A traf2 inhibitor and its use in the preparation of an anti-tumor drug

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a TRAF2 inhibitor and application thereof in preparation of an anti-tumor drug. It is found in the application that knocking out TRAF2 can significantly improve the sensitivity of liver cancer to NK cell killing, and TRAF2 is a therapeutic target for overcoming NK immune escape of hepatocellular carcinoma. Further, a deep learning-based prediction model is used for drug screening and experimental verification, and an effective TRAF2 small molecule antagonist is obtained, which is expected to be used as a lead compound for treatment of tumors with high TRAF2 expression.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a TRAF2 inhibitor and application thereof in preparation of an antitumor drug. BACKGROUND

[0002] In the past decade, immunotherapy represented by immune checkpoint inhibitors and chimeric antigen receptor T cells has brought a breakthrough in the treatment of tumors, and has completely changed the treatment strategy for many cancers. CD8 + T cells recognize tumor cell-presented neoantigens through major histocompatibility complex class I molecules (MHC-I), and the inhibitory molecules expressed on the surface of tumor cells can cause dysfunction of T cells by interacting with them. Immune checkpoint inhibitors can block the restriction of these inhibitory molecules on T cell function, thereby achieving the purpose of treatment. However, due to DNA mutations, RNA expression down-regulation, epigenetic silencing or autophagy-mediated protein degradation of tumor cells, more than 65% of tumors have MHC-I deficiency, leading to primary and / or acquired resistance to immune checkpoint inhibitors, which is manifested as "immune cold" tumors. Therefore, only a small number of patients benefit from immune checkpoint inhibitor therapy. Clarifying the key molecules and their regulatory networks in tumor immune escape, and finding new therapeutic targets and drugs to overcome tumor immune escape, are key scientific problems to be solved in the field of tumor treatment, which are expected to bring a revolutionary breakthrough in tumor treatment and have important theoretical and clinical value.

[0003] Natural killer cells (NK cells) are cytotoxic lymphocytes in the innate immune system, which have the ability to spontaneously monitor and kill infected or malignant cells. NK cells use a series of activating receptors to recognize up-regulated, embryonically encoded ligands on the surface of cancer cells, without the need for MHC molecules to present tumor neoantigens like T cells. In theory, its non-antigen restricted killing mechanism has a double advantage: it can evade tumor escape caused by antigen drift and overcome the limitations of adaptive immunity, and it has broad killing potential for heterogeneous tumors, so it may be suitable for patients who are not sensitive to T cell therapy. More and more evidence shows that NK immunotherapy is a safe, feasible and promising treatment strategy. However, NK cells in the tumor microenvironment show impaired cytotoxicity, and the number of infiltrating NK cells in tumor tissue is extremely low. The core molecular mechanism of NK cell dysfunction in the tumor microenvironment is still unclear, and the inhibition of NK cell anti-tumor activity by the tumor microenvironment is still the main obstacle for its breakthrough in solid tumor treatment. Therefore, finding key targets and candidate drugs that can overcome NK immune escape is expected to provide new strategies for cancer immunotherapy to overcome NK immune escape.

[0004] TRAF2 (TNF receptor associated factor 2) is a core member of the TRAF protein family, which contains N-terminal RING finger domain, central ZINC finger domain and C-terminal TRAF domain. The N-terminal RING finger domain can interact with E3 protein, participate in ubiquitination, signal transduction, apoptosis and other biological processes; the ZINC finger domain is mainly involved in the interaction between TRAF2 and other proteins; the C-terminal TRAF domain binds to the intracellular region of the TNF receptor superfamily (such as TNFR2, CD40, BAFF-R) to form a signal complex, mediate downstream signal transmission, and activate NF-κB, JNK / AP-1 and other signaling pathways to precisely regulate cell survival, death, inflammatory response and immune response. In hepatocellular carcinoma, TRAF2 inhibits caspase-8-dependent apoptosis by stabilizing the RIPK1 complex and promotes tumor cell resistance to chemotherapy drugs (such as sorafenib). In T cells, the CD27-TRAF2-SHP-1 signaling axis regulates memory differentiation after initial T cell activation by inhibiting Lck phosphorylation, and promotes the expression of memory-related genes (such as TCF7, LEF1). In acute myeloid leukemia (AML), LILRB3 inhibits leukemia cell apoptosis through the TRAF2-cFLIP-NF-κB pathway, while weakening the anti-tumor activity of T cells. There is also evidence that TRAF2 differentially regulates the activation of classical and non-classical NF-κB signaling pathways in mature B cells. These evidences suggest that TRAF2 plays an important role in the occurrence and evolution of tumors, and is a key hub for immune cell fate determination and inflammatory response, and its dysfunction may lead to immune escape or excessive inflammatory response. Currently, there is no research report on the role of TRAF2 in regulating tumor NK cell immune escape, and there is also a lack of research on its use as a therapeutic target, and there is no ideal TRAF2 small molecule antagonist on the market, which is a significant technical gap. SUMMARY

[0005] Currently, there is no report on the role of TRAF2 in regulating tumor NK cell immune escape, and there is also a lack of research on its use as a therapeutic target, and there is no ideal TRAF2 small molecule antagonist on the market, which is a significant technical gap. The technical problem to be solved by the present application is to determine whether TRAF2 is a key molecule and therapeutic target for mediating tumor NK immune escape, and to find small molecule antagonists targeting TRAF2, providing drugs for the treatment of TRAF2 positive tumors.

