Application of MSC1094308 in preparation of medicine for preventing and / or treating inflammasome-related diseases

Abstract

The invention provides application of MSC1094308 in preparation of a medicine for preventing and / or treating inflammasome-related diseases, and belongs to the technical field of biological medicine preparation. The Vps4B ATPase inhibitor MSC1094308 can be used for remarkably reducing the activation of cysteine proteinase-1 (Caspase-1) and the interaction between ASC protein and Caspase-1 protein in a mouse bone marrow-derived macrophage (BMDM). Therefore, the invention proves that the MSC1094308 can effectively inhibit the activation of the inflammasome, thereby providing a new means for developing medicines for treating inflammasome-related diseases.

Description

Technical Field

[0001] This application belongs to the field of biomedical preparation technology, specifically relating to the application of MSC1094308 in the preparation of drugs for the prevention and / or treatment of inflammasome-related diseases. Background Technology

[0002] Inflammatory body activation plays a crucial role in host inflammation, resistance to pathogen invasion, and the development of autoimmune diseases. One classic inflammatory body is the NLRP3 inflammatory body, whose activation promotes the binding of apoptosis-associated speckle-like protein (ASC) to caspase-1 by recognizing different stimuli. This leads to the activation of cellular caspase-1, which in turn cleaves gastrointestinal dermatin D (GSDMD), ultimately resulting in pyroptosis. Abnormal inflammatory body activation is associated with the development of various diseases, and the research and development of drugs targeting inflammatory body activation is increasing.

[0003] MSC1094308 is a small molecule compound that reversibly and noncompetitively binds to the allosteric site of the Vps4B protein, inducing an unfavorable conformational change that disrupts the necessary cooperative ATP hydrolysis cycle and conformational rearrangement. There are currently no reports on the use of MSC1094308 to inhibit inflammasome activation. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for MSC1094308, specifically the application of MSC1094308 in the preparation of drugs that inhibit the activation of inflammasomes.

[0005] This invention provides the use of MSC1094308 in the preparation of inhibitors or drugs that inhibit the activation of inflammasomes.

[0006] This invention provides the use of MSC1094308 in the preparation of medicaments for the prevention and / or treatment of diseases related to inflammasome activation.

[0007] Preferably, the inflammasome is NLRP3.

[0008] Preferably, the inflammasome activation includes activation by exogenous molecules and / or endogenous molecules.

[0009] Preferably, the exogenous molecule includes at least one of the following: Gram-negative bacterial cell wall components, nucleic acids, adenosine triphosphate, and perforin; The endogenous molecules include Nigerian styracin and / or uric acid crystals.

[0010] Preferably, the activation of the inflammasome includes co-activation by lipopolysaccharide and adenosine triphosphate.

[0011] Preferably, the inflammasome-related diseases include at least one of the following inflammatory diseases: Alzheimer's disease, gout, type II diabetes, and systemic lupus erythematosus.

[0012] Preferably, the drug comprises oral and / or injectable formulations.

[0013] Preferably, the oral medication includes at least one of the following dosage forms: tablets, powders, capsules, and oral liquids.

[0014] Preferably, the injectable formulation includes injectable powder and / or injectable solution.

[0015] This invention provides the application of MSC1094308 in the preparation of inhibitors or drugs that inhibit inflammasome activation. The embodiments of this invention analyze the effects of MSC1094308 on NLRP3 inflammasome activation and the interaction between ASC protein and Caspase-1 protein in primary mouse BMDM cells. The results show that MSC1094308 can significantly inhibit inflammasome activation in mouse BMDM cells and significantly reduce the interaction between ASC protein and Caspase-1 protein. This invention is the first to demonstrate that the Vps4BATPase inhibitor MSC109430 can be used to inhibit inflammasome activation, indicating that MSC1094308 has potential pharmaceutical value in treating diseases with abnormal inflammasome activation (such as gout, type II diabetes, systemic lupus erythematosus, and other inflammatory diseases), and broadens the pharmaceutical applications of MSC1094308. Attached Figure Description

[0016] Figure 1 A comparison of pyroptosis in primary BMDM cells from mice in the MSC1094308 treatment group and the control group; Figure 2 Caspase-1 protein p20 and GSDMD protein GSDMD were activated in primary BMDM cells of mice in the MSC1094308 treatment group and the control group. NT A comparison diagram of the expressions; Figure 3 This is a comparison of the interaction between ASC protein and Caspase-1 protein in primary BMDM cells of mice in the MSC1094308 treatment group and the control group. Detailed Implementation

[0017] This invention provides the use of MSC1094308 in the preparation of inhibitors or drugs that inhibit the activation of inflammasomes.

[0018] In this invention, MSC1094308 is a non-competitive and reversible allosteric inhibitor of Vps4B, with the molecular formula C. 29 H29 F3N2, with the structural formula shown in Formula I, was purchased from MedChemExpress, part number HY-123872.

