Anti-tumor small molecule compounds targeting FGL2 and their applications

Abstract

The present invention relates to the field of biomedical technology, and discloses anti-tumor small molecule compounds targeting FGL2, including any one of the small molecule compounds C16H17N3O2 and C14H16N2O2S2; and also discloses the application thereof. The present invention has discovered a small molecule compound C16H17N3O2 that can specifically bind to FGL2. 16 H 17 N3O2 and C 14 H 16 N2O2S2, the above two small molecule compounds can insert into the pocket of the fibrinogen-related domain composed of 233 amino acids at the C-terminus of the FGL2 protein and bind tightly; the above two small molecule compounds interfere with the binding of FGL2 to its receptor FcγRIIB, thereby promoting the proliferation and differentiation of T cells induced by anti-CD3 antibodies + anti-CD28 antibodies, thereby promoting the function of T cells and interfering with the activation of myeloid-derived immunosuppressive cells (MDSCs), thereby inducing tumor clearance, and can be used in tumor diseases with high expression of FGL2, such as liver cancer, severe hepatitis and other related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to an anti-tumor small molecule compound targeting FGL2 and its application. Background Art

[0002] Fibrinogen-like protein 2 (FGL2) belongs to the fibrinogen-related protein superfamily and is also known as FGL2 prothrombinase. In the absence of coagulation factors FVII or FX, FGL2 can directly cleave prothrombin into thrombin, thereby promoting thrombosis. There are two forms of FGL2 protein. Transmembrane FGL2 (mFGL2) is only expressed in macrophages, endothelial cells and intestinal trophoblasts. However, under viral infection or cytokine induction, endothelial cells, T cells, especially CD4 + CD25 + Regulatory T cells (Treg) and macrophages can highly express secretory FGL2 (sFGL2). Structurally, the C-terminus of the FGL2 protein has a 233-amino acid fibrinogen-related domain (FRED), which is the recognition region of FGL2 and the FcγRIIb receptor; it is also the site where FGL2 binds to calcium and substrates; and its N-terminus is mainly composed of some linearly arranged hydrophobic amino acids and contains a transmembrane region and serine Ser89 that can promote coagulation activity, which can quickly initiate the coagulation process. It has been confirmed that mFGL2 has procoagulant activity and sFGL2 mainly mediates T cell damage and even promotes CD8 + T cell apoptosis. HBV, HCV and murine hepatitis virus-3 (MHV-3) can stimulate infected hepatocytes or macrophages to upregulate the expression of mFGL2, causing intrahepatic thrombosis and subsequently tissue necrosis. In acute transplant rejection, mFGL2 induces coagulation events in the transplanted organ, so targeting mFGL2 is one of the effective strategies to intervene in transplant rejection. In addition, studies have found that sFGL2 can inhibit dendritic cell maturation (downregulating CD80 and MHC-II expression) and induce CD4 + T cell anergy; sFGL2-FcγRIIb signaling pathway can activate the downstream BTK-JNK pathway to promote CD8 + Apoptosis of T and B cells. Furthermore, studies have found that sFgl2 secreted by Tregs is the molecular basis for their immunosuppressive function. FcγRIIB is expressed on the surface of various cells and can mediate the regulation of various immune cell functions, such as inhibiting macrophage killing, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and T cell proliferation and activation.

[0003] The inventors' experimental results indicate that sFgl2, upon binding to its ligand FcγRIIB, inhibits neutrophil infiltration, promotes neutrophil apoptosis, and macrophage clearance, accelerating the resolution of peritonitis in mice. These results suggest that FGL2 plays a key role in virally induced severe hepatitis and provide a new target for the treatment of inflammatory diseases. Studies have found that FGL2 is abundantly expressed in tumor tissues, including melanoma, ovarian cancer, cervical cancer, lung cancer, kidney cancer, liver cancer, colorectal cancer, breast cancer, and prostate cancer. In human malignant tumors, FGL2 is highly expressed primarily in tumor parenchymal cells, interstitial infiltrating cells, tumor vascular endothelial cells, and extracellular matrix (EMC), and this high expression correlates anatomically with fibrin deposition. It has also been found that its ligand FcγRIIB is highly expressed primarily on myeloid-derived immunosuppressive cells (MDSCs) and T cells. Subcutaneous tumor growth in Fgl2-knockout mice is inhibited, with an increase in tumor-infiltrating M1 macrophages and DCs, and a decrease in MDSC infiltration. Further studies found that the infiltration of granulocyte-derived MDSCs subsets in tumor tissues of the Fgl2 KO group was significantly reduced. In addition, the ability of Fgl2 KO HPCs to differentiate into granulocyte / macrophage precursor cells (GMPs) was weakened. In particular, the study found that FGL2 neutralizing antibodies could significantly reduce the level of MDSCs in tumors and increase CD8 + At the same time, the use of Fgl2 neutralizing antibodies and PD-1 neutralizing antibodies can further reduce the content of MDSCs in tumors and activate anti-tumor immunity. The results show that Fgl2 plays a key role in tumor immune escape.