[0006] To solve the above problems, the present application first proves by experiments that knocking out TRAF2 can significantly improve the sensitivity of liver cancer to NK cell killing, and TRAF2 is expected to become a therapeutic target for overcoming NK immune escape of hepatocellular carcinoma. On this basis, according to the structure of TRAF2 protein, through intelligent screening of molecular docking (AutoDock Vina) and experimental verification, it is found that the compound ZB-020 can bind to the RING finger and the first ZINC finger domain (1st ZINC finger) of the N-terminal of TRAF2 protein, and the interaction between the two has been verified by in vitro SPR experiment, indicating that the compound ZB-020 inhibits the biological function of TRAF2 by binding to the key domain of TRAF2 protein. Therefore, the present application provides a small molecule antagonist targeting TRAF2.

[0007] In a first aspect of the present application, a small molecule inhibitor targeting TRAF2 is provided, which has a structure as shown in formula I:

[0008]

[0009] Formula I.

[0010] In a second aspect of the present application, the small molecule inhibitor targeting TRAF2 provided has the ability to bind to the RING finger and the first ZINC finger domain (1st ZINC finger) of the N-terminal of TRAF2.

[0011] In a third aspect of the present application, the use of the above-mentioned inhibitor targeting TRAF2 in the preparation of an antitumor drug is provided.

[0012] In a fourth aspect of the present application, the use of the small molecule inhibitor targeting TRAF2 in the preparation of a TRAF2 high-expression tumor such as a hepatocellular carcinoma treatment drug is provided.

[0013] In a fifth aspect of the present application, a drug for treating hepatocellular carcinoma is provided, and the active ingredient of the drug includes an antitumor drug of the above-mentioned inhibitor targeting TRAF2.

[0014] Further, the drug includes a pharmaceutically acceptable adjuvant or carrier.

[0015] Further, the drug form includes injection, tablet, capsule, aerosol, suppository, film, controlled or sustained release preparation or nano preparation.

[0016] The present application has the following beneficial effects:

[0017] (1) The present application firstly proves that knocking out TRAF2 can significantly improve the killing sensitivity of hepatocellular carcinoma to NK cells, and TRAF2 can become a potential therapeutic target for overcoming NK immune escape of hepatocellular carcinoma.

[0018] (2) The present application provides an inhibitor targeting TRAF2, and its application in the preparation of a drug targeting NK immune escape of cancer. The small molecule inhibitor can effectively synergize with NK cells to play an anti-tumor effect in vivo, has great prospects for the development of cancer immunotherapy drugs, lays a foundation for subsequent clinical trials, and provides new ideas and methods for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Flow cytometry detection of the purity of NK cells isolated from PBMC.

[0020] Figure 2 : Flow cytometry detection of the purity of mouse NK cells isolated from mouse spleen.

[0021] Figure 3 : Western blot detection of TRAF2 knockout efficiency in Hep-12 human hepatocellular carcinoma cells.

[0022] Figure 4 : Western blot detection of Traf2 knockout efficiency in Hepa 1-6 mouse hepatocellular carcinoma cells.

[0023] Figure 5 : Schematic diagram of the killing effect of NK-92MI cells on Hep-12 cells after TRAF2 knockout.

[0024] Figure 6 : Schematic diagram of the killing effect of human NK cells on Hep-12 cells after TRAF2 knockout.

[0025] Figure 7 : Schematic diagram of the killing effect of mouse NK cells on Hepa 1-6 cells after Traf2 knockout.

[0026] Figure 8 : Schematic diagram of the cytotoxicity of small molecule inhibitors on NK-92MI cells, spontaneous LDH release of target cells, and spontaneous LDH release of effector cells in the present application.

[0027] Figure 9 : Schematic diagram of the cytotoxicity of small molecule inhibitors on human NK cells, spontaneous LDH release of target cells, and spontaneous LDH release of effector cells in the present application.

[0028] Figure 10 : AlphaFold prediction of TRAF2 protein structure.

[0029] Figure 11 Structure of small molecule inhibitor in the present application.

[0030] Figure 12 Prediction of binding site of small molecule inhibitor in the present application and N-terminal RING finger + 1st ZINC finger domain of TRAF2 protein.

[0031] Figure 13 Purification of full-length and truncated TRAF2 protein.

[0032] Figure 14 Affinity of small molecule inhibitor in the present application and full-length TRAF2 protein.

[0033] Figure 15 Affinity of small molecule inhibitor in the present application and TRAF2 (1-130 aa) truncated protein.

[0034] Figure 16 Growth curve of mouse tumor after administration of small molecule inhibitor in the present application.

[0035] Figure 17 Changes of mouse body weight after administration of small molecule inhibitor in the present application.

