TRAF2 inhibitor and application thereof in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a TRAF2 inhibitor and application thereof in preparation of antitumor drugs. The invention finds that the knockout of the TRAF2 can significantly improve the sensitivity of liver cancer to NK cell killing, and the TRAF2 is a therapeutic target for overcoming hepatocellular carcinoma NK immune escape. Furthermore, drug screening and experimental verification are carried out based on a deep learning prediction model, and an effective TRAF2 small-molecule antagonist is obtained and is expected to be used as a lead compound for treating TRAF2 high-expression tumors.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and particularly relates to a TRAF2 inhibitor and its application in the preparation of antitumor drugs. Background Technology

[0002] Over the past decade, immunotherapy, represented by immune checkpoint inhibitors and chimeric antigen receptor T cells, has brought groundbreaking progress to cancer treatment, fundamentally changing the treatment strategies for many cancers. CD8 + T cells recognize neoantigens presented by tumor cells through major histocompatibility complex class I (MHC-I) molecules. Inhibitory molecules expressed on the surface of tumor cells can interact with T cells, leading to their dysfunction. Immune checkpoint inhibitors can block the restriction of T cell function by these inhibitory molecules, thereby achieving therapeutic goals. However, due to DNA mutations, downregulated RNA expression, epigenetic silencing, or autophagy-mediated protein degradation in tumor cells, over 65% of tumors exhibit MHC-I deficiency, leading to primary and / or acquired resistance to immune checkpoint inhibitor therapy, manifesting as "immune cold" tumors. Therefore, only a small percentage of patients clinically benefit from immune checkpoint inhibitor therapy. Elucidating the key molecules and regulatory networks involved in tumor immune escape, and identifying new therapeutic targets and drugs to overcome tumor immune escape, are critical scientific problems urgently needing to be solved in the field of cancer treatment. This holds promise for revolutionary breakthroughs in cancer therapy and has significant theoretical and clinical value.

[0003] Natural killer (NK) cells are cytotoxic lymphocytes in the innate immune system, capable of spontaneously monitoring and killing infected or malignant cells. NK cells utilize a series of activating receptors to recognize germline-encoded ligands regulated on the surface of cancer cells, unlike T cells which present tumor neoantigens via MHC molecules. Theoretically, their non-antigen-restricted killing mechanism has a dual advantage: it can evade tumor escape caused by antigen drift, overcoming the limitations of adaptive immunity, and possesses broad-spectrum killing potential against heterogeneous tumors, thus potentially being applicable to patients insensitive to T-cell therapy. Increasing evidence suggests that NK immunotherapy is a safe, feasible, and promising treatment strategy. However, NK cells in the tumor microenvironment exhibit impaired cytotoxicity, and the number of NK cells infiltrating tumor tissue is extremely low. The core molecular mechanisms of NK cell dysfunction in the tumor microenvironment remain unclear, and the inhibition of NK cell antitumor activity by the tumor microenvironment remains a major obstacle to its breakthrough in the treatment of solid tumors. Therefore, identifying key targets and candidate drugs that can overcome NK immune escape holds promise for providing new strategies for immunotherapy in cancer to overcome NK immune escape.

[0004] TRAF2 (TNF receptor-associated factor 2) is a core member of the TRAF protein family. Its structure includes an N-terminal RING finger domain, a central ZING finger domain, and a C-terminal TRAF domain. The N-terminal RING finger domain interacts with E3 proteins, participating in various biological processes such as ubiquitination, signal transduction, and apoptosis. The ZING finger domain primarily participates in the interaction between TRAF2 and other proteins. The C-terminal TRAF domain binds to the intracellular regions of the TNF receptor superfamily (such as TNFR2, CD40, and BAFF-R) to form signaling complexes, mediating downstream signal transduction and activating signaling pathways such as NF-κB and JNK / AP-1 to precisely regulate cell survival, death, inflammatory responses, and immune responses. In liver cancer, TRAF2 inhibits caspase-8-dependent apoptosis by stabilizing the RIPK1 complex, promoting tumor cell resistance to chemotherapeutic drugs (such as sorafenib). In T cells, the CD27-TRAF2-SHP-1 signaling axis regulates memory differentiation after naïve T cell activation by inhibiting Lck phosphorylation, promoting the expression of memory-related genes (such as TCF7 and LEF1). In acute myeloid leukemia (AML), LILRB3 inhibits leukemia cell apoptosis through the TRAF2-cFLIP-NF-κB pathway, while simultaneously weakening the anti-tumor activity of T cells. There is also evidence that TRAF2 differentially regulates the activation of both classical and non-classical NF-κB signaling pathways in mature B cells. This evidence suggests that TRAF2 plays a crucial role in tumorigenesis and progression, and is a key hub for immune cell fate determination and inflammatory responses; its dysfunction may lead to immune escape or excessive inflammatory responses. Currently, there are no studies reporting the role of TRAF2 in regulating tumor NK cell immune escape, and there is a lack of research clearly identifying it as a therapeutic target. Furthermore, there are no commercially available small-molecule TRAF2 antagonists with ideal efficacy, indicating a significant technological gap. Summary of the Invention

