USP9X protein allosteric activator T7150 and application thereof

Abstract

The invention provides a USP9X protein allosteric activator T7150 and application of the USP9X protein allosteric activator T7150. It is found for the first time that T7150 induces allosteric deubiquitination enzyme USP9X, enhances the activity of the deubiquitination enzyme USP9X, meanwhile, it is found that T7150 inhibits absorption of macrophages to ox-LDL and reduces formation of foam cells, it is indicated that T7150 can prevent formation of foam cells, and therefore T7150 can be used for inhibiting the progress of atherosclerotic plates. On the other hand, the invention also finds that the T7150 obviously reduces lipid deposition in atherosclerotic plaques and obviously reduces the lipid content in the plaques, which also shows that the T7150 can effectively inhibit the progress of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a USP9X protein allosteric activator, and specifically, to a USP9X protein allosteric activator T7150 and an application thereof. Background Art

[0002] Atherosclerosis is a chronic inflammatory disease characterized by the formation of atherosclerotic plaques on the arterial wall. Rupture of atherosclerotic plaques is the main cause of acute cardiovascular events, such as coronary artery disease (CAD), myocardial infarction and stroke. Macrophages are the main component of atherosclerotic plaques and play an important role in the development of atherosclerosis. During the formation of atherosclerotic plaques, monocytes migrate to the subintimal layer and differentiate into macrophages, which then phagocytize a large amount of oxidized low-density lipoproteins and secrete chemokines and adhesion factors to recruit more macrophages to lipid deposits on the inner wall of the blood vessel. Therefore, analyzing the specific mechanism of macrophage recruitment in the formation of atherosclerotic plaques has important scientific value and clinical significance.

[0003] The deubiquitinase USP9X is involved in regulating many cellular biological activities, such as endocytosis, immune response, and neurogenesis. In addition, USP9X is also involved in regulating the occurrence and development of diseases such as neurodegenerative diseases, neurodevelopmental disorders, and malignant tumors. However, the role of USP9X in macrophages in foam cell formation and atherosclerosis is still unclear. Therefore, screening antagonists or activators targeting USP9X protein and exploring its role in atherosclerosis is one of the important topics in this field. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a USP9X protein allosteric activator T7150 and applications thereof.

[0005] In the first aspect of the present invention, a use of a small molecule compound T7150 in the preparation of a USP9X protein allosteric activator is provided, wherein the structural formula of the small molecule compound T7150 is:

[0006]

[0007] In the second aspect of the present invention, there is provided a use of a small molecule compound T7150 in the preparation of a drug for inhibiting the absorption of ox-LDL by macrophages, wherein the structural formula of the small molecule compound T7150 is:

[0008]

[0009] In the third aspect of the present invention, there is provided a use of a small molecule compound T7150 in the preparation of a drug for preventing the formation of foam cells, wherein the structural formula of the small molecule compound T7150 is:

[0010]

[0011] In a fourth aspect of the present invention, there is provided a use of a small molecule compound T7150 in the preparation of a drug for preventing and / or treating atherosclerosis, wherein the structural formula of the small molecule compound T7150 is:

[0012]

[0013] Furthermore, the drug for preventing and / or treating atherosclerosis reduces lipid deposition in atherosclerotic plaques.

[0014] Furthermore, the drug for preventing and / or treating atherosclerosis reduces the lipid content in plaques.

[0015] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0016] Compared with the prior art, the present invention has the following advantages and progress:

[0017] The present invention provides a USP9X protein allosteric activator T7150 and its application. The present invention first discovered that T7150 induces the allosteric change of the deubiquitinase USP9X and enhances the deubiquitinase activity of USP9X. Specifically, according to the results of the macrophage Oil Red O staining experiment, the present invention discovered that T7150 inhibits the absorption of ox-LDL by macrophages and reduces the formation of foam cells, indicating that T7150 can prevent the formation of foam cells, and thus can be used to inhibit the progression of atherosclerotic plaques.

[0018] On the other hand, the present invention uses a mouse atherosclerosis model and finds that T7150 significantly reduces lipid deposition in atherosclerotic plaques based on the results of atherosclerotic plaque assessment. Specifically, the present invention finds that T7150 can significantly reduce the lipid content in plaques based on the results of Oil Red O staining in atherosclerotic plaque assessment, which also shows that T7150 can effectively inhibit the progression of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 : Chemical structure formula of small molecule T7150.

[0021] Figure 2 : Molecular docking diagram of USP9X and T7150 small molecules.

