Application of ACOD1 gene activity inhibitor in the preparation of drugs for treating cancer

Abstract

The present invention belongs to the technical field of cancer drugs, and specifically relates to the use of an ACOD1 gene activity inhibitor in the preparation of drugs for treating cancer. The ACOD1 gene activity inhibitor can significantly inhibit the occurrence and development of tumors, and will not affect blood routine indexes and liver function, and has no obvious toxic side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of cancer drugs, and specifically relates to the use of an ACOD1 gene activity inhibitor in the preparation of drugs for treating cancer. Background Art

[0002] Malignant tumors pose a serious threat to human health, and the tumor microenvironment (TME) plays an important regulatory role in the development and progression of malignant tumors. TME is a complex network composed of immune cells, blood vessels, and extracellular matrix, which interact with malignant tumor cells and play an important role in tumor growth, infiltration, invasion, and metastasis.

[0003] Tumor-associated macrophages (TAMs), the most abundant immune cells in the TME, have an important impact on tumor development and immunotherapy. First, TAMs can secrete a variety of inhibitory cytokines, such as TGF-β and IL-10, which inhibit the functions of other immune cells and reduce the body's immune surveillance and resistance to tumors. Second, TAMs can promote tumor growth and infiltration. For example, they can secrete a variety of growth factors, angiogenic factors and cytokines, such as tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF), etc. These molecules can directly act on malignant tumor cells to promote their proliferation and growth, while also promoting the formation of tumor blood vessels to provide sufficient nutrients and oxygen for the tumor. Third, TAMs can also promote the invasion and metastasis of tumor cells. For example, they can secrete a series of proteases, such as matrix metalloproteinases (MMPs), which can degrade the matrix and destroy the structure of normal tissues, thereby providing tumor cells with a pathway for invasion and metastasis. In addition, tumor-associated macrophages can also promote the adhesion and migration of tumor cells to other cells by increasing the expression of surface adhesion molecules (such as ICAM-1, VCAM-1, etc.) of tumor cells.

[0004] However, TAMs have high heterogeneity. They can transform from M1 macrophages with anti-tumor activity to M2 macrophages with immunosuppressive effects (M2 macrophages can produce signaling molecules that inhibit immune responses, inhibit the effects and activation of T cells, and promote immune tolerance). Among them, IFNγ and LPS can activate TAMs and promote the transformation of TAMs into M1 macrophages with pro-inflammatory ability. M1 macrophages secrete pro-inflammatory cytokines TNFα and interleukins (IL-1β, IL-2, IL-6, IL-12, and IL-23), and at the same time produce nitric oxide by upregulating inducible nitric oxide synthase (iNOS) to kill tumors. Therefore, reprogramming TAMs may be a strategy to control cancer. Summary of the invention

[0005] The present invention uses bioinformatics technology and transcriptome sequencing analysis to find that ACOD1 plays an important role in TAMs metabolic reprogramming, can promote TAMs reprogramming, and inhibit tumor growth; and ACOD1 + TAMs are closely related to the poor prognosis of cancer patients.

[0006] Therefore, one of the purposes of the present invention is to provide an inhibitor of ACOD1 activity for use in the preparation of a drug for treating cancer. The inhibitor of ACOD1 activity can effectively inhibit the occurrence and development of tumors.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides an application of an ACOD1 gene activity inhibitor in the preparation of a drug for treating cancer.

[0009] Another aspect of the present invention provides a use of a compound or a pharmaceutically acceptable salt thereof in preparing a preparation for inhibiting ACOD1 gene activity. The compound has a structural formula as shown in Formula I:

[0010]

[0011] The beneficial effects of the present invention include at least:

[0012] (1) The compound that inhibits the activity of the ACOD1 gene in the present invention can significantly inhibit the occurrence and development of tumors without affecting blood routine indexes and liver function and without obvious toxic side effects.

