Effect of small molecule inhibitor of GSDME protein in tumor and immunotherapy thereof

Abstract

The invention discloses an effect of a small-molecule inhibitor of GSDME protein in tumor and immunotherapy thereof. The invention provides application of Eliprodil or an Eliprodil combined immune checkpoint inhibitor in preparation of anti-tumor drugs, a direct action target of Eliprodil is a PDPK1 binding region in a non-N-terminal fragment of GSDME protein, GSDME is prevented from playing a downstream PI3K-AKT pathway activation function, so that M2-like polarization of macrophages is inhibited, in-vivo anti-tumor experiments show that Eliprodil significantly inhibits proliferation of tumors, and the Eliprodil and the immune checkpoint inhibitor can be used for preparing anti-tumor drugs. Compared with the prior art, the compound has the advantages that the quantity of TAMs (M2 sample) infiltrated by tumors is reduced, the infiltration of toxic CD8 + T cells is increased, the growth of the tumors is remarkably inhibited when the compound is combined with an immune checkpoint inhibitor alphaPD-1, and the compound has a very good synergistic sensitization effect, is low in toxicity and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the role of small molecule inhibitors of GSDME protein in tumors and immunotherapy thereof, and belongs to the technical field of biomedicine. Background Art

[0002] In recent years, immune checkpoint inhibitor therapy has been the focus of clinical translation and basic research, showing significant anti-tumor effects in some cancer patients. However, the prevalence of drug resistance hinders its efficacy in most cases. Therefore, the combination of immune checkpoint inhibitors with other drugs or treatments is expected to become an innovative strategy for tumor treatment. M1 (pro-inflammatory) and M2 (anti-inflammatory) are the two main subtypes of macrophages. In solid tumor tissues, tumor-associated macrophages (TAMs) often exhibit an M2-like phenotype, which helps promote tumor development. Because TAMs not only directly affect the survival and death pathways of tumor cells, but also have significant immunosuppressive effects, mediate immunotherapy resistance, and indirectly promote tumor growth. Studies have shown that TAMs have a direct or indirect inhibitory effect on the activity of CD8+T cells, so the depletion of macrophages can enhance the anti-tumor efficacy of CD8+T cells. Due to the regulatory role of TAMs in the tumor microenvironment, the study of drug targeting TAMs to improve tumor immunotherapy has attracted widespread attention. Therefore, it is of great significance to develop new small molecule drugs that can reprogram or repolarize TAMs, regain anti-tumor properties, and combine with immune checkpoint inhibitors (αPD1) to improve immunotherapy.

[0003] GSDME, an N-terminal fragment generated by caspase-3 protein cleavage, localizes on the membrane and forms pores, triggering cell pyroptosis. New evidence shows that GSDME can promote the development of pancreatic adenocarcinoma and colitis-associated colorectal cancer, which is independent of the pyroptosis mechanism mediated by its N-terminal fragment. However, the regulatory role and mechanism of its non-N-terminal fragment on the immune microenvironment in solid tumors have not been reported. Summary of the invention

[0004] The purpose of the present invention is to address the low efficacy of current immune checkpoint inhibitors in cancer treatment and the existence of problems such as drug resistance. The present invention provides the role of Eliprodil, a small molecule inhibitor of GSDME protein, in tumors and their immunotherapy.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect of the present invention, use of Eliprodil or Eliprodil combined with an immune checkpoint inhibitor in the preparation of an anti-tumor drug is provided, wherein the chemical structural formula of the Eliprodil is as follows:

[0007]

[0008] The drug treats liver cancer by improving the tumor immune microenvironment and enhancing cytotoxic CD8+T cells; the drug can sensitize immune checkpoint inhibitors; the drug can reverse the immune escape of tumors or prepare products that reverse the immune escape of tumors; the drug can inhibit the resistance of tumors to immune checkpoint inhibitors.

[0009] Preferably, the tumor comprises liver cancer.

[0010] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is Eliprodil or a combination of Eliprodil and an immune checkpoint inhibitor.

[0011] Preferably, the immune checkpoint inhibitor is αPD-1.

