CN111358793B

CN111358793B - Novel application of HUWE1 inhibitor BI8622

Info

Publication number: CN111358793B
Application number: CN202010176486.XA
Authority: CN
Inventors: 齐晓朋; 徐涛; 李艺辉; 郭晓敏
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2021-08-10
Anticipated expiration: 2040-03-13
Also published as: CN111358793A; WO2021179396A1

Abstract

The invention provides a new application of HUWE1 inhibitor BI8622 in inhibiting activation of inflammasome, and researches show that: the HUWE1 inhibitor BI8622 can remarkably reduce the activation of cysteine protease-1 (Caspase-1) and the expression of interleukin 1 beta (IL-1 beta) in mouse bone marrow-derived macrophages (BMDM), and reduce the occurrence of cell death events; meanwhile, the BI8622 is proved to be capable of remarkably reducing the activation of Caspase-1 and the expression of IL-1 beta and remarkably reducing the cell death in a human monocyte/macrophage line THP1 and human primary Peripheral Blood Mononuclear Cells (PBMC), so that the BI8622 can remarkably inhibit the activation of inflammatory corpuscles.

Description

Novel application of HUWE1 inhibitor BI8622

Technical Field

The invention relates to the technical field of medicines, and in particular relates to a new application of HUWE1 inhibitor BI 8622.

Background

Activation of the inflammasome plays an important role in the development of inflammation, resistance to pathogen invasion, and development of autoimmune diseases in the host. Classical inflammatory bodies include NLRP3, AIM2 and NLRC4 inflammatory bodies, which are activated by recognition of different stimuli, respectively, and ultimately all result in the activation of cellular cysteine protease-1 (Caspase-1), thereby inducing the production of mature interleukin 1 beta (IL-1 beta), interleukin 18(IL-18), activated Caspase-1 cleavable gut dermatan D (GSDMD), leading to the occurrence of cellular apoptosis (Inflamamosomes: mechanism of action, role in disease, and thermoeutics Haitao Guo, Justin B Callaway & Jenny P-Y Ting; Nature medicinal volume 21, pages 677-687 (2015)). Abnormal activation of inflammasome is associated with the development of a variety of diseases, and more drugs are developed by targeting activation of inflammasome.

HUWE1 inhibitor BI8622 is a class of small molecule compounds having the following structural formula:

BI8622 is obtained by screening by Stefanie Peter et al, can specifically inhibit ubiquitin ligase HUWE1 through ubiquitination modification function, and finds that the inhibitor has a certain inhibiting effect on tumorigenesis and development (Peter S, Bumultick J, Myant K, Jaenicke LA, Walz S, Muller J, et al. Tumor cell-specific inhibition of MYC functional using small molecular inhibitors of the HUWE1ubiquitin ligand. EMBO Mol. Med. 2014; 6: 5-.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of HUWE1 inhibitor BI8622 in preparing a medicine for inhibiting activation of inflammatory corpuscles.

The chemical structural formula of BI8622 of the invention is as follows:

in the present invention, the inflammasome includes NLRP3, AIM2 and NLRC 4.

In the invention, the NLRP3 inflammasome is activated by lipopolysaccharide and adenosine triphosphate together, the AIM2 inflammasome is activated by Francisella or dsDNA, the NLRC4 inflammasome is activated by Salmonella, and the NLRP3 and AIM2 inflammasome are simultaneously activated by Listeria.

The invention provides a medicament for inhibiting activation of an inflammasome, which comprises an active ingredient BI8622 and a pharmaceutically acceptable carrier.

The invention provides application of HUWE1 inhibitor BI8622 in preparation of medicines for treating gout, type II diabetes mellitus or inflammatory diseases of systemic lupus erythematosus.

The invention provides a medicament for treating inflammatory diseases such as gout, type II diabetes mellitus or systemic lupus erythematosus, which comprises an active ingredient BI8622 and a pharmaceutically acceptable carrier.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

according to the invention, the BI8622 has effects on activated NLRP3, AIM2 and NLRC4 inflammatory corpuscles and cell death on mouse primary BMDM cells, human monocyte macrophage line THP1 cells and human primary PBMC cells respectively, and the results show that the BI8622 can remarkably inhibit the activation of the inflammatory corpuscles and remarkably reduce the occurrence of cell death events in the mouse BMDM cells, the human monocyte macrophage line THP1 cells and the human primary PBMC cells.