[0005] Currently, there are no reports on TRAF2 regulating tumor NK cell immune escape, and even fewer reports explicitly proposing it as a therapeutic target. Furthermore, there are no commercially available small-molecule antagonists for TRAF2 with ideal efficacy, creating a technological gap. The technical problem this invention aims to solve is to clarify whether TRAF2 is a key molecule mediating tumor cell NK cell immune escape and a therapeutic target, and to identify small-molecule antagonists targeting TRAF2 to provide drugs for the treatment of TRAF2-positive tumors.

[0006] To address the aforementioned issues, this invention first experimentally demonstrates that knocking out TRAF2 significantly enhances the sensitivity of hepatocellular carcinoma to NK cell killing, suggesting that TRAF2 holds promise as a therapeutic target for overcoming NK cell immune escape in hepatocellular carcinoma. Based on this, and using AutoDock Vina intelligent screening and experimental verification, a compound code-named ZB-020 was discovered to bind to the RING finger and the first ZINC finger domain at the N-terminus of the TRAF2 protein. This interaction was validated by in vitro SPR experiments, indicating that compound ZB-020 inhibits the biological function of TRAF2 by binding to key domains of the TRAF2 protein. Therefore, this invention provides a small molecule antagonist targeting TRAF2.

[0007] A first aspect of the present invention provides a small molecule inhibitor targeting TRAF2, said small molecule inhibitor having a structure as shown in Formula I:

[0008] Formula I.

[0009] In a second aspect, the provided small molecule inhibitor targeting TRAF2 has the ability to bind to the RING finger and the first ZINC finger domain at the N-terminus of TRAF2.

[0010] A third aspect of the present invention provides the use of the above-mentioned TRAF2-targeting inhibitor in the preparation of antitumor drugs.

[0011] A fourth aspect of the invention provides the use of the aforementioned small molecule inhibitor targeting TRAF2 in the preparation of a therapeutic agent for tumors with high TRAF2 expression, such as liver cancer.

[0012] In a fifth aspect of the present invention, there is a medicament for treating hepatocellular carcinoma, wherein the active ingredient of the medicament comprises the above-mentioned antitumor medicament targeting TRAF2 inhibitor.

[0013] Furthermore, the drug includes pharmaceutically acceptable excipients or carriers.

[0014] Furthermore, drug forms include injections, tablets, capsules, aerosols, suppositories, films, controlled-release or sustained-release formulations, or nanoformulations.

[0015] The beneficial effects of this invention are as follows: (1) This invention is the first to demonstrate through research that knocking out TRAF2 can significantly improve the killing sensitivity of liver cancer to NK cells, and TRAF2 can become a potential therapeutic target to overcome NK immune escape in hepatocellular carcinoma.

[0016] (2) This invention provides an inhibitor targeting TRAF2 and its application in the preparation of drugs targeting cancer NK immune evasion. This small molecule inhibitor can effectively synergize with NK cells to exert anti-tumor effects in vivo, and has great potential for the development of cancer immunotherapy drugs, laying the foundation for subsequent clinical trials and providing new ideas and methods for clinical treatment. Attached Figure Description

[0017] Figure 1 Flow cytometry was used to detect the purity of NK cells isolated from PBMCs.

[0018] Figure 2 Flow cytometry was used to detect the purity of mouse NK cells isolated from mouse spleen.

[0019] Figure 3 Western blot analysis of TRAF2 knockout efficiency in Hep-12 human liver cancer cells.

[0020] Figure 4 Western blot analysis of Traf2 knockout efficiency in Hepa 1-6 mouse hepatocellular carcinoma cells.

[0021] Figure 5 Schematic diagram of the killing effect of NK-92MI cells on Hep-12 cells after TRAF2 knockout.

[0022] Figure 6 Schematic diagram of the killing effect of human NK cells on Hep-12 cells after TRAF2 knockout.

[0023] Figure 7 Schematic diagram of the killing effect of Traf2 knockout mouse NK cells on Hepa 1-6 cells.