[0004] Liver studies have found that hFgl2 levels in liver tissue from patients with severe chronic hepatitis B are higher than those in patients with mild or moderate chronic hepatitis B, suggesting that hFgl2 may play a role in exacerbating liver damage in chronic hepatitis B. Furthermore, hFgl2 expression levels in liver tissue from patients with severe hepatitis were positively correlated with serum bilirubin. Furthermore, fibrin deposition and microthrombosis were observed in the tissue adjacent to cells with high hFgl2 expression in liver tissue from patients with severe hepatitis B. Notably, studies have also found that hFgl2 is highly expressed in liver cancer tissue, at levels higher than those in patients with mild or moderate chronic hepatitis B. This suggests that hFgl2 may be involved in the development, progression, and activity of liver cancer and severe hepatitis caused by HBV infection. Therefore, screening for small molecule compounds that interfere with FGL2 function could provide new therapeutic approaches. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to use FGL2 as a target and screen small molecule compounds that can specifically bind to it. These small molecule compounds can interfere with the Fgl2-FcγRIIB inhibitory signaling pathway, thereby promoting the function of T cells and interfering with the activation of myeloid-derived immunosuppressive cells (MDSCs), thereby inducing tumor clearance.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] An anti-tumor small molecule compound targeting FGL2, wherein the small molecule compound is any one of C16H17N3O2 and C14H16N2O2S2.

[0008] Furthermore, the tumor is a liver tumor.

[0009] Furthermore, the small molecule compound is used at a concentration of 1 μM.

[0010] Furthermore, the small molecule compound tightly binds to the FGL2 protein by inserting into the pocket of the fibrinogen-related domain composed of 233 amino acids at the C-terminus of the FGL2 protein.

[0011] To achieve the above objectives, the present invention also provides the use of small molecule compounds in the preparation of drugs for treating tumor diseases with high expression of FGL2.

[0012] Furthermore, the drug interferes with the binding of FGL2 to its receptor FcγRIIB by binding to FGL2, thereby promoting T cell proliferation and differentiation.

[0013] Furthermore, the drug interferes with the binding of FGL2 to its receptor FcγRIIB by binding to FGL2, thereby interfering with the activation of myeloid-derived immunosuppressive cells, thereby inducing tumor clearance.

[0014] Furthermore, the drug can also be used to treat severe hepatitis.

[0015] The beneficial effect of the present invention is that: the present invention discovered a small molecule compound C that can specifically bind to FGL2 16 H 17 N3O2 and C 14 H 16N2O2S2, the above two small molecule compounds can insert into the pocket of the fibrinogen-related domain composed of 233 amino acids at the C-terminus of the FGL2 protein and bind tightly; the above two small molecule compounds interfere with the binding of FGL2 to its receptor FcγRIIB, thereby promoting the proliferation and differentiation of T cells induced by anti-CD3 antibodies + anti-CD28 antibodies, thereby promoting the function of T cells and interfering with the activation of myeloid-derived immunosuppressive cells (MDSCs), thereby inducing tumor clearance, and can be used in tumor diseases with high expression of FGL2, such as liver cancer, severe hepatitis and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0017] Figure 1 Figure 1 is a protein structure model diagram of hFGL2 constructed in Example 1;

[0018] Figure 2 This is a list of potential small molecule compounds that can bind to FGL2 and insert into the FGL2 pocket in Example 1;

[0019] Figure 3 The small molecule compounds in Example 1 interfere with the sFGL2-FcγRIIB pathway on CD3 + Results of the regulatory effect on T cell proliferation;

[0020] Figure 4 The figure shows the drugability analysis results of the small molecule compound in Example 1;

[0021] Figure 5 C in Example 1 14 H 16 Figure 2 shows the binding effect of N2O2S2 compounds on mouse FGL2;

[0022] Figure 6 This is a picture of the tumor size observed by in vivo imaging in Example 2;

[0023] Figure 7 This is a graph showing the inhibitory effects of the two small molecule compounds in Example 2 on the liver cancer transplant tumor model. DETAILED DESCRIPTION

[0024] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0025] Example 1:

[0026] Since there is no report on the structure of human and mouse FGL2 proteins, the inventors used Discovery studio and alphafold 2 to predict the structure of human fibroblast hFGL2 protein, focusing on its FRED domain; further constructed a protein structure model of the hFGL2 catalytic site, and predicted a volume of approximately The average depth is The cavity has the potential to bind small molecules.