[0013] (2) The compound represented by the above formula I can effectively inhibit the expression of ACOD1 gene and is significantly better than other small molecules. The compound can inhibit the ACOD1 metabolite Itaconate to a concentration of only 2 nM, which is much lower than the inhibitory concentration of other small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a photo of the tumor growth after ACOD1 knockout in MC38 subcutaneous tumors;

[0015] Figure 2 This is the time-volume curve of tumor growth after MC38 subcutaneous tumor formation after ACOD1 knockout;

[0016] Figure 3 ACOD1 - / - -Tumor weight of mice in the MC3 group;

[0017] Figure 4 Schematic diagram of the active pocket of ACOD1 protein;

[0018] Figure 5The schematic diagram of the docking of the top 18 compounds with strong binding ability with the active pocket of ACOD1 protein;

[0019] Figure 6 It is a 2D diagram of the action of the small molecule compound AE-848 / 32800028;

[0020] Figure 7 It is a 2D diagram of the action of the small molecule compound AE-641 / 01080004;

[0021] Figure 8 It is a 2D diagram of the action of the small molecule compound AA-516 / 33243012;

[0022] Fig. 9 It is a 2D diagram of the action of the small molecule compound AE-641 / 01080005;

[0023] Fig.10 It is a 2D diagram of the action of the small molecule compound AE-848 / 32324022;

[0024] Fig.11 It is a 2D diagram of the action of the small molecule compound AE-848 / 06858005;

[0025] Fig.12 It is a 2D diagram of the action of the small molecule compound AE-641 / 01213026;

[0026] Fig.13 It is a 2D diagram of the action of the small molecule compound AE-641 / 00644014;

[0027] Fig.14 This is a 2D diagram of the action of the small molecule compound AE-641 / 30110001;

[0028] Fig.15 It is a 2D diagram of the action of the small molecule compound AE-848 / 36287024;

[0029] Fig.16 It is a 2D diagram of the action of the small molecule compound AE-641 / 11289519;

[0030] Fig.17 It is a 2D diagram of the action of the small molecule compound AE-848 / 02714008;

[0031] Fig.18 It is a 2D diagram of the action of the small molecule compound AE-641 / 40197985;

[0032] Fig.19 It is a 2D diagram of the action of the small molecule compound AB-323 / 13887458;

[0033] Fig. 20 It is a 2D diagram of the action of the small molecule compound AE-473 / 30080064;

[0034] Fig.21 It is a 2D diagram of the action of the small molecule compound AE-641 / 02349027;

[0035] Fig. 22 It is a 2D diagram of the action of the small molecule compound AE-641 / 40791360;

[0036] Fig.23 It is a 2D diagram of the action of the small molecule compound AE-848 / 32313029;

[0037] Fig.24 This is a graph showing the effect of the top 18 compounds with the strongest binding ability on the concentration of itaconic acid, a metabolite of ACOD1;

[0038] Fig.25 This is a diagram of the docking model of the small molecule compound SP10 (AE-848 / 36287024) and ACOD1;

[0039] Fig.26 The SPR (surface plasmon resonance) experiment shows the interaction between SP10 and ACOD1;

[0040] Fig. 27 This is a picture showing the actual tumor growth after treatment with 20 mg / kg SP10 small molecule;

[0041] Fig.28 This is the time-volume graph of tumor growth after treatment with 20 mg / kg SP10 small molecule;

[0042] Fig.29 This is a graph showing the tumor weight after treatment with 20 mg / kg SP10 small molecule;

[0043] Fig.30 This graph shows the effect of 20 mg / kg SP10 small molecule on the blood routine indexes (RBC, WBC, HGB and PLT) and liver function indexes (ASL and ALT) of mice. DETAILED DESCRIPTION

[0044] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0045] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0046] An embodiment of the present invention provides an application of an ACOD1 gene activity inhibitor in the preparation of a drug for treating cancer.

[0047] It should be noted that the present invention found through bioinformatics technology and transcriptome sequencing analysis that ACOD1 plays an important role in TAMs metabolic reprogramming, can promote TAMs reprogramming, and inhibit tumor growth; and ACOD1 + TAMs are closely related to the poor prognosis of cancer patients. Therefore, inhibitors that inhibit the activity of the ACOD1 gene can be used in drugs to treat tumors.

[0048] It should also be noted that the full name of ACOD1 gene is aconitate decarboxylase 1, ID: 730249; Ensembl: ENSG00000102794. In addition, the ACOD1 gene activity inhibitor mentioned above refers to any substance that can inhibit the activity of ACOD1 gene, including but not limited to polypeptides or compounds.