[0022] Figure 3 : Oil Red O staining was used to detect the effect of T7150 on the phagocytosis of oxLDL by bone marrow-derived macrophages (Means±SEM, *p<0.05; two-way ANOVA followed by Bonferroni post hoc test, n=6).

[0023] Figure 4 : Effects of T7150 on the progression of atherosclerotic plaques in mice (Means±SEM, *p<0.05; unpaired two-tailed t-test, n=6). DETAILED DESCRIPTION

[0024] The present invention provides USP9X protein allosteric activator T7150 and its application. The present invention is specifically described below in conjunction with the examples to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the examples, and they are carried out in accordance with the general technical conditions described in the literature of the field or the relevant product instructions.

[0025] Example

[0026] Part I Experimental Materials and Methods

[0027] 1. Isolation and culture of mouse bone marrow-derived macrophages (BMDM)

[0028] 1. Remove leg bones from mice

[0029] After anesthesia, the mice were killed by dislocation. The mice were placed in a beaker containing sufficient 75% ethanol and soaked for disinfection for 5 minutes. The soaked animals were used to absorb excess alcohol. A small cut was made on the back of the mouse with scissors, and the skin was directly torn open to the joint of the mouse's calf by hand to remove the mouse's foot joint and skin. The hind limbs were removed along the greater trochanter of the mouse's thigh root with scissors, and the muscle tissue was removed and placed in a culture dish containing 75% ethanol and soaked for 5 minutes. A new culture dish containing 75% ethanol was replaced and moved to the clean bench.

[0030] 2. BMDM cell extraction and induction

[0031] Transfer the leg bones soaked in ethanol to cold PBS to wash off the ethanol on the surface of the tibia and femur. This process can be repeated 3 times. Separate the cleaned femur and tibia, and cut the two ends of the femur and tibia with scissors. Use a 1mL syringe to absorb the cold induction culture medium to blow the bone marrow out of the femur and tibia, and blow and wash repeatedly 3 times until no obvious red color is seen in the leg bones. Use a 5mL pipette to repeatedly blow the culture medium containing bone marrow cells to disperse the cell clumps, then use a 70μm cell filter to sieve the cells, transfer them to a 15mL centrifuge tube, centrifuge at 1500rpm / min for 5min, discard the supernatant, add red blood cell lysis buffer to resuspend, let stand for 5min, and then centrifuge at 1500rpm / min for 5min, discard the supernatant, resuspend with cold prepared bone marrow macrophage induction culture medium, and plate. The culture medium is not changed during cell culture. Half of the bone marrow macrophage induction culture medium is replaced after the third day of culture, and all the culture medium is replaced on the fifth day. It can be used for subsequent experiments on the seventh day.

[0032] 2. Oil Red O Staining

[0033] Dissolve Oil Red O in isopropanol to make a 0.5% stock solution, mix the stock solution with double distilled water in a ratio of 3:2 to make Oil Red O working solution, filter and store at 4°C. Fix the cultured cell samples with 4% paraformaldehyde for 20 minutes. Then wash three times with PBS buffer, 5 minutes each time. Before staining, you can choose to rinse with 60% isopropanol for 30 seconds. Add Oil Red O staining solution and stain for 30 minutes in the dark. After staining, you can choose to rinse with 60% isopropanol to remove excess Oil Red O staining solution. Wash three times with PBS buffer and then take pictures or seal the slides with glycerol.

[0034] 3. Assessment of Atherosclerotic Plaques in Mice

[0035] Apoe- / - mice were given placebo (PBS) or T7150 10 mg / kg / 2d, and fed a high-fat diet for 4 weeks. Atherosclerosis markers were evaluated. First, the mice were weighed, and 300 μL of avertin was injected intraperitoneally for every 10 g of the mice's body weight to deeply anesthetize them; after anesthesia, the chest and abdomen of the mice were moistened with 75% alcohol to fully expose the mouse's abdominal cavity, and blood was collected from the right apex of the mouse using a 1 mL syringe soaked in sodium heparin. The extracted mouse blood was transferred to a 1.5 mL EP tube soaked in sodium heparin, centrifuged at 5000 rpm at room temperature for 10 minutes, and the upper plasma portion was taken to a new 1.5 mL EP tube, which was stored in a -80°C refrigerator for later use; the right auricle of the mouse was cut, and about 5 mL of PBS buffer was slowly perfused from its left ventricle; the mouse heart, liver, kidney and other tissues were removed and placed in a 2 mL EP tube, quickly placed in liquid nitrogen, and then stored in a 80°C refrigerator for later use; the mouse heart was embedded with an embedding agent (OCT), quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for cardiac outflow tract frozen sections.

