Use of doramectin as an inhibitor of nlrp3 inflammasome activation

Abstract

The application provides application of doramectin in an NLRP3 inflammasome activation inhibitor, and belongs to the technical field of medicines.The application first discloses that doramectin can directly target NLRP3 protein, inhibit the interaction between NLRP3 and NEK7, inhibit NLRP3 inflammasome assembly, and thus inhibit NLRP3 inflammasome activation; meanwhile, doramectin can improve the occurrence of NLRP3 inflammasome related inflammatory diseases at an animal level.The application provides a theoretical basis and experimental basis for treating NLRP3 inflammasome related inflammatory diseases by using doramectin, and also expands the use field of the drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and specifically to the use of doramectin as an inhibitor of NLRP3 inflammasome activation. Background Technology

[0002] The innate immune system, as the body's first line of defense in the immune response, has always been at the forefront of immunological research. Inflammasomes, as one of the innate immune molecules, play a crucial role in the body's inflammatory response induced by exogenous pathogens and endogenous danger signals. Overactivation or dysregulation of inflammasomes often exacerbates the body's inflammatory response, leading to damage.

[0003] As an important component of innate immunity, the NLRP3 inflammasome is mainly composed of the cytoplasmic sensing protein NLRP3, apoptosis-associated speckle-like protein (ASC), and the cysteine ​​protease Caspase-1. Upon activation, the NLRP3 inflammasome forms a complex with NLRP3, ASC, and Caspase-1, which then activates Caspase-1. Subsequently, activated Caspase-1 mediates the maturation and secretion of the inflammatory cytokine IL-1β, which in turn participates in the body's inflammatory response. Simultaneously, activated Caspase-1 can mediate pyroptosis through the cleavage effector protein GSDMD. The NLRP3 inflammasome is one of the most extensively studied inflammasomes, capable of recognizing 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, among others. The NLRP3 inflammasome plays an important role in the body's resistance to pathogen infection and recognition of danger signals, but its overactivation or dysregulation is closely related to many inflammatory diseases, such as gout, type 2 diabetes, atherosclerosis, non-alcoholic steatohepatitis, Parkinson's disease, Alzheimer's disease, and various autoimmune diseases.

[0004] Currently, several NLRP3 inflammasome activation inhibitors (primarily targeting the NLRP3 protein) have been developed, including MCC950, CY-09, MNS, and OLT1177. The efficacy of these inhibitors in NLRP3 inflammasome-related inflammatory disease models has been validated. 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 inhibits NLRP3 inflammasome activation and also has neuroprotective effects, effectively reducing motor deficits, dopaminergic degeneration of the substantia nigra and striatum, and the accumulation of α-synuclein aggregates; CY-09 shows good therapeutic effects in mouse models of cryopyrin-related autoinflammatory syndrome and type 2 diabetes. Although many NLRP3 inflammasome activation inhibitors have shown good efficacy in animal models, very few have been approved for marketing, with most remaining in clinical trials or discontinued. Therefore, finding novel and effective drugs to inhibit NLRP3 inflammasome activation remains an urgent need and a key research focus. 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 drug options for the treatment of NLRP3 inflammasome-related inflammatory diseases.

[0006] The specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of Doramectin as an inhibitor of NLRP3 inflammasome activation.

[0008] Doramectin is a new generation of macrolide antiparasitic drugs, considered one of the most effective antiparasitic drugs in the avermectin family. However, its function and role in innate immunity are rarely reported. This invention is the first to discover that doramectin can significantly inhibit NLRP3 inflammasome activation in both cell and animal experiments.

[0009] Secondly, this invention provides the application of Doramectin in the preparation of reagents that inhibit the interaction between NLRP3 and NEK7. This invention reveals for the first time that Doramectin can directly target the NBD domain of the NLRP3 protein, thereby inhibiting the interaction between NLRP3 and NEK7, and consequently inhibiting NLRP3 inflammasome activation.

[0010] Thirdly, the present invention provides the use of Doramectin in the preparation of medicaments for treating NLRP3 inflammasome-related inflammatory diseases, including but not limited to colitis and sepsis.

[0011] The experiments of this invention demonstrate that Doramectin can improve the development of NLRP3 inflammasome-related inflammatory diseases at the animal level, providing effective reference value for the clinical treatment of related inflammatory diseases; at the same time, it expands the application field of this drug and provides new ideas for the development of anti-inflammatory drugs.