[0049] In some specific embodiments, the ACOD1 gene activity inhibitor is a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0050]

[0051] It should be noted that the above-mentioned pharmaceutically acceptable salts are known in the art, including all salts that can be acidified from the compound represented by the above formula I, and the commonly used salt is hydrochloride. In addition, the term "gene activity" is known in the art, has no specific meaning, and generally refers to the expression of the ACOD1 gene.

[0052] In addition, the present invention uses the Specs library small molecule compounds for screening, determines the molecular active site by determining the ACOD1 active pocket, docks the small molecules finally screened, sorts the results by docking score, selects the top 18 compounds with high scores for synthesis, verifies that the small molecule inhibitor blocks the activity of the ACOD1 enzyme through liquid chromatography-mass spectrometry experiments, verifies the effectiveness and safety of the drug through in vivo experiments, and finally selects the compound shown in the above formula I.

[0053] In addition, the compound represented by the above formula I is also referred to as AE-848 / 36287024, or SP10 for short.

[0054] In some specific embodiments, the above application includes: application of ACOD1 gene activity inhibitor in the preparation of a drug for promoting TAMs reprogramming. It should be noted that ACOD1 gene activity inhibitor can promote TAMs reprogramming, thereby inhibiting the occurrence and development of tumors.

[0055] In some specific embodiments, the above application includes: the use of an ACOD1 gene activity inhibitor in the preparation of a drug for inhibiting ACOD1 from metabolizing itaconic acid. It should be noted that the ACOD1 gene activity inhibitor can inhibit the activity of the ACOD1 enzyme, thereby inhibiting ACOD1 from catalyzing the decarboxylation of aconitic acid to produce itaconic acid (Itaconate), promoting TAMs reprogramming, and inhibiting tumor growth; and the compound has low toxicity to normal cells and does not cause obvious side effects at the in vivo level. In addition, itaconic acid, a metabolite of ACOD1, promotes the transformation of macrophages to M2 type, thereby exerting an anti-inflammatory effect.

[0056] In some specific embodiments, the cancer may be one or more of breast cancer, colorectal cancer and ovarian cancer.

[0057] In some specific embodiments, the dosage form of the above-mentioned drug includes tablets, powders, capsules, injections, oral liquids, suspensions or gels. It should be noted that, as described above, the above-mentioned compounds can be added with excipients to prepare different drugs, and the drugs can be selected according to specific clinical needs, such as tablets, powders, capsules, injections, oral liquids, suspensions or gels. In addition, the excipients are all known in the art, such as the preparation of tablets mainly using diluents (such as starch, dextrin, sucrose or mannose, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (polyvidone, syrup or hypromellose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvidone, etc.); for example, the preparation of liquids mainly uses expanders, suspending agents, emulsifiers or colorants, etc. In addition, the above-mentioned compounds can also be prepared into tablets after being combined with other active ingredients to increase the therapeutic effect.

[0058] Another embodiment of the present invention provides a use of a compound or a pharmaceutically acceptable salt thereof in preparing a preparation for inhibiting ACOD1 gene activity. The compound has a structural formula as shown in Formula I:

[0059]

[0060] It should be noted that, as described above, the present invention uses the Specs library small molecule compounds for screening, determines the molecular active site by determining the ACOD1 active pocket, docks the small molecules finally screened, and sorts the results by docking score. The top 18 compounds with high scores are selected for synthesis, and the small molecule inhibitors are verified by liquid chromatography-mass spectrometry experiments to block the activity of the ACOD1 enzyme, and the effectiveness and safety of the drug are verified by in vivo experiments.

[0061] In addition, ACOD1 is an important molecular marker that is associated with poor patient prognosis in various types of tumors. The above-mentioned compound or its salt can effectively inhibit the activity of ACOD1, thereby affecting behaviors related to ACOD1 gene activity, such as macrophage reprogramming, immune escape and tumor prognosis.

