Application of Roxitinib in preparation of NLRP3 inflammasome inhibitor

Abstract

The invention discloses application of Roxitinib in preparation of an NLRP3 inflammasome inhibitor, and belongs to the technical field of biological medicines. The invention specifically provides any one of the following applications of Rociletinib: 1, an application of Rociletinib for non-treatment purpose in in-vitro inhibition of activation of NLRP3 inflammasomes, and 2, an application of Rociletinib for non-treatment purpose in vitro inhibition of activation of NLRP3 inflammasomes; 2, application in preparation of an anti-inflammatory drug targeting NLRP3; 3, application in preparation of drugs for prevention and / or treatment of NLRP3 inflammasome abnormal activation related diseases; 4, application in preparation of drugs for inhibiting interaction of NLRP3 and NEK7; 5, application in preparation of drugs for inhibiting secretion of inflammatory cytokines IL-1beta; and 6, application in preparation of drugs for inhibiting pyroptosis caused by Gasdermin D protein cleavage. The invention has the beneficial effects that a new medical application of the rocetinib is explored, and a new application field is opened up. By establishing an NLRP3 inflammasome related disease model, it is found that the rocetinib also has a good effect of inhibiting activation of the NLRP3 inflammasome in vivo. Good application prospects are realized.

Description

Technical Field

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

[0002] As the first line of defense for the human body, innate immunity is centered on the precise recognition of various pathogenic microorganisms, environmental irritants and endogenous danger signals by innate immune receptors. Among them, NLRP3 (a protein containing LRR and pyrin domains 3) is a key innate immune receptor that can not only recognize pathogen-associated molecular patterns (PAMPs), but also recognize a variety of damage-associated molecular patterns, thereby triggering the formation of a multi-protein complex - inflammasome, which regulates a wide range of inflammatory responses.

[0003] The NLRP3 inflammasome is composed of the NLRP3 sensor protein, the apoptosis-associated speck-like protein ASC (containing the caspase recruitment domain), and the effector protease caspase-1. The addition of the NIMA-related kinase NEK7 further promotes the assembly of the inflammasome. When stimulated by damage-associated molecular patterns (DAMPs) or PAMPs, a series of complex upstream signals are activated, including changes in ion flow, lysosomal and organelle damage, and mitochondrial reactive oxygen species (mtROS) generation, all of which promote the interaction between NLRP3 and NEK7 and the assembly of the inflammasome. Activated caspase-1 not only catalyzes the maturation and secretion of inflammatory factors such as IL-1β and IL-18, but also mediates the cleavage of gas protein D (GSDMD) and initiates cell pyroptosis. However, when the activation of the NLRP3 inflammasome is out of control, excessive inflammatory responses may induce a variety of inflammatory diseases including sepsis, gout, neurodegenerative diseases, atherosclerosis, and type 2 diabetes. Therefore, pharmacological inhibition strategies targeting the NLRP3 inflammasome are considered to be a potential effective means of treating the above inflammatory diseases.

[0004] Rociletinib (ROC) is an oral third-generation EGFR kinase inhibitor with the following chemical structure: It is composed of a piperazine ring, an aminopyrimidine group, and an active acrylamide group. This drug is used in the Phase III clinical trial to study and treat patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC). It forms a covalent bond with the cysteine ​​797 residue of EGFR through its acrylamide group, thereby irreversibly inhibiting the activity of EGFR. However, the specific pharmacological effects of ROC in the NLRP3 inflammasome and inflammatory diseases are still unknown, and its exact target for the NLRP3 inflammasome has not yet been revealed. Therefore, exploring the role of ROC in the activation of the NLRP3 inflammasome is of great significance for the treatment of NLRP3 inflammasome-related inflammatory diseases and the preparation of drugs for the treatment of related diseases. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to solve the problem of the current lack of drugs that can effectively inhibit NLRP3 inflammasomes.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention proposes any of the following uses of Rociletinib:

[0008] (1) Application for non-therapeutic purposes in inhibiting NLRP3 inflammasome activation in vitro;

[0009] (2) Application in the preparation of anti-inflammatory drugs targeting NLRP3;

[0010] (3) Use in the preparation of drugs for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasomes;

[0011] (4) Application in the preparation of drugs for inhibiting the interaction between NLRP3 and NEK7;

[0012] (5) Application in the preparation of drugs for inhibiting the secretion of inflammatory cytokine IL-1β;

[0013] (6) Application in the preparation of drugs for inhibiting gasdermin D protein cleavage-induced cell pyroptosis.