[0027] The application website of Alphafold 2 is: (AlphaFold Protein Structure Database, http: / / alphafold.com), and the application website of Discovery studio is: BIOVIA Discovery Studio | Dassault Systèmes (3ds.com https: / / www.3ds.com / products / biovia / discovery-studio). Figure 1 Figure A shows that the structure of human FGL2 protein differs significantly from that of chicken fibronogen, as chicken FGL2 lacks the FcrIIR receptor binding domain. Figure 2 shows the structure of human FGL2 protein predicted using Alphafold2 software. Figure 3 shows the characteristics of the FRED domain, which was the focus of the study.

[0028] Next, the inventors used Schrodinger Glide software to perform virtual screening using molecular docking technology based on the pocket structure (cavity) in the hFGL2 protein structure. They evaluated protein-small molecule binding affinity for approximately 3 million small molecules from compound libraries such as Specs and ChemDiv, and screened for drug-like structures based on the five rules of drug development and the PAINS rules. The enriched structures were analyzed based on structural diversity and binding mode, ultimately selecting 50 to 100 compounds for purchase and subsequent evaluation.

[0029] The inventors established a method using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), enzyme activity assays, and crystallography to clarify the binding activity of small molecules obtained through virtual screening against hFGL2. Based on the binding capacity of the small molecules and structure-activity relationship data suggested by molecular docking, they used medicinal chemistry methods to modify the small molecules and discovered new, specific hFGL2 lead compounds with excellent activity and strong selectivity, providing new chemical entities for subsequent functional research and drug development of hFGL2.

[0030] Through the above research, the inventors screened 25 candidate molecules from the small molecule compound library, see attached Figure 2 .

[0031] Attachment Figure 2 The candidate molecules obtained in the experiment may not actually bind to FGL2, or even if they do, their interference effect may be weak or even non-functional. Therefore, it is necessary to further enrich the number of candidate small molecule compounds and verify their interference effect.

[0032] The inventors extracted mouse spleen tissue, ground the tissue to obtain a single cell suspension, and used flow cytometry or magnetic bead sorting technology to obtain purified mouse naive CD3 + T cells.

[0033] Stimulation with anti-CD3 antibody combined with CD28 antibody CD3 + T cells were induced to activate and proliferate, and the cells were labeled with CFSE. After 48 hours, the cell proliferation was detected by flow cytometry.

[0034] In the above operation, mouse T lymphocytes were obtained by taking lymphocytes from mouse spleen and obtaining mouse T lymphocytes (magnetic bead negative selection). The steps for CFSE labeling of T lymphocytes are as follows:

[0035] 1. Gently resuspend the cells in DPBS preheated at 37°C containing 1 μmol / mL CFSE to a cell concentration of 1x10 6 cell / ml (the original concentration of CFSE is 10 mM and needs to be diluted 10,000 times);

[0036] 2. Incubate at 37°C in the dark for 5 minutes, then add 5 times the volume of 37°C preheated 1640 complete medium, mix well, incubate at 37°C for 5 minutes (to terminate staining), centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in preheated 1640 complete medium, incubate at 37°C for 10 minutes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the cells in preheated 1640 complete medium, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0037] 3. Then use 1640 complete component culture medium containing 10% fetal bovine serum to adjust the cell concentration to 1x10 6 cell / ml.

[0038] The steps for T cell activation (CD3 / CD28 beads activation method) are as follows:

[0039] 1. Wash the magnetic beads: Resuspend the magnetic beads in the vial (vortex > 30 seconds), transfer the required volume of magnetic beads to the tube, add an equal amount of MACS buffer, or at least 200ul, mix (vortex 5 seconds, or pipette to mix), and place the tube on the magnetic rack for 1 minute;

[0040] 2. Add magnetic beads (the ratio of magnetic beads to cells is 1:1, as shown in the table below) to the cell suspension and mix well.

