Application of Doramectin as NLRP3 inflammasome activation inhibitor

Abstract

The invention provides an application of Doramectin as an NLRP3 inflammasome activation inhibitor, and belongs to the technical field of medicines. The invention discloses that Doramectin can directly target the NLRP3 protein, inhibit the interaction between the NLRP3 and NEK7 and inhibit the assembly of NLRP3 inflammasomes for the first time, thereby inhibiting the activation of the NLRP3 inflammasomes; at the same time, Doramectin at an animal level can improve the occurrence of inflammatory diseases related to NLRP3 inflammasome. The invention provides a theoretical basis and an experimental basis for treating NLRP3 inflammasome related inflammatory diseases by using Doramectin, and also expands the application field of the medicine.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of Doramectin as an inhibitor for activating NLRP3 inflammasome. Background Art

[0002] As the first line of defense in the body's immune response, the innate immune system has always been a frontier area of immunological research. Among them, inflammasome, as one of the innate immune molecules, plays an important role in the body's inflammatory response caused by exogenous pathogens and endogenous danger signals. The over-activation or out-of-control regulation of inflammasome often exacerbates the body's inflammatory response, leading to body damage.

[0003] As an important part of innate immunity, NLRP3 inflammasome mainly consists of cytoplasmic sensor protein NLRP3, apoptosis-associated speck-like protein (ASC), and cysteine protease Caspase-1. After the activation of NLRP3 inflammasome, NLRP3, ASC, and Caspase-1 form a complex to activate Caspase-1; subsequently, the activated Caspase-1 mediates the maturation and secretion of inflammatory factor IL-1β, and the inflammatory factor IL-1β further participates in processes such as the body's inflammatory response; at the same time, the activated Caspase-1 can mediate pyroptosis by cleaving effector protein GSDMD. NLRP3 inflammasome is one of the most widely studied inflammasomes at present. It can recognize pathogen-associated molecular patterns (PAMPs) and danger signal-associated molecular patterns (DAMPs), including viruses, bacteria, Nigericin, monosodium urate, ATP, silica crystals, and aluminum salt crystals, etc. NLRP3 inflammasome plays an important role in the body's resistance to pathogen infection and recognition of danger signals, but its over-activation or out-of-control regulation is closely related to many inflammatory diseases, such as gout, type 2 diabetes, atherosclerosis, non-alcoholic fatty liver disease, Parkinson's disease, Alzheimer's disease, and various autoimmune diseases, etc.

[0004] A variety of NLRP3 inflammasome activation inhibitors have been developed (mainly targeting the NLRP3 protein), including MCC950, CY-09, MNS, OLT1177, and so on. The effects of these inhibitors in NLRP3 inflammasome-related inflammatory disease models have been verified. For example, in mice, MCC950 can reduce the severity of experimental autoimmune encephalomyelitis; in a mouse model of Parkinson's disease, oral administration of MCC950 can inhibit NLRP3 inflammasome activation, and also has a neuroprotective effect, which can effectively reduce motor deficits, nigrostriatal dopaminergic degeneration, and the accumulation of α-synuclein aggregates; CY-09 has good therapeutic effects in cryopyrin-associated periodic syndrome and type 2 diabetes mouse models. Although a variety of NLRP3 inflammasome activation inhibitors have good effects in animal models, there are few inhibitors approved for marketing at present, and most inhibitors are in the clinical trial stage or have stopped at the clinical trial stage. Therefore, finding new and effective drugs to inhibit NLRP3 inflammasome activation remains an urgent need and research focus at present. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention aims to develop new NLRP3 inflammasome activation inhibitors to provide new alternative drugs for the treatment of NLRP3 inflammasome-related inflammatory diseases.

[0006] The technical solution of the present invention is specifically as follows: In the first aspect, the present invention provides the use of Doramectin as an NLRP3 inflammasome activation inhibitor.