The invention proves the new application of HUWE1 inhibitor BI8622 for the first time: can be used for inhibiting and verifying activation of corpuscles, and shows that BI8622 has potential medicinal value in treating inflammatory corpuscle abnormal activation diseases (such as gout, II type diabetes, systemic lupus erythematosus and other inflammatory diseases).

Drawings

FIG. 1 is a graph comparing the expression of activated caspase 1P20 in mouse primary BMDM cells after treatment by BI8622 treatment and control;

FIG. 2 is a graph comparing IL-1 β expression levels in mouse primary BMDM cells after treatment with BI8622 treatment and control;

FIG. 3 is a graph comparing TNF expression levels in mouse primary BMDM cells after treatment with BI8622 and a control;

FIG. 4 is a graph comparing IL-6 expression levels in mouse primary BMDM cells after treatment with BI8622 treatment and control;

FIG. 5 is a graph comparing the rate of cell death in primary mouse BMDM after treatment with BI8622 and a control;

FIG. 6 is a graph comparing the IL-1. beta. expression levels of human THP1 cells after treatment with BI8622 and a control group;

FIG. 7 is a graph comparing TNF expression levels in human THP1 cells after treatment with BI8622 and a control;

FIG. 8 is a graph comparing the IL-6 expression levels of human THP1 cells after treatment with BI8622 and a control group;

FIG. 9 is a graph comparing the rate of human THP1 cell death after treatment with BI8622 and a control;

FIG. 10 is a graph comparing the expression levels of IL-1 β in human primary PBMC cells after treatment with BI8622 and a control group;

FIG. 11 is a graph comparing TNF expression levels in human primary PBMC cells after treatment with BI8622 and a control group;

FIG. 12 is a graph comparing the expression levels of IL-6 in human primary PBMC cells after treatment with BI8622 and a control group;

FIG. 13 is a graph comparing the proportion of human primary PBMC cell death following treatment with BI8622 and control.

Detailed Description

In a specific embodiment of the present invention, healthy C57/BL mice were obtained from kunming animal research institute of chinese academy of sciences [ experimental animal license number: SYXK (Dian) K2013-0013 ].

The HUWE1 inhibitor BI8622 of the present invention was purchased from MedChemexpress, Inc. under the designation HY-120929.

The human monocyte-macrophage line THP1 used in the present invention is derived from Kunming animal research institute cell bank, Chinese academy of sciences, and other monocyte-macrophage cells in the art can also be used.

NLRP3 inflammasome is typically activated by exogenous molecules such as gram negative bacterial cell wall components, nucleic acids, perforin, endogenous molecules such as Adenosine Triphosphate (ATP), uric acid crystals, etc. (case s.l., Eisenbarth s.c., i yer s.s., et al, The nap 3 fluorescence is an infection for The development of silicon, proc.natl.acad.sci.usa,2008,105(26):9035-9040. kannegatroni t.d., Body-Malapel m, Amer a., 2006, critical roll for Cryopropyrin/nap 3 in activation of pase-1 response to viral infection and 19. biological chemical rna.281 j.281, 3648).

The NLRC4 inflammasome is activated by Salmonella (Salmonella) infection (Mariatasan S., Newton K., Monack D.M., et al. differential activation of the inflamasome by caspase-1adapt ASC and Ipaf. Nature,2004,430(6996): 213-218.).

AIM2 inflammasome is activated by Francisella and double-stranded DNA (dsDNA) induction (Fernandes-Alnemri T., Yu J. -W., Juliana C., et al, The AIM2 inflamasome is criterial for in not natured to Francisella tularensis. Nat. Immunol.2010,11:385-393. Brckst. hammer T., Baumann C., Blu ml S., et al, an organic proteinaceous-genetic materials AIM2 a cytoplasmic DNA sensor for The in not natrumame. Immunol.2009,10: 272).

In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

BI8622 was tested for the effect of three activated inflammasome and cell death in mouse primary BMDM cells NLRP3, AIM2, NLRC 4.

The material and the method are as follows:

preparation of primary mouse BMDM cells: euthanizing adult (e.g. 4-6 week old) C57/BL mice, separating the femur and tibia under sterile conditions and placing them in PBS buffer, scraping off the muscle, cartilage and epiphysis on the bone surface with a scalpel, cutting the bone to expose the medullary cavity, flushing the medullary cavity with 1mL DMEM-F12 medium aspirated by a 1mL syringe, collecting cells, uniformly blowing the cells, counting and adjusting the cell density to 1-2Million/mL, adding 20ng/mL M-CSF growth factor to the DMEM-F12 complete medium, plating the cells on a cell culture plate, inoculating the cells in 5% CO 5%₂37 ℃ CO₂Culturing in an incubator, and collecting adherent cells for corresponding experiments after 5 days of static culture.