[0014] Preferably, the diseases associated with abnormal activation of NLRP3 inflammasomes in (3) include any one or more of hereditary NLRP3-dependent spontaneous inflammatory diseases, metabolic diseases caused by abnormal activation of NLRP3 inflammasomes, neuroinflammatory diseases, arthritis, acute tissue damage, and chronic inflammation.

[0015] Preferably, the diseases associated with abnormal activation of NLRP3 inflammasome described in (3) include any one or more of acute inflammatory diseases caused by abnormal activation of NLRP3 inflammasome, acute peritonitis, cold pyrin-associated periodic syndrome, inflammatory bowel disease, type 2 diabetes, obesity, non-alcoholic fatty liver disease, aging, tumors, atherosclerosis, gout, renal dysfunction, Alzheimer's disease, Parkinson's syndrome, sepsis, endotoxemia, peritonitis, war trauma, cardiovascular stress, and myocardial hypertrophy.

[0016] Preferably, the type 2 diabetes is one of streptozotocin (STZ)-induced type 2 diabetes and high-fat diet (HFD)-induced type 2 diabetes.

[0017] Preferably, the abnormal activation of NLRP3 inflammasome described in (3) includes classical NLRP3 inflammasome activation and non-classical NLRP3 inflammasome activation.

[0018] Preferably, the classical NLRP3 inflammasome activation is NLRP3 inflammasome activation induced by an NLRP3 inflammasome activation activator;

[0019] The NLRP3 inflammasome activation activator includes at least one of nigericin, alum, ATP, urate crystals, silicon dioxide, imiquimod and dextran sulfate sodium salt.

[0020] Preferably, the non-classical NLRP3 inflammasome activation is activation of NLRP3 inflammasome induced by intracellular lipopolysaccharide.

[0021] Preferably, the cells in (5) are macrophages.

[0022] Preferably, the macrophages are bone marrow-derived macrophages.

[0023] Preferably, in (2), (3), (4), (5) and (6), the drug further comprises a pharmaceutically acceptable carrier.

[0024] Preferably, the dosage form of the drug is any one of tablets, granules, capsules, injections, and granules.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention explores new medical uses for ROC and opens up new application fields.

[0027] 2. ROC has been applied to phase III clinical trials for advanced EGFR-mutated non-small cell lung cancer (NSCLC). The adverse reactions are known, indicating strong medicinal prospects.

[0028] 3. ROC can specifically inhibit the activation of NLRP3 inflammasome in vitro.

[0029] 4. Rociletinib inhibited IL-1β release induced by relevant NLRP3 agonists (ATP, alum and R837) in vitro.

[0030] 5. ROC blocks the assembly of NLRP3 inflammasome by binding to NEK7

[0031] 6. By establishing a NLRP3 inflammasome-related disease model, it was found that rociletinib also has a good effect in inhibiting NLRP3 inflammasome activation in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the results of ROC inhibiting NLRP3 inflammasome activation in Example 1 of the present invention. Figure 1 A is the analysis of LPS-induced BMDMs treated with different concentrations of ROC by ELISA. ROC dose-dependently reduced IL-1β release. Figure 1 B is immunoblotting analysis of IL-1β and caspase-1 (P20) in the culture supernatant of BMDMs pretreated with LPS at different concentrations of ROC, as well as Pro-IL-1β and Pro-caspase-1 (Pro-casp1) in the cell lysate (Input). ROC dose-dependently reduced caspase-1 cleavage; Figure 1 C is ELISA analysis of TNF-α content in culture supernatants obtained from BMDM pretreated with LPS treated with different concentrations of ROC. ROC did not affect the release of NLRP3-independent cytokine TNF-α; Figure 1 D: LDH in the culture supernatant obtained from LPS-pretreated BMDMs treated with different concentrations of ROC was analyzed by ELISA, followed by stimulation with 5 μM nigericin for 30 min. ROC inhibited the release of LDH and reduced the activation of NLRP3 inflammasome and pyroptosis.

