Application of ML345 in preparation of medicine for treating inflammatory diseases activated and induced by NLRP3 inflammasome

Abstract

The invention discloses application of ML345 in preparation of a medicine for treating inflammatory diseases activated and induced by NLRP3 inflammasome, and belongs to the technical field of biological medicine. The invention finds that ML345 can inhibit the activation of NLRP3 inflammasome by inhibiting the interaction between endogenous NEK7 and NLRP3, and can effectively treat NLRP3 related inflammatory diseases, such as septicopyemia. The invention finds that the ML345 can effectively inhibit the activation of the NLRP3 inflammasome, and has great significance for developing a new NLRP3 inflammasome inhibitor and preparing a medicine for treating NLRP3 inflammasome related inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the use of ML345 in inhibiting the activation of NLRP3 inflammasome, and the use of ML345 in the preparation of drugs for treating NLRP3 inflammasome-related inflammatory diseases. Background Art

[0002] NLRP3 (NACHT, LRR and PYD domains-containing protein 3) is an important member of the NOD-like pattern recognition receptor family, mainly expressed in the cytoplasm of natural immune cells such as macrophages. When cells receive specific stimuli, NLRP3 can recruit the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) and the effector protein pro-caspase-1, and assemble into a multi-protein complex called inflammasome. The activation of NLRP3 inflammasome promotes the autoproteolysis of pro-caspase-1, generating the enzymatically active caspase-1. Caspase-1 further cleaves the precursor proteins of IL-1β and IL-18 (pro-IL-1β and pro-IL-18), promoting their maturation and secretion, and mediating pyroptosis at the same time.

[0003] The NLRP3 inflammasome plays a key role in innate immune-mediated inflammation, and its abnormal activation is closely related to a variety of major human diseases, such as: sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome and cryopyrin-associated periodic syndrome (CAPS), etc. Therefore, the NLRP3 inflammasome is a key target for the treatment of these inflammatory diseases. However, there is still a lack of intervention drugs that specifically target the NLRP3 inflammasome in clinical practice.

[0004] ML345 is a small molecule inhibitor of insulin degrading enzyme (IDE), and its Chinese name is 5-fluoro-2-[2-(1,4-oxazepan-4-yl)-5-[(1,4-oxazepan-4-yl)dioxo-λ 6 -sulfanyl]phenyl]-2,3-dihydrobenzo[2,1-d][1,2]thiazepin-3-one, and its molecular formula is C21 H 22 FN3O5S2, the chemical structural formula of which is shown in Formula I. ML345 is generated from ultra-high throughput screening (uHTS) activities and has been proven to be a potent small molecule inhibitor of IDE. It inhibits the activity of IDE by targeting the Cys819 site of IDE. ML345 has good cell permeability and chemical stability. The inhibition of IDE by ML345 is reversible and can be reversed by a variety of reducing agents, such as β-mercaptoethanol or dithiothreitol. Since IDE is the main protease responsible for insulin degradation in vivo, ML345 and its subsequent derivatives are considered to be able to enhance insulin signaling and have the potential for the treatment of diabetes. However, the role of ML345 in inflammatory diseases has not been reported. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides the use of ML345 in the preparation of a drug for treating NLRP3 inflammasome activation-induced inflammatory diseases, which can effectively inhibit NLRP3 inflammasome activation and is of great significance for the development of new NLRP3 inflammasome inhibitors and the preparation of drugs for treating NLRP3 inflammasome-related inflammatory diseases.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0007] The use of ML345 or any one or more of its pharmaceutically acceptable salts, stereoisomers, and precursor compounds as an inhibitor of NLRP3 inflammasome activation, the structural formula of which is shown in Formula (I).

[0008]

[0009] Furthermore, ML345 inhibits the activation of NLRP3 inflammasome by inhibiting the interaction between endogenous NEK7 and NLRP3.

[0010] Furthermore, ML345 can inhibit the maturation and secretion of IL-1β and IL-18, as well as the autoproteolytic maturation of caspase-1.

[0011] Furthermore, the agonists that induce NLRP3 inflammasome activation include: Nigericin, ATP, MSU, and / or LPS.