[0062] In some specific embodiments, the above application includes: the use of the compound or its pharmaceutically acceptable salt in the preparation of a drug for inhibiting ACOD1 from metabolizing itaconic acid. It should be noted that, as mentioned above, the above compound or its pharmaceutically acceptable salt can inhibit the activity of ACOD1 enzyme, thereby inhibiting ACOD1 from catalyzing the decarboxylation of aconitic acid to produce itaconic acid (Itaconate), promoting TAMs reprogramming, and inhibiting tumor growth; and the compound has low toxicity to normal cells and does not cause obvious side effects at the in vivo level.

[0063] In some specific embodiments, the dosage form of the above-mentioned preparation includes tablets, powders, capsules, injections, oral liquids, suspensions or gels. It should be noted that the above-mentioned compounds can be added with excipients to prepare different preparations, and the preparations can be selected according to specific clinical needs, such as tablets, powders, capsules, injections, oral liquids, suspensions or gels. In addition, the excipients are all known in the art, such as the preparation of tablets mainly using diluents (such as starch, dextrin, sucrose or mannose, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (polyvidone, syrup or hydroxypropyl methylcellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvidone, etc.); for example, the preparation of liquids mainly uses expanders, suspending agents, emulsifiers or colorants, etc. In addition, the above-mentioned compounds can also be prepared into tablets after being combined with other active ingredients to increase the therapeutic effect.

[0064] In addition, the above-mentioned preparations do not specifically refer to drugs, but can also be other forms of products, such as food or health products.

[0065] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0066] 1. Knockout of ACOD1 can effectively inhibit tumor growth verification

[0067] In the examples of the present invention, it was verified that blocking ACOD1 activity can effectively inhibit tumor growth, as follows:

[0068] (I) Construction of subcutaneous colon cancer model in mice

[0069] In the present invention, the 6-8 week old wild-type (WT) C57BL / 6J mice used were purchased from the Experimental Animal Center of Chongqing Medical University; ACOD1 - / - (C57BL / 6N-Acod1 tm1cyagen ) Mice were purchased from Saiye Biotechnology Co., Ltd.

[0070] (1) Mouse colon cancer cells MC38 (purchased from American Type Culture Collection) were cultured in advance, digested, centrifuged and counted, and 15×10 4 / 100μl / mouse;

[0071] (2) In WT and ACOD1 - / - The cells were inoculated subcutaneously on the right side of the midline of the lower abdomen of mice, 100 μl / mouse;

[0072] (3) After inoculation, mice were kept in an SPF environment. Starting from the 7th day, the tumor diameters were measured every two days, and the volume was calculated and the growth curve was drawn. Tumor volume (mm 3 ) = 0.5 × major diameter × minor diameter 2 ;

[0073] (4) After measuring for 5 time points, the mice were killed, the tumors were removed, photographed, and weighed.

[0074] (II) Test results

[0075] The tumors of mice in each group are shown in Figure 1 As shown (the upper row is the WT group, the lower row is the ACOD1 - / - The results showed that ACOD1 - / - The tumor volume of the WT group was significantly smaller than that of the WT group.

[0076] The changes in the volume of the tumors of each group of mice from day 7 to day 15 are shown in the figure. Figure 2As shown, the results showed that ACOD1 - / - The growth of MC38 tumor in the WT group was significantly slower than that in the WT group.

[0077] The tumor weights of mice in each group after 15 days are shown in Table 1 and Figure 3 As shown, the results showed that ACOD1 - / - The MC38 tumor weight of the group was significantly smaller than that of the control group.

[0078] Table 1 WT and ACOD1 - / - Mouse tumor weight

[0079]

[0080] 2. Finding small molecule inhibitors through virtual molecular screening

[0081] (I) Selection and preparation of protein structure

[0082] The ACOD1 protein file (PDB ID: 7br9) was found in the Protein Data Bank (www.rcsb.org); it was used in the docking analysis. The Prep Wiz module in the software package prepares the protein by performing pretreatment operations such as hydrogenation and dehydration.

[0083] (II) Preparation of small molecule database

[0084] The SPESC small molecule library was used in this virtual screening. All compounds were screened for drug-likeness using the Lipinski's rule of five and Veber rule modules in DS4.0 to eliminate molecules with poor drug-likeness. The LigPrep module in the software package was used for preparation, the OPLS_2005 force field was used, and the protonation of the molecule was operated using the Epik module at pH 7.4.