[0012] Eliprodil is called Eliprodil in Chinese, a highly effective and selective inhibitor of NR2B-NMDA receptor. It has a weak effect on NR2A- and NR2C-NMDA receptors and can be used in trials to study the treatment of Parkinson's disease and movement disorders. The present invention found that Eliprodil has the effect of reversing the resistance of immune checkpoint inhibitors and has low toxicity. At the same time, in vitro and in vivo experiments have confirmed that Eliprodil is a new small molecule inhibitor that inhibits macrophage M2-like polarization and enhances the killing activity of CD8+T cells, and has application prospects in reversing tumor immune escape and sensitizing immune checkpoint inhibitors.

[0013] The Eliprodil is a drug or an experimental reagent.

[0014] The pharmaceutically acceptable carrier may include excipients and diluents, etc. Moreover, according to the usual method, the drug can be prepared into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0015] The pharmaceutically acceptable carriers are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0016] The medicine of the present invention can be applied to the body by known means. For example, it is delivered to the tissue of interest by intravenous systemic delivery or local injection. Alternatively, it is applied via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0017] Preferably, the drug administration subjects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0018] The second aspect of the present invention provides the use of a reagent for detecting GSDME protein expression in macrophages in the preparation of a product for detecting liver cancer or liver cancer prognosis.

[0019] Preferably, the reagent for detecting the expression of GSDME protein in macrophages comprises a reagent for detecting the expression of macrophage marker CD68 and a reagent for detecting the expression of GSDME protein.

[0020] Preferably, the reagent for detecting the expression of the macrophage marker CD68 is a CD68 antibody, and the reagent for detecting the expression of the GSDME protein is a GSDME antibody.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention performs computer virtual screening of non-N-segment fragments of GSDME protein and verifies through in vitro molecular docking experiments that Eliprodil mainly acts on the PDPK1 protein binding region of GSDME protein. In addition, through protein immunoblotting experiments, cell flow cytometry, multiple fluorescent immunohistochemistry experiments and other experiments, the results show that Eliprodil can inhibit the M2-like polarization of macrophages and enhance the killing activity of CD8+T cells.

[0023] (2) The present invention has demonstrated through in vivo and in vitro experiments that the direct target of Eliprodil is pyroconazole E (GSDME), and has determined that high expression of GSDME in macrophages is negatively correlated with the prognosis of liver cancer, and inhibits the activation of the PI3K-AKT pathway of macrophages, making it a new target for tumor treatment; Eliprodil also enhances the toxic function of CD8+T cells, and has a significant synergistic effect when used in combination with the immune checkpoint inhibitor αPD-1, significantly improving the immunotherapy effect of tumors.

[0024] (3) The present invention has been confirmed through in vitro and in vivo experiments that Eliprodil significantly inhibits macrophage M2-like polarization and enhances the killing function of CD8+T cells. It is a new type of small molecule inhibitor that can improve the tumor immune microenvironment and has application prospects in reversing tumor immune escape and sensitizing immune checkpoint inhibitors.

[0025] (4) The in vivo anti-tumor experiments of the present invention show that Eliprodil significantly inhibits tumor proliferation, reduces the number of tumor-infiltrating TAMs (M2-like), increases the infiltration of toxic CD8+T cells, and significantly inhibits tumor growth when combined with the immune checkpoint inhibitor αPD-1. It has a good synergistic sensitization effect and low toxicity, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the expression and prognosis of GSDME and CD68 in human liver cancer and adjacent tumors in Example 1; wherein: A is the expression of GSDME and CD68 in human liver cancer and adjacent tumors, B is the correlation analysis of GSDME and CD68 expression, and C is the relationship between the expression of GSDME and CD68 and the prognosis of liver cancer.

[0027] Figure 2 Schematic diagram of virtual screening of small molecule inhibitors of GSDME in Example 2; wherein: A is the chemical structure of Eliprodil; B is the docking pattern diagram of Eliprodil and GSDME molecule (281-512aa fragment); C is the top 100 small molecules with the most binding potential to GSDME molecule (281-512aa fragment) screened by in vitro differential scanning fluorimetry (DSF) experiment; D is the affinity result of Eliprodil and GSDME molecule (281-512aa fragment) verified by in vitro plasma resonance (SPR) experiment.