[0012] It is understandable that, in the above applications, the drug may also contain pharmaceutically acceptable excipients, such as preservatives, diluents, excipients, etc.; the dosage form of the drug may be any one of tablets, granules, injections, or capsules.

[0013] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0015] Figure 1 This is the molecular structural formula of Doramectin in this invention;

[0016] Figure 2 Figure 1 shows the experimental results (A) of the small molecule drug screening model in Example 1 of this invention and the toxicity test results (B) of Doramectin.

[0017] Figure 3 The graph shows the results of detecting the effects of different concentrations of Doramectin on IL-1β secretion and IL-1β and NLRP3 transcription levels in Example 1 of this invention.

[0018] Figure 4 This is a diagram showing the experimental results in Embodiment 2 of the present invention, which verify that Doramectin inhibits the interaction between NLRP3 and NEK7 by targeting the NBD domain of NLRP3.

[0019] Figure 5The figure shows the experimental results of Doramectin inhibiting LPS-induced septic shock in Example 3 of this invention.

[0020] Figure 6 This is a diagram showing the experimental results of Doramectin alleviating DSS-induced colitis in mice in Example 4 of the present invention. Detailed Implementation

[0021] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion.

[0023] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0024] In the following embodiments, THP-1 induced differentiated macrophages were induced to differentiate using phorbol ester (TPA). Those skilled in the art can implement this method according to existing technology, and the present invention will not elaborate further.

[0025] Example 1

[0026] This example demonstrates the establishment of a small molecule drug screening model at the cellular level to inhibit NLRP3 inflammasome activation. Using this model, the ability of Doramectin as an inhibitor of NLRP3 inflammasome activation in macrophages was obtained and validated. The specific experiments included the following:

[0027] (1) Construction of small molecule drug screening model.

[0028] In THP-1-induced differentiated macrophages, lipopolysaccharide (LPS, 100 ng / ml) and nigericin (10 μM) were added as stimulants for the NLRP3 inflammasome, with LPS and nigericin stimulation times of 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. The cell culture supernatant was collected, and the content of mature IL-1β was detected by ELISA. Results are as follows: Figure 2 As shown in Figure A, LPS and Nigericin can activate the NLRP3 inflammasome, while MCC950 can effectively inhibit the activation of the NLRP3 inflammasome, indicating that the experimental model was successfully constructed.

[0029] (2) Doramectin can inhibit the activation of NLRP3 inflammasome.

[0030] In THP-1-induced differentiated macrophages, LPS (100 ng / ml, 6 h) and Nigericin (10 μM, 1 h) were added as stimulants for the NLRP3 inflammasome, followed by the addition of Doramectin (10 μM, 12 h). MCC950 (10 μM, 12 h) was added as a positive control to inhibit NLRP3 inflammasome activation. Cell culture supernatant was collected, and the level of mature IL-1β was measured. The results suggest that Doramectin can effectively inhibit NLRP3 inflammasome activation compared to other small molecule drugs.

[0031] (3) Regulation of pro-IL-1β mRNA by Doramectin.

[0032] The classic NLRP3 inflammasome activation mainly involves two steps: upregulating the mRNA levels of NLRP3 and pro-IL-1β, resulting in the high expression of NLRP3 and pro-IL-1β proteins; and assembling components such as NLRP3, ASC, and Caspase-1 into a complex. After the NLRP3 inflammasome assembly is activated, pro-IL-1β is cleaved into mature IL-1β.

[0033] Therefore, this study clarified the regulatory role of Doramectin on pro-IL-1β mRNA using 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 for the NLRP3 inflammasome, followed by Doramectin (10 μM, 12 h). MCC950 (10 μM, 12 h) was added as a positive control to inhibit NLRP3 inflammasome activation. Cells were collected, and RNA was extracted using Trizol. The mRNA levels of pro-IL-1β were then detected. The results showed that Doramectin effectively inhibited the mRNA level of pro-IL-1β.

[0034] (4) Detection of cytotoxicity of Doramectin.

[0035] 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 using a CCK8 assay kit. The results are as follows: Figure 2 As shown in B, when the concentration of Doromectin exceeds 10 μM, the drug exhibits significant toxicity to cells.