[0012] An inhibitor of NLRP3 inflammasome activation, which comprises any one or more of the above-mentioned ML345 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds.

[0013] Use of any one or more of the above-mentioned ML345 or its pharmaceutically acceptable salts, stereoisomers, precursor compounds, or NLRP3 inflammasome activation inhibitors in the preparation of a drug for inhibiting NLRP3 inflammasome activation.

[0014] A drug for inhibiting NLRP3 inflammasome activation, which comprises any one or more of the above-mentioned ML345 or its pharmaceutically acceptable salts, stereoisomers, precursor compounds, or NLRP3 inflammasome activation inhibitors.

[0015] Use of any one or more of the above-mentioned ML345 or its pharmaceutically acceptable salts, stereoisomers, precursor compounds, NLRP3 inflammasome activation inhibitors, or drugs for inhibiting NLRP3 inflammasome activation in the preparation of a drug for preventing and / or treating NLRP3 inflammasome activation-induced inflammatory diseases.

[0016] Furthermore, the NLRP3 inflammasome activation-induced inflammatory disease is any one of hereditary NLRP3-dependent autoinflammatory diseases, NLRP3-induced metabolic diseases, diseases caused by crystal and protein aggregation, acute tissue injury, and chronic inflammation.

[0017] Furthermore, the NLRP3 inflammasome activation-induced inflammatory diseases include sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome, and cryopyrin-associated periodic syndrome (CAPS).

[0018] Furthermore, the NLRP3 inflammasome activation-induced inflammatory disease is sepsis, more preferably lipopolysaccharide (LPS)-induced sepsis.

[0019] A drug for preventing and / or treating NLRP3 inflammasome activation-induced inflammatory diseases, which comprises any one or more of the above-mentioned ML345 or its pharmaceutically acceptable salts, stereoisomers, precursor compounds, NLRP3 inflammasome activation inhibitors, or drugs for inhibiting NLRP3 inflammasome activation;

[0020] And its pharmaceutically acceptable adjuvants.

[0021] Furthermore, the drug is an injection, granule, tablet, capsule, or pill.

[0022] A pharmaceutical composition, which comprises ML345 or its active metabolite, supplemented with a pharmaceutically acceptable carrier, for the treatment of NLRP3 inflammasome-related inflammatory diseases.

[0023] The above NLRP3 inflammasome-related inflammatory diseases include sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome, and cold urticaria-associated periodic syndrome (CAPS), etc.;

[0024] Preferably, the NLRP3 inflammasome-related inflammatory disease is sepsis, more preferably lipopolysaccharide (LPS)-induced sepsis.

[0025] A preparation, which comprises the pharmaceutical composition defined above, and optionally a container and an instruction manual, for the treatment of NLRP3 inflammasome-related inflammatory diseases;

[0026] The above NLRP3 inflammasome-related inflammatory diseases include sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome, and cold urticaria-associated periodic syndrome (CAPS), etc.;

[0027] Preferably, the NLRP3 inflammasome-related inflammatory disease is sepsis, more preferably lipopolysaccharide (LPS)-induced sepsis.

[0028] Advantages of the present invention:

[0029] 1. The small molecule ML345 provided by the present invention can efficiently inhibit the activation of NLRP3 inflammasome in murine BMDM cells at a relatively low concentration (nanomolar), inhibit the autoproteolytic activation of caspase-1, and inhibit the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18. ML345 specifically inhibits the activation of NLRP3 inflammasome without affecting the activation of other inflammasomes. ML345 inhibits the assembly of NLRP3 inflammasome by inhibiting the interaction between NEK7 and NLRP3, thereby inhibiting the activation of NLRP3 inflammasome. More importantly, ML345 has a good therapeutic effect on LPS-induced sepsis. Therefore, ML345 plays an important role in inhibiting the activation of NLRP3 inflammasome and in the preparation of drugs for treating NLRP3 inflammasome-related inflammatory diseases.