[0028] Figure 3 The present invention is the regulation of macrophage polarization and the effect of macrophages on tumor proliferation by Eliprodil in Example 3; wherein: A is the in vitro promotion of the proliferation inhibition of liver cancer cells by the supernatant of macrophages Raw264.7 by Eliprodil; B is the in vitro promotion of the proliferation inhibition of liver cancer cells by the supernatant of bone marrow-derived macrophages (mouse BMDMs) by Eliprodil; C is the in vivo inhibition of liver tumor proliferation by Eliprodil; D is an in vitro experiment proving that Eliprodil inhibits the activation of the PI3K-AKT pathway of macrophages.

[0029] Figure 4Eliprodil inhibits the proliferation of liver cancer (two spontaneous hepatocellular carcinomas) in vivo and has a synergistic therapeutic effect with αPD-1 in Example 4; wherein: A means Eliprodil inhibits the proliferation of spontaneous liver cancer overexpressing C-MYC in vivo and has a synergistic therapeutic effect with αPD-1; B means Eliprodil inhibits the proliferation of liver cancer induced by tail vein injection of plasmid in vivo and has a synergistic therapeutic effect with αPD-1.

[0030] Figure 5 Eliprodil inhibits macrophage M2-like polarization and enhances CD8+T cell effector function in Example 5; wherein: A is that Eliprodil reduces the proportion of CD206+ macrophages; B is that Eliprodil enhances the secretion of PFN and GZMB by CD8+T cells. DETAILED DESCRIPTION

[0031] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0032] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used were conventional commercially available products unless otherwise specified.

[0033] Example 1: Expression of GSDME and CD68 in liver cancer and adjacent tissues and their prognostic value for liver cancer patients

[0034] In this example, about 250 clinical specimens were collected, and liver cancer tissue samples were collected from Zhongshan Hospital Affiliated to Fudan University. Immunohistochemistry was used to detect the expression of GSDME and CD68; the tissue was fixed in 10% neutral formalin overnight, dehydrated, and then embedded in low-melting paraffin. 4 μm thick tissue sections were cut continuously and fixed on siliconized glass slides. Immunohistochemical staining used the streptavidin-peroxidase coupling method. Each tissue section was deparaffinized, rehydrated, immersed in antigen retrieval solution, boiled at 100°C for 10 minutes, and then incubated with peroxidase inhibitors. After blocking nonspecific binding with normal goat serum, the tissue sections were incubated with the following primary antibodies at 4°C overnight: anti-GSDME (Abcam, ab215191) and anti-CD68 (CST, 76437); the antibodies were used at a dilution of 1:100 or 1:200, respectively. Finally, according to the clinical data and prognosis corresponding to the specimens, the relationship between GSDME and CD68 expression and clinical pathological characteristics and the prognosis of liver cancer patients was analyzed. Statistical methods: The results of at least three independent experiments are shown as mean ± standard deviation. GraphPadPrism software (version 9.0) was used for statistical analysis. When only two groups were analyzed, the differences between the groups were evaluated using two Student t tests; when more than two groups were compared, one-way analysis of variance was performed. The Gehan-Breslow-Wilcoxon test was performed to analyze the correlation between the expression of the target protein and the overall survival time.

[0035] Results: Figure 1 As shown in A, the expression of GSDME and CD68 in human liver cancer is significantly higher than that in adjacent liver cancers, and there is a significant correlation between their expressions, such as Figure 1 As shown in B; Survival analysis showed that high expression of GSDME and CD68 was associated with poor prognosis of liver cancer. The prognosis and survival rate of liver cancer patients in the group with high expression of GSDME and CD68 was significantly improved (P = 0.025). Figure 1 As shown in C.

[0036] Example 2: Screening of small molecule inhibitors of GSDME

[0037] In this embodiment, in order to perform molecular docking on more than 20,000 small molecule compounds in the TargetMol database, a two-step screening mode was used, starting with high-throughput virtual screening (HTVS) and then entering standard precision (SP). According to the docking score, the top 20% of the compounds were retained in each step, and 461 compounds with affinity ranging from -9.95kcal / mol to -5.39kcal / mol were finally obtained. These 461 compounds were then clustered and divided into 342 groups when the similarity threshold was 70%. In each group, a compound with good affinity was screened out. A total of 342 compounds were retained, and their interaction patterns with the target were subsequently analyzed using the protein-ligand interface fingerprint (PLIF) method. Through this analysis, 144 suitable candidate compounds were identified. To further verify the binding of the top 100 compounds to mouse GSDME protein in vitro, differential scanning fluorimetry (DSF) experiments were performed. From this experiment, 43 compounds were found to be potentially effective based on the protein Tm values, and all increased the temperature by more than 5°C. In vitro, surface plasmon resonance (SPR) technology was further used to verify the direct interaction between mouse GSDME protein and the small molecule Eliprodil.