[0024] Figure 8 : Schematic diagram of the cytotoxicity of small molecule inhibitors on NK-92MI cells, spontaneous LDH release from target cells, and spontaneous LDH release from effector cells in this invention.

[0025] Figure 9 : A schematic diagram of the cytotoxicity of small molecule inhibitors on human NK cells, spontaneous LDH release from target cells, and spontaneous LDH release from effector cells in this invention.

[0026] Figure 10 AlphaFold predicts the structure of the TRAF2 protein.

[0027] Figure 11 The structural formula of the small molecule inhibitor in this invention.

[0028] Figure 12 This invention predicts the binding sites of small molecule inhibitors to the N-terminal RING finger + 1st ZINC finger domain of the TRAF2 protein.

[0029] Figure 13 Purification of full-length and truncated TRAF2 protein.

[0030] Figure 14 The affinity of the small molecule inhibitor in this invention for the full-length TRAF2 protein.

[0031] Figure 15 The affinity of the small molecule inhibitor in this invention for the truncated form of TRAF2 (1-130 aa) is shown in the figure.

[0032] Figure 16 The growth curve of tumors in mice after administration of the small molecule inhibitor in this invention.

[0033] Figure 17 The change in mouse body weight after administration of the small molecule inhibitor in this invention.

[0034] Figure 18 : Anatomical diagram of mouse tumors after administration of the small molecule inhibitor in this invention.

[0035] Figure 19 : A statistical chart of tumor weight in mice after administration of the small molecule inhibitor in this invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0039] Experimental materials: 1. Cell lines: Hep-12 human hepatocellular carcinoma cell line, Hepa 1-6 mouse hepatocellular carcinoma cell line, NK-92MI cells, human primary NK cells, and mouse primary NK cells, provided by the Beijing Cancer Prevention and Treatment Institute Laboratory. Among them, The steps for isolating and purifying primary human NK cells are as follows: (1) Incubate cells at room temperature (15–25°C) with DNase I solution at a concentration of 100 μg / mL (diluted in Hanks' balanced salt solution) for at least 15 min.

[0040] (2) The polymer suspension was filtered through a 37 μm cell filter to obtain single PBMC cells. The cells were then resuspended in EasySep™ Buffer to a density of 5 × 10⁻⁶ cells. 7 cells / mL.

[0041] (3) Human primary NK cells were isolated using the EasySep™ Human NK Cell Enrichment Kit and activated and cultured according to the instructions.

[0042] (4) The purity of the isolated human NK cells was determined by flow cytometry. The cells were resuspended in 100 μL of PBS buffer (1×10⁶ cells / mL). 6 PBMC and 1×10 6 After isolating and purifying NK cells, 5 μL of APC-anti-human CD3 antibody and PE-anti-human CD56 antibody were added to both PBMCs and NK cells. After incubation at 37°C for 30 min, the cells were washed three times with PBS buffer. Finally, the cells were resuspended in 400 μL of PBS buffer and filtered through a flow cytometry tube with a 35 μm cell filter. Similarly, isotype control antibodies against APC and PE were used to stain PBMCs and NK cells as gating for subsequent flow cytometry.

[0043] like Figure 1 As shown, PBMCs and purified NK cells were stained with APC-anti-human CD3 antibody and PE-anti-human CD56 antibody. Flow cytometry analysis showed that the purity of the purified NK cells was as high as 96%.

[0044] The steps for isolating and purifying primary mouse NK cells are as follows: (1) Obtain fresh spleens from 8-week-old female C57BL / 6 mice.

[0045] (2) Place the mouse spleen in a 10 cm culture dish containing 5 mL of Hanks' balanced salt solution.

[0046] (3) Using a razor or scalpel blade, carefully cut the spleen into small pieces (~0.2 cm).2 ).

[0047] (4) Myeloid cells were prepared as follows and crudely separated: small pieces of spleen were digested at 37°C using 5 mL of HBSS solution containing type IV collagenase (100 U / mL) and 1% FBS Dnase (20 μg / mL) for 20-30 min.

[0048] (5) Add 1 mM EDTA solution and let stand at room temperature for 5 min to terminate the enzymatic reaction.

[0049] (6) Place a 70 μm cell sieve on top of a 50 mL conical tube.

[0050] (7) Use a disposable pipette to transfer the digested spleen into a cell sieve.

[0051] (8) Use the plunger end of a syringe to crush or break up the spleen so that it passes through a sieve. If necessary, rinse with 5-10 mL of PBS.