[0007] Doramectin is a new generation of macrolide anti-parasitic drugs and is considered to be one of the most excellent anti-parasitic drugs in the avermectin family. However, there are few reports on the function and role of Doramectin in innate immunity at present. The present invention first discovers that Doramectin can significantly inhibit NLRP3 inflammasome activation in both cell experiments and animal experiments.

[0008] In the second aspect, the present invention provides the use of Doramectin in the preparation of a reagent for inhibiting the interaction between NLRP3 and NEK7. The present invention first reveals that Doramectin can directly target the NBD domain of the NLRP3 protein, thereby inhibiting the interaction between NLRP3 and NEK7, and further inhibiting NLRP3 inflammasome activation.

[0009] In the third aspect, the present invention provides the use of Doramectin in the preparation of a drug for treating NLRP3 inflammasome-related inflammatory diseases, wherein the NLRP3 inflammasome-related inflammatory diseases include but are not limited to colitis and sepsis.

[0010] Experiments of the present invention show that Doramectin can improve the development of NLRP3 inflammasome-related inflammatory diseases at the animal level, providing effective reference value for the treatment of related inflammatory diseases in clinical practice; at the same time, it expands the application field of this drug and provides new ideas for the development of anti-inflammatory drugs.

[0011] It can be understood that in the above application, the drug may also contain pharmaceutically acceptable excipients, such as preservatives, diluents, excipients, etc.; the dosage form of the drug can be any one of tablets, granules, injections, capsules.

[0012] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are given below. Brief Description of the Drawings

[0013] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0014] Figure 1 It is the molecular structural formula of Doramectin in the present invention; Figure 2 It is the experimental result (A) of the small molecule drug screening model and the toxicity detection result (B) of Doramectin in Example 1 of the present invention; Figure 3 It is the detection result diagram of the effect of different concentrations of Doramectin on IL-1β secretion and the transcriptional levels of IL-1β and NLRP3 in Example 1 of the present invention; Figure 4 It is the relevant experimental result diagram for verifying that Doramectin inhibits the interaction between NLRP3 and NEK7 by targeting the NBD domain of NLRP3 in Example 2 of the present invention; Figure 5 It is the experimental result diagram of Doramectin inhibiting LPS-induced septic shock in Example 3 of the present invention; Figure 6 It is the experimental result diagram of Doramectin relieving DSS-induced murine colitis in Example 4 of the present invention. Detailed Description of the Invention

[0015] The embodiments of the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "comprising" and any variation thereof herein is intended to cover non-exclusive inclusion.

[0017] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention and cannot be understood as a limitation of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0018] In the following embodiments, THP-1-induced differentiated macrophages are induced and differentiated by phorbol ester (TPA), which can be implemented by those skilled in the art according to the existing technology, and the present invention will not elaborate further.

[0019] Example 1 In this example, a small molecule drug screening model for inhibiting the activation of NLRP3 inflammasome was established at the cellular level, and Doramectin's ability as an NLRP3 inflammasome activation inhibitor in macrophages was obtained and verified using this model, specifically including the following experiments: (1) Construction of the small molecule drug screening model.

[0020] In THP-1-induced differentiated macrophages, lipopolysaccharide (LPS, 100 ng / ml) and Nigericin (10 μM) were added as stimulants for the NLRP3 inflammasome, and the stimulation culture times for LPS and Nigericin were 6 h and 1 h, respectively; then, MCC950 (1 μM) or MCC950 (10 μM) was added as an inhibitor of NLRP3 and cultured for 12 h, and then the cell culture supernatant was collected, and the content of mature IL-1β was detected by ELISA. The results are as Figure 2 shown in A, LPS and Nigericin can activate the NLRP3 inflammasome, and MCC950 can effectively inhibit the activation of the NLRP3 inflammasome, that is, the experimental model was successfully constructed.

[0021] (2) Doramectin can inhibit the activation of NLRP3 inflammasome.