[0030] 2. The present invention discovers that ML345 has a good therapeutic effect on LPS-induced murine sepsis model. Therefore, ML345 has the potential to treat NLRP3 inflammasome-related inflammatory diseases (including sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome and cryopyrin-associated periodic syndromes (CAPS), etc.) clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It shows the effect of ML345 in inhibiting the activation of NLRP3 inflammasome induced by Nigericin in Example 1 of the present invention; wherein, Figure 1 A shows the secretion of IL-1β, Figure 1 B shows the secretion of IL-18, Figure 1 C shows the mature forms of IL-1β and caspase-1 (P20).

[0032] Figure 2 It shows the effect of ML345 in inhibiting the activation of NLRP3 inflammasome induced by multiple agonists in Example 1 of the present invention; wherein, Figure 2 A shows the secretion of IL-1β, Figure 2 B shows the mature forms of IL-1β and caspase-1.

[0033] Figure 3 It shows the effect of ML345 in inhibiting the activation of non-classical NLRP3 inflammasome induced by LPS in Example 1 of the present invention; wherein,Figure 3 A shows the secretion of IL-1β, Figure 3 B shows the status of mature IL-1β and caspase-1.

[0034] Figure 4 It shows the effect that in Example 2 of the present invention, ML345 does not affect the activation of AIM2 inflammasome induced by PolyA:T and the activation of IPAF inflammasome induced by Salmonella; wherein, Figure 4 A shows the secretion of IL-1β, Figure 4 B shows the status of mature IL-1β and caspase-1.

[0035] Figure 5 It shows the effect that in Example 3 of the present invention, ML345 does not affect the activation of NF-κB signaling pathway induced by LPS during the priming stage of NLRP3 inflammasome activation; wherein, Figure 5 A shows the secretion of IL-6, Figure 5 B shows the secretion of TNF-α.

[0036] Figure 6 It shows the effect that in Example 3 of the present invention, ML345 does not affect the up-regulation of NLRP3 inflammasome component protein expression induced by LPS.

[0037] Figure 7 It shows the effect that in Example 4 of the present invention, ML345 inhibits NLRP3 inflammasome activation independent of its IDE protein inhibitor function; wherein, Figure 7 A shows the secretion of IL-1β, Figure 7 B shows the status of mature IL-1β and caspase-1.

[0038] Figure 8 It shows the effect that in Example 5 of the present invention, ML345 inhibits NLRP3 inflammasome assembly by inhibiting the interaction between NEK7 and NLRP3; wherein, Figure 8 A shows the interaction between endogenous NEK7 and NLRP3, Figure 8 B shows the interaction between exogenous NEK7 and NLRP3.

[0039] Figure 9 It shows the effect that in Example 6 of the present invention, ML345 inhibits LPS-induced sepsis in mice; wherein, Figure 9 A shows the survival rate of mice, Figure 9 B shows the secretion of IL-1β in mouse serum, Figure 9 C shows the secretion of IL-18 in mouse serum. Detailed implementation manners

[0040] The specific embodiments of the present invention will be described below to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0041] The "pharmaceutically acceptable carrier" described in the present invention specifically refers to other components in a pharmaceutical preparation except the active ingredient, and these components do not produce toxic effects on the subject. Specifically, such carriers include, but are not limited to, conventional pharmaceutical excipients such as buffers, excipients, stabilizers, and preservatives.

[0042] The "activation of NLRP3 inflammasome" described in the present invention means that under the action of NLRP3 agonists (such as LPS + Nigericin), NLRP3, ASC, and pro-caspase1 assemble to form a multi-protein complex, and then pro-caspase1 forms biologically active caspase1 through self-cleavage. Caspase1 further cleaves pro-IL-1β and pro-IL-18 to promote the maturation and secretion of IL-1β and IL-18.

[0043] The activation of NLRP3 inflammasome can be divided into two pathways: classical and non-classical. Among them, the classical activation pathway includes two key steps: the first stage is pre-stimulated with LPS, and the second stage is activated with agonists such as Nigericin. The non-classical activation pathway includes: the first stage is pre-stimulated with Pam3CSK4, and the second stage is activated by intracellular transport of LPS.

[0044] The pre-stimulatory signal in the process of NLRP3 inflammasome activation refers to that NF-κB pathway agonists (such as LPS) induce the secretion of inflammatory factors IL-6 and TNF-α by activating the NF-κB pathway, and at the same time induce the expression of NLRP3 and pro-IL-1β.