[0036] Figure 18 Dissection diagram of mouse tumor after administration of small molecule inhibitor in the present application.

[0037] Figure 19 Statistical diagram of mouse tumor weight after administration of small molecule inhibitor in the present application. DETAILED DESCRIPTION

[0038] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0039] In the following examples, the experimental methods used are conventional methods, and those not specifically described are conventional methods.

[0040] In the following examples, the materials, reagents and the like used, if not specifically described, can be obtained from commercial channels.

[0041] Experimental materials:

[0042] 1. Cell lines: Hep-12 human hepatocarcinoma cell line, Hepa 1-6 murine hepatocarcinoma cell line, NK-92MI cells, human primary NK cells, mouse primary NK cells, provided by the laboratory of Beijing Cancer Prevention and Treatment Research Institute. Among them,

[0043] The purification steps of human primary NK cells are as follows:

[0044] (1) Incubate the cells with DNase I solution at a concentration of 100 μg / mL (diluted in Hanks' balanced salt solution) at room temperature (15-25°C) for at least 15 min.

[0045] (2) Filter the aggregated suspension through a 37 μm cell strainer to obtain individual PBMC cells. Subsequently, resuspend the cells using EasySep™ Buffer to a density of 5 × 10 7 cells / mL.

[0046] (3) Isolate human primary NK cells using EasySep™ Human NK Cell Enrichment Kit and activate the culture of human NK cells according to the instructions.

[0047] (4) Use flow cytometry to determine the purity of the isolated human NK cells. Resuspend 1 × 10 6 PBMC and 1 × 10 6 purified NK cells. Add 5 μL APC-anti-human CD3 antibody and PE-anti-human CD56 antibody to each of PBMC and NK cells, incubate at 37°C for 30 min, then wash three times with PBS buffer, finally resuspend the cells with 400 μL PBS buffer and filter using a flow tube with a 35 μm cell strainer. Similarly, use the same isotype control antibodies of APC and PE to stain PBMC and NK cells as the later flow cytometry gating.

[0048] As shown in Figure 1 , PBMC and purified NK cells were double-stained with APC-anti-human CD3 antibody and PE-anti-human CD56 antibody, and flow cytometry analysis showed that the purity of the purified NK cells was as high as 96%.

[0049] The purification steps of mouse primary NK cells are as follows:

[0050] (1) Obtain fresh spleen of 8-week-old C57BL / 6 female mice.

[0051] (2) Place the mouse spleen in a 10 cm culture dish containing 5 mL Hanks' balanced salt solution.

[0052] (3) Using a razor or surgical blade, carefully cut the spleen into small pieces (~0.2 cm 2 ).

[0053] (4) Myeloid cells were prepared as follows for bulk isolation: Small pieces of spleen were digested at 37°C for 20-30 min using 5 mL of HBSS solution containing collagenase type IV (100 U / mL) and 1% FBS with Dnase (20 μg / mL).

[0054] (5) The enzymatic reaction was stopped by adding 1 mM EDTA solution and resting for 5 min at room temperature.

[0055] (6) Place a 70 μm cell strainer over a 50 mL conical tube.

[0056] (7) Using a disposable pipette, transfer the digested spleen into the cell strainer.

[0057] (8) Using the plunger end of a syringe, crush or press the spleen through the strainer. If necessary, add 5-10 mL of PBS to rinse.

[0058] (9) Rinse the cells through the strainer with a large volume of PBS. Repeat steps (5) and (6) if necessary.

[0059] (10) Centrifuge the cells at 400-600 x g for 5 min at 4°C and discard the supernatant.

[0060] (11) Resuspend the cells with 2-5 mL of pre-chilled lx red blood cell lysis solution (10x red blood cell lysis solution diluted with double distilled water).

[0061] (12) Place the resuspension on ice for 5 min.

[0062] (13) Wash the cell suspension with 10-20 mL of ice-cold PBS.

[0063] Note: Perform steps (1)-(8) at room temperature and use pre-chilled buffers for steps (9)-(13) performed on ice.

[0064] (14) Centrifuge the cells at 400-600 x g for 5 min at 4°C and discard the supernatant. Subsequently, resuspend the cells using EasySep™ Buffer to a density of 1 x 10 8 cells / mL.

[0065] (15) Isolate mouse primary NK cells using EasySep™ Mouse NK Cell Isolation Kit and activate the mouse NK cells according to the instructions.

[0066] (16) Purified NK cells were cultured in 1640 medium containing 10% FBS, L-glutamine (2 mM), non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), recombinant mouse IL-2 (500 U / mL) and recombinant mouse IL-15 (10 ng / mL) at 37°C and 5% CO. After 6 or 7 days of culture, the cells were used as effector cells.

[0067] (17) Measure the purity of the isolated and purified mouse NK cells using flow cytometry. Resuspend 1 x 10 6 Mouse splenocytes and 1 x 10 6 Isolated and purified mouse NK cells. Add 2 μL PE-anti-mouse CD49b (pan-NK cells) antibody and 5 μL APC-anti-mouse CD3 antibody to each of the mouse splenocytes and mouse NK cells, incubate at 37°C for 30 min, then wash three times with PBS buffer, finally resuspend the cells with 400 μL PBS buffer and filter using a flow tube with a 35 μm cell strainer. Similarly, use the same isotype control antibodies for APC and PE to stain the splenocytes and mouse NK cells as a later flow cytometry gating control.