[0052] (9) Rinse the cells with plenty of PBS to pass them through a sieve. Repeat steps (5) and (6) if necessary.

[0053] (10) Centrifuge the cells at 400-600 xg for 5 min at 4℃ and discard the supernatant.

[0054] (11) Resuspend the cells in 2-5 mL of pre-cooled 1x red blood cell lysis buffer (dilute 10× red blood cell lysis buffer with double distilled water).

[0055] (12) Place the resuspended solution on ice and let it stand for 5 minutes.

[0056] (13) Wash the cell suspension with 10-20 mL of ice-cold PBS.

[0057] Note: Perform steps (1)-(8) at room temperature, and perform steps (9)-(13) on ice using pre-cooled buffer.

[0058] (14) Centrifuge the cells at 400-600 xg for 5 min at 4°C and discard the supernatant. Then resuspend the cells in EasySep™ Buffer to a density of 1 × 10⁻⁶ cells / min. 8 cells / mL.

[0059] (15) Primary mouse NK cells were isolated using the EasySep™ Mouse NK Cell Isolation Kit and activated and cultured mouse NK cells according to the instructions.

[0060] (16) The isolated and 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% CO2. After 6 or 7 days of culture, they were used as effector cells.

[0061] (17) The purity of the isolated and purified mouse NK cells was determined by flow cytometry. 1×10⁻⁶ cells were resuspended in 100 μL of PBS buffer. 6 Mouse spleen cells and 1×10 6 Purified mouse NK cells were isolated. 2 μL of PE-anti-mouse CD49b (pan-NK cell) antibody and 5 μL of APC-anti-mouse CD3 antibody were added to both mouse spleen cells and mouse NK cells. After incubation at 37°C for 30 min, the cells were washed three times with PBS buffer. Finally, the cells were resuspended in 400 μL of PBS buffer and filtered through a flow cytometry tube with a 35 μm cell filter. Similarly, isotype control antibodies against APC and PE were used to stain the spleen cells and mouse NK cells as gating agents for subsequent flow cytometry.

[0062] like Figure 2 As shown, mouse spleen cells and purified mouse NK cells were stained with APC-anti-mouse CD3 antibody and PE-anti-mouse CD49b antibody. Flow cytometry analysis showed that the purity of the purified NK cells was as high as 82.6%.

[0063] 2. Experimental animals: 8-week-old female C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. and raised by the Animal Center of Beijing Cancer Prevention and Control Institute. All animal experimental procedures were approved by the Beijing Cancer Prevention and Control Institute Laboratory Animal Protection and Use Association.

[0064] 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.

[0065] Example 1: TRAF2 was knocked out in Hep-12 cells using CRISPR / Cas9 technology, and the knockout efficiency was verified by Western blot experiments.

[0066] 1. Construction of TRAF2 knockout expression vector (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.

[0067] (2) Hybrid oligonucleotides (gRNAs) are ligated to DNA using T4-DNA ligase. BsmBI An sgRNA expression vector was constructed in a linearized lentiCRISPR-V2 vector.

[0068] (3) Electroporation was performed using competent stbl4 cells. After plating the bacterial culture, the culture was incubated for 12-16 h. Single colonies were selected for plasmid extraction, enzyme digestion identification, and verification. Positive clones that were correctly identified were sequenced. Plasmids were extracted from the samples of clones that were correctly sequenced.

[0069] 2. The sgRNA expression vector was transiently transfected into Hep-12 cells using PEI reagent for expression, and the cells were screened for three days using puromycin. Single-clonal cells were then sorted and transferred to 96-well plates. Once the single-clonal cells in the 96-well plates reached a certain density, they were transferred to 24-well plates for further growth. When the confluence of single-clonal cell lines in the 24-well plates reached over 90%, some cells were collected for whole-protein extraction, and TRAF2 knockout cell lines were identified and screened using Western blot experiments.

[0070] 3. After washing the collected cells three times with cooled PBS, total cell protein was extracted, and the protein samples were subjected to electrophoresis and membrane transfer. Further, the cells were incubated with TRAF2 antibody and the corresponding secondary antibody, and a chemiluminescent solution was prepared for color development.

[0071] The identification results are shown in 3-4. Western blot results indicate that Hep-12 ( Figure 3 ) and Hepa 1-6 cells ( Figure 4 TRAF2 (Traf2) knockout was successful. TRAF2 knockout Hep-12 monoclonal cell lines and Hepa 1-6 monoclonal cell lines were obtained.

[0072] Example 2: To verify the effect of TRAF2 knockout on the sensitivity of liver cancer cells to NK cell killing.

