown in Figure 10, IL-1β was not released without the addition of Alum. The addition of Alum resulted in extracellular release of IL-1β, and a similar level of IL-1β was released in 3-MST knockdown cells. Regardless of the knockdown level of 3-MST, the addition of I3MT-3 reduced the amount of IL-1β released, demonstrating its suppression. This result suggests that I3MT-3 can suppress IL-1β release through a mechanism independent of 3-MST function inhibition.

Claims

1. An inflammasome inhibitor containing an inhibitor of 3-mercaptopyruvate sulfotransferase as an active ingredient.

2. The inflammasome inhibitor according to claim 1, wherein the inhibitor of 3-mercaptopyruvate sulfotransferase comprises a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: 【Chemical 1】 (wherein A is an aromatic ring, B is a moiety having a pyrimidone skeleton, and X is a direct bond or -NH-).

3. The inflammasome inhibitor according to claim 1, wherein the inhibitor of 3-mercaptopyruvate sulfotransferase comprises a compound represented by the following formula (II): 【Chemistry 2】

4. The inflammasome inhibitor of claim 1, wherein the inflammasome inhibitor comprises an AIM2 inflammasome inhibitor or an NLRP3 inflammasome inhibitor.

5. The inflammasome inhibitor according to claim 1, which is used for the treatment of an inflammatory disease.

6. A method for inhibiting ASC speck formation in cells, comprising administering an inhibitor of 3-mercaptopyruvate sulfotransferase to cells derived from a non-human animal or to cells derived from a mammal in vitro.

7. A method for inhibiting the formation of inflammasomes in cells, comprising administering an inhibitor of 3-mercaptopyruvate sulfotransferase to cells derived from a non-human animal or to cells derived from a mammal in vitro in an amount effective to suppress the formation of ASC specks.

8. A method for inhibiting the release and / or production of inflammatory cytokines from cells, comprising administering to the cells an inhibitor of 3-mercaptopyruvate sulfotransferase in an amount effective to inhibit inflammasome formation.

9. The method for inhibiting 3-mercaptopyruvate sulfotransferase according to any one of claims 6 to 8, wherein the inhibitor of 3-mercaptopyruvate sulfotransferase comprises a compound represented by the following formula (II): 【Chemistry 3】

10. An inflammatory cytokine inhibitor containing an inhibitor of 3-mercaptopyruvate sulfotransferase as an active ingredient.

Images