[0045] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0046] Sources of materials and reagents used in the examples:

[0047] C57BL / 6 wild-type mice: SPF (Beijing) Biotechnology Co., Ltd.

[0048] BMDM cells (mouse bone marrow-derived macrophages): Isolated and cultured from the bone marrow of C57BL / 6 wild-type mice.

[0049] Mouse M-CSF: Novoprotein, CB34.

[0050] HEK293T: ATCC (American Type Culture Collection)

[0051] Pam3CSK4: Sigma, 112208-00-1.

[0052] LPS: Invitrogen, tlrl-peklps.

[0053] DMSO: Sangon Biotech, A100231.

[0054] ML345: MCE, HY-117878.

[0055] Nigericin: Sigma, N7143.

[0056] MSU: Sigma, U0881.

[0057] ATP: Sigma, A2383.

[0058] PolyA:T: Sigma, P0883.

[0059] Anti-NLRP3 antibody: AdipoGen, AG-20B-0014.

[0060] Anti-mouse IL-1β antibody: R&D, AF-401-NA.

[0061] Anti-mouse caspase-1 antibody: AdipoGen, AG-20B-0042.

[0062] Anti-NEK7 antibody: Abcam, ab133514.

[0063] Anti-β-actin antibody: Proteintech Group, 66009-1-Ig.

[0064] Anti-ASC antibody: Cell Signaling Technology, 67824.

[0065] Anti-Flag antibody: Sigma, F2555.

[0066] Anti-VSV antibody: Sigma, V4888.

[0067] Anti-Flag beads: Sigma, A2220.

[0068] Protein G beads: Millipore, 16-266.

[0069] Transfection reagent Lipo2000: Invitrogen, 11668027.

[0070] Transfection reagent Pei: Polysciences, 23966.

[0071] 6bK: A small molecule inhibitor of IDE protein.

[0072] Example 1 Inhibition of NLRP3 inflammasome activation by ML345

[0073] 1. Differentiation and culture of BMDM cells: Prepare female CBA / J mice at 8-10 weeks of age, sacrifice the mice, and cut off the hind leg bones of the mice. Isolate the bone marrow cells from the hind leg bones and lyse them thoroughly in erythrocyte lysate to remove erythrocytes. Culture in DMEM medium containing 20 ng / mL M-CSF for 4-6 days.

[0074] 2. Transfer the differentiated BMDM cells into a 12-well plate. The next day, remove the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS. After 3 h, add different concentrations of ML345 (0 μM, 0.25 μM, 0.5 μM, 1 μM) and treat for 0.5 h, then add 3 μM Nigericin and stimulate for 0.5 h. Collect the cell supernatant and cell lysate, and detect the level of IL-1β in the cell supernatant by ELISA. The results are shown in Figure 1 A; Detect the level of IL-18 in the cell supernatant by ELISA. The results are shown in Figure 1 B; Detect the levels of mature IL-1β and caspase-1 by Western blot. The results are shown in Figure 1 C.

[0075] As Figure 1 shown, ML345 can efficiently inhibit Nigericin-induced NLRP3 inflammasome activation in vitro, inhibit the maturation and secretion of IL-1β and IL-18, and the autoproteolytic maturation of caspase-1, and shows a dose-dependent manner.

[0076] 3. Transfer the differentiated BMDM cells into a 12-well plate. The next day, remove the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS. After 3 h, add different concentrations of ML345 (0 μM, 1 μM) and incubate for 0.5 h, then add 3 μM Nigericin and stimulate for 0.5 h; or add 2.5 mM ATP and stimulate for 0.5 h; or add 150 μg / mL MSU and stimulate for 4 h. Collect the cell supernatant and cell lysate, and detect the level of IL-1β in the cell supernatant by ELISA. The results are shown as Figure 2 shown in Figure 2 A; Detect the levels of mature IL-1β and caspase-1 by Western blot. The results are shown as

[0077] shown in Figure 2 shown in. ML345 can efficiently inhibit the activation of NLRP3 inflammasome induced by various agonists in vitro, inhibit the maturation and secretion of IL-1β, and the autoproteolytic maturation of caspase-1.

