New use, compound, and new use of FASN inhibitor

Abstract

The present invention belongs to the field of biotechnology, and discloses the use of FASN inhibitors in the preparation of drugs for treating blood tumors. The present invention knocks down the FASN gene of blood tumor-related cells, and finds that the growth and clone formation ability of cells are significantly inhibited, and cell apoptosis and cell cycle progression are blocked. At the same time, by constructing a mouse model and knocking out the FASN gene, it is found that the knockout of the FASN gene has no effect on the normal hematopoietic function of mice. At the same time, the proliferation effects of various blood tumor-related cells are evaluated by a variety of FASN inhibitors, and it is found that different FASN inhibitors can achieve the purpose of inhibiting cell growth and clone formation, inducing cell apoptosis and cell cycle progression to varying degrees. At the same time, the present invention also screens out a new FASN inhibitor, which has better effects on the growth inhibition and apoptosis induction of blood tumor cells than traditional FASN inhibitors; at the same time, the present invention also discloses various potential applications of the FASN inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a new use of a FASN inhibitor, a compound, and a new use of the compound. Background Art

[0002] Fatty acids are components of many important substances in living organisms, including cell membranes, energy storage substances, and signaling molecules. During fatty acid synthesis, FASN catalyzes a series of condensation, reduction, dehydration, and re-reduction steps, which is crucial for normal metabolism and the resynthesis of fatty acids after starvation. In certain types of cancers, the expression and activity of FASN may be abnormally upregulated. For example, FASN is overexpressed in human ovarian cancer cells and has a tendency to be more highly expressed in more invasive and dedifferentiated tumors. FASN has also been detected overexpressed in HER2+ breast cancer patients. In addition, overexpression of FASN has been detected in cancers such as hepatocellular carcinoma, prostate cancer, gastric cancer, and ovarian cancer. Because of its overexpression and its association with tumorigenesis, development, invasion and metastasis, and the formation of drug resistance, FASN is considered an anti-tumor target. Studies have shown that FASN promotes the energy supply and membrane biosynthesis of tumor cells in these solid tumors, supporting the rapid proliferation of tumor cells.

[0003] In current studies on FASN in tumors, most researchers have focused on the field of solid tumors, and excellent anti-tumor effects have also been shown in the study of solid tumors, and inhibitors targeting FASN have been developed, including C75, TVB series, and Orlistat, etc.

[0004] As a gastrointestinal lipase inhibitor, the main function of Orlistat is to inhibit lipid metabolism enzymes. It has been widely recognized as an effective weight loss drug, which can significantly reduce the weight of obese patients and reduce the visceral fat content. However, the mechanism of action of Orlistat is not limited to the field of weight loss. Multiple studies have shown that it can inhibit FASN activity, thus expanding its application in tumor treatment. Experiments have proved that Orlistat can effectively inhibit the growth of tumor cells by inducing ferroptosis and lipid peroxidation processes in lung cancer cells, and shows significant anti-tumor effects in lung cancer animal models. At the same time, it can also induce apoptosis of pancreatic cancer cells and inhibit the proliferation of breast cancer cells. In breast cancer, Orlistat achieves anti-tumor effects by blocking the cell cycle process and inhibiting Her2 gene transcription. In addition, in an AKT / c-Met-induced liver cancer mouse model, Orlistat delays the occurrence and development of liver cancer by upregulating PTEN expression and inhibiting AKT phosphorylation. In summary, Orlistat exhibits significant anti-proliferative and anti-cancer effects in the treatment of multiple types of tumors and specific disease model mice.

[0005] C75 is an effective inhibitor of FASN. Extensive studies have confirmed its anti-tumor activity. It has shown the ability to inhibit fatty acid synthesis, promote cell apoptosis and inhibit tumor growth in various cancer models. Including but not limited to: reducing cell viability and tumor growth in breast cancer cell lines and mouse models, inhibiting cell proliferation and tumor progression in prostate cancer xenograft models, and showing anti-tumor effects by preventing the conversion of acetyl CoA to fatty acids in pancreatic cancer; inhibiting tumors by downregulating FASN expression and inhibiting AKT phosphorylation in ovarian cancer; effectively inhibiting the growth of tumor xenografts in human mesothelioma models; and in different hepatocellular carcinoma cell lines, C75 exerts anti-cancer effects by affecting the cell cycle and activating the p38 MAPK signaling pathway.