[0022] In THP-1-induced differentiated macrophages, LPS (100 ng / ml, 6 h) and Nigericin (10 μM, 1 h) were added as stimulants of the NLRP3 inflammasome, and then Doramectin (10 μM, 12 h) was added. MCC950 (10 μM, 12 h) was added as a positive control for inhibiting NLRP3 inflammasome activation. The cell culture supernatant was collected to detect the content of mature IL-1β. The results showed that Doramectin could effectively inhibit NLRP3 inflammasome activation compared with other small molecule drugs.

[0023] (3)Regulation of pro-IL-1β mRNA by Doramectin.

[0024] The activation of the classical NLRP3 inflammasome mainly includes two steps: upregulating the mRNA levels of NLRP3 and pro-IL-1β to allow for the abundant expression of NLRP3 and pro-IL-1β proteins; assembling components such as NLRP3, ASC, and Caspase-1 into a complex, and after the assembly and activation of the NLRP3 inflammasome, cleaving pro-IL-1β into mature IL-1β.

[0025] Therefore, in this example, the regulatory effect of Doramectin on pro-IL-1β mRNA was clarified by RT-PCR. The specific experiment was as follows: In THP-1-induced differentiated macrophages, LPS (100 ng / ml, 6 h) and Nigericin (10 μM, 1 h) were added as stimulants of the NLRP3 inflammasome, and then Doramectin (10 μM, 12 h) was added. MCC950 (10 μM, 12 h) was added as a positive control for inhibiting NLRP3 inflammasome activation. Cells were collected, Trizol was added to extract RNA, and the mRNA of pro-IL-1β was detected. The results showed that Doramectin could effectively inhibit the mRNA level of pro-IL-1β.

[0026] (4)Detection of the cytotoxicity of Doramectin.

[0027] Different concentrations (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20 μM) of Doramectin were added to THP-1-induced differentiated macrophages, and its toxicity was detected by a CCK8 kit. The results were as Figure 2 shown in Figure B. When the concentration of Doramectin exceeded 10 μM, the drug had obvious toxicity to the cells.

[0028] (5)Effect of Doramectin concentration on the secretion and transcriptional level of IL-1β.

[0029] In THP-1-induced differentiated macrophages, after adding Doramectin (1.25, 2.5, 5 μM) for 12 hours, LPS (100 ng / ml, 6 h) and Nigericin (10 μM, 1 h) were added as stimulants of the NLRP3 inflammasome. The cell supernatant was collected to detect the content of mature IL-1β. The results were as follows Figure 3 shown in Figure A. Doramectin could inhibit the secretion of IL-1β with the change of concentration, thereby inhibiting the activation of the NLRP3 inflammasome.

[0030] In THP-1-induced differentiated macrophages, after adding Doramectin (1.25, 2.5, 5 μM) for 12 hours, LPS (100 ng / ml, 6 h) and Nigericin (10 μM, 1 h) were added as stimulants of the NLRP3 inflammasome. Finally, the cells were collected, Trizol was added to extract RNA, and the mRNA levels of IL-1β and NLRP3 were detected. The results were as follows Figure 3 shown in Figures B and 3C. Doramectin could also inhibit the mRNA level of IL-1β with the change of concentration, but had little effect on the mRNA level of NLRP3.

[0031] Example 2 This example elucidated the mechanism by which Doramectin targets the NBD domain of NLRP3 to inhibit the interaction between NLRP3 and NEK7, specifically including the following experiments The immunoprecipitation method (Co-IP) was used to verify the effect of Doramectin (10 μM) on the interaction between NLRP3 and NEK7: NLRP3 and NEK7 plasmids were transfected into 293T cells in a 6-cm dish. After 24 hours of transfection, the cells were lysed with 1 ml of RIPA lysis buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol). After lysis, 100 μl was taken as Input, and 25 μl of 5×SDS was added and boiled for 10 minutes; 900 μl of the lysis reaction solution was taken for immunoprecipitation (IP), and Flag antibody was added and incubated with the lysis reaction solution overnight; the next day, Pierce protein A / G magnetic beads were added and incubated for 2 hours, and then washed 6 times with RIPA elution buffer (0.05 M Tris-HCl, 0.3 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol). After adding 60 μl of 2×SDS and boiling for 10 minutes, the protein content in the sample was detected by Western Blot. The results were as follows Figure 4As shown in Figure A, Doramectin significantly inhibited the interaction between NLRP3 and NEK7.