[0033] Figure 2 This is a result diagram showing that ROC can specifically block the activation of NLRP3 inflammasome in Example 2 of the present invention. Figure 2 A is the ELISA analysis of IL-1β in the culture supernatant of BMDM pretreated with LPS treated with ROC, followed by stimulation with 2.5 mM ATP for 30 min, 300 μg / ml alum for 6 h, or 15 μg / mL imiquimod for 3 h. ROC was able to inhibit the IL-1β release induced by these stimulants. Figure 2B: BMDMs pretreated with LPS were treated with ROC and then stimulated with 2.5 mM ATP for 30 min, 300 μg / ml alum for 6 h or 15 μg / mL imiquimod for 3 h. Immunoblot analysis of IL-1β and P20, Pro-IL-1β and Pro-casp1 in the culture supernatant. ROC showed a dose-dependent inhibition of IL-1β release and caspase-1 cleavage induced by these stimulants.

[0034] Figure 3 This is a result diagram showing that ROC blocks the assembly of NLRP3 inflammasome by binding to NEK7 in Example 3 of the present invention. Figure 3 A is the immunoprecipitation (IP) analysis of ROC treatment in LPS-pretreated BMDMs followed by stimulation with 5 μM nigericin for 30 min. ROC inhibits endogenous ASC-NLRP3 interaction. Figure 3 B is immunoblot analysis of ROC in LPS-pretreated BMDMs followed by stimulation with 5 μM nigericin for 30 min, where ROC blocked the binding of NEK7 to NLRP3. Figure 3 C is the docked complex of ROC and NEK7, with ENB shown as sticks and NEK7 as the bottom panel.

[0035] Figure 4 This is a result diagram showing that the binding of ROC to NEK7 and inhibition of NLRP3 inflammasome activation depends on reactive α, β-unsaturated carbonyl groups in Example 3 of the present invention. Figure 4 A is the chemical structure of reduced rociletinib (r-ROC). The carbon-carbon double bond of ROC is reduced to obtain r-ROC. Figure 4 B is the DARTS analysis of r-ROC treated BMDMs followed by pronase degradation of NEK7, and r-ROC could not interact with NEK7. Figure 4 C is the immunoblot analysis of IL-1β and P20 in the culture supernatant and Pro-IL-1β and Pro-casp1 in the cell lysate after LPS-induced BMDM were treated with different concentrations of ROC or r-ROC and then stimulated with 5 μM nigericin for 30 min. r-ROC did not affect nigericin-induced IL-1β release and caspase-1 cleavage. Figure 4 D is the ELISA analysis of IL-1β in the culture supernatant obtained after LPS-pretreated BMDM were treated with different compounds and then stimulated with 5 μM nigericin for 30 min.

[0036] Figure 5 It is the ROC covalently targeting cysteine ​​79 on NEK7 in Example 3 of the present invention. Figure 5A: BMDM pretreated with LPS were treated with ROC for 15 min and then stimulated with 5 μM nigericin for 30 min. The IL-1β content in the culture supernatant was analyzed by ELISA. Figure 5 B, BMDM pretreated with LPS were treated with ROC for 15 min and stimulated with 5 μM nigericin for 30 min. Immunoblotting analysis was performed for IL-1β and P20 in the culture supernatant, and Pro-IL-1β and Pro-casp1 in the cell lysate. ROC was able to inhibit the release of IL-1β and the cleavage of caspase-1 before and after washing. Figure 5 C, IL-1β and P20 in the culture supernatant and Pro-IL-1β and Pro-casp1 in the cell lysates after knockdown of NEK7 in iBMDMs transfected with WT or mutant Flag-NEK7. Figure 5 D is ELISA analysis of IL-1β in NEK7 knockout iBMDMs 326 transfected with WT or mutant Flag-NEK7. ROC covalently binds to the cysteine ​​79 residue of NEK7 and inhibits NLRP3-NEK7 interaction and inflammasome activation.