[0006] Currently, there are no clear reports in the literature and relevant experimental data to confirm that FASN can play a role in hematological tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a new use of FASN inhibitors. The present invention knocks down the FASN gene of blood tumor-related cells, and finds that the growth and clone formation ability of cells are significantly inhibited, and cell apoptosis and cell cycle progression are blocked. At the same time, after constructing a mouse model and knocking out the FASN gene, it is found that the knockout of the FASN gene has no effect on the normal hematopoietic function of mice. At the same time, the proliferation effects of various FASN inhibitors on various blood tumor-related cells are evaluated, and it is found that different FASN inhibitors can achieve the purpose of inhibiting cell growth and clone formation, inducing cell apoptosis and cell cycle progression to different degrees.

[0008] At the same time, the present invention also screened out a new FASN inhibitor, which has better effects on inhibiting the growth of blood tumor cells and inducing apoptosis than traditional FASN inhibitors;

[0009] At the same time, the invention also discloses various potential applications of the FASN inhibitor.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] Use of FASN inhibitors in the preparation of drugs for treating blood tumors.

[0012] In the above-mentioned use, the blood tumor is leukemia, lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm or multiple myeloma.

[0013] At the same time, the present invention also discloses a compound MS-C19, whose structural formula is shown in Formula 1;

[0014]

[0015] The chemical formula of the compound of the present invention is: C34H36N4O7.

[0016] The black bold part in the above formula 1 means that the direction of the carbon atom is out of the paper surface.

[0017] The invention relates to the use of the above-mentioned compound in preparing a medicine for treating blood tumors.

[0018] In the above-mentioned use, the blood tumor is leukemia, lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm or multiple myeloma.

[0019] At the same time, the present invention also discloses the use of the above compound in preparing medicine for treating solid tumors.

[0020] In the above-mentioned use, the solid tumor is hepatocellular carcinoma, prostate cancer, gastric cancer, ovarian cancer, breast cancer or lung cancer.

[0021] Theoretical studies on the mechanism of action and cell pathways of FASN inhibitors in solid tumors are already very sufficient. Therefore, the compound MS-C19 of the present invention, as a FASN inhibitor, undoubtedly has a good therapeutic effect.

[0022] At the same time, the present invention also discloses the use of the above-mentioned compounds in the preparation of drugs for preventing and treating obesity-related diseases, type 2 diabetes or fatty liver disease. Studies have shown that 1004 obese patients lost significantly more weight than the control group after receiving Orlistat treatment for 6 months, and Orlistat can significantly reduce the fat content of viscera during the treatment of obese patients. In addition, when Orlistat is combined with a low-calorie diet to treat obese patients, the results after one year of treatment show that patients taking Orlistat lost 2-5 kilograms of weight compared with the control group, and in the second year of Orlistat combined with diet, the weight continued to decrease. The U.S. Food and Drug Administration (FDA) approved Orlistat for obesity treatment in 1999. The MS-C19 and Orlistat of the present application are both typical FASN inhibitors, so the MS-C19 of the present invention has the potential to be used as a drug for the above-mentioned purpose.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] In the present invention, the real-time fluorescence quantitative PCR technique is used to confirm that fatty acid synthase (FASN) is expressed at an elevated level in patients with acute myeloid leukemia (AML) and can be used as a target for detecting AML;

[0025] Based on the discovery of the target, a new FASN inhibitor was synthesized. When the inhibitor was used to treat leukemia cell lines, the percentage of apoptotic cells increased and the cell cycle progression was blocked.

[0026] The new FASN inhibitor synthesized by the present invention has better therapeutic effects on leukemia cells than the existing FASN inhibitors;

[0027] In the present invention, gene editing of leukemia cells and knocking down the expression of FASN can also increase the apoptosis of leukemia cells and significantly prolong the survival time of mice in the AML mouse model.

[0028] In the present invention, the normal hematopoietic function of FASN knockout mice was studied, and the results showed that knockout of FASN did not affect the normal hematopoiesis of mice;

[0029] In the present invention, transcriptome sequencing was used to find that the downstream target of FASN in the pathogenesis of AML is granulin (GRN), which affects the signaling pathway related to lysosomes and inflammation.