[0078] 4. Transfer the differentiated BMDM cells into a 12-well plate. The next day, remove the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 200 ng / mL Pam3CSK4. After 3 h, add different concentrations of ML345 (0 μM, 0.25 μM, 0.5 μM, 1 μM) and incubate for 0.5 h, then transfect the cells with 1 μg LPS using Lipo2000 for 16 h. Collect the cell supernatant and cell lysate, and detect the level of IL-1β in the cell supernatant by ELISA. The results are shown as Figure 3 shown in Figure 3 A; Detect the levels of mature IL-1β and caspase-1 by Western blot. The results are shown as

[0079] shown in Figure 3 shown in. ML345 can efficiently inhibit the activation of non-classical NLRP3 inflammasome induced by LPS in vitro, inhibit the maturation and secretion of IL-1β, and the autoproteolytic maturation of caspase-1.

[0080] Based on Figures 1 to 3 the detection results, it can be known that ML345 can efficiently inhibit the activation of NLRP3 inflammasome in vitro.

[0081] Example 2 ML345 specifically inhibits the activation of NLRP3 inflammasome

[0082] 1. Differentiation and culture of BMDM cells: The same as in Example 1.

[0083] 2. Transfer the differentiated BMDM cells into 12-well plates. The next day, discard the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS. After 3 h, add different concentrations of ML345 (0 μM, 1 μM) and incubate for 0.5 h, then add 3 μM Nigericin and stimulate for 0.5 h; or transfect 0.5 μg / mL Poly A:T using Lipo2000 and stimulate for 4 h; or add Salmonella (multiplicity of infection (MOI) = 10) and stimulate for 4 h. Collect the cell supernatant and cell lysate, and detect the level of IL-1β in the cell supernatant by ELISA. The results are as Figure 4 shown in Figure 4 A; Detect the levels of mature IL-1β and caspase-1 by Western blot. The results are as

[0084] shown in Figure 4 B. As Figure 4 shown, ML345 can effectively inhibit the activation of NLRP3 inflammasome induced by Nigericin in vitro, inhibit the maturation and secretion of IL-1β, and the autoproteolytic maturation of caspase-1, but does not affect the activation of AIM2 inflammasome induced by Poly A:T and the activation of IPAF inflammasome induced by Salmonella. The results indicate that ML345 does not affect the activation of AIM2 inflammasome and IPAF inflammasome, that is, the inhibitory effect of ML345 on NLRP3 inflammasome activation in vitro is specific.

[0085] Example 3 Effect of ML345 on the priming signal in the process of NLRP3 inflammasome activation

[0086] 1. Differentiation and culture of BMDM cells: The same as in Example 1.

[0087] 2. Transfer the differentiated BMDM cells into 12-well plates. The next day, discard the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS, and incubate for 3 h. Meanwhile, add different concentrations of ML345 (0 μM, 0.25 μM, 0.5 μM, 1 μM) before and after LPS treatment respectively, and incubate for 0.5 h. Collect the cell supernatant and cell lysate, and detect the level of IL-6 in the cell supernatant by ELISA. The results are as Figure 5 shown in Figure 5 A; Detect the level of TNF-α in the cell supernatant by ELISA. The results are as Figure 5 shown in Figure 6 B; Detect the levels of NLRP3 inflammasome component proteins (NLRP3, pro-IL-1β, pro-caspase-1, ASC, and NEK7) by Western blot. The results are as Figure 6 shown.

[0088] As Figure 5 shown, whether ML345 is added before or after LPS treatment, ML345 does not affect the secretion of IL-6 and TNF-α induced by LPS. Similarly, whether ML345 is added before or after LPS treatment, ML345 does not affect the expression of NLRP3, pro-IL-1β, pro-caspase-1, ASC, and NEK7 ( Figure 6 ).

[0089] The above detection results indicate that ML345 does not affect the priming signal in the process of NLRP3 inflammasome activation, that is, the inhibitory effect of ML345 on NLRP3 inflammasome activation is not achieved by inhibiting the priming signal.