[0068] As shown in Figure 2, the mouse splenocytes and the isolated and purified mouse NK cells were double-stained with APC-anti-mouse CD3 antibody and PE-anti-mouse CD49b antibody, and the flow cytometry analysis showed that the purity of the isolated and purified NK cells was as high as 82.6%. Figure 2

[0069] 2. Experimental animals: 8-week-old female C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. and were raised by the Animal Center of Beijing Cancer Prevention and Treatment Research Institute. All animal experiments were approved by the Beijing Cancer Prevention and Treatment Research Institute Animal Protection and Use Association.

[0070] ​3. Major experimental reagents: Small molecule compound ZB-020 (publicly available at https: / / go.drugbank.com / ) was purchased from MedChem Express. Opti-MEM, RPMI-1640, DMEM, MEMα (nucleoside-free) media, type IV collagenase, DNase, Hanks' balanced salt solution, 10× erythrocyte lysis buffer, L-glutamine, non-essential amino acids, sodium pyruvate, trypsin, and stbl4 electroporated competent E. coli cells were purchased from Gibco. CytoTox 96 Non-Radioactive Cytotoxicity Assay and T4-DNA ligase were purchased from Promega. EasySep™ Human NK Cell Enrichment Kit, ImmunoCult™ NK Cell Expansion Kit, EasySep™ Mouse NK Cell Isolation Kit; recombinant mouse IL-2 and IL-15 were purchased from STEMCELL; restriction endonucleases were purchased from NEB; PEI was purchased from Sigma; TRAF2 antibody was purchased from CST; E. coli competent strains DH5α and BL21(RES) were purchased from TransGen; and Matrigel was purchased from Corning. Flow cytometry antibodies APC-anti-human CD3 antibody, APC-anti-mouse CD3 antibody, and PE-anti-mouse CD49b (pan-NK cells) antibody were purchased from Biolegend; PE-anti-human CD56 (HCD56) antibody was purchased from STEMCELL.

[0071] Example 1:

[0072] TRAF2 was knocked out in Hep-12 cells using CRISPR / Cas9 technology, and the knockout efficiency was verified by Western blot experiments.

[0073] 1. Construction of TRAF2 knockout expression vector

[0074] (1) Based on human and rodent sources on the NCBI website TRAF2 Gene sequences were used to design sgRNA sequences targeting human and mouse TRAF2 gene knockout using the CRISPR online tool (https: / / crispor.gi.ucsc.edu / ), and sgRNA sequences were added to both ends of the sequences. BsmBI Enzyme cleavage site.

[0075] (2) Hybrid oligonucleotides (gRNAs) are ligated to DNA using T4-DNA ligase. BsmBIConstruction of sgRNA expression vector in linearized lentiCRISPR-V2 vector.

[0076] (3) Use competent stbl4 for electroporation. After plating the bacterial solution and culturing for 12-16 h, single colony is selected for plasmid extraction, enzyme digestion identification, verification, and sequencing of the positive clone identified correctly. The plasmid of the correctly sequenced clone sample is extracted.

[0077] 2. The sgRNA expression vector is transiently transfected into Hep-12 cells using PEI reagent for expression, and is screened by puromycin for three days. Then, the single clone cells are sorted into 96-well plates. After the single clone cells in the 96-well plates grow to a certain density, they are transferred to 24-well plates for further growth. When the confluence of the single clone cell lines in the 24-well plates reaches more than 90%, part of the cells are collected for total protein extraction, and Western blot experiment is used to identify and screen the TRAF2 knockout cell lines.

[0078] 3. The collected cells are washed with cold PBS for 3 times, total protein is extracted, and the protein sample is subjected to electrophoresis and membrane transfer. Further, antibody incubation is performed using TRAF2 antibody and corresponding secondary antibody, and color development is performed by configuring chemiluminescence solution.

[0079] The identification results are shown in 3-4, and the Western Blot detection results show that TRAF2 (Traf2) knockout is successful in Hep-12 ( Figure 3 ) and Hepa 1-6 cells ( Figure 4 ).

[0080] Example 2:

[0081] Verify the effect of TRAF2 knockout on the sensitivity of hepatocarcinoma cells to NK cell killing.

[0082] 1. Take the tumor target cells in the logarithmic growth phase (Hep-12 as the target cells of NK-92MI and human NK, and Hepa 1-6 as the target cells of mouse NK), wash with 1x CMF washing solution, digest with 0.25% trypsin, resuspend the cells with complete medium containing 10% serum and transfer to a 15 mL centrifuge tube. After centrifugation at 1000 rpm for 5 min, resuspend the cell pellet with complete medium containing 10% serum, and filter through a 400-mesh cell sieve to prepare a single cell suspension.