[0030] In summary, the core innovation of this case lies in:

[0031] 1. The relationship between blood cancer and FASN gene was discovered, and the potential application of FASN inhibitors in the treatment of blood cancer was demonstrated;

[0032] 2. A new compound was discovered with excellent performance. It has very good inhibitory and apoptosis-promoting effects on solid tumors and blood tumors. It is a compound with great medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A The graph is a cell proliferation graph of MOLM-13 cells;

[0034] Figure 1B The graph is a cell proliferation graph of K562 cells;

[0035] Figure 1C The graph is a cell proliferation graph of NB-4 cells;

[0036] Figure 1D This is a picture of apoptosis in MOLM-13 cells;

[0037] Figure 1E This is a picture of apoptosis in K562 cells;

[0038] Figure 1F This is a picture of apoptosis in NB-4 cells;

[0039] Figure 1GThis is the cell cycle diagram of MOLM-13 cells;

[0040] Figure 1H This is the cell cycle diagram of K562 cells;

[0041] Fig. 1I This is the cell cycle diagram of NB-4 cells;

[0042] Figure 1J This is a diagram showing the formation of MOLM-13 cell clones;

[0043] Figure 1K This is a diagram showing the formation of K562 cells;

[0044] Figure 1L This is a diagram showing the formation of NB-4 cells;

[0045] Figure 2A This is a graph showing the cell proliferation curves of the control group and the seven groups of MOLM-13 cells with FASN knockout using a hemocytometer;

[0046] Figure 2B The figure shows the results of analyzing the cell cycle progression of MOLM-13 cells with FASN knockout;

[0047] Figure 2C This is a diagram showing the cell apoptosis of MOLM-13 cells with FASN knockout detected by AnnexinV / PI staining on the fourth and sixth days;

[0048] Figure 2D The clone formation experiment of MOLM-13 in the control group and knockout group was performed, and the statistical results of the clone number after 7 days;

[0049] Figure 3A The survival time of mice was shown in the figure after MOLM-13 cells with FASN knockdown and control group were transplanted into NSGS mice by tail vein injection. There were 10 mice in each group of shNC and shFASN#1, 9 mice in the shFASN#4 group, and 5 mice in each group of shFASN#5 and shFASN#6.

[0050] Figure 3B This is a graph showing the survival time of NSGS mice after FASN knockout and control MOLM-13 cells were transplanted into the body through tail vein injection;

[0051] Figure 4A This is a statistical chart of the number of white blood cells in the peripheral blood of mice;

[0052] Figure 4B This is a statistical diagram of the number of red blood cells in the peripheral blood of mice;

[0053] Figure 4C This is a statistical chart of the number of neutrophils in the peripheral blood of mice;

[0054] Figure 4D This is a statistical diagram of the number of monocytes in the peripheral blood of mice;

[0055] Figure 5A It is the ratio diagram between spleen weight and body weight of mice;

[0056] Figure 5B This is a comparison chart of the percentage of mature myeloid cells and erythrocytes in the bone marrow and spleen of mice;

[0057] Figure 5C This is a statistical chart of the total number of mature myeloid cells and red blood cells in the bone marrow of mice;

[0058] Figure 5D This is a statistical chart of the total number of mature myeloid cells and red blood cells in the spleen of mice;

[0059] Figure 5E This is a comparison chart of the percentage of HSC cells in the bone marrow and spleen of mice;

[0060] Fig. 5F This is a statistical diagram of the number of HSC cells in the bone marrow and spleen of mice;

[0061] Figure 5G The figure is a comparison of the cell percentages of LT-HSC, ST-HSC and MPP in the bone marrow and spleen of mice;

[0062] Figure 5H The figure is a statistical diagram of the number of LT-HSC, ST-HSC and MPP cells in the bone marrow and spleen of mice;

[0063] Fig.5I The figure is a comparison of the cell percentages of CMP, GMP and MEP in the bone marrow and spleen of mice;

[0064] Figure 5J The figure is a statistical chart of the number of CMP, GMP and MEP cells in the bone marrow and spleen of mice;

[0065] Figure 6 This is a graph showing the results of competitive transplantation assay of CD45.1 / CD45.2 in FASN knockout mice;

[0066] Fig. 7A This is a statistical diagram of the gene analysis results of MOLM-13 cells with FASN knocked down;