[0085] (III) Finding active sites

[0086] use The SiteMap module in the software package found the active site of ACOD1 protein, which is as follows Figure 4 As shown, the active site is defined as the docking pocket.

[0087] (IV) Filtration of compounds with PAINS properties

[0088] In order to identify whether the screened compounds are PAINS structures, the present embodiment uses the Canvas 1.1 program for filtering. It should be noted that pan-assay interference compounds (PAINS) refer to the "activity" of some compounds in the new drug screening process that is not based on the specific interaction between the compound molecule and the protein, which is a false positive result.

[0089] (V) ADME property prediction and cluster analysis

[0090] In order to predict the pharmacokinetic properties of the compounds screened in the present invention, the present invention uses The QikProp 3.2 program in the software package calculated the corresponding properties. It should be noted that the QikProp3.2 program mainly calculates whether the compound violates Lipinski's five rules and Jorgensen's three rules. These two properties can preliminarily evaluate the ADME characteristics of the compound. The screening compound must meet at least four of the Lipinski five rules to have good oral bioavailability. During this period, the molecules obtained by different pharmacophore screening and docking were merged, the same molecules were eliminated, and the remaining small molecules were subjected to structural clustering analysis using FCFP_6 fingerprints.

[0091] (VI) Molecular docking

[0092] Select the optimal docking condition Glide force field, and use the ACOD1 protein file as the receptor for docking research. Before docking, the ACOD1 protein receptor needs to prepare and define a size of After all molecules and receptor proteins were prepared, the Glide algorithm was used for docking at SP docking accuracy, and other parameters were kept as default (refer to Figure 5 ).

[0093] (VII) Analysis of docking results

[0094] The SPECS small molecule library was used for screening, and the small molecules finally screened were docked. The results were sorted by docking score, and the ones with high scores were selected to synthesize the 18 drugs with the highest scores in the database (as shown in Table 2).

[0095] Table 2 2D diagram of small molecule effects obtained by protein screening of the SPECS library

[0096] No. ID Dockingscore Structure SP1 AE-848 / 32800028 -7.922 Figure 6 SP2 AE-641 / 01080004 -7.918 Figure 7 SP3 AA-516 / 33243012 -7.462 Figure 8 SP4 AE-641 / 01080005 -7.282 Fig. 9 SP5 AE-848 / 32324022 -7.094 Fig.10 SP6 AE-848 / 06858005 -7.075 Fig.11 SP7 AE-641 / 01213026 -7.059 Fig.12 SP8 AE-641 / 00644014 -7.055 Fig.13 SP9 AE-641 / 30110001 -7.002 Fig.14 SP10 AE-848 / 36287024 -6.967 Fig.15 SP11 AE-641 / 11289519 -6.943 Fig.16 SP12 AE-848 / 02714008 -6.928 Fig.17 SP13 AE-641 / 40197985 -6.837 Fig.18 SP14 AB-323 / 13887458 -6.823 Fig.19 SP15 AE-473 / 30080064 -6.815 Fig. 20 SP16 AE-641 / 02349027 -6.794 Fig.21 SP17 AE-641 / 40791360 -6.766 Fig. 22 SP18 AE-848 / 32313029 -6.724 Fig.23

[0097] 3. Screening and effect verification of small molecule inhibitor SP10

[0098] In the embodiment of the present invention, the parameters for detecting small molecule compounds using liquid chromatography-mass spectrometry include: mass spectrometry analysis using a Waters XEVOTQ-SMicro tandem quadrupole mass spectrometer system; ion source voltage 3.0 kV, temperature 150°C; desolvation temperature 350°C, desolvation gas flow rate 1000 L / h; cone gas flow rate 10 L / h.

[0099] (I) Screening of the best drugs to inhibit ACOD1 activity

[0100] In the embodiment of the present invention, the 18 small molecule compounds (synthesized by Taoshu Biotechnology Co., Ltd.) were tested by liquid chromatography-mass spectrometry (LC-MS) method, and the method for determining the best drug for inhibiting ACOD1 activity was as follows:

[0101] (1) RAW 264.7 cells were seeded in a 15 cm cell culture dish. When the cell density was approximately 70%, they were treated with 10 μM of different compounds for 24 h. The cells were then scraped and transferred to a 1.5 ml EP tube and centrifuged at 1000 rpm for 5 min.