[0037] Figure 6 This is a result diagram showing that ROC has a protective effect on the HFD-induced diabetic mouse model in Example 4 of the present invention.

[0038] in Figure 6 A: The body weights of mice were measured at the indicated time points. Figure 6 B is the plasma glucose concentration at the indicated time points in mice treated with or without ROC, which can control the increase in blood glucose concentration. Figure 6 C is a representative H&E and Oil Red staining of the livers of HFD-fed mice and mice treated or not with ROC for 16 weeks. ROC treatment can reduce lipid accumulation in the livers of mice. Figure 6 D, ELISA analysis of IL-1β in mouse serum, liver, and fat. Figure 6 E is ELISA analysis of IL-18 in mouse serum, liver, and fat. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0041] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0042] In the present invention, the term "pharmaceutically acceptable carrier" refers to ingredients other than active ingredients in a pharmaceutical preparation that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0043] In the present invention, the term "NLRP3 inflammasome activation" refers to the fact that under the action of activators (such as nigericin, ATP and MSU), NLRP3, ASC and Pro-Caspase1 are combined and assembled to form a multi-protein functional complex, Pro-Caspase 1 self-splices to the active form Caspase 1 (p20), and the activated Caspase 1 splices cytokine precursors such as Pro-IL-1β and Pro-IL-18 into active factors, which are released outside the cell.

[0044] The activation process of NLRP3 inflammasome can be divided into the classical pathway and the non-classical pathway. In the classical pathway, the activation of NLRP3 inflammasome is divided into two main stages: the first is the initial stage, which is pre-treated by lipopolysaccharide (LPS) as the first signal, and then agonists such as nigericin or ATP are used as the second signal to directly stimulate and trigger the activation of NLRP3 inflammasome. In the non-classical pathway, Pam3CSK4 is first used as the first signal for pre-treatment; then, lipopolysaccharide (LPS) is transported into the cell by endocytosis as the second signal to stimulate and induce the non-classical activation of NLRP3 inflammasome.

[0045] In the present invention, ROC can specifically inhibit NLRP3 inflammasome activation.

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

[0047] The mice used in the examples are C57BL / 6 wild-type mice purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[0048] The cells used in the examples are BMDM cells (mouse bone marrow-derived macrophages), which are isolated and cultured from the bone marrow of C57BL / 6 wild-type mice.

[0049] Human monocytic THP-1 cell line: purchased from the American Type Culture Collection (ATCC).

[0050] Rociletinib (T2369), WZ4002 (T6238), Avitinib (T3024), Spebrutinib (T2603), CC-90003 (T14894) and canertinib (T6136) were purchased from TargetMol.

[0051] Nigerianmycin (HY-100381) and R 837 (Imiquimod, HY-B0180) were obtained from MedChemExpress. Protein G agarose was from Millipore.

[0052] MSU, ATP, poly(A:T), PMA (phorbol-12-myristate-13-acetate), glucose and insulin were obtained from Sigma.

[0053] LPS, Pam3CSK4 and MitoSOX were obtained from InvivoGen.

[0054] Alum was purchased from Pierce Biochemicals.

[0055] Anti-mouse IL-1β antibody: R&D, AF-401-NA.

[0056] Anti-NLRP3 antibody: AdipoGen, AG-20B-0014.

[0057] Anti-mouse caspase-1 antibody: AdipoGen, AG-20B-0042.

[0058] Anti-β-actin antibody: Abmart, P30002.

[0059] Anti-human IL-1β antibody: Proteintech, 60136-1-Ig.

[0060] Anti-human caspase-1 antibody: CST, #2225.

[0061] Anti-ASC antibody: CST, #67824.

[0062] Anti-VSV antibody: Sigma, V4888.

[0063] Anti-Flag antibody: Sigma, F2555.

[0064] DMSO (dimethyl sulfoxide): Shanghai Bioengineering, A10023.

[0065] Example 1: Rociletinib (ROC) inhibits caspase-1 activation and inflammatory cytokine IL-1β secretion in vitro

[0066] (1) Obtaining and differentiating mouse bone marrow-derived macrophages (BMDM): The bone marrow of C57BL / 6 mice of about 8 weeks old was obtained and differentiated for 4-5 days using DMEM medium + 10% fetal bovine serum + 25 ng / ml MSCF (macrophage colony-stimulating molecule).