[0036] (5) Effects of Doramectin concentration on the secretion and transcription levels of IL-1β.

[0037] 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 for the NLRP3 inflammasome. Cell supernatant was collected, and the content of mature IL-1β was detected. The results are as follows. Figure 3 As shown in Figure A, Doramectin can inhibit the secretion of IL-1β by varying its concentration, thereby inhibiting the activation of the NLRP3 inflammasome.

[0038] 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 for the NLRP3 inflammasome. Cells were then collected, and RNA was extracted using Trizol. The mRNA levels of IL-1β and NLRP3 were then detected. Results are as follows: Figure 3As shown in B and 3C, Doramectin can also inhibit the mRNA level of IL-1β with changes in concentration, but has little effect on the mRNA level of NLRP3.

[0039] Example 2

[0040] This example elucidates the mechanism by which Doramectin targets the NBD domain of NLRP3 to inhibit the interaction between NLRP3 and NEK7, specifically including the following experiments:

[0041] The effect of Doramectin (10 μM) on the interaction between NLRP3 and NEK7 was verified using this co-immunoprecipitation (Co-IP) method: NLRP3 and NEK7 plasmids were transfected into 293T cells in 6 cm dishes. 24 h after transfection, 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). 100 μl of the lysed sample was used as input, and 25 μl of 5×SDS was added for boiling for 10 min. 900 μl of the lysis buffer was then used for immunoprecipitation (IP), and Flag antibody was added and incubated overnight. The next day, Pierce protein A / G magnetic beads were added and incubated for 2 h, followed by elution with RIPA buffer (0.05 M Tris-HCl, 0.3 M NaCl, 0.001 M EDTA, 1% NP-40). Wash 6 times with 5% glycerol, add 60 μl of 2×SDS and boil for 10 minutes, then detect the protein content in the sample by Western blot. Results are as follows: Figure 4 As shown in Figure A, Doramectin significantly inhibits the interaction between NLRP3 and NEK7.

[0042] NLRP3 and NEK7 plasmids were transfected into 293T cells, and concentration gradients of Doramectin (1 μM, 5 μM, and 10 μM) were established. Co-IP was used to verify the effect of different concentrations of Doramectin on the interaction between NLRP3 and NEK7. Results are as follows: Figure 4 As shown in Figure B, Doramectin inhibits the interaction between NLRP3 and NEK7 in a concentration-dependent manner.

[0043] NLRP3 and NEK7 plasmids were transfected into 293T cells. Drug affinity response targeting stability (DARTS) technology was used to identify specific component proteins that bind to Doramectin. Specifically, cells were lysed 24 hours after transfection, and the cell lysates were divided into two portions. One portion was added to DMSO, and the other to Doramectin (100 μM), and incubated overnight at 4°C. The next day, each portion was divided again. One portion was added to protease pronase and incubated at 37°C for 4 minutes, followed immediately by boiling with 5×SDS for 10 minutes. The other portion was directly boiled with 5×SDS for 10 minutes. Finally, the protein content in the samples was detected by Western blotting. Results are as follows: Figure 4 As shown in Figure C, NLRP3 in the group with added Doramectin was not hydrolyzed by streptocytoxin, indicating that Doramectin may inhibit the interaction between NLRP3 and NEK7 by binding to NLRP3.

[0044] In mouse bone marrow-derived macrophages (BMDMs), after stimulation with LPS (100 ng / ml, 6 h) followed by cell lysis, DARTS technology revealed that the effect of Pronase hydrolysis on NLRP3 was weakened in the group with the addition of Doramectin (100 μM). Figure 4 (D) This further verifies that Doramectin inhibits the interaction between NLRP3 and NEK7 by binding to NLRP3.

[0045] The NLRP3 protein contains three main domains: the pyrin domain (PYD), the central nucleotide binding and oligomerization domain (NACHT domain), and the leucine-rich repeat domain (LRR domain). Therefore, after transfecting 293T cells with NBD, LRR, and Pyrin plasmids, and using DARTS experiments, it was determined that Doramectin binds to the NBD domain of the NLRP3 protein. Figure 4 EG).