[0090] Example 4 ML345 inhibits NLRP3 inflammasome activation independently of its IDE protein inhibitor function

[0091] 1. Differentiation and culture of BMDM cells: The same as in Example 1.

[0092] 2. Transfer the differentiated BMDM cells into a 12-well plate. The next day, remove the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS. After 3 h, add different concentrations of ML345 (0 μM, 1 μM) and treat for 0.5 h, or add different concentrations of 6bK (100 μM, 200 μM) and treat for 0.5 h, and then add 3 μM Nigericin and stimulate for 0.5 h. Collect the cell supernatant and cell lysate, and detect the level of IL-1β in the cell supernatant by ELISA. The results are as Figure 7 shown in Figure 7 A; Detect the levels of mature IL-1β and caspase-1 by Western blot. The results are as

[0093] As Figure 7 shown, ML345 can effectively inhibit Nigericin-induced NLRP3 inflammasome activation in vitro, inhibit the maturation and secretion of IL-1β, and the autoproteolytic maturation of caspase-1. However, 6bK does not affect Nigericin-induced NLRP3 inflammasome activation. This result indicates that the inhibitory effect of ML345 on NLRP3 inflammasome activation is not achieved by inhibiting the function of IDE protein.

[0094] Example 5 ML345 inhibits the interaction between NEK7 and NLRP3

[0095] 1. Differentiation and culture of BMDM cells: The same as in Example 1.

[0096] 2. Transfer the differentiated BMDM cells into a 6-well plate. The next day, remove the culture supernatant and replace it with 500 μL of Opti-MEM medium containing 1% fetal bovine serum and 50 ng / mL LPS. After 3 h, add different concentrations of ML345 (0 μM, 0.25 μM, 1 μM) and treat for 0.5 h, then add 3 μM Nigericin and stimulate for 0.5 h. Remove the supernatant, add 1 mL of PBS, and repeat 3 times to thoroughly wash the cells. After the washing is completed, add 300 μL of NP-40. After sufficient lysis, take 50 μL of the lysate as the cell lysate sample, and mix the remaining 250 μL of the lysate with an appropriate amount of Protein G magnetic beads and anti-NEK7 antibody, and incubate overnight with rotation on a rotary shaker at 4 °C. After the incubation is completed, wash the magnetic beads 5 times with NP-40. After the washing is completed, detect the levels of NEK7 and NLRP3 by Western blot, and the results are as Figure 8 shown in

[0097] 3. Seed 293T cells into a 6-well plate, and use the transfection reagent Pei to transfect Flag-NEK7 and VSV-NLRP3 plasmids. After 8 h, add different concentrations of ML345 (0 μM, 0.25 μM, 1 μM), and continue to culture for 16 h. Remove the supernatant, add 300 μL of NP-40. After sufficient lysis, take 50 μL of the lysate as the cell lysate sample, and mix the remaining 250 μL of the lysate with an appropriate amount of anti-Flag magnetic beads, and incubate overnight with rotation on a rotary shaker at 4 °C. After the incubation is completed, wash the magnetic beads 5 times with NP-40. After the washing is completed, detect the levels of Flag-NEK7 and VSV-NLRP3 by Western blot, and the results are as Figure 8 shown in

[0098] As Figure 8 shown, ML345 can effectively inhibit the interaction between endogenous NEK7 and NLRP3, as well as the interaction between exogenous NEK7 and NLRP3. That is to say, ML345 inhibits NLRP3 inflammasome activation by inhibiting the interaction between NEK7 and NLRP3.

[0099] Example 6 Inhibition of LPS-induced sepsis in mice by ML345

[0100] Prepare 10-week-old adult male mice and divide them into two groups. Intraperitoneally inject the same volume of vehicle solvent or ML345 (10 mg / kg) into the mice. After 30 minutes, intraperitoneally inject LPS (20 mg / kg) into the mice. For one group, record the survival rate of the mice, and the results are as Figure 9 shown in shown in A. For the other group, 6 h after LPS injection, collect blood by eye puncture and sacrifice the mice, and detect the level of IL-1β in the serum by ELISA, and the results are asFigure 9 as shown in B; the level of IL-18 in serum was detected by ELISA, and the results are as Figure 9 shown in C.