[0067] (2) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 The next day, cells were pretreated with opti-MEM medium + 1% fetal bovine serum + 50ng / ml LPS for 3-6 hours. Then, different concentrations of ROC (0μM, 0.5μM, 1μM, 2μM) were added for half an hour, and then the cells were stimulated with 3.5μM nigericin for 15 minutes, and the cell supernatant and cell lysate were collected. At the same time, the pretreated cells were selected, and the cell supernatant and cell lysate were directly collected as a control. ELISA detected the secretion of IL-1β, and the results are as follows Figure 1 As shown in A; Western blot was used to detect IL-1β and caspase-1 (P20) in the culture supernatant, and Pro-IL-1β and Pro-caspase-1 (Pro-casp1) in the cell lysate (Input). The results are shown in Figure 1 As shown in B.

[0068] (3) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 The next day, cells were pretreated with opti-MEM medium + 1% fetal bovine serum + 50ng / ml LPS for 3-6 hours. Then, different concentrations of ROC (0μM, 0.5μM, 1μM, 2μM) were added for half an hour, and then the cells were stimulated with 3.5μM nigericin for 15 minutes, and the cell supernatant and cell lysate were collected. At the same time, the pretreated cells were selected and the cell supernatant was directly collected as a control. ELISA was used to detect the secretion of TNF-α and LDH. The results are as follows Figure 1 C and 1D.

[0069] Figure 1 The results of A and 1B indicate that ROC reduces IL-1β release and caspase-1 cleavage in a dose-dependent manner and does not affect the release of NLRP3-independent cytokine TNF-α ( Figure 1 C) Figure 1 ROC also inhibits LDH release as shown in D. In summary, ROC can reduce NLRP3 inflammasome activation and pyroptosis.

[0070] Example 2: ROC specifically inhibits the activation of NLRP3 inflammasome in vitro

[0071] (1) Differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The next day, cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50 ng / ml LPS) for 3 h. BMDM were treated with or without ROC for 30 min and then stimulated with the indicated inducers: 5 μM nigericin or 3 mM ATP for 30 min; infected with Salmonella typhimurium (MOI=5) for 3 h; 15 μg / ml R 837 or transfected with 0.5 μg / ml poly(A:T) for 4 h; 150 μg / ml MSU or 300 μg / ml alum for 6 h. Cell supernatants and cell lysates were collected, and the secretion of IL-1β and p20 in the supernatant and Pro-IL-1β and Pro-caspase-1 (Pro-casp1) in the cell lysate (Input) were detected by Western Blot and ELISA, respectively.

[0072] like Figure 2 As shown in A and 2B, the results of ELISA and Western Blot showed that ROC inhibited the secretion of IL-1β and p20 and caspase-1 cleavage induced by NLRP3 inflammasome agonists. In summary, ROC can inhibit the activation of NLRP3 inflammasome induced by various NLRP3 inflammasome agonists.

[0073] Example 3: ROC inhibits inflammasome assembly by binding to NEK7.

[0074] (1) Differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50 ng / ml LPS) for 3 hours. BMDM were treated with or without ROC for 30 minutes, and then with 5 μM nigericin for 15 minutes; the cell supernatant and cell lysate were collected, and the expression of NLRP3 and ASC in the supernatant and cell lysate was detected by Western Blot.

[0075] (2) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50 ng / ml LPS) for 3 hours. BMDM were treated with or without ROC for 30 minutes, and then with 5 μM nigericin for 15 minutes; the cell supernatant and cell lysate were collected, and the expression of NLRP3 and NEK7 in the supernatant and cell lysate was detected by Western Blot.

[0076] like Figure 3 As shown in Figures A and 3B, ROC not only inhibited endogenous ASC-NLRP3 interaction but also blocked the binding of NEK7 to NLRP3, indicating that ROC inhibited NLRP3 inflammasome assembly. Figure 3 C shows a simulated molecular model showing that ROC can be easily docked into NEK7.