[0032] NLRP3 and NEK7 plasmids were transfected into 293T cells, and concentration gradients of 1 μM, 5 μM, and 10 μM Doramectin were set. Co-IP was used to verify the effect of Doramectin at different concentrations on the interaction between NLRP3 and NEK7. The results are as Figure 4 shown in Figure B, and Doramectin inhibited the interaction between NLRP3 and NEK7 in a concentration-dependent manner.

[0033] NLRP3 and NEK7 plasmids were transfected into 293T cells, and the drug affinity responsive target stability (DARTS) technique was used to find the specific component proteins that bind to Doramectin. Specifically: after 24 h of transfection, the cells were lysed, and the cell lysate was divided into two parts. One group was added with DMSO, and the other group was added with Doramectin (100 μM). They were incubated overnight at 4 °C; the next day, each group was further divided into two parts. One part was added with protease pronase, incubated at 37 °C for 4 minutes, and then immediately added with 5×SDS and boiled for 10 minutes. The other part was directly added with 5×SDS and boiled for 10 minutes; finally, the protein content in the samples was detected by Western Blot. The results are as Figure 4 shown in Figure C. NLRP3 in the group added with Doramectin was not hydrolyzed by pronase, indicating that Doramectin may inhibit the interaction between NLRP3 and NEK7 by binding to NLRP3.

[0034] After stimulating mouse bone marrow-derived macrophages (BMDMs) with LPS (100 ng / ml, 6 h), the cells were lysed. The DARTS technique was used to find that the effect of pronase hydrolysis on NLRP3 in the group added with Doramectin (100 μM) was weakened ( Figure 4 Figure D), further verifying that Doramectin inhibits the interaction between NLRP3 and NEK7 by binding to NLRP3.

[0035] The NLRP3 protein contains three main domains, namely the pyrin domain (PYD), the central nucleotide binding and oligomerization domain (NACHT domain), and the leucine-rich repeat domain (LRR domain). Therefore, by separately transfecting NBD, LRR, and Pyrin plasmids into 293T cells and performing DARTS experiments, it was found that Doramectin binds to the NBD domain on the NLRP3 protein ( Figure 4 E-G).

[0036] Example 3 Given the close relationship between the LPS-induced sepsis model and the activation mechanism of the NLRP3 inflammasome, in this example, the inhibitory ability of Doramectin on the LPS-induced sepsis model was detected. The specific experiment is as follows: The mice were divided into 4 groups. The first group was intraperitoneally injected with normal saline (n = 4), the second group was intraperitoneally injected with Doramectin (2.5 mg / kg) (n = 4), the third group was intraperitoneally injected with LPS (20 mg / kg, 4 h) (n = 6), and the fourth group was intraperitoneally injected with LPS (20 mg / kg, 4 h) plus Doramectin (2.5 mg / kg, 12 h) (n = 6); where n represents the number of mice in each group. The fourth group was injected with LPS 12 h after injecting Doramectin. Blood was collected from the eyeballs 4 h after injecting LPS, and the contents of IL-1β and TNF-α in the blood were measured. The results are as Figure 5 shown in A and 5B. Doramectin can effectively inhibit the secretion of IL-1β and inhibit the activation of the NLRP3 inflammasome, but has no inhibitory effect on the secretion of TNF-α.