[0073] 1. Take tumor target cells in the logarithmic growth phase (Hep-12 as target cells for NK-92MI and human NK, Hepa 1-6 as target cells for mouse NK), wash with 1×CMF wash buffer, digest with 0.25% trypsin, resuspend the cells in complete culture medium containing 10% serum, and transfer to 15 mL centrifuge tubes. After centrifugation at 1000 rpm for 5 min, resuspend the cell pellet in complete culture medium containing 10% serum, and filter through a 400-mesh cell sieve to prepare a single-cell suspension.

[0074] 2. Count the cells using a cell counter. Use a constant number of Hep-12 cells (50 μL, 1×10⁻⁶ cells). 4 (10 cells) were seeded in a 96-well plate.

[0075] 3. NK-92MI Cell Processing Method: NK-92MI cells with high clumping rates were precipitated using a precipitation method, discarding the supernatant and any cell debris contained therein. The cell pellet was resuspended in 1×CMF and centrifuged at 900 rpm for 5 min. The cell pellet was then resuspended in RPMI-1640 medium containing 10% serum and filtered through a 400-mesh cell strainer to prepare a single-cell suspension.

[0076] 4. Primary human / mouse NK cell processing method: Collect activated and cultured human NK / mouse NK cells into 15 mL centrifuge tubes and centrifuge at 300 xg for 10 min. Resuspend the cell pellet using ImmunoCult™ NK Cell Expansion Medium (human NK cell culture medium) or specially formulated RPMI-1640 (mouse NK culture medium) and filter through a 400-mesh cell sieve to prepare a single-cell suspension.

[0077] 5. Use a cell counter to count the cells. Incubate a constant number of NK-92MI cells (50 μL) or human / mouse NK cells (50 μL) with the target cells for 6 h. The cell ratio of NK-91MI cells or human / mouse NK cells to target cells is 1:1, 2:1, or 5:1.

[0078] 6. The killing effect of NK-92MI cells or human NK cells on tumor cells was determined using the Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay. Simultaneously, the spontaneous LDH release from NK-92MI cells, human NK cells, mouse NK cells, and tumor cells, as well as the maximum LDH release from tumor cells, were measured. The specific lysis rate of tumor cells was calculated according to the manufacturer's instructions.

[0079] like Figure 5-7 As shown, compared with Hep-12 sg ctrl cells, the lysis rate of Hep-12 sg TRAF2 cells after co-incubation with NK-92MI or human NK cells was significantly increased. Figure 5-6 This indicates that knocking out the TRAF2 gene significantly increased the in vitro killing sensitivity of Hep-12 cells to NK-92MI cells and human NK cells. Similarly, knocking out Traf2 in mouse hepatocellular carcinoma cells Hepa 1-6 significantly enhanced the killing effect of mouse NK cells on Hepa 1-6 cells. Figure 7 ).

[0080] Example 3: Virtual screening of TRAF2 small molecule antagonists.

[0081] The TRAF2 inhibitor crystal complex structure based on TRAF2 was downloaded from the PDB database (PDB ID: 1ca4); its full-length structure was used to dock with small molecules; a deep learning-based protein-compound interaction prediction model (including DeepCPI, DeepConvDTI and DeepDTA) was used to virtually screen the Drugbank commercial compound database; after screening, the top 1000 compound molecules were further screened based on Lipinski drug-likeness rules and cluster analysis to obtain the top 100 most diverse molecules for subsequent experimental testing.

[0082] Example 4: To verify the effect of treatment of Hep-12 cells with top-ranked small molecule compounds on the NK cell killing sensitivity.

[0083] 1. Take Hep-12 cells in the logarithmic growth phase and prepare a single-cell suspension as described in Example 2. 2. Count the cells using a cell counter, using a constant number of Hep-12 cells (50 μL, 1×10⁻⁶ cells). 4 Cells were seeded in 96-well plates, with four final concentrations of each compound: 0 nM, 1 nM, 10 nM, and 100 nM, in triplicate per group. Cells were incubated at 37°C for 24 h.

[0084] 3. As described in Example 2, NK-92MI cells and human NK cells were prepared into a single-cell suspension.

[0085] 4. Count the cells using a cell counter. Use a constant number of NK-92MI cells (50 μL, 5 × 10⁻⁶ cells). 4 (50 μL, 2.5 × 10⁶ cells) or human NK cells (50 μL, 2.5 × 10⁶ cells). 4 (1 cell) was co-incubated with Hep-12 cells that had been treated with the compound for 24 h for 6 h.