[0083] 2. Count using a cell counter, and inoculate a constant number of Hep-12 cells (50 μL, 1x10 4 cells) in a 96-well plate.

[0084] 3. NK-92MI cell processing method: The NK-92MI cells with high clumping rate were precipitated using the precipitation method, and the cell debris contained in the supernatant was discarded. After resuspending the cell precipitate with 1x CMF, centrifugation was performed at 900 rpm for 5 min. The cell precipitate was resuspended with RPMI-1640 medium containing 10% serum, and filtered through a 400-mesh cell sieve to prepare a single cell suspension.

[0085] 4. Primary human / mouse NK cell processing method: The activated cultured human NK / mouse NK cells were collected into a 15 mL centrifuge tube and centrifuged at 300 x g for 10 min. The cell precipitate was resuspended with ImmunoCult™ NK Cell Expansion Medium (human NK cell culture medium) or specially formulated RPMI-1640 (mouse NK cell culture medium), and filtered through a 400-mesh cell sieve to prepare a single cell suspension.

[0086] 5. A constant number of NK-92MI cells (50 μL) or human / mouse NK cells (50 μL) were co-incubated with target cells for 6 h using a cell counter, and the cell number ratio of NK-92MI cells or human / mouse NK cells to target cells was 1:1, 2:1, and 5:1.

[0087] 6. The killing effect of NK-92MI cells or human NK cells on tumor cells was determined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay of Promega. The spontaneous LDH release amount of NK-92MI cells or human / mouse NK cells and tumor cells, and the maximum LDH release amount of tumor cells were also determined, and the specific lysis rate of tumor cells was calculated according to the instructions.

[0088] As shown in Figures 5-7 , the lysis rate of Hep-12 sg TRAF2 cells after co-incubation with NK-92MI or human NK cells was significantly higher than that of Hep-12 sg ctrl cells ( Figures 5-6 ), indicating that after knocking out the TRAF2 gene, the in vitro killing sensitivity of Hep-12 cells to NK-92MI cells and human NK cells was significantly increased. Similarly, after knocking out Traf2 in mouse hepatoma cells Hepa 1-6, the killing effect of mouse NK cells on Hepa 1-6 cells was significantly enhanced ( Figure 7 ).

[0089] Example 3:

[0090] Virtual screening of TRAF2 small molecule antagonists.

[0091] TRAF2 inhibitor The crystal complex structure of TRAF2 was downloaded from the PDB database (PDB ID: 1ca4); the full-length structure thereof was used for docking with small molecules; a prediction model for protein-compound interactions based on deep learning (including DeepCPI, DeepConvDTI and DeepDTA) was used to perform virtual screening on the Drugbank commercial compound database; after screening, the top 1000 compounds were screened based on Lipinski's drug rules, cluster analysis, etc., and more than 100 diversity molecules ranked first were obtained for subsequent experimental detection.

[0092] Example 4:

[0093] Verify the effect of the top-ranking small molecule compounds on the killing sensitivity of NK cells after treating Hep-12 cells.

[0094] 1. Take the logarithmic phase growth of Hep-12 cells, and prepare a single cell suspension as described in Example 2. 2. Count using a cell counter, and inoculate a constant number of Hep-12 cells (50 μL, 1×10 4 cells) in a 96-well plate, with four final concentrations of 0 nM, 1 nM, 10 nM and 100 nM for each compound, with 3 replicate wells for each group. Incubate in a 37°C cell incubator for 24 h.

[0095] 3. Prepare a single cell suspension of NK-92MI cells and human NK cells as described in Example 2.

[0096] 4. Count using a cell counter, and co-incubate a constant number of NK-92MI cells (50 μL, 5×10 4 cells) or human NK cells (50 μL, 2.5×10 4 cells) with Hep-12 cells that have been treated with compounds for 24 h for 6 h.

[0097] 5. Use the CytoTox 96 Non-Radioactive Cytotoxicity Assay of Promega to determine the killing effect of NK-92MI cells or human NK cells on tumor cells. At the same time, the spontaneous LDH release amount of NK-92MI cells or human NK cells and tumor cells, and the maximum LDH release amount of tumor cells are determined, and the specific lysis rate of tumor cells is calculated according to the instructions.

[0098] As Figures 8-9 shown, we performed functional verification on the top ten non-anticancer drugs screened through in vitro killing experiments, and the results showed that Figure 8As shown, the small molecule inhibitor of this invention enhances the killing sensitivity of Hep-12 cells to NK-92MI cells. Furthermore, the compound itself does not exhibit significant toxicity to either Hep-12 or NK-92MI cells; Figure 9 As shown, the small molecule inhibitor of this invention enhances the killing sensitivity of Hep-12 against human NK cells. Furthermore, the compound itself does not exhibit significant toxicity to either Hep-12 cells or human NK cells. Additionally, ZB-020 at concentrations of 1 nM and 10 nM enhances the killing sensitivity of Hep-12 against NK-92MI cells, and the compound itself does not exhibit significant toxicity to either Hep-12 cells or NK-92MI cells. ZB-020 at concentrations of 10 nM and 100 nM also enhances the killing sensitivity of Hep-12 against human NK cells.