[0067] Figure 7B This is a statistical graph of the gene analysis results of cells treated with MS-C19;

[0068] Fig. 8AComparison diagram of pathway enrichment of significantly upregulated genes between the KD group and the control group;

[0069] Figure 8B Comparison diagram of pathway enrichment of significantly downregulated genes between the KD group and the control group;

[0070] Figure 8C This is a comparison diagram of pathway enrichment of genes significantly upregulated between DMSO groups after MS-C19 treatment;

[0071] Fig.8D .Comparison of pathway enrichment of significantly downregulated genes between MS-C19 and DMSO groups;

[0072] Fig.9A Overlap diagram of significantly up-regulated genes between KD group and MS-C19 group;

[0073] Fig. 9B Overlap diagram of significantly down-regulated genes between KD group and MS-C19 group;

[0074] Fig. 9C This is the pathway enrichment statistics of genes significantly upregulated after Overlap;

[0075] Fig.9D This is the pathway enrichment statistics of genes significantly upregulated after Overlap;

[0076] Fig. 10A This is the result of qPCR verification of the expression levels of differentially expressed genes related to lysosomes and inflammation in the knockdown group cells;

[0077] Fig. 10B This is the result of qPCR verification of the expression levels of differentially expressed genes related to lysosomes and inflammation in cells of the MS-C19 treatment group;

[0078] Fig.11 This is a statistical chart of the survival time of AML patients with high expression of GRN;

[0079] Fig. 12A The figure shows the cell apoptosis of MOLM-13 cells with FASN and GRN knocked down simultaneously on the fourth and sixth days detected by AnnexinV / PI staining;

[0080] Fig. 12B This is a diagram of the clone formation test of nine groups of cells in methylcellulose semi-solid medium. After 7 days of culture, the clone formation was detected.

[0081] Fig. 12C . Record the survival curve of cells through the blood cell counting plate;

[0082] Fig.13Ais the reaction equation of plate mycin derivatives;

[0083] Fig. 13B The structural diagrams of the characteristic groups of 70 plate mycin derivatives;

[0084] Fig. 13C The figure shows the results of screening 70 inhibitors in K562 cells;

[0085] Fig.13D To verify the interaction map between MS-C19 and FASN protein for thermal shift experiments;

[0086] Fig.13E The figure shows the protein expression of FASN detected by WB in MOLM-13 cells treated with MS-C19;

[0087] Fig.14A It is a statistical graph showing the effects of different FASN inhibitors on inducing apoptosis of MOLM-13 cells;

[0088] Fig. 14B This is a statistical graph showing the effects of different FASN inhibitors on inducing apoptosis in K562 cells;

[0089] Fig. 14C It is a statistical diagram of the effects of different FASN inhibitors on inducing apoptosis of NB-4 cells;

[0090] Fig.14D It is a statistical diagram of the effects of different FASN inhibitors on the induction of MOLM-13 cell cycle;

[0091] Fig.14E It is a statistical diagram of the effects of different FASN inhibitors on the induction of K562 cell cycle;

[0092] Fig.14F It is a statistical diagram of the effects of different FASN inhibitors on the induction of NB-4 cell cycle;

[0093] Figure 14G This is a statistical graph showing the effects of different FASN inhibitors on the ability of inducing MOLM-13 cell clone formation;

[0094] Fig.14H This is a statistical graph showing the effects of different FASN inhibitors on the ability of K562 cells to induce clone formation;

[0095] Fig.14I This is a statistical graph showing the effects of different FASN inhibitors on the ability of inducing NB-4 cell clone formation. DETAILED DESCRIPTION

[0096] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0097] Part I: Functional study of leukemia cell lines by knocking down FASN

[0098] Using RNA interference technology, short hairpin RNA was designed to knock down the expression of FASN in different leukemia cell lines, confirming the role of this target in the pathogenesis of leukemia. The three cell lines were infected with four shFASNs respectively. After puromycin screening, the cell lines that stably expressed low levels of FASN were obtained. The proliferation curves of MOLM-13, K562 and NB-4 were recorded within 6 days after infection, and the apoptosis and cycle of cells were detected by flow cytometry antibody staining, and the feasibility of FASN as a therapeutic target was evaluated by tumor cell cloning experiments.