[0019] Formula I.

[0020] In this invention, the inflammasome preferably includes at least one of the following inflammasomes: NLRP3, AIM2, and NLRC. The activation of the inflammasome preferably includes activation by exogenous and / or endogenous molecules. The exogenous molecules preferably include at least one of the following: Gram-negative bacterial cell wall components, nucleic acids, adenosine triphosphate (ATP), and perforin. The Gram-negative bacterial cell wall components preferably include lipopolysaccharide (LPS). The endogenous molecules preferably include nigericin and / or uric acid crystals. The activation of the inflammasome preferably includes co-activation by LPS and ATP.

[0021] In this invention, lipopolysaccharide (LPS) combined with ATP is preferably used to activate the NLRP3 inflammasome in BMDM cells. In BMDM cells, the interaction between ASC protein and Caspase-1 protein is a key step in inflammasome activation. When cells are stimulated, inflammasome sensor proteins (such as NLRP3) are activated, subsequently recruiting ASC protein to form a multimeric complex. This complex further recruits Caspase-1, leading to Caspase-1 self-cleavage and activation. Activated Caspase-1 can cleave precursors IL-1β and IL-18, releasing mature inflammatory cytokines, thereby triggering an inflammatory response. In one embodiment of this invention, the working concentration of LPS during NLRP3 inflammasome activation is preferably 500 ng / mL. The final concentration of ATP is 5 mM. After treatment of the NLRP3 inflammasome with LPS combined with ATP, macrophages exhibit pyroptosis, and the expression levels of p20 and GSDMD are significantly increased. NT The expression of both proteins significantly increased, indicating successful activation of the NLRP3 inflammasome. Simultaneously, MSC1094308 significantly inhibited the interaction between ASC and Caspase-1 proteins. This suggests that MSC1094308 can preferentially inhibit inflammasome activation and reduce the inflammatory response.

[0022] This invention provides the use of MSC1094308 in the preparation of medicaments for the prevention and / or treatment of diseases related to inflammasome activation.

[0023] In this invention, the inflammasome-related diseases preferably include at least one of the following inflammatory diseases: Alzheimer's disease, gout, type II diabetes, and systemic lupus erythematosus. In the brains of Alzheimer's patients, immune cells—primarily microglia and astrocytes—are overactivated under this pathological environment, releasing large amounts of pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6), triggering a chronic inflammatory response. This leads to neuronal damage, synaptic dysfunction, and accelerates the pathological progression of Aβ plaques and Tau protein tangles. Significant progress has been made in research on treating Alzheimer's disease by reducing the inflammatory response. The subjects of the disease preferably include animals and humans. The animals preferably include rodents, dogs, etc.

[0024] In this invention, the drug preferably comprises an oral dosage form and / or an injectable formulation. The oral dosage form includes at least one of the following dosage forms: tablets, powders, capsules, and oral liquids. The injectable formulation preferably comprises injectable powders and / or injectable solutions. The drug also preferably includes pharmaceutically acceptable excipients. The type of excipient is selected according to different dosage forms to prepare the drug. This invention does not impose any particular limitation on the preparation method of the drug; any drug preparation method well known in the art can be used.

[0025] The following detailed description, in conjunction with embodiments, illustrates the application of MSC1094308 provided by the present invention in the preparation of medicaments for the prevention and / or treatment of inflammasome-related diseases, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 Effects of MSC1094308 on NLRP3-activated inflammasomes in primary mouse BMDM cells.

[0027] 1. Preparation method of primary mouse BMDM cells Adult C57 / BL mice of appropriate age (e.g., 4-6 weeks old) were euthanized. The femur and tibia were isolated under aseptic conditions and placed in PBS buffer. The muscle, cartilage, and epiphysis on the bone surface were scraped off with a scalpel. The two ends of the bone were cut to expose the medullary cavity. The medullary cavity was flushed with DMEM-F12 medium using a 1 mL syringe. Cells were collected, and after being pipetted and homogenized, the cells were counted and the cell density was adjusted to 1 Million / mL. 20 ng / mL of M-CSF growth factor was added, and the cells were seeded in cell culture plates and cultured in a CO2 incubator at 37°C with 5% CO2. After 5 days of static culture, adherent cells were harvested for the corresponding experiments.

[0028] 2. Activation of inflammasomes The preparation method for the drug used to treat MSC1094308 cells in the experimental group was as follows: MSC1094308 was prepared into a 10 mM stock solution using dimethyl sulfoxide (DMSO), and then diluted to a working concentration of 10 μM using DMEM-F12 complete medium before cell treatment. The control group was treated with 0.1% DMSO. The treatment time for both the experimental and control groups was 2 h, followed by subsequent inflammasome stimulation and detection experiments.