[0086] 5. The killing effect of NK-92MI cells or human NK cells on tumor cells was determined using the Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay. Simultaneously, the spontaneous LDH release from NK-92MI cells or human NK cells and tumor cells, as well as the maximum LDH release from tumor cells, were measured. The specific lysis rate of tumor cells was calculated according to the manufacturer's instructions.

[0087] like Figure 8-9 As shown, we validated the function of the top ten non-anticancer drugs selected through in vitro killing experiments. The results showed that, as 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.

[0088] Example 5: 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.

[0089] 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.

[0090] Table 1. Docking results of the RING finger+1st ZINC finger domain with compound ZB-020

[0091] Example 6: Affinity was verified by surface plasmon resonance (SPR) assay between the small molecule compound ZB-020 and TRAF2.

[0092] 1. Construction of pCDNA3.0-TRAF2 full-length plasmid (1) Search on NCBI TRAF2The mRNA sequence of the gene (Accession: NM_021138.4) was obtained, and its CDS region was retrieved. Primer sequences were designed upstream and downstream of the CDS sequence, and the primer sequences were sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0093] The primer sequences are as follows: Upstream primer (F): 5'-CTCGGATCCATGGCTGCAGCTAGCGTGAC-3' Downstream primer (R): 5'-CGAGCGGCCGCCGGAGCCCTGTCAGGTCCACAA-3' (2) The full-length TRAF2 sequence was obtained by PCR amplification using cDNA from Hep-12 cells as a template.

[0094] (3) Use competent DH5α cells to transform homologous recombination products. After plating the bacterial culture, incubate for 12-16 h, select single colonies to extract plasmids, identify and verify them by enzyme digestion, and sequence the positive clones that are correctly identified.

[0095] (4) Extract plasmids from the correctly sequenced clone samples to obtain pCDNA3.0-TRAF2 plasmid.

[0096] 2. Construction of PGEX-TRAF2 truncated somatic plasmid (1) Primer sequences were designed upstream and downstream of the genes corresponding to the two predicted domains of the TRAF2 protein, namely amino acids 1-130 and 334-501, and 15-20 bp homologous sequences were introduced. The primer sequences were sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0097] The primer sequences are as follows: 1) 1-130 structural domain: Upstream primer (F): 5'-CCGCGTGGATCCATGGCTGCAGCTAGCGT-3' Downstream primer (R): 5'-ATGCGGCCGCTCGAGCGGGCAGCGGCCTTCGTG-3' 2) 334-501 structural domain: Upstream primer (F): 5'-GTTCCGCGTGCGATGGCTGACTTGGAGCA-3' Downstream primer (R): 5'-ATGCGGCCGCTCGAGGAGCCCTGTCAGGT-3' (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.

[0098] (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.

[0099] (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.

[0100] (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.

[0101] 3. TRAF2 protein expression and purification (1) pCDNA3.0-TRAF2 was transfected into 293FT cells and the cells were collected 48 hours after transfection.

[0102] (2) Wash the cells twice with PBS, add 1% NP-40 lysis buffer and rotate at 4°C for 1 h to lyse.

[0103] (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.

[0104] (4) Centrifuge at 13000 xg for 15 min at 4℃, collect the supernatant and place it in an ice water bath or on ice.

[0105] (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.

[0106] (6) Add the cell supernatant to the above-mentioned agar beads and incubate at 4°C for 16 h.

[0107] (7) Centrifuge to remove liquid, wash the beads three times with TBS buffer, add 200 ng / μL of 3xFlag short peptide for competitive elution for 1 h, and concentrate the TRAF2 protein by centrifugation using an ultrafiltration tube.

[0108] 4. Expression and purification of TRAF2 truncated protein (1) Use BL21(RES) competent cells to transform the prokaryotic plasmids PGEX-TRAF2 1-130 aa and PGEX-TRAF2 334-501 aa.

[0109] (2) Select expression clones and inoculate them into 4 mL of LB medium containing ampicillin. Incubate overnight at 37°C and 220 rpm.

[0110] (3) Take the bacterial culture that has been cultured overnight at a ratio of 1:100 and inoculate it into 200 mL of LB medium containing ampicillin.

[0111] (4) Incubate the bacteria at 37℃ and 220 rpm for about 3.5 h until the OD600 of the bacterial solution reaches about 0.6. Then add IPTG to the final concentration of 0.2 mM and continue to incubate at 16℃ and 220 rpm for 16 h.

[0112] (5) Collect the bacterial culture into a centrifuge tube, centrifuge at 4℃ and 4500 xg for 15 min, discard the supernatant and collect the precipitate.