[0038] result: Figure 2 A is the general formula of Eliprodil; Figure 2 B shows the docking mode diagram of Eliprodil and mouse GSDME molecule; Figure 2 C is the potential of the top 100 small molecules to bind to recombinant mouse GSDME protein screened by in vitro differential scanning fluorescence (DSF) experiment, and Eliprodil has the highest Tm difference of 36.7°C; Figure 2 In vitro surface plasmon resonance (SPR) experiments of D showed that Eliprodil had a high binding affinity to GSDME, with a dissociation constant KD of 2.13E-07.

[0039] Example 3: Eliprodil regulates macrophage polarization and macrophage effects on tumor proliferation

[0040] Cell proliferation and colony formation assay: 1×10 7Mouse macrophage cell lines Raw264.7 or BMDMs cells (bone marrow-derived macrophages) were cultured in 10 cm culture dishes with 15 ml DMEM complete medium (containing 10 μM Eliprodil or control reagent) for 48 hours. Subsequently, the culture medium was collected and filtered with a 0.22 μm filter. The filtrate was mixed with an equal volume of fresh culture medium to form the final culture medium. The cells were attached to 96-well plates and 6-well plates, and different culture media were added to perform CCK8 and clone formation experiments on tumor cells. Cell counting kit-8 (Epizyme Biotech, China) was used to determine cell proliferation ability. Hep1-6 was inoculated in a 96-well plate at a density of 2000 cells per well, and 100 μL of cell suspension was used per well for culture. The test was performed for a total of five days, and the absorbance was measured at 450 nm using an ELISA reader every 24 hours. Each experiment was repeated six times, and a total of three independent experiments were performed. Cell proliferation was then calculated. For colony formation experiments, Hep1-6 cells were seeded in 6-well plates at a density of 500 cells per well and cultured in two complete growth media until visible colonies were formed (about two weeks). Cell colonies were fixed with tissue fixative, stained with 1% crystal violet and counted. All experiments were performed independently three times. In vivo animal experiments: All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996) and the Ethical Guidelines for Animal Experimentation Institutions formulated by Fudan University. Animals were kept under pathogen-free conditions in the experimental animal room of Zhongshan Hospital Affiliated to Fudan University. 1×10 6 Hep1-6 cells were suspended in 50 μl PBS solution and directly injected into the liver of 6-week-old C57BL / 6J mice to form orthotopic HCC. Tumor-bearing mice were randomly divided into two groups of 6 on the 5th day after Hep1-6 cell implantation and were treated with Eliprodil monotherapy (1 mg / kg) or placebo, and then given Eliprodil (1 mg / kg) or placebo every 2 days until the end of the experiment (a total of three weeks). The liver weight / body weight ratio was finally measured to evaluate the effect of GSDME inhibitor Eliprodil on HCC growth in mice.

[0041] Western blotting: After treating macrophages with different concentrations of Eliprodil (0-20 μM) for 48 hours, the phosphorylation level of PI3K-AKT pathway proteins was detected. For Western blotting, cells were lysed using RIPA lysis buffer (Biyuntian, China) and protease and phosphatase inhibitors were added. The protein concentration was quantified using BCA reagent, and then equal amounts of protein were separated using SDS-PAGE and subsequently transferred to Immobilon-P membrane (Millipore, Billerica). QuickBlockTM The membrane was blocked by Western blotting (Biyuntian, China) for 20 min and then incubated with the corresponding antibodies. The protein bands were visualized and analyzed using a gel image analysis system (Tanon).

[0042] result: Figure 3 A and Figure 3 B is CCK8 and clone formation experiments that prove Eliprodil promotes macrophages to inhibit the proliferation of liver tumor cells in vitro; Figure 3 C is an in vivo experiment proving that Eliprodil inhibits the proliferation of tumor cells; Figure 3 D is an in vitro immunoblotting experiment demonstrating that Eliprodil inhibits the phosphorylation levels of PDPK1 and AKT proteins in macrophages.