[0046] Example 3

[0047] Given the close relationship between LPS-induced sepsis models and the NLRP3 inflammasome activation mechanism, this study investigated the inhibitory effect of Doramectin on LPS-induced sepsis models. The specific experiments are as follows:

[0048] Mice were divided into four groups: Group 1 received intraperitoneal injection of saline (n=4); Group 2 received intraperitoneal injection of Doramectin (2.5 mg / kg) (n=4); Group 3 received intraperitoneal injection of LPS (20 mg / kg, 4 h) (n=6); and Group 4 received intraperitoneal injection of LPS (20 mg / kg, 4 h) plus Doramectin (2.5 mg / kg, 12 h) (n=6). Here, n represents the number of mice in each group. In Group 4, LPS was injected 12 h after Doramectin injection. Blood samples were collected from the eyeballs 4 h after LPS injection to measure the levels of IL-1β and TNF-α in the blood. The results are as follows: Figure 5 As shown in A and 5B, Doramectin can effectively inhibit the secretion of IL-1β and inhibit the activation of NLRP3 inflammasome, but it has no inhibitory effect on the secretion of TNF-α.

[0049] Mice were randomly divided into 5 groups. Group 1 received intraperitoneal injection of saline (n=8); Group 2 received intraperitoneal injection of LPS (20 mg / kg) (n=12); Group 3 received intraperitoneal injection of Doramectin (2.5 mg / kg) (n=8); Group 4 received intraperitoneal injection of LPS (20 mg / kg) plus Doramectin (2.5 mg / kg) (n=12); and Group 5 received intraperitoneal injection of LPS (20 mg / kg) plus Doramectin (1.25 mg / kg) (n=12). Here, n represents the number of mice in each group. In Groups 4 and 5, LPS (20 mg / kg) was injected 2 hours after the intraperitoneal injection of Doramectin (1.25 mg / kg) or Doramectin (2.5 mg / kg). Mice were observed and their weight was recorded daily at 8:00 AM, 2:00 PM, and 8:00 PM for a total of 72 hours. Results are as follows: Figure 5 As shown in C and 5D, Doramectin effectively inhibited LPS-induced sepsis, improved mouse survival, and alleviated weight loss in mice.

[0050] Example 4

[0051] This example demonstrates that Doramectin can be used to relieve colitis, as follows:

[0052] Seven-week-old female C57BL / 6 mice were divided into five groups. The control group (n=8) was given normal drinking water for 10 days, while the experimental group (n=12) was fed dextran sulfate sodium (DSS) for 7 days, followed by normal drinking water for 3 days. Starting from day one, mice were treated with intraperitoneal injections of 1.25 mg / kg or 2.5 mg / kg of Doramectin every other day. Intraperitoneal injections of physiological saline or 20 mg / kg of MCC950 served as controls. After the experiment, the colon length of each group was measured. Furthermore, the mice were scored based on fecal characteristics and the degree of bloody stool, and their disease index was calculated. The results showed that Doramectin effectively alleviated the severity of loose and bloody stools in the mice.

[0053] The results are as follows Figure 6 As shown, Doramectin can alleviate DSS-induced colonic shortening in mice (A and B), and also alleviate the degree of loose stools and bloody stools in mice.

[0054] In summary, Doramectin significantly inhibits NLRP3 inflammasome activation, and its mechanism is as follows: Doramectin targets the NLRP3 protein, affecting NLRP3 function, inhibiting NLRP3 inflammasome assembly, thereby suppressing NLRP3 inflammasome activation and subsequently inhibiting downstream inflammatory responses. At the animal level, Doramectin can improve the occurrence of NLRP3 inflammasome-related inflammatory diseases (such as sepsis and colitis). This invention provides a theoretical basis and experimental foundation for the use of Doramectin to treat NLRP3 inflammasome-related inflammatory diseases.

[0055] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. The use of Doramectin as the sole active ingredient in the preparation of a medicament for treating NLRP3 inflammasome-related inflammatory diseases, wherein the NLRP3 inflammasome-related inflammatory diseases are colitis.

2. The application according to claim 1, characterized in that, The Doramectin inhibits NLRP3 inflammasome activation.

3. The application according to claim 1, characterized in that, The Doramectin inhibits the interaction between NLRP3 and NEK7.

4. The application according to claim 1, characterized in that, The drug contains pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The drug is any one of tablets, granules, injections, or capsules.

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