[0113] (6) Add 10 mL of lysis buffer containing protease inhibitors and add lysozyme to a final concentration of 1 mg / mL. Incubate in an ice water bath or ice bath for 30 min.

[0114] (7) Sonicate bacteria on ice with an ultrasonic power of 200-300 W, sonicate for 10 s each time, and let stand on ice for 10 s until the bacterial solution is clear.

[0115] (8) Centrifuge at 13000 xg for 15 min at 4℃, collect the supernatant and place it in ice water or on ice.

[0116] (9) Take 300 μL of well-mixed GST-bound beads, centrifuge at 1000 rpm at 4℃ and discard the storage solution. Resuspend the beads with PBS washing solution, centrifuge and discard the liquid.

[0117] (10) Add the supernatant of the bacterial solution to the above-mentioned beads and incubate at room temperature for 2 h.

[0118] (11) Centrifuge to remove liquid and wash the beads three times with PBS buffer. Add thrombin directly to the beads to digest the GST tag for 16 hours to obtain TRAF2 1-130 aa and TRAF2 334-501 aa truncated proteins.

[0119] 5. Surface Plasmon Resonance (SPR) Affinity Detection The purified truncated prokaryotic proteins TRAF2, TRAF2 1-130 aa, and TRAF2 334-501 aa were analyzed for their affinity for the small molecule compound ZB-020 using surface plasmon resonance (SPR) assay. The specific steps are as follows: (1) Protein chip immobilization The Series S Sensor Chip CM5 was mounted into the Biacore 8K biomolecular interaction analysis system. The coupling buffer was PBS-P+ solution (pH 7.4), filtered to remove air bubbles. Then, based on the isoelectric point of protein 20, the optimal pH (4.0, 4.5, 5.0, 5.5) and concentration (25, 50, 100 μg / mL) for protein immobilization on the COOH chip were selected. Protein pre-enrichment was performed to ensure sufficient protein immobilization on the chip, and pH 4.5 was ultimately determined to be the optimal coupling condition. The protein coupling involved three steps: EDC / NHS activation; protein coupling; and ethanolamine blocking of excess protein binding sites. (2) Preparation of compound detection solution Dissolve the small molecule compound ZB-020 in dimethyl sulfoxide (DMSO) solution. Take each reference standard stock solution, dilute 100 times with PBS-P solution, and then dilute sequentially with PBS solution containing 1% DMSO (pH=7.4).

[0120] (3) Affinity test The flow cell binding to the target protein was set as the detection channel, and the flow cell not binding to the target protein was set as the reference channel. The flow was initially run at the maximum flow rate (150 μL / min) with PBS-P as the detection buffer. After the signal baseline stabilized, the sample loop was rinsed with buffer to remove air. After the signal reached the baseline, the buffer flow rate was adjusted to 20 μL / min. EDCNNHS (1:1) solution was loaded to activate the chip. 200 μL of ligand diluted in activation buffer was loaded and run for 4 min. After binding stabilized, the sample loop was rinsed with buffer. 200 μL of blocking solution was loaded, and the sample loop was rinsed with buffer to remove air. To ensure stability, the baseline was observed for 5 min. Analytes were diluted with buffer and loaded at different concentrations at 20 μL / min. The protein-ligand binding time was 240 s, and the spontaneous dissociation time was 360 s. (4) Affinity curve fitting After sample injection, data processing was performed. Solvent correction results were added to the Evaluation software to obtain the response unit (RU) at the binding saturation point. The binding affinity of TRAF2, TRAF2 1-130 aa, and TRAF2 334-501 aa proteins to the small molecule compound ZB-020 was calculated using a single-site interaction model fitted with Biacore T200 Evaluation software.

[0121] like Figure 13 As shown, the full-length TRAF2 protein was expressed in eukaryotes, and the GST-tagged TRAF2 truncated protein was expressed in prokaryotes. The purified TRAF2 truncated prokaryotic protein was obtained by enzymatic digestion. Figure 14 and Figure 15 As shown, surface plasmon resonance (SPR) was used to detect affinity. The results showed that TRAF2 has a micromolar affinity for compound ZB-020, with KD(M) = 25.9 μM ( Figure 14 Furthermore, it exhibits a micromolar affinity for its N-terminal RING finger + 1st ZINC finger domain (TRAF2 truncated form 1-130 aa). Figure 15 The KD(M) was 17.1 μM, indicating that compound ZB-020 can bind to a key domain of the TRAF2 protein, thereby inhibiting the biological function of TRAF2. This result provides a strong experimental basis for further drug development.