[0036] Take out the frozen tissue sections at -80℃, and after the frozen sections return to room temperature, wash them with PBS three times, 5 minutes each time, to remove the OCT in the tissues; prepare fresh working solution of Oil Red O; soak the sections in the Oil Red O working solution for 30 minutes; wash off the floating color with ultrapure water, and wash the floating color with 60% isopropanol for 5 minutes; prepare 90% glycerol with PBS as a sealing agent for sealing; take pictures of the tissues with a Leica microscope, and analyze the area of ​​Oil Red O positive area with Image Pro Plus 6.0.

[0037] 4. Data Statistical Analysis

[0038] Cell and molecular experiments were independently repeated at least three times. Animal experiments were repeated at least six times. Data from repeated experiments were averaged and expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 5.0 software. Unpaired t-test was used to determine the differences between the two groups, and analysis of variance (ANOVA) was used for multiple group comparisons, followed by Bonferroni post hoc test to analyze the comparisons between groups. P < 0.05 was considered statistically significant.

[0039] Part II Experimental Results and Conclusions

[0040] 1. Molecular docking

[0041] The small molecule ligand (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea, BTdCPU, T7150, CAS No.: 1257423-87-2, Figure 1 )’s 2D structure, input the 2D structure into Chem Office 20.0 software to create its 3D structure, and save it as a mol2 file. Then, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen the protein target and the crystal structure with high resolution as the molecular docking receptor, and the protein was dehydrated, dephosphorylated, and saved as a PDB file using PyMOL 2.6.0 software. The Molecular Operating Environment 2019 software was used to minimize the energy of the compound, pre-treat the target protein, and search for active pockets. Finally, MOE 2019 was run for molecular docking, and the number of operations was set to 50. The binding activity of the two was evaluated according to the size of the binding energy, and the results were visualized using PyMOL 2.6.0 and Discovery studio 2019 software ( Figure 2). According to the binding results, from the perspective of molecular docking, T7150 has a high affinity with USP9X protein ( Figure 2 ).

[0042] II. Biological function experiments of T7150 as an allosteric activator of the deubiquitinating enzyme USP9X

[0043] T7150 small molecule (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea, CAS number: 1257423-87-2) was purchased from TargetMol, product number T7150.

[0044] Macrophage Oil Red O staining experiment divided BMDM into 4 groups, Control group, ox-LDL group, Control+PBS group and ox-LDL+T7150 group. The Control group and ox-LDL group were treated with PBS or ox-LDL for 24 hours, respectively. The Control+PBS group and ox-LDL+T7150 group were treated with 10μM T7150 for 1 hour, followed by PBS or ox-LD for 24 hours. According to the results of the macrophage Oil Red O staining experiment, T7150 inhibited the absorption of ox-LDL by macrophages and reduced the formation of foam cells ( Figure 3 ). This indicates that T7150 can prevent the formation of foam cells and thus be used to inhibit the progression of atherosclerotic plaques.

[0045] At the same time, using the mouse atherosclerosis model, it was found that T7150 significantly reduced lipid deposition in atherosclerotic plaques ( Figure 4 ). Specifically, the experiment divided Apoe- / - mice into two groups: PBS group and T7150 group (the two groups were given PBS or T7150 (100 mg / kg / 2d), respectively, and fed a high-fat diet for 4 weeks), and the atherosclerotic plaques were evaluated using the method described in the third part (assessment of atherosclerotic plaques in mice) of the above experimental materials and methods. According to the results of Oil Red O staining, it was found that T7150 can significantly reduce the lipid content in the plaques. The experimental results show that T7150 inhibits the progression of atherosclerosis.

[0046] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of a small molecule compound T7150 in the preparation of a USP9X protein allosteric activator, characterized in that: The structural formula of the small molecule compound T7150 is:

2. Use of a small molecule compound T7150 in the preparation of a drug for inhibiting the absorption of ox-LDL by macrophages, characterized in that: The structural formula of the small molecule compound T7150 is:

3. Use of a small molecule compound T7150 in the preparation of a drug for preventing foam cell formation, characterized in that: The structural formula of the small molecule compound T7150 is:

4. Use of a small molecule compound T7150 in the preparation of a drug for preventing and / or treating atherosclerosis, characterized in that: The structural formula of the small molecule compound T7150 is:

5. The use according to claim 4, characterized in that: The drug for preventing and / or treating atherosclerosis reduces lipid deposition in atherosclerotic plaques.

6. The use according to claim 4, characterized in that: The drug for preventing and / or treating atherosclerosis reduces the lipid content in plaques.

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