Application of a small molecule compound dBET1 in the treatment of psoriasis

Abstract

The present invention discloses that a small molecule compound dBET1 can play a role in the treatment of psoriasis by inhibiting the expression of TNF receptor-associated factor 1 (TRAF1) in macrophages, belonging to the field of biotechnology. By screening out a small molecule compound that inhibits the expression of TRAF1, it is verified on macrophages that this compound can inhibit the expression of TRAF1, and when this compound acts on the skin of mice, it can relieve the skin lesions of psoriasis, which has important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of dBET1. Specifically, it relates to the application of dBET1 in the treatment of psoriasis. Background Art

[0002] Psoriasis is a chronic, autoimmune skin disease, and its onset is related to multiple factors, including genetic and environmental factors. In addition to skin involvement, psoriasis can also affect joints and other parts, bringing great harm to the health of patients. TRAF1 is a signaling ligand that can negatively regulate TNFR2 signaling. Studies have shown that TRAF1 is associated with various diseases and can exacerbate lung cancer and skin cancer. It is highly expressed in patients with rheumatoid arthritis and is considered to be related to the severity of the disease. However, there is no study on the role of TRAF1 in cutaneous psoriasis. dBET1 is a PROTAC composed of a Cereblon ligand and a BRD4 ligand. Previous studies have shown that it plays a role in the treatment of ischemic brain injury and neoplastic diseases such as acute myeloid leukemia and colorectal cancer, but there is no exploration on its relationship with TRAF1 and the treatment of psoriasis. Summary of the Invention

[0003] In view of the above problems, the object of the present invention is to provide a new small molecule drug that can target TRAF1 and play a role in the treatment of psoriasis.

[0004] To achieve the above object, the present invention provides the following technical solutions.

[0005] The present invention provides the use of a small molecule compound dBET1 and its pharmaceutically acceptable salts in the preparation of a drug for treating psoriasis. The structural formula of the small molecule compound dBET1 is:

[0006] 。

[0007] Further, the drug comprises an effective dose of the small molecule compound dBET1 and a pharmaceutically acceptable carrier or excipient.

[0008] Further, the dosage form of the drug includes tablets, capsules, granules, pills, powders, powders for external use, ointments, suspensions, oral liquids or injections.

[0009] Further, the drug plays a role in the treatment of psoriasis by inhibiting the expression of TNF receptor-associated factor 1 in macrophages.

[0010] The present invention also provides the use of a small molecule compound dBET1 and its pharmaceutically acceptable salts in the preparation of a reagent for inhibiting the expression of TNF receptor-associated factor 1. The small molecule compound is dBET1, and its structural formula is:

[0011] 。

[0012] Furthermore, the reagent for inhibiting the expression of TNF receptor-associated factor 1 comprises an effective dose of the small molecule compound dBET1 and a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the dosage forms of the reagent for inhibiting the expression of TNF receptor-associated factor 1 include tablets, capsules, granules, pills, powders, powders for external use, ointments, suspensions, oral liquids or injections.

[0014] The beneficial effects of the present invention compared with the prior art.

[0015] The present invention for the first time discovers that the small molecule compound dBET1 can treat psoriasis, and this therapeutic effect exerts its function by inhibiting the expression of TNF receptor-associated factor 1, providing a new method for the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a molecular docking diagram of TRAF1 and dBET1.

[0017] Figure 2 It is the expression of TRAF1 in macrophages. D5 represents a dBET1 concentration of 5 nM, D50 represents a dBET1 concentration of 50 nM, and D500 represents a dBET1 concentration of 500 nM.

[0018] Figure 3 It is the establishment of a psoriasis model in mice with imiquimod, and the skin lesions of psoriasis after dBET1 acts on the skin of mice and imiquimod acts for 7 days. Among them, A is the process of model establishment; B is the comparison diagram of dBET1 application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] In order to more clearly understand the above objects, features and advantages of the present invention, the following further describes the present invention with specific embodiments. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the present invention is not limited by the specific embodiments disclosed in the following specification.

[0020] 1. Experimental methods.

[0021] 1. Molecular docking: Use the Protein Preparation Wizard module in Schrödinger software to optimize the bond order, add hydrogen, assign disulfide bonds to the structure of the MATH domain of TRAF1_MOUSE protein, and perform protonation at pH 7.0 using the PROPKA method; perform restrained energy optimization with the OPLS4 force field to eliminate atomic conflicts in the structure, converge the RMSD of heavy atoms to 0.3 Å, and perform side-chain position optimization to obtain a good side-chain structure. Perform molecular docking using the HTVS and SP modes.

[0022] 2. Cells: RAW264.7 macrophages (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in high-glucose DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics at 37 °C and 5% CO 2 in an incubator. When the cell density reached 70 - 90%, trypsin was used for digestion at 37 °C, and the cells were passaged at a ratio of 1:4 or plated according to the corresponding required number for subsequent experiments. Dbet1 (purchased from TargetMol) was dissolved in DMSO and added to the cells at concentrations of 5 nM, 50 nM, and 500 nM, and then cultured for 12 hours for subsequent experiments.

[0023] 3. Protein extraction and Western blotting.