[0122] Example 7: The therapeutic effect of the small molecule compound ZB-020 on Hepa 1-6 allogeneic xenograft tumors was verified by administering the compound to tumor-bearing, immunocompetent mice (C57BL / 6).

[0123] 1. Model establishment: Hepa 1-6 cells in logarithmic growth phase were digested, resuspended, washed twice with PBS, and then resuspended in an appropriate amount of serum-free PRMI-1640 medium. After counting the cells, the cell density was adjusted to 2 × 10⁶ cells / year. 7 / mL, use a 1mL sterile syringe to draw up the cell suspension and inoculate it subcutaneously into the back of the right upper limb of nude mice, with each mouse inoculated with 100 µL of cell suspension; 2. Animal grouping: When the tumor grows to approximately 100 mm 3 Mice with excessively large or small tumors were removed, and the remaining mice were divided into three groups: a low-dose group (10 mg / kg) of the small molecule compound ZB-020, a high-dose group (30 mg / kg) of the small molecule compound, and a control group, with 5 mice in each group.

[0124] 3. Administration and Observation: The small molecule compound ZB-020 was administered intraperitoneally at two doses: 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% physiological saline; the control group used the solvent). On the day of the first administration, the mouse's body weight, tumor volume, and condition were recorded, and the mouse was observed. The small molecule compound was administered intraperitoneally daily, and the mouse tumors were measured and recorded every two days, along with the mouse's body weight. When the maximum tumor volume in the control group mice reached 1.5 cm... 3 The experiment was terminated around 10:00 AM. Mice were euthanized by cervical dislocation, and the tumors were dissected, photographed, and weighed. Finally, the tumors were divided into two parts, one fixed with paraformaldehyde and stored at room temperature, and the other flash-frozen in liquid nitrogen and stored at -80°C.

[0125] 4. Statistical Analysis of Experimental Data: After the experiment, the body weight change curves, tumor volume change curves, and final tumor weight distribution maps of each group of mice were plotted. The tumor inhibition rate was calculated. Tumor inhibition rate IR (%) = (1 - TTW / CTW) × 100% TTW: Tumor weight in the treatment group; CTW: Tumor weight in the control group.

[0126] like Figure 16-19 As shown, the inhibitory effect of intraperitoneal injection of ZB-020 (10 mg / kg and 30 mg / kg) on ​​tumor growth was investigated in a Hepa 1-6 xenograft syngeneic mouse model. The results showed that the small molecule compound ZB-020 effectively inhibited tumor growth in Hepa 1-6 xenograft syngeneic mice at both high and low doses, with an inhibition rate of 89.58% in the low-dose group and 100% in the high-dose group. Furthermore, the small molecule compound showed no significant toxic side effects in mice.

[0127] The small molecule inhibitor ZB-020 exhibits a strong affinity for TRAF2 at the micromolar level. This inhibitor significantly enhances the in vitro killing effect of NK-92MI or human NK cells on HCC cells and demonstrates good inhibitory effects on tumor growth in immunocompetent mouse models. This evidence suggests the potential therapeutic efficacy of ZB-020 for tumor types that highly express TRAF2.

[0128] In summary, this invention verifies that compound ZB-020, a non-antitumor drug, can enhance the killing effect of NK-92MI or human NK cells on liver cancer cells in vitro, and exhibits good therapeutic effects on syngeneic mouse tumors in in vivo. It is a promising candidate drug for targeting immune escape by liver cancer NK cells.

[0129] The above description is only a simpler and preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a TRAF2 inhibitor in the manufacture of an antitumor medicament, characterized in that having a structure represented by Formula I: Formula I.

2. Use according to claim 1, characterized in that: The TRAF2 inhibitor has the ability to bind to the RING finger and the first ZINC finger domain of the N-terminal of TRAF2.

3. An antitumor drug comprising the TRAF2 inhibitor of claim 1.

4. The antitumor drug according to claim 3, wherein: The TRAF2 inhibitor has the ability to bind to the RING finger and the first ZINC finger domain of the N-terminal of TRAF2.

5. The antitumor drug according to claim 3, wherein: ###0001### The antitumor drug comprises a pharmaceutically acceptable adjuvant or carrier.

6. The antitumor drug according to claim 3, wherein: ###0002### The tumor is a tumor that highly expresses TRAF2.

7. The antitumor drug according to claim 3 or 6, wherein: The tumor is liver cancer.

8. The antitumor drug according to claim 3, wherein: ###0002### The preparation forms include injections, tablets, capsules, aerosols, suppositories, films, controlled or sustained release agents, or nano-preparations.

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