The test compound BI8622 is prepared into a stock solution with the concentration of 10mM by using dimethyl sulfoxide (DMSO), the cell is diluted to a working concentration of 10uM by using DMEM-F12 complete culture medium before being treated, a control group is treated by using 0.1% DMSO, the treatment time of the test group BI8622 and the control group is 3 hours, and then subsequent inflammatory corpuscle stimulant stimulation and detection experiments are carried out.

The inflammasome stimulators are: 1) lipopolysaccharide (LPS) + Adenosine Triphosphate (ATP) that activates NLRP3 inflammasome; 2) francisella (f. novicida) and double-stranded dna (dsdna) that activate AIM2 inflammasome; 3) salmonella (Salmonella) which activates NLRC4 inflammasome; 4) listeria (Listeria) can activate both NLRP3 and AIM2 inflammasome.

The specific stimulation mode is as follows: stimulating with LPS with a final concentration of 500ng/mL for 1h and ATP with a final concentration of 5mM for 15min, thereby activating NLRP3 inflammasome; activating AIM2 inflammasome by stimulating dsDNA transfected with 1.5ug X-fect for 1h or f.novicida at 200MOI for 12 h; stimulating with Salmonella at a final concentration of 3MOI for 2h, thereby activating NLRC4 inflammasome; stimulation with Listeria at a final concentration of 30MOI for 3h activated NLRP3 and AIM2 inflammasome simultaneously.

Meanwhile, culture supernatant (medium) without added stimulus was selected as a 0h control group.

Detecting the expression level of a cysteine protease-1 (Caspase-1) activated cleavage product P20 in culture supernatant and the expression level of a Caspase-1 precursor in cell lysate by using western blot; simultaneously detecting the expression levels of interleukin 1 beta (IL-1 beta), Tumor Necrosis Factor (TNF) and interleukin 6(IL-6) by using an enzyme-linked immunosorbent assay (ELISA); cell death detection (LDH method) was performed by measuring the level of Lactate Dehydrogenase (LDH) secretion in the culture supernatant.

The results showed that in mouse primary BMDM cells, BI8622 treated group cells showed significantly reduced expression of activated caspase-1P20 after LPS + ATP, dsDNA, f. novicida, Salmonella, and Listeria stimulation treatments compared to DMSO control group cells (fig. 1, where Med group represents culture supernatant control without added stimulus and caspase1 represents caspase-1). ELISA test results show that after LPS + ATP, F.novicida, Salmonella and Listeria stimulation treatment, IL-1 beta secretion of cells in BI8622 treated group is obviously lower than that of cells in control group, IL-1 beta expression level of cells in dsDNA treated group is lower, and no difference exists between two groups of cells (FIG. 2); the expression level of TNF was not significantly different in both groups of cells in the above treatment regimes (FIG. 3); IL-6 expression was not significantly different in cells from the other stimulation treated groups except that BI8622 treated group was reduced compared to the control group after f.novicida, Salmonella stimulation (fig. 4). LDH assay results indicated that cell death was lower in BI8622 treated group than in DMSO control group cells in all of the above stimulation regimens (FIG. 5). The above results show that BI8622 can significantly inhibit the activation of NLRP3, AIM2 and NLRC4 inflammasome in mouse BMDM cells, and reduce cell death.

Example 2

BI8622 was tested for its effect on three inflammasome and cell death activated in human THP1 cells, NLRP3, AIM2, NLRC 4.

The THP1 cell is human monocyte macrophage, the culture condition is that 1640 culture medium is added with 10% FBS, and the specific culture method is according to the conventional method. The test compound BI8622 is prepared into a stock solution with the concentration of 10mM by using dimethyl sulfoxide (DMSO), the stock solution is diluted into a working concentration of 10uM by using 1640 complete culture medium before treating cells, a control group is treated by using 0.1% DMSO, the treatment time of the test group BI8622 and the control group is 3 hours, and then subsequent inflammatory corpuscle stimulant stimulation and detection experiments are carried out.

The inflammasome stimulators are: 1) lipopolysaccharide (LPS) + Adenosine Triphosphate (ATP) that activates NLRP3 inflammasome; 2) double stranded dna (dsdna) that activates AIM2 inflammasome; 3) salmonella (Salmonella) which activates the NLRC4 inflammasome.