[0037] Another group of mice was divided into 5 groups. The first group was intraperitoneally injected with normal saline (n = 8), the second group was intraperitoneally injected with LPS (20 mg / kg) (n = 12), the third group was intraperitoneally injected with Doramectin (2.5 mg / kg) (n = 8), the fourth group was intraperitoneally injected with LPS (20 mg / kg) plus Doramectin (2.5 mg / kg) (n = 12), and the fifth group was intraperitoneally injected with LPS (20 mg / kg) plus Doramectin (1.25 mg / kg) (n = 12); where n represents the number of mice in each group. In the fourth and fifth groups, LPS (20 mg / kg) was injected 2 hours after intraperitoneal injection of Doramectin (1.25 mg / kg) or Doramectin (2.5 mg / kg); The survival of the mice was observed every day (at 8:00, 14:00, and 20:00) and the body weight was recorded for a total of 72 hours. The results are as Figure 5 shown in Figures 5C and 5D. Doramectin can effectively inhibit the LPS-induced sepsis model, improve the survival rate of mice and relieve the decrease in body weight of mice.

[0038] Example 4 This example shows that Doramectin can be used to relieve colitis, specifically as follows: Female C57BL / 6 mice (7 weeks old) were divided into 5 groups. The control group (n = 8) was given normal water to drink for 10 days. The experimental group (n = 12) was fed with dextran sulphate sodium (DSS) for 7 days and then changed to normal water for 3 days. From the first day, the mice were treated with intraperitoneal injection of 1.25 mg / kg or 2.5 mg / kg of Doramectin every other day, and intraperitoneal injection of normal saline or 20 mg / kg of MCC950 was set as the control. After the experiment was completed, the colon length of each group of mice was measured; in addition, according to the stool characteristics and the degree of bloody stool of the mice, scores were given, and the disease index was counted. It can be seen that Doramectin can relieve the loose stool and bloody stool of the mice.

[0039] The results are as Figure 6 shown. Doramectin can relieve the shortening of the colon in DSS-induced mice (Figures 5A and 5B), and can also relieve the loose stool and bloody stool of the mice.

[0040] In summary, Doramectin can significantly inhibit the activation of NLRP3 inflammasome, and its mechanism is as follows: Doramectin can target NLRP3 protein, affect the function of NLRP3, inhibit the assembly of NLRP3 inflammasome, thereby inhibiting the activation of NLRP3 inflammasome, and further inhibiting the downstream inflammatory response. At the animal level, Doramectin can improve the occurrence of NLRP3 inflammasome-related inflammatory diseases (such as sepsis and colitis). The present invention provides a theoretical basis and experimental foundation for using Doramectin to treat NLRP3 inflammasome-related inflammatory diseases.

[0041] It should be noted that the present invention is not limited to the above embodiments. The above embodiments are only examples, and embodiments having the same composition and playing the same effect within the scope of the technical solution of the present invention are included in the technical scope of the present invention. In addition, within the scope of not departing from the gist of the present invention, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways constructed by combining some constituent elements of the embodiments are also included in the scope of the present invention.

Claims

1. Use of doramectin as an inhibitor of NLRP3 inflammasome activation.

2. Use of doramectin in the preparation of a reagent for inhibiting the interaction between NLRP3 and NEK7.

3. Use of doramectin in the preparation of a drug for treating NLRP3 inflammasome-related inflammatory diseases.

4. The application according to claim 3, characterized in that, The NLRP3 inflammasome-related inflammatory disease is colitis.

5. The application according to claim 3, wherein The NLRP3 inflammasome-related inflammatory disease is sepsis.

6. The application according to claim 3, wherein The drug contains pharmaceutically acceptable excipients.

7. The application according to claim 3, wherein The drug is any one of tablets, granules, injections, and capsules.

8. A drug for inhibiting the activation of NLRP3 inflammasome, characterized in that, The drug contains doramectin.

9. The drug according to claim 8, wherein The drug contains pharmaceutically acceptable excipients.

10. The medicament according to claim 8, characterized in that, The drug is any one of tablets, granules, injections, and capsules.

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