[0099] The method for preparing a cell line that stably expresses low levels of FASN is as follows:

[0100] Step 1: Generate RNAi sequences targeting the FASN CDS region using the website http: / / rnaidesigner.lifetechnologies.com / rnaiexpress / , select 4 of them, and combine the sequences with the restriction sites and hairpin structure sequences of the pLKO.1 vector to obtain two upstream and downstream shRNA primers;

[0101] The RNAi sequences are shown in Table 1;

[0102] Table 1 Sequence Listing

[0103]

[0104]

[0105] Step 2: shRNA Oligo annealing;

[0106] Step 3: After double restriction digestion to obtain the linearized pLKO.1 vector, the vector was digested with T4 enzyme for ligation and transformation;

[0107] Step 4: Extract the plasmid, package the plasmid using a lentiviral packaging system, and infect cells by centrifugation infection;

[0108] Step 5: After 48 hours of infection, cells were screened for puromycin resistance to obtain FASN knockdown cell lines.

[0109] refer to Figures 1A to 1L , Figures 1A to 1C Meaning: The cell proliferation curves of MOLM-13, K562 and NB-4 cells after knockdown of FASN were recorded using a blood cell counting plate; Figure 1A .MOLM-13 cells; Figure 1B .K562 cells; Figure 1C .NB-4 cells;

[0110] Figure 1D-1F Representative: After knocking down FASN in cells, cell apoptosis was detected by AnnexinV / PI antibody staining; Figure 1D :MOLM-13 cells; Figure 1E : K562 cells; Figure 1F : NB-4 cells;

[0111] Figure 1G-1I Representative: After knocking down FASN in cells, the cell cycle was detected by PI staining and flow cytometry; Figure 1G :MOLM-13 cells; Figure 1H : K562 cells; Fig. 1I : NB-4 cells;

[0112] Figure 1J-1L Representative: The FASN knockdown cells and control cells were subjected to a clone formation experiment, and the clone formation was detected after seven days of culture. Figure 1J :MOLM-13 cells; Figure 1K : K562 cells; Figure 1L : NB-4 cells;

[0113] The results showed that knocking down FASN significantly inhibited cell growth and clone formation ability, induced cell apoptosis and blocked cell cycle progression.

[0114] 1.1 Functional study of FASN knockout in AML cell lines

[0115] CRISPR Cas9 technology was used to design guide RNA to knock out FASN in the AML cell line MOLM-13, confirming the role of this target in the pathogenesis of AML. We infected MOLM-13 with four sgFASN by lentivirus, obtained the MOLM-13 cell line with FASN knockout by single-cell clone selection technology, recorded the MOLM-13 proliferation curve, detected cell apoptosis and cycle by flow cytometry antibody staining, and evaluated the feasibility of FASN as a therapeutic target by tumor cell clone formation experiments.

[0116] The method for constructing the FASN knockout MOLM-13 cell line is as follows:

[0117] Step 1: Generate sgRNA sequences targeting the FASN genome using the website https: / / www.benchling.com / academic / , select 4 of them, and combine the sequences with the restriction sites and hairpin structures according to the restriction sites of the CRISPR V2 vector to obtain two upstream and downstream shRNA primers.

[0118] The sgRNA sequences are shown in Table 2;

[0119] Table 2 Sequence Listing

[0120]

[0121] Step 2: sgRNA Oligo annealing;

[0122] Step 3: After double enzyme digestion, the linearized CRISPR V2 vector is digested with T4 enzyme for ligation and transformation;

[0123] Step 4: Extract the plasmid, package the plasmid using a lentiviral packaging system, and infect cells by centrifugation infection;

[0124] Step 5: 48 hours after infection, the cells were screened for puromycin resistance; 2 days after screening, the cells were diluted to the limit and the cell density was adjusted to only 100 cells in 10 mL of culture medium;

[0125] Step 6: After inverting and mixing 10 mL of the cell suspension containing 100 cells, add 100 μL per well into a 96-well plate and culture in a cell culture incubator;

[0126] Step 7: After one week of culture, observe the cell growth in the well plate, and transfer the wells with obvious cell proliferation to a 24-well plate for culture for one week;

[0127] Step 8: Collect the cultured cells, detect the expression level of FASN in the cells by Western blot, and analyze the results to obtain cells with FASN knockout.