[0077] ROC binds to NEK7 and inhibits NLRP3 inflammasome activation depending on the reactive α,β-unsaturated carbonyl group.

[0078] (like Figure 4 (shown)

[0079] (1) Differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50ng / ml LPS) for 3 hours. BMDM were treated with r-ROC without carbon-carbon double bonds for 30 minutes, and then with 5μM nigericin for 15 minutes; the cell supernatant and cell lysate were collected, and the expression of NEK7 in the supernatant was detected by Western Blot.

[0080] (2) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50 ng / ml LPS) for 3 hours. BMDM were treated with ROC or r-ROC for 30 minutes, and then with 5 μM nigericin for 15 minutes; the cell supernatant and cell lysate were collected, and Western blot was used to detect IL-1β and caspase-1 (P20) in the culture supernatant, and Pro-IL-1β and Pro-caspase-1 (Pro-casp1) in the cell lysate (Input). The results are shown in Figure 4 As shown in C.

[0081] (3) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 cells. The cells were pretreated with opti-MEM medium (containing 1% fetal bovine serum and 50 ng / ml LPS) for 3 hours. BMDM were treated with WZ4002, avitinib, spebrutinib, CC-90003, canertinib, CO-1686 intermediate and N-phenylacrylamide or ROC for 30 minutes, and then with 5 μM nigericin for 15 minutes; the cell supernatant was collected and the IL-1β content in the supernatant was detected by ELISA. The results are shown in Figure 4D.

[0082] like Figure 4 A shows the structure of r-ROC obtained by reducing the carbon-carbon double bond in ROC. Figure 4 B shows that r-ROC cannot interact with NEK7. Figure 4 C indicates that r-ROC neither inhibits the interaction between NLRP3 and NEK7 nor affects nigericin-induced IL-1β release and caspase-1 cleavage. Figure 4 D shows that ROC can inhibit NLRP3 inflammasome activation. In summary, ROC targets NEK7 and blocks NLRP3 inflammasome activation depending on the reactive α,β-unsaturated carbonyl group.

[0083] ROC covalently targets cysteine ​​79 on NEK7. Figure 5 (shown)

[0084] (1) Differentiated BMDM cells were placed in 12-well plates, with 5×10 cells per well. 5 The next day, cells were treated with opti-MEM medium + 1% fetal bovine serum + 50ng / ml LPS for 3-6 hours, and then treated with ROC for 15 minutes. Cell supernatants and cell lysates were collected without washing or three times. At the same time, cells that had been pretreated were selected and cell supernatants and cell lysates were directly collected as controls. ELISA was used to detect the secretion of IL-1β. The results are shown in Figure 5 As shown in A; Western blot was used to detect IL-1β and caspase-1 (P20) in the culture supernatant, and Pro-IL-1β and Pro-caspase-1 in the cell lysate (Input). The results are shown in Figure 5 As shown in B.

[0085] (2) The differentiated BMDM cells were divided into 12-well plates, with 5×10 cells per well. 5 After 24 hours, the cells were divided into three groups: Vector, WT and C79A. The cells were treated with opti-MEM medium + 1% fetal bovine serum + 50 ng / mL LPS for 3-6 hours, and then treated with ROC for 15 minutes. Western blot was used to detect caspase-1 (P20), Pro-caspase-1 and NEK7 in the culture supernatant. The results are shown in Figure 2. Figure 5 C. ELISA was used to detect the secretion of IL-1β. The results are shown in Figure 5 D

[0086] like Figure 5 A and 5B show that ROC can still inhibit the release of IL-1β and the cleavage of caspase-1 after washing, while Figure 5C and 5D show that the C79A mutation blocks ROC inhibition of NLRP3-NEK7 interaction and inflammasome activation. In summary, ROC covalently binds to the cysteine ​​79 residue of NEK7.

[0087] Example 4: ROC alleviates NLRP3 inflammasome-driven metabolic inflammation and type 2 diabetes in vivo

[0088] (1) Eight-week-old C57BL / 6j mice were divided into two groups (HFD+Vehicle group and HFD+ROC group), with 8 mice in each group. They were fed with a 60 kcal% high-fat diet (HFD, China) and orally administered with ROC (10 mg / kg body weight) for 16 weeks (once every two days). The weight changes of the mice were recorded once a month. The results are shown in Table 1. Figure 6 As shown in A.