[0041]

[0042] Activated CD3 + T cells were labeled with CFSE and analyzed by flow cytometry 48 hours later for the inhibition of CD3 T cells by sFGL2 through its receptor FcγRIIB. + T cell proliferation.

[0043] In the study, it was confirmed that sFGL2 inhibited CD3 through its receptor FcγRIIB. + Under the premise of T cell proliferation, anti-CD3 antibody + CD28 antibody + sFGL2 stimulation CD3 + T cells were added to the Figure 2The small molecule compounds were 10 μM each, and the cells were labeled with CFSE. After 48 hours, the effects of the small molecule compounds on CD3 by interfering with the sFGL2-FcγRIIB pathway were analyzed by flow cytometry. + Regulation of T cell proliferation.

[0044] The steps for plating sFGL2 and compound addition are as follows:

[0045] 1. Take the U-bottom 96-well plate as an example, the plate needs to be rinsed with PBS first, and 8×10 4 cells, 150ul RPMI-1640 complete medium;

[0046] 2. Blank group: blank control cells + 0.15ul DMSO

[0047] Stim group: CFSE-stained cells + 2ul beads + 0.15ul DMSO

[0048] fgl2 group: CFSE-stained cells + 5ug / mL fgl2 + 0.15ul DMSO

[0049] Stim+fgl2 group: CFSE-stained cells+2ul beads+5ug / mL fgl2+0.15ul DMSO

[0050] Stim+fgl2+compound group: CFSE-stained cells + 2ul beads + 5ug / mL fgl2 + 10uM compound (the original concentration of the compound is 10mM, which needs to be diluted 1000 times)

[0051] 3. Spread evenly into the well plate and place in the incubator for 48 hours.

[0052] See attached Figure 3 The experimental results showed that treatment with 8 drugs, including 4, 10, 11, 16, 17, 21, 22 and 23, could restore T cell proliferation ability by interfering with FGL2 activity.

[0053] To further verify the drugability of the small molecule compound obtained above, the inventors further diluted the concentration of the compound from 10 μM to 1 μM, as follows:

[0054] 1. Take the U-bottom 96-well plate as an example, the plate needs to be rinsed with PBS first, and 8×10 4 cells, 150ul RPMI-1640 complete culture medium;

[0055] 2. Solution:

[0056] Blank group: blank control cells + 0.15ul DMSO

[0057] Stim group: CFSE-stained cells + 2ul beads + 0.15ul DMSO

[0058] fgl2 group: CFSE-stained cells + 5ug / mL fgl2 + 0.15ul DMSO

[0059] Stim+fgl2 group: CFSE-stained cells+2ul beads+5ug / mL fgl2+0.15ul DMSO

[0060] stim+fgl2+compound groups (4, 10, 11, 16, 17, 21, 22, 23): CFSE-stained cells+2ul beads+5ug / mL fgl2+1uM compound;

[0061] 3. Evenly spread the cells into the well plate and place them in the incubator for 48 hours;

[0062] 4. After incubation, add 150ul FACS solution to each well, mix well to terminate staining, centrifuge at 1700rpm for 5min, discard the supernatant, add 200ul FACS solution to resuspend the cell pellet; centrifuge at 1700rpm for 5min, discard the supernatant, add 200ul FACS solution to resuspend the cell pellet, transfer to a flow cytometer, and detect on the flow cytometer.

[0063] See attached Figure 4 , it can be seen that No. 16 (C 16 H 17 N3O2 (ChemDiv Product No.: Y206-1674)) and No. 17 (C 14 H 16 Treatment with N2O2S2 (ChemDiv product number: Y206-8392) at a concentration of 1 μM may restore T cell proliferation ability by interfering with FGL2 activity.

[0064] Based on the above results, the inventors further used molecular docking technology to analyze the 16 H 17 N3O2) compounds and No. 17 (C 14 H 16 The binding effect of N2O2S2) compounds on mouse FGL2 is shown in the attached Figure 5 The results showed that both compounds could stably bind to mouse FGL2.

[0065] Example 2: Treatment with small molecule compounds inhibits the progression of liver cancer transplant models

[0066] Since FGL2 is expressed in liver cancer and liver with the highest abundance, the inventors established a liver cancer transplant model and analyzed the expression of FGL2 in 16 (C 16 H 17 N3O2) and No. 17 (C 14 H 16 The regulatory effect of small molecule compounds (N2O2S2) on the progression of liver cancer. The Hepa1-6-Luc-puro cell line used in this example was purchased from Yimo Biotechnology, and SPF-grade C57BL / 6 (WT) mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd., all of which were 6 to 8 weeks old and weighed 17 to 22 g. The animal experiments involved in this example were in accordance with the animal research protocol approved by the Experimental Animal Welfare and Ethics Committee of the Army Medical University.