[0101] As Figure 9 shown, in the LPS-induced sepsis model, ML345 could effectively improve the survival rate of mice and reduce the levels of inflammatory factors IL-1β and IL-18. It is indicated that ML345 can be used to treat NLRP3-related inflammatory diseases. Therefore, ML345 has the potential to prepare drugs for treating NLRP3 inflammasome-related inflammatory diseases.

[0102] According to the above detection results of the embodiments of the present invention, ML345 can efficiently inhibit the activation of the classical NLRP3 inflammasome induced by Nigericin, ATP, and MSU in vitro, and inhibit the activation of the non-classical NLRP3 inflammasome induced by intracellular LPS. Moreover, ML345 can specifically inhibit the activation of the NLRP3 inflammasome in vitro without affecting the activation of the AIM2 inflammasome and the IPAF inflammasome. In addition, the inhibition of NLRP3 inflammasome activation by ML345 does not depend on its function as an IDE protein inhibitor. At the same time, the inhibitory effect of ML345 on NLRP3 inflammasome activation is not achieved by inhibiting the pre-stimulatory signal, but by inhibiting the interaction between NEK7 and NLRP3 to inhibit the assembly and activation of the NLRP3 inflammasome complex. In addition, ML345 can effectively treat LPS-induced sepsis.

[0103] These results further indicate that ML345 has strong NLRP3 inflammasome inhibitory activity, can be used to treat NLRP3-related inflammatory diseases, and provides a new theoretical basis for preparing drugs for treating NLRP3 inflammasome-related inflammatory diseases.

[0104] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of any one or more of ML345 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds as an inhibitor of NLRP3 inflammasome activation.

2. The use according to claim 1, wherein The ML345 inhibits NLRP3 inflammasome activation by inhibiting the interaction between endogenous NEK7 and NLRP3.

3. An NLRP3 inflammasome activation inhibitor, characterized in that, It includes any one or more of ML345 described in claim 1 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds.

4. Use of any one or more of ML345 described in claim 1 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds, or the NLRP3 inflammasome activation inhibitor described in claim 3 in the preparation of a drug for inhibiting NLRP3 inflammasome activation.

5. A drug for inhibiting the activation of NLRP3 inflammasome, characterized in that, It includes any one or more of ML345 described in claim 1 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds, or the NLRP3 inflammasome activation inhibitor described in claim 3.

6. Use of any one or more of ML345 described in claim 1 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds, the NLRP3 inflammasome activation inhibitor described in claim 3, or the drug for inhibiting NLRP3 inflammasome activation described in claim 5 in the preparation of a drug for preventing and / or treating inflammatory diseases induced by NLRP3 inflammasome activation.

7. The use according to claim 6, wherein The inflammatory diseases induced by NLRP3 inflammasome activation are any one of hereditary NLRP3-dependent autoinflammatory diseases, NLRP3-induced metabolic diseases, diseases caused by crystal and protein aggregation, acute tissue injury, and chronic inflammation.

8. The use according to claim 7, wherein The inflammatory diseases induced by NLRP3 inflammasome activation include sepsis, atherosclerosis, contact hypersensitivity, hepatitis, pneumonia, asbestosis, depression, peritonitis, amyotrophic lateral sclerosis, Behcet's disease, silicosis, silicosis, Alzheimer's disease, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis, asthma or acute respiratory distress syndrome, obesity, gout, type II diabetes, Parkinson's disease, arthritis, familial cold autoinflammatory syndrome, myocardial infarction, ultraviolet-induced skin sunburn, Muckle-Wells syndrome, and cryopyrin-associated periodic syndrome.

9. A drug for preventing and / or treating inflammatory diseases induced by NLRP3 inflammasome activation, characterized in that, It includes any one or more of ML345 described in claim 1 or its pharmaceutically acceptable salts, stereoisomers, and precursor compounds, the NLRP3 inflammasome activation inhibitor described in claim 3, or the drug for inhibiting NLRP3 inflammasome activation described in claim 5; and its pharmaceutically acceptable adjuvants.

10. The drug according to claim 9, characterized in that, The drug is an injection, granule, tablet, capsule, or pill.

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