The specific stimulation mode is as follows: stimulating with LPS with a final concentration of 500ng/mL for 1h and ATP with a final concentration of 5mM for 15min, thereby activating NLRP3 inflammasome; the dsDNA transfection was stimulated for 1h with a final concentration of 1.5ug X-fect, activating AIM2 inflammasome; stimulation with Salmonella at a final concentration of 3MOI for 2h activated NLRC4 inflammasome.

ELISA method is used for detecting the expression of IL-1 beta, TNF and IL-6, and LDH method is used for detecting the cell death.

The results showed that after THP1 cells were treated with LPS + ATP, dsDNA, Salmonella stimulation, BI 8622-treated cells secreted significantly less IL-1 β than control cells (fig. 6); BI8622 treated cells also showed lower expression levels of TNF and IL-6 than control cells (FIG. 7); the expression level of TNF in the cells did not differ significantly in any of the above-described treatment modalities (fig. 8); LDH assay also revealed that BI 8622-treated THP1 cells died significantly less after LPS + ATP, dsDNA, Salmonella treatment than did control cells (FIG. 9). The results show that, similar to mouse BMDM cells, BI8622 can remarkably inhibit the activation of NLRP3, AIM2 and NLRC4 inflammasome in human THP1 cells, and can obviously reduce the death of the cells.

Example 3

BI8622 was tested for the effect of three inflammasome and cell death activated in human primary PBMC cells NLRP3, AIM2, NLRC 4.

Peripheral Blood Mononuclear Cells (PBMCs) were obtained by density gradient centrifugation of 10mL of peripheral blood taken from normal donors [ approved by the ethical committee of life science of kunming institute of science and technology, code: SMKX-20191201-01 ]. The PBMC cells obtained by separation are cultured in 1640 medium + 10% FBS, and the specific culture method can be according to the conventional method. The test compound BI8622 is prepared into a stock solution with the concentration of 10mM by using dimethyl sulfoxide (DMSO), the stock solution is diluted into a working concentration of 10uM by using 1640 complete culture medium before treating cells, a control group is treated by using 0.1% DMSO, the treatment time of the test group BI8622 and the control group is 3 hours, and then subsequent inflammatory corpuscle stimulant stimulation and detection experiments are carried out. The stimulus and the specific stimulation mode were specifically selected in example 2, and the culture supernatant (medium) without the stimulus was used as a 0h control group. Expression of IL 1-beta, TNF and IL-6 was measured by ELISA and cell death was measured by LDH.

The results showed that after PBMC cells were stimulated with LPS + ATP, dsDNA, Salmonella, the secretion of IL-1. beta. was significantly lower in BI 8622-treated cells than in control cells (FIG. 10); expression of TNF in cells of BI8622 treated group was lower than control PBMC cells after LPS + ATP and Salmonella stimulation, there was no significant difference in TNF expression in cells of both groups stimulated by dsDNA (fig. 11); IL-6 expression levels in PBMCs from BI8622 treated groups were significantly lower after Salmonella stimulation than in control groups, but not significantly different after LPS + ATP and dsDNA stimulation (FIG. 12). LDH detection also found that cellular LDH secretion levels were significantly lower in the BI 8622-treated human primary PBMCs after LPS + ATP, dsDNA, Salmonella treatment than in the control PBMCs, suggesting that the BI 8622-treated human primary PBMCs died significantly lower than in the control (fig. 13). The above results show that BI8622 can significantly inhibit the activation of NLRP3, AIM2 and NLRC4 inflammasome in human primary PBMC, and can significantly reduce cell death.

From the results in the examples, the present invention demonstrates for the first time the novel use of HUWE1 inhibitor BI 8622: BI8622 can both significantly inhibit the activation of inflammatory bodies and significantly reduce the occurrence of cell death events.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Use of an HUWE1 inhibitor BI8622 for the manufacture of a medicament for the treatment of inflammatory diseases such as gout, type II diabetes, or systemic lupus erythematosus, wherein the HUWE1 inhibitor BI8622 inhibits the activation of inflammasome, said inflammasome being NLRP3, AIM2, and NLRC 4.
2. The use of claim 1, wherein said BI8622 has the following chemical structure:
3. the use of claim 1, wherein said NLRP3 inflammasome is activated by both lipopolysaccharide and adenosine triphosphate, said AIM2 inflammasome is activated by francisella or dsDNA, said NLRC4 inflammasome is activated by salmonella, and said NLRP3 and AIM2 inflammasome are simultaneously activated by listeria.