[0089] (2) The mice were fasted for 14 h (no food and free access to water), and then glucose (1.5 g / kg body weight) was injected intraperitoneally. Blood was collected from the tail tip of the mice at 15, 30, 60, 90, and 120 min using a glucometer to measure blood glucose levels. The results are shown in Table 1. Figure 6 As shown in B.

[0090] (3) The liver tissue of the mouse was fixed with 4% paraformaldehyde at 4°C for 24 hours and then embedded in paraffin and sliced. The slices were stained with H&E or Oil Red O. The results were as follows: Figure 6 As shown in C.

[0091] (4) The production of proinflammatory cytokines IL-1β and IL-18 in mouse serum, liver, and white adipose tissue (WAT) was detected by ELISA. Figure 6 D, E

[0092] like Figure 6 A shows that ROC treatment significantly attenuated HFD-induced weight gain. Figure 6 B shows that ROC can control the increase of blood glucose concentration. In addition, Figure 6 C shows that ROC treatment can reduce the body weight and lipid accumulation in the liver of mice. Figure 6 The results in D and E showed that ROC treatment could inhibit the production of IL-1β and IL-18 in mouse tissues. In summary, ROC protected HFD-induced T2D by inhibiting the activation of NLRP3 inflammasome and interstitial inflammation in mice.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Rociletinib is used in any of the following applications: (1) Application for non-therapeutic purposes in inhibiting NLRP3 inflammasome activation in vitro; (2) Application in the preparation of anti-inflammatory drugs targeting NLRP3; (3) Use in the preparation of drugs for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasomes; (4) Application in the preparation of drugs for inhibiting the interaction between NLRP3 and NEK7; (5) Application in the preparation of drugs for inhibiting the secretion of inflammatory cytokine IL-1β; (6) Application in the preparation of drugs for inhibiting gasdermin D protein cleavage-induced cell pyroptosis.

2. The use according to claim 1, characterized in that: The diseases associated with abnormal activation of NLRP3 inflammasomes described in (3) include any one or more of hereditary NLRP3-dependent autoinflammatory diseases, metabolic diseases caused by abnormal activation of NLRP3 inflammasomes, neuroinflammatory diseases, arthritis, acute tissue damage, and chronic inflammation.

3. The use according to claim 2, characterized in that: The diseases associated with abnormal activation of NLRP3 inflammasomes described in (3) include inflammatory diseases caused by abnormal activation of NLRP3 inflammasomes, including any one or more of peritonitis, cryopyrin-associated periodic syndrome, inflammatory bowel disease, type 2 diabetes, obesity, non-alcoholic fatty liver disease, aging, tumors, atherosclerosis, gout, renal dysfunction, Alzheimer's disease, Parkinson's syndrome, sepsis, endotoxemia, peritonitis, war trauma, cardiovascular stress, and myocardial hypertrophy.

4. The use according to claim 3, characterized in that: The type 2 diabetes is one of streptozotocin (STZ)-induced type 2 diabetes and high-fat diet (HFD)-induced type 2 diabetes.

5. The use according to claim 1, characterized in that: The abnormal activation of NLRP3 inflammasome described in (3) includes classical NLRP3 inflammasome activation and non-classical NLRP3 inflammasome activation.

6. The use according to claim 5, characterized in that: The classical NLRP3 inflammasome activation is NLRP3 inflammasome activation induced by an NLRP3 inflammasome activation activator; the NLRP3 inflammasome activation activator includes at least one of nigericin, alum, ATP, urate crystals, silica, imiquimod and dextran sulfate sodium salt.

7. The use according to claim 5, characterized in that: The non-classical NLRP3 inflammasome activation is induced by intracellular lipopolysaccharide.

8. The use according to claim 1, characterized in that: The cells in (5) are macrophages; the macrophages are bone marrow-derived macrophages.

9. The use according to claim 1, characterized in that: In (2), (3), (4), (5) and (6), the drug further comprises a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that: in, The dosage form of the medicine is any one of tablets, granules, capsules, injections and granules.

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