[0099] Example 5:

[0100] The structure of the full-length human TRAF2 protein was predicted using the AlphaFold database, and the structure of ZB-020 was searched in the Durgbank database. Molecular docking techniques were used to predict the binding sites of TRAF2 and small molecules.

[0101] like Figure 10 As shown, the TRAF2 protein has 501 amino acids and contains three domains: the RING finger domain—which interacts with the E3 protein and participates in various biological processes such as ubiquitination, signal transduction, and apoptosis; the ZING finger domain—which interacts with other proteins; and the TRAF domain—which binds to the tumor necrosis factor (TNF) receptor superfamily. Figure 11 , Figure 12 As shown in Table 1, the binding sites of TRAF2 and small molecules were predicted using molecular docking technology. The prediction results showed that compound ZB-020 mainly binds to the N-terminal RING finger +1st ZINC finger domain of the TRAF2 protein.

[0102] Table 1. Docking results of the RING finger+1st ZINC finger domain with compound ZB-020

[0103]

[0104] Example 6:

[0105] Affinity was verified by surface plasmon resonance (SPR) assay between the small molecule compound ZB-020 and TRAF2.

[0106] 1. Construction of pCDNA3.0-TRAF2 full-length plasmid

[0107] (1) Query mRNA sequence of gene (Accession: NM_021138.4) on NCBI, and call its CDS region, design primer sequences respectively at upstream and downstream of CDS sequence, send primer sequences to Beijing Genesee Biotechnology Co., Ltd. for synthesis. TRAF2

[0108] Primer sequences are as follows:

[0109] Upstream primer (F): 5'-CTCGGATCCATGGCTGCAGCTAGCGTGAC-3'

[0110] Downstream primer (R): 5'-CGAGCGGCCGCCGGAGCCCTGTCAGGTCCACAA-3'

[0111] (2) Use cDNA of Hep-12 cell as template to obtain TRAF2 full-length sequence by PCR amplification.

[0112] (3) Use competent DH5a to transform homologous recombination product. After plating of bacterial liquid, culture for 12-16 h, select single colony to extract plasmid, perform enzyme digestion identification and verification, and perform sequencing on positive clone identified correctly.

[0113] (4) Perform plasmid extraction on clone sample with correct sequencing to obtain pCDNA3.0-TRAF2 plasmid.

[0114] 2. Construction of PGEX-TRAF2 truncated plasmid

[0115] (1) Design primer sequences respectively at upstream and downstream of gene corresponding to two predicted domains 1-130 amino acids and 334-501 amino acids of TRAF2 protein, and introduce 15-20 bp homologous sequences, send primer sequences to Beijing Genesee Biotechnology Co., Ltd. for synthesis.

[0116] Primer sequences are as follows:

[0117] 1) 1-130 domain:

[0118] Upstream primer (F): 5'-CCGCGTGGATCCATGGCTGCAGCTAGCGT-3'

[0119] Downstream primer (R): 5'-ATGCGGCCGCTCGAGCGGGCAGCGGCCTTCGTG-3'

[0120] 2) 334-501 domain:

[0121] ​Upstream primer (F): 5'-GTTCCGCGTGCGATGGCTGACTTGGAGCA-3'

[0122] Downstream primer (R): 5'-ATGCGGCCGCTCGAGGAGCCCTGTCAGGT-3'

[0123] (2) The TRAF2 truncated sequence was obtained by PCR amplification using cDNA from Hep-12 cells as a template; the linearized vector was obtained by reverse PCR amplification using PGEX-4T1 vector as a template.

[0124] (3) The purified linearized vector and target fragment were recovered by agarose gel and multi-fragment homologous recombination was performed using Vazyme’s ClonExpress® Ultra One Step Cloning Kit.

[0125] (4) Transformation of homologous recombination products using competent DH5α cells. After plating the bacterial culture, culture for 12-16 h, select single colonies to extract plasmids, identify them by enzyme digestion, verify them, and sequence the positive clones that are correctly identified.

[0126] (5) Plasmids were extracted from the correctly sequenced clone samples to obtain PGEX-TRAF2 1-130 aa and PGEX-TRAF2 334-501 aa truncated plasmids.

[0127] 3. TRAF2 protein expression and purification

[0128] (1) pCDNA3.0-TRAF2 was transfected into 293FT cells and the cells were collected 48 hours after transfection.

[0129] (2) Wash the cells twice with PBS, add 1% NP-40 lysis buffer and rotate at 4°C for 1 h to lyse.

[0130] (3) Lyse cells on ice with ultrasound at a power of 200-300 W, for 5 seconds each time, and then let stand on ice for 5 seconds until the cells are clear.

[0131] (4) Centrifuge at 13000 xg for 15 min at 4℃, collect the supernatant and place it in an ice water bath or on ice.

[0132] (5) Take 100 μL of well-mixed Flag-bound beads, centrifuge at 4℃ and 1000 rpm to discard the storage liquid, resuspend the beads with TBS washing solution, centrifuge and discard the liquid.