[0029] The inflammasome stimulant is lipopolysaccharide (LPS) + ATP, which activates the NLRP3 inflammasome.

[0030] The specific stimulation method was as follows: the NLRP3 inflammasome was stimulated with LPS at a final concentration of 500 ng / mL for 4 h, followed by stimulation with ATP at a final concentration of 5 mM for 60 min, thereby activating the NLRP3 inflammasome. Culture supernatant without added stimulants (Media) was used as a 0 h control group.

[0031] Western blotting was used to detect the expression levels of caspase-1 activation cleavage product p20 in culture supernatant, as well as the expression levels of caspase-1 precursor and GSDMD protein and its activator GSDMD in cell lysate. NT The expression.

[0032] The results showed that, in primary mouse BMDM cells, compared with the DMSO control group, the MSC1094308-treated group had significantly fewer cells exhibiting pyroptosis after LPS+ATP treatment, as indicated by optical microscopy. Figure 1 Western blot analysis showed that LPS+ATP treatment increased the expression of activated caspase-1 p20 and GSDMD. NT The expression of all of them decreased significantly ( Figure 2 In the diagram, the Media group represents the control group of culture supernatant without added stimulants, L+A represents lipopolysaccharide (LPS) + ATP, and casp1 represents caspase-1. These results indicate that MSC1094308 can significantly inhibit the activation of the NLRP3 inflammasome and reduce cell death in mouse BMDM cells.

[0033] Example 2 Effects of MSC1094308 on the interaction between ASC protein and Caspase-1 protein in primary mouse BMDM cells.

[0034] 1. Preparation method of primary mouse BMDM cells For details on the culture method of primary mouse BMDM cells, please refer to Example 1.

[0035] 2. Activation of inflammasomes For the preparation method and concentration of MSC1094308, please refer to Example 1. The inflammasome stimulant is lipopolysaccharide (LPS) + ATP, which activates the NLRP3 inflammasome.

[0036] The specific stimulation method was as follows: the NLRP3 inflammasome was stimulated with LPS at a final concentration of 500 ng / mL for 4 h, followed by stimulation with ATP at a final concentration of 5 mM for 20 min to activate the NLRP3 inflammasome. Culture supernatant without added stimulants (Media) was used as a 0 h control group.

[0037] 3. Co-immunoprecipitation assay (co-IP) The interaction between ASC protein and Caspase-1 protein was detected using co-immunoprecipitation (co-IP). Specific experimental procedures were as follows: After lysing cells, total protein was obtained, and the lysis supernatant was collected by centrifugation. An appropriate amount of Protein A / G magnetic beads (Thermoscientific, 88802) was washed with lysis buffer and then incubated overnight at 4°C with the protein sample and antibody (anti-ASC: AdipoGen, AG-25B-0006) to allow the target protein and its interacting proteins to bind to the magnetic beads. The magnetic beads were washed 3-5 times with pre-chilled lysis buffer to remove non-specific binding. Loading buffer was added, and the mixture was boiled to elute the protein. SDS-PAGE and Western blotting analyses were performed to detect the target protein and potential interacting proteins.

[0038] The results showed that after LPS+ATP stimulation of mouse BMDM cells, the binding of ASC protein to Caspase-1 protein was significantly reduced in the MSC1094308 treatment group. Figure 3 This indicates that MSC1094308 can significantly inhibit the interaction between ASC protein and Caspase-1 protein.

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of MSC1094308 in the preparation of inhibitors or drugs that inhibit the activation of inflammasomes.

2. Application of MSC1094308 in the preparation of drugs for the prevention and / or treatment of diseases related to inflammasome activation.

3. The application according to claim 1 or 2, characterized in that, The inflammasome is NLRP3.

4. The application according to claim 1 or 2, characterized in that, The activation of the inflammasome includes activation by exogenous and / or endogenous molecules.

5. The application according to claim 4, characterized in that, The exogenous molecules include at least one of the following: Gram-negative bacterial cell wall components, nucleic acids, adenosine triphosphate, and perforin; The endogenous molecules include Nigerian styracin and / or uric acid crystals.

6. The application according to claim 5, characterized in that, The activation of the inflammasome includes co-activation by lipopolysaccharide and adenosine triphosphate.

7. The application according to claim 2, characterized in that, The inflammasome-related diseases include at least one of the following inflammatory diseases: Alzheimer's disease, gout, type II diabetes, and systemic lupus erythematosus.

8. The application according to claim 1 or 2, characterized in that, The drugs include oral and / or injectable formulations.

9. The application according to claim 8, characterized in that, The oral medication includes at least one of the following dosage forms: tablets, powders, capsules, and oral liquids.

10. The application according to claim 8, characterized in that, The injectable formulation includes injectable powder and / or injectable solution.

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