[0067] Prepare 20% urethane anesthetic solution with normal saline and inject it intraperitoneally at 1500 mg / kg. Adjust the Hepa1-6-Luc-puro cells to 5×10 7 After the mouse is fully anesthetized, the abdominal cavity of the mouse is exposed, the liver lobe of the mouse is squeezed out, and 20 μl of the cell suspension (about 10 6 cells / each) and injected into the exposed liver lobe. At the same time, mice were intraperitoneally injected with 16 (C 16 H 17 N3O2)(1mg / kg); and No. 17(C 14 H 16 N2O2S2)(1mg / kg).

[0068] In vivo imaging of tumor size: After 7 days (observed every three days), when the mouse wounds were essentially healed, tumor size was observed by in vivo imaging. In vivo imaging results were analyzed using Living Image 4.4 software, and immunohistochemistry results were quantified using Image J software. Experimental data were expressed as mean ± standard deviation, and GraphPad Prism 8.0 software was used for difference analysis and graphing. The unpaired t-test was used to compare the two samples; p < 0.05 was considered statistically significant. The mice were weighed and labeled.

[0069] In the process of tracking phenotypic differences in the liver cancer model constructed by the above-mentioned Hepa1-6-Luc-puro cell line, it was found that the model mice showed abnormal behaviors such as hunched back, isolation, and slow activity about three days after surgery, while the mice treated with the small-molecule compound C 16 H 17 N3O2 and small molecule compound C 14 H 16 Mice treated with N2O2S2 did not experience similar phenomena, their activities were normal, and there were no other abnormalities.

[0070] See attached Figure 6 In vivo imaging results showed that the small fraction compound C 16 H 17 N3O2 and small molecule compound C 14 H 16 N2O2S2 can inhibit the progression of liver transplant tumor model. Figure 7 On the tenth day after treatment, the 16 H 17 N3O2 and small molecule compound C 14 H 16 The size of liver cancer treated with N2O2S2 was smaller, indicating that the small molecule compound C 16 H 17 N3O2 and small molecule compound C 14 H 16 N2O2S2 has a significant inhibitory effect on the progression of liver cancer in vivo.

[0071] Example 3: Single-cell transcriptome sequencing analysis of the potential mechanism by which small molecule compound treatment regulates liver cancer progression

[0072] The inventors performed single-cell sequencing analysis on liver cancer tissues of C57BL / 6 mice, grouped the cells, and then continued to perform differential analysis, GO function enrichment, and KEGG pathway enrichment on the main affected cell populations to further analyze the potential mechanism by which small molecule compound treatment affects the occurrence and development of liver cancer.

[0073] The results showed that endothelial cells and B cells accounted for a large proportion in normal liver tissue, while T cells and macrophages were dominant in cancer tissue. In addition, the immune cells infiltrating the cancer tissue after modeling were significantly higher than those in the Mock-treated mice. 16 H 17 N3O2 and small molecule compound C 14 H 16 N2O2S2 treatment can lead to CD8 + T cells, macrophages, monocytes and neutrophils increased significantly in vivo, which indicated that the small molecule compound C 16 H 17 N3O2 and small molecule compound C 14 H 16 Both N2O2S2 can antagonize tumor progression by blocking the sFGL2 signaling pathway.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of a small molecule compound in the preparation of a drug for treating tumor diseases with high expression of FGL2, characterized in that: The small molecule compound is C 14 H 16 N2O2S2, the tumor disease with high expression of FGL2 is liver tumor, the small molecule compound C 14 H 16 The structure of N2O2S2 is: 。 2. The use according to claim 1, characterized in that The drug interferes with the binding of FGL2 to its receptor FcγRIIB by binding to FGL2, thereby promoting T cell proliferation and differentiation.

3. The use according to claim 1, characterized in that The drug binds to FGL2 and interferes with the binding of FGL2 to its receptor FcγRIIB, thereby interfering with the activation of myeloid-derived immunosuppressive cells, thereby inducing tumor clearance.

4. The use according to claim 1, characterized in that The medicine can also be used to treat severe hepatitis.

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