[0133] (6) Add the cell supernatant to the above-mentioned agar beads and incubate at 4°C for 16 h.

[0134] (7) Remove liquid by centrifugation, and wash the beads three times with TBS buffer, add 200 ng / μL of 3xFlag short peptide to compete for elution for 1 h, and concentrate the TRAF2 protein by centrifugation using an ultrafiltration tube.

[0135] 4. Expression and purification of TRAF2 truncated protein

[0136] (1) Transform the prokaryotic plasmids PGEX-TRAF2 1-130 aa and PGEX-TRAF2 334-501 aa into BL21 (RES) competent cells.

[0137] (2) Pick a single colony expressing the monoclonal and inoculate it into 4 mL of LB medium containing ampicillin, and incubate it at 37°C, 220 rpm overnight.

[0138] (3) Take the overnight culture at a ratio of 1:100, and inoculate it into 200 mL of LB medium containing ampicillin.

[0139] (4) Incubate it at 37°C, 220 rpm for about 3.5 h until the OD600 of the bacterial solution reaches about 0.6, then add IPTG to a final concentration of 0.2 mM, and continue to incubate it at 16°C, 220 rpm for 16 h.

[0140] (5) Collect the bacterial solution into a centrifuge tube, centrifuge it at 4°C, 4500 x g for 15 min, discard the supernatant, and collect the precipitate.

[0141] (6) Add 10 mL of lysis buffer containing protease inhibitors, and add lysozyme to a final concentration of 1 mg / mL, and incubate it in an ice water bath or ice bath for 30 min.

[0142] (7) Sonicate the bacteria on ice, with a sonication power of 200-300 W, each sonication for 10 s, and let it stand on ice for 10 s, until the bacterial solution is clear.

[0143] (8) Centrifuge it at 4°C, 13000 x g for 15 min, collect the supernatant, and place it in ice water or on ice.

[0144] (9) Take 300 μL of the mixed GST binding beads, centrifuge them at 4°C, 1000 rpm to discard the storage solution, resuspend the beads with PBS wash solution, centrifuge and discard the liquid.

[0145] (10) Add the bacterial supernatant to the above beads, and incubate it at room temperature for 2 h.

[0146] (11) centrifugation to remove liquid, and PBS buffer is used to wash the coagulation beads three times, and thrombin is directly added to the coagulation beads to cut the GST tag for 16 h to obtain TRAF2 1-130 aa and TRAF2 334-501 aa truncated protein.

[0147] 5. Surface plasmon resonance (SPR) affinity detection

[0148] The purified TRAF2, TRAF2 1-130 aa, and TRAF2 334-501 aa truncated prokaryotic protein is detected for affinity with small molecule compound ZB-020 by surface plasmon resonance (SPR). The specific steps are as follows:

[0149] (1) Protein chip fixation

[0150] The Series S Sensor Chip CM5 chip is installed into the Biacore 8K biomolecular interaction analysis system, and the coupling buffer is PBS-P+ solution (pH 7.4), and bubbles are filtered out. Then, according to the isoelectric point of the protein, the optimal pH value (4.0, 4.5, 5.0, 5.5) and concentration (25, 50, 100 μg / mL) of the protein fixed on the COOH chip are selected, and the protein is pre-enriched to ensure that enough protein is fixed on the protein chip, and finally the pH 4.5 is determined as the optimal coupling condition. The protein coupling includes three steps: EDC / NHS activation; protein coupling; and ethanolamine blocking of the excess binding sites of the protein.

[0151] (2) Preparation of compound detection solution

[0152] The small molecule compound ZB-020 is dissolved in dimethyl sulfoxide (DMSO) solution. The control stock solution is taken, diluted 100 times with PBS-P solution, and then sequentially diluted with PBS solution containing 1% DMSO (pH=7.4).

[0153] (3) Affinity detection

[0154] The flow cell with the target protein bound was set as the detection channel, and the flow cell without the target protein bound was set as the reference channel. The running started at the maximum flow rate (150 μL / min) with PBS-P as the detection buffer. After the signal baseline was stable, the sample loop was flushed with the buffer for emptying. After the signal reached the baseline, the buffer flow rate was adjusted to 20 μL / min. The EDCNNHS (1:1) solution was loaded to activate the chip. After 200 μL of the ligand diluted with the activation buffer was loaded and ran for 4 min, and the binding was stable, the sample loop was flushed with the buffer. 200 μL of the blocking solution was loaded, the sample loop was flushed with the buffer, and the air was emptied. To ensure stability, the baseline was observed for 5 min. The analyte was diluted with the buffer, and was loaded at different concentrations at 20 μL / min. The protein binding time with the ligand was 240 s, and the natural dissociation time was 360 s.

[0155] (4) Affinity curve fitting

[0156] After the sample injection was completed, data processing was performed. The solvent correction result was added to the Evaluation software to obtain the resonance unit (RU) at the binding saturation period. Through the Biacore T200 Evaluation software, the binding affinity of TRAF2, TRAF2 1-130 aa, and TRAF2 334-501 aa protein to the small molecule compound ZB-020 was calculated by using a single site interaction model.