[0102] (2) Take the sample, place it at 4°C, thaw it, and vortex it for 10 seconds;

[0103] (3) Add 300 μL of 50% methanol, sonicate for 30 min, and oscillate for 30 min;

[0104] (4) Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4°C and 12,000 rpm for 5 min;

[0105] (5) Take 50 μL of the supernatant, add 50 μL of succinate isotope internal standard (5 μg / mL), then add 50 μL of 3-nitrophenylhydrazine (3-NPH) (250 mM, prepared with 50% methanol / water solution), add 50 μL of EDC (150 mM, prepared with 75% methanol / water solution (containing 7.5% pyridine), i.e., methanol: water: pyridine = 69.375: 23.125: 7.5), place in a shaking mixer, and derivatize at 30°C for 30 min;

[0106] (6) Add 50 μL of 2,6-di-tert-butyl-p-cresol (BHT) methanol solution (2 mg / mL) and 250 μL of 75% methanol aqueous solution;

[0107] (7) Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4°C and 12,000 rpm for 5 min;

[0108] (8) Take 200 μL of the supernatant and place it in a sampling bottle for mass spectrometry detection.

[0109] According to the above method, the 18 compounds shown in Table 2 were tested for their ability to inhibit the activity of ACOD1 enzyme in the macrophage cell line RAW264.7. The higher the activity of ACOD1 enzyme, the stronger the catalytic production of Itaconate. Fig.24 As shown, in RAW264.7 cells, the small molecule compound SP10 has the most significant effect in inhibiting the concentration of ACOD1 metabolite Itaconate, which is better than other small molecule compounds, indicating that SP10 can significantly inhibit the activity of ACOD1 enzyme.

[0110] (II) Interaction of inhibitor SP10 with ACOD1

[0111] The small molecule compound SP10 (AE-848 / 36287024) was docked with ACOD1, specifically including:

[0112] (1) Preparation of proteins and small molecules: Download the corresponding protein (PDB ID: 7br9) of ACOD1 from the protein database (www.rcsb.org) and the small molecule SP10 from PubChem. The Prep Wiz module in the software package performs pretreatment operations such as hydrogenation and dehydration on the protein to prepare the protein, and the small molecule SP10 is used in the docking process. The Protein Preparation module in the software package hydrogenates it, adds charges, adds protonation states, and assigns it the OPLS-2005 force field;

[0113] (2) Molecular docking: Select the optimal docking condition Glide force field and conduct docking research using the ACOD1 protein file as the receptor. Before docking, the ACOD1 protein receptor needs to prepare and define a size of After the pocket of SP10 and receptor protein were prepared, the Glide algorithm was used for docking at the SP docking accuracy, and other parameters remained default.

[0114] The docking model of the small molecule compound SP10 (AE-848 / 36287024) and ACOD1 (detailed diagram of the interaction between compound SP10 and ACOD1 protein) is shown in Fig.25 As shown in Figure 2, SP10 generates three hydrogen bonds with amino acids Glu234 and Arg322 on ACOD1 protein, and the interaction distances are and

[0115] In addition, the interaction between SP10 small molecule compound and ACOD1 was analyzed using SPR (surface plasmon resonance) experiments, including:

[0116] (1) Start the Biacore T200 instrument according to standard operation;

[0117] (2) Prepare 500 ml of deionized water (filtered through a 0.22 μm membrane) for buffer and cleaning the injection needle;

[0118] (3) Start installing the chip and install the CM5 chip according to the standard process;

[0119] (4) Prepare to start the formal experiment. The buffer will flush the flow system inside the entire system at a high flow rate;

[0120] (5) Select appropriate procedures based on sample size;

[0121] (6) Start activating the chip, prepare a sufficient volume of ligand protein ACOD1, EDC / NHS, and blocking buffer; start the coupling procedure, the coupling time is 7 minutes, the flow rate is 10 μl / min, and the final ligand coupling amount is about 8200 RU;

[0122] (7) After the coupling is completed, the sample detection is started, and the analyte binding time is set to 120 s and the flow rate is 30 μl / min; the dissociation time is set to 200 s and the flow rate is 30 μl / min; the regeneration time is set to 30 s and the flow rate is 30 μl / min;

[0123] (8) Prepare the corresponding samples to be tested as required and start the automatic running program for testing;

[0124] (9) Result analysis: according to the running results, perform data fitting analysis to obtain the final affinity fitting KD value;

[0125] The SPR results are shown in Figure 26, which show that compound SP10 has a strong affinity for ACOD1 and is dose-dependent, with an affinity (KD) of 7.92 μM.