[0043] Example 4: Exploration of the effect of Eliprodil combined with immune checkpoint inhibitors in mice;

[0044] In this example, HCC was induced by tail vein injection of plasmids. Specifically, a mixture of 10 μg pX330-p53 (Addgene59910), 10 μg pt3-n90-β-catenin (Addgene 31785) and 10 μg CMV-SB13 transposase plasmids was mixed with 2 ml (about 10% of the mouse body weight) of 0.9% Nacl sterile solution. Six-week-old C57BL / 6J mice were injected via the lateral tail vein within 5-7 seconds. Five days after plasmid injection, mice were given Eliprodil (1 mg / kg) or placebo as an initial injection, and then mice were alternately given anti-PD1 monoclonal antibodies (5 mg / kg) or IgG homologous controls and Eliprodil (1 mg / kg) or placebo every 2 days until the mice were killed 21 days later. Liver weight / body weight was statistically measured to analyze the therapeutic effect of Eliprodil or combined PD1 monoclonal antibodies on liver cancer in mice. CRISPER gene-edited c-Myc+ / +; Alb-cre+ / + mice were obtained from Shanghai South Model Organisms Company. Drug administration began after the 8th week of birth, and mice were intraperitoneally injected with anti-PD1 monoclonal antibody (5 mg / kg) or IgG homologous control and Eliprodil (1 mg / kg) or placebo once a week until 13 weeks of age. Liver weight / body weight indicators were measured to evaluate the therapeutic effect of Eliprodil alone or in combination with PD1 monoclonal antibody on liver cancer in mice.

[0045] Results: Eliprodil was able to inhibit the proliferation of spontaneous hepatocellular carcinoma in c-Myc+ / +; Alb-cre+ / + mice in vivo and had a synergistic therapeutic effect with αPD-1. Figure 4As shown in A; Eliprodil inhibits the proliferation of HCC induced by tail vein injection of plasmid in vivo and has a synergistic therapeutic effect with αPD-1, as shown in Figure 4 As shown in B.

[0046] Example 5: Flow cytometry detection of tumor-infiltrating macrophages and T lymphocytes

[0047] In this implementation, the experimental mice were killed by dislocation, the tumor tissue was peeled off, photographed and weighed, and then the tumor tissue was cut into 2-4 mm fragments. The tumor tissue was dissociated into a single cell suspension using Miltenyi tumor tissue dissociation solution and an eight-channel tissue processor for subsequent detection and analysis. The above-dissociated tumor single cell suspension was tested on the machine using flow cytometry antibodies labeled with CD45, CD11b / F480, CD206, CD86, CD8, PFN, and GzmB.

[0048] Results: In the HCC model induced by orthotopic injection of Hep1-6 cells in C57BL / 6J mice, Eliprodil inhibited the M2-like polarization of macrophages (the proportion of CD206+ tumor-associated macrophages decreased). Figure 5 As shown in A; in the HCC model induced by orthotopic injection of Hep1-6 cells in C57BL / 6J mice, Eliprodil increased the proportion of PFN+ and GZMB+CD8+T cells, as shown in Figure 5 As shown in B.

[0049] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Eliprodil or Eliprodil combined with immune checkpoint inhibitors in the preparation of anti-tumor drugs, characterized in that: The chemical structural formula of Eliprodil is shown below:

2. The use according to claim 1, characterized in that The tumor includes liver cancer.

3. The use according to claim 1, characterized in that The drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is Eliprodil or a combination of Eliprodil and an immune checkpoint inhibitor.

4. The use according to any one of claims 1 to 3, characterized in that: The immune checkpoint inhibitor is αPD-1.

5. Use of a reagent for detecting GSDME protein expression in macrophages in the preparation of a product for detecting liver cancer or liver cancer prognosis.

6. The use according to claim 5, characterized in that The reagent for detecting the expression of GSDME protein in macrophages comprises a reagent for detecting the expression of macrophage marker CD68 and a reagent for detecting the expression of GSDME protein.

7. The use according to claim 6, characterized in that The reagent for expressing the macrophage marker CD68 is a CD68 antibody, and the reagent for detecting the expression of the GSDME protein is a GSDME antibody.

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