[0157] As shown in Figure 13 , the full-length TRAF2 protein was expressed in eukaryotic cells, and the TRAF2 truncated protein containing a GST tag was expressed in prokaryotic cells. The purified TRAF2 truncated protein was obtained by enzyme digestion. As shown in Figure 14 and Figure 15 , the surface plasmon resonance method (SPR) was used for affinity detection. The results showed that TRAF2 had micromolar affinity with the compound ZB-020, KD(M)=25.9 μM ( Figure 14 ), and had micromolar affinity with the N-terminal RING finger + 1st ZINC finger domain (TRAF2 truncated body 1-130 aa) ( Figure 15 ), KD(M)=17.1 μM. It was shown that the compound ZB-020 could bind to the key domain of the TRAF2 protein, thereby inhibiting the biological function of TRAF2. This result provided a strong experimental basis for further drug development.

[0158] Example 7:

[0159] The therapeutic effect of the small molecule compound ZB-020 on Hepa 1-6 homotransplantation tumor was verified by administering the tumor-bearing immunocompetent mice (C57BL / 6).

[0160] 1. Model establishment: the Hepa 1-6 cells in logarithmic growth phase were taken, digested, resuspended, washed twice with PBS, and resuspended with an appropriate amount of serum-free PRMI-1640 medium; after counting with a cell counter, the cell density was adjusted to 2 x 10 7 / mL, and 1 mL of the cell suspension was taken with a sterile syringe and subcutaneously inoculated on the back of the right upper limb of the nude mice, 100 μL of the cell suspension was inoculated for each mouse;

[0161] 2. Animal grouping: when the tumor grew to about 100 mm 3 , the mice with too large or too small tumors were picked out, and the remaining mice were evenly grouped, and were divided into three groups. The small molecule compound ZB-020 low-dose group (10 mg / kg), the small molecule compound high-dose group (30 mg / kg), and the control group, each group had 5 mice.

[0162] 3. Drug administration and observation: the small molecule compound ZB-020 was administered by intraperitoneal injection at a low dose of 10 mg / kg and a high dose of 300 mg / kg (the small molecule compound ZB-020 was dissolved in a solvent composed of 40% PEG300, 5% Tween 80 and 55% normal saline, and the control group was the solvent), the body weight and tumor volume of the mice were recorded on the first day of administration, and the state of the mice was observed. The small molecule compound needed to be administered by intraperitoneal injection every day, the tumor of the mice was measured and recorded every two days, and the body weight of the mice was weighed. When the maximum tumor volume of the control group mice reached 1.5 cm 3 , the experiment was terminated, the mice were sacrificed by cervical dislocation, the tumor was dissected, photographed and weighed. Finally, the tumor was divided into two parts, which were fixed with polyformaldehyde and stored at room temperature, and frozen in liquid nitrogen and stored at -80°C, respectively.

[0163] 4. Statistical analysis of experimental data: after the experiment, the body weight change curve of each group of mice, the tumor volume change curve, and the final tumor weight distribution graph were drawn. The tumor inhibition rate was calculated:

[0164] Tumor inhibition rate IR (%) = (1-TTW / CTW) x 100%

[0165] TTW: tumor weight of the treatment group (Treated Tumor Weight); CTW: tumor weight of the control group (Control Tumor Weight).

[0166] For example, Figures 16-19As shown, the inhibitory effect of tumor growth was investigated by intraperitoneal administration of ZB-020 (10 mg / kg and 30 mg / kg) at two doses in a Hepa 1-6 tumor xenograft mouse model. The experimental results show that the small molecule compound ZB-020 can effectively inhibit the growth of Hepa 1-6 tumor xenograft mice at high and low doses, and the tumor inhibition rate of the low dose group is 89.58%, and the tumor inhibition rate of the high dose group reaches 100%. In addition, the small molecule compound has no obvious toxic side effects on mice.

[0167] The small molecule inhibitor ZB-020 has a strong affinity with TRAF2 at the micromolar level. The inhibitor can significantly enhance the in vitro killing effect of NK-92MI or human NK on HCC cells, and has a good inhibitory effect on tumor growth in an immune-competent mouse model. The above evidence suggests that ZB-020 has a good prospect for the treatment of tumor types with high expression of TRAF2.

[0168] In summary, the present application verifies that the non-antitumor drug compound ZB-020 can enhance the killing effect of NK-92MI or human NK cells on hepatoma cells in in vitro experiments, and has a good therapeutic effect on tumor in in vivo experiments. It is a promising candidate drug for targeting hepatoma NK cell immune escape.

[0169] The above only describes the relatively simple and preferred embodiments of the present application, and is not intended to limit the present application. Any modification, replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The use of a TRAF2 inhibitor in the preparation of an anti-hepatocellular carcinoma drug, wherein the TRAF2 inhibitor has the structure shown in Formula I: Formula I.

2. The application as described in claim 1, characterized in that: The TRAF2 inhibitor has the ability to bind to the RING finger and the first ZINC finger domain at the N-terminus of TRAF2.

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