[0024] Add 60 μL of freshly prepared RIPA lysis buffer to each well of a six-well plate, which contains 47.4 μL of RIPA (strong) lysis buffer, 6 μL of protease inhibitor, 6 μL of protein phosphatase inhibitor, and 0.6 μL of PMSF. Lyse on ice for 10 minutes, then scrape the sample into an EP tube with a pre-chilled cell scraper. Then centrifuge at 4 °C and 15,000 g for 15 minutes, carefully aspirate the supernatant, and transfer it into a new pre-chilled 1.5 mL EP tube to obtain the protein sample. The protein sample was quantified using the BCA standard curve method, and after adjusting the concentration, it was aliquoted and frozen at -80 °C. Use the Bio-Rad SDS-PAGE gel preparation kit to prepare the gel for Western blot detection. Specifically, for each gel: Mix the separating gel solution A and B, and the stacking gel solution A and B at a ratio of 1:1 (6 mL of separating gel and 2 mL of stacking gel in total). Add 6 μL and 4 μL of TEMED to the separating gel and stacking gel respectively, mix well, and place it in a cool and dark place to let the air out for 10 minutes. During this period, assemble the relevant equipment for making vertical gels and set it aside. After the air is exhausted, add 30 μL and 10 μL of 30% ammonium persulfate to the separating gel and stacking gel respectively, gently pipette and mix well, and then immediately pour the gels into the gel glass tank in sequence (separating gel at the bottom, stacking gel on top). Then slowly insert a 1.0 mm sample comb, let it stand at room temperature for 30 minutes, and after the gel is completely solidified, remove the glass plate, rinse the residual gel on the surface with tap water, and soak it in deionized water for storage at 4 °C for later use.

[0025] Install a vertical electrophoresis device and an SDS-PAGE gel. Pour 1 L of freshly prepared electrophoresis buffer (14.4 g glycine, 3.03 g Tris, 1 g SDS, made up to 1000 mL with deionized water) into the electrophoresis tank. Carefully remove the sample comb, and gently pipette the sample wells to expel air bubbles and residual gel. Mix the protein solution extracted from the above experiment with 5×SDS loading buffer (4:1), and heat at 95 °C for 10 minutes to denature the protein. Then load the samples at 20 μg of protein per sample well, and simultaneously add 10 μL of prestained protein molecular weight marker protein, and electrophorese at a constant voltage of 200 V for 60 minutes. After the protein samples are fully separated, remove the vertical electrophoresis tank, cut the SDS-PAGE gel, and transfer it to the protein transfer tank. The installation order of the transfer tank (from bottom to top) is: sponge pad - thickened filter paper - gel - PVDF membrane (0.45 μm, pre-activated with methanol) - thickened filter paper - sponge pad. Pour 1 L of freshly prepared electrotransfer buffer (14.4 g glycine, 3.03 g Tris, 200 mL methanol, made up to 1000 mL with deionized water) into the transfer tank, and place the transfer tank in ice, and transfer at a constant current of 300 mA for 1 hour.

[0026] After the transfer is completed, remove the transfer device, cut the PVDF membrane according to the molecular weight of the protein to be detected, rinse with TBST, and block with 5% skim milk at room temperature for 2 hours. After blocking, rinse the PVDF membrane with TBST, add the primary antibody prepared with 5% skim milk to the corresponding PVDF membrane, and incubate on a horizontal shaker at 4 °C with slow shaking overnight. On the second day, recover the primary antibody, rinse the PVDF membrane 3 times with TBST, 10 minutes each time. Then add the secondary antibody prepared with 5% skim milk, incubate with shaking at room temperature for 2 hours, rinse the PVDF membrane 3 times with TBST, 10 minutes each time. After washing, soak the PVDF membrane in a new TBST solution for later use.

[0027] Mix equal proportions of ECL luminescent solution A and B to prepare ECL developing solution and keep it in the dark for standby. Take out the PVDF membrane, blot it dry with absorbent paper, and place it in the gel imager. Drop a little ECL developing solution on the surface of the PVDF membrane so that the membrane is just wetted by the developing solution. Change the imager to chemiluminescence (CL) mode, expose to observe the results, and judge the corresponding protein expression level.

[0028] 4. Mouse model: Shave the back skin of 6-8-week-old C57BL / 6 mice, with an area of 2 cm * 3 cm. Apply 62.5 mg of imiquimod cream to the back skin of the mice and apply it continuously for 7 days. Among them, apply 50 μL of a total of 1 mg of dBET1 to the back on the 2nd and 4th days of modeling.

[0029] II. Experimental results.

[0030] AsFigure 1 As shown, it is the molecular docking diagram of TRAF1 and dBET1. The carbon atoms of dBET1 are shown in magenta, oxygen atoms in red, nitrogen atoms in blue, chlorine atoms in green, and the protein carbon atoms in green.

[0031] As Figure 2 shown, dBET1 acting on cells can inhibit the expression of TRAF1 in cells. D5 represents a dBET1 concentration of 5 nM, D50 represents a dBET1 concentration of 50 nM, and D500 represents a dBET1 concentration of 500 nM.

[0032] As Figure 3 shown in A, it is the time axis of the mouse experiment, and IMQ represents imiquimod cream.

[0033] As Figure 3 shown in B, dBET1 acting on the mouse skin can relieve psoriasis lesions, and three mice are shown in each group.

[0034] In summary, the present invention screens out a small molecule compound that inhibits the expression of TRAF1, verifies on macrophages that this compound can inhibit the expression of TRAF1, and this compound acting on the mouse skin can relieve psoriasis lesions.

Claims

1. Use of a small molecule compound dBET1 and its pharmaceutically acceptable salts in the preparation of a medicament for treating psoriasis, wherein the structural formula of the small molecule compound dBET1 is: 。 2. The use according to claim 1, wherein, the medicament comprises an effective dose of the small molecule compound dBET1 and a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 2, wherein, the dosage form of the medicament is a topical preparation.

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