[0126] IV. Experiment on the inhibition of tumor growth and toxic side effects of small molecule inhibitor SP10

[0127] (I) Construction of mouse MC38 subcutaneous transplant tumor model

[0128] In the examples of the present invention, 6-8 week old SPF grade C57BL / 6J mice were purchased from the Animal Center of Chongqing Medical University.

[0129] (1) MC38 cells were cultured in advance, digested and centrifuged, and counted. 15×10 4 / 100μl / mouse;

[0130] (2) Cells were subcutaneously inoculated on the right side of the midline of the lower abdomen of mice, 100 μl / mouse;

[0131] (3) After the inoculation, mice were housed in an SPF environment and given SP10 by gavage every two days after tumor formation, 20 mg / kg / mouse / time; in addition, the blank control group (Vehicle) was given corn oil by gavage;

[0132] (4) Starting from the 7th day, the tumor length and short diameter were measured every two days, and the volume was calculated and the growth curve was drawn; tumor volume (mm 3 ) = 0.5 × major diameter × minor diameter 2 ;

[0133] (5) After measuring 5 time points, the blood of the mice was taken out and divided into two parts, one for routine blood test and the other for liver function test (preparation of working reagents: single reagent method directly used; double reagent method using R1 and R2 separately; setting the corresponding parameters (primary and secondary wavelengths: 340 / 405nm) on the fully automatic biochemical microplate reader (Mindray veterinary fully automatic blood cell analyzer model: BC-2800vet); loading the sample, and the fully automatic biochemical analyzer (Shenzhen Raydu Life Science fully automatic biochemical analyzer: Chemray 240) automatically measured; the experimental results were exported after being tested by the fully automatic biochemical analyzer, and graphed using Graphpad);

[0134] (6) The mice were killed, the tumors were removed, photographed, and weighed.

[0135] (II) Test results

[0136] The tumors of mice in each group are shown in Fig. 27 As shown, the results showed that the MC38 tumor volume in the SP10 group was significantly smaller than that in the control group.

[0137] The changes in tumor volume of mice in each group from day 7 to day 15 are shown in Figure 2. Fig.28 As shown, the results showed that the growth of MC38 tumors in the SP10 group was significantly slower than that in the control group.

[0138] The tumor weights of mice in each group after 15 days are shown in Table 3 and Fig.29 As shown, the results showed that the tumor weight of the SP10 group was significantly lower than that of the control group.

[0139] Table 3 Tumor weights of mice in different groups

[0140]

[0141] The blood routine indexes (RBC, WBC, HGB and PLT) and AST and ALT indexes of each group of mice were as follows: Fig.30 As shown, the results showed that there were no obvious abnormalities in the blood routine and liver function of mice after SP10 treatment, that is, the small molecule compound SP10 had no obvious toxic side effects on the blood routine indexes, ASL and ALT of mice.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Use of an ACOD1 gene activity inhibitor in the preparation of a drug for treating colon cancer; the ACOD1 gene activity inhibitor is a compound represented by formula I or a pharmaceutically acceptable salt thereof, 2. The use according to claim 1, characterized in that: Applications include: Use of ACOD1 gene activity inhibitors in the preparation of drugs that promote TAMs reprogramming.

3. The use according to claim 1 or 2, characterized in that: Applications include: application of the ACOD1 gene activity inhibitor in the preparation of a drug for inhibiting ACOD1 from metabolizing itaconic acid.

4. The use according to claim 1 or 2, characterized in that: The dosage forms of the drug include tablets, powders, capsules, injections, oral solutions, suspensions or gels.

5. The use according to claim 3, characterized in that: The dosage forms of the drug include tablets, powders, capsules, injections, oral solutions, suspensions or gels.

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