[0128] The cell cycle detection method is:

[0129] Step 1: Take 100 μL of cells on the 4th and 6th day after infection screening into a 1.5 mL centrifuge tube, add 1 mL PBS to wash once, and centrifuge at 5000 rpm, 4°C for 5 minutes;

[0130] Step 2: Discard the supernatant, retain the cell pellet, and add 500 μL of the prepared cell cycle staining solution (1 mL of staining solution includes 0.5 μL of 10% NP40 solution; 2 μL of RNaseA; 10 μL of PI (1 mg / mL); 1 mL of PBS);

[0131] Step 3: 37°C, place for more than 30 minutes;

[0132] Step 4: Detect the PI value in cells by flow cytometry;

[0133] The detection method of cell apoptosis is:

[0134] Step 1: Take 100 μL of cells on the 4th and 6th day after infection screening into a 1.5 mL centrifuge tube, add 1 mL PBS to wash once, and centrifuge at 5000 rpm, 4°C for 5 minutes;

[0135] Step 2: Discard the supernatant, retain the cell pellet, and add 50 μL of the prepared cell apoptosis staining solution. The apoptosis staining solution is prepared by adding 0.25 μL of APC-Annexin V antibody to 50 μL of Annexin V Binding buffer.

[0136] Step 3: Stain at room temperature in the dark for 15 minutes;

[0137] Step 4: Before testing, add 100 μL of PI solution prepared with Annexin V Binding buffer, with the ratio of PI to Annexin V Binding buffer being 1:1000.

[0138] Step 5: After mixing, flow cytometry was performed to detect the expression of Annexin V and PI in the cells;

[0139] Step 6: The result data was analyzed by Flow jo.

[0140] The clone formation assay method is:

[0141] Step 1: Prepare a 24-well plate in advance and draw a tic-tac-toe grid at the bottom of the wells where the experiment is to be conducted for easy counting later;

[0142] Step 2: After 48 hours of puromycin selection, count the cells and adjust the number of cells per ml of cell suspension to 3×10 4 indivual;

[0143] Step 3: Add 1500 cells, i.e. 50 μL cell suspension, 5 μL double antibody, and 1 μL 1 mg / mL puromycin to a 1.5 mL centrifuge tube and shake to mix;

[0144] Step 4: Add 56 μL of cell suspension containing penicillin-streptomycin and puromycin to the center of the well in the 24-well plate, then add 450 μL of methylcellulose semisolid culture medium, and shake the well plate to evenly distribute the cells in the culture medium;

[0145] Step 5: Add 1-2 mL of PBS to the 16 wells outside the semi-solid medium to prevent the medium from drying out.

[0146] Step 6: After culturing in the cell culture incubator for 7 days, record the number and morphology of clones. Cell groups larger than 50 cells are considered clones.

[0147] Step 7: Use Prism software to plot the number of cell clones.

[0148] refer to FIG. 2A to FIG. 2D ; Figure 2A .Use a blood cell counting plate to record the cell proliferation curves of the control group and the 7 groups of MOLM-13 cells with FASN knockout; Figure 2B .Analysis of cell cycle progression in FASN knockout MOLM-13 cells; Figure 2C . Annexin V / PI staining was used to detect cell apoptosis in MOLM-13 cells with FASN knockout on the fourth and sixth days; Figure 2D .The clone formation experiment was performed on MOLM-13 in the control group and the knockout group, and the number of clones was counted after 7 days.

[0149] The results showed that knocking out FASN significantly inhibited cell growth and clone formation ability, induced cell apoptosis and blocked cell cycle progression.

[0150] 1.2 Functional study of FASN target inhibition in AML mouse model

[0151] An AML mouse model was constructed using immunodeficient mice and MOLM-13 cell lines. FASN was knocked down or knocked out in MOLM-13 using short hairpin RNA and CRISPR Cas9 technology, and then transplanted into immunodeficient mice, and the survival curve of the mice was recorded.

[0152] The ML mouse model was constructed as follows:

[0153] Step 1: Adjust the density of MOLM-13 cells to contain 2 × 10 cells per ml. 5 cells;

[0154] Step 2: Cell resuspension: Centrifuge the cell suspension at 1800 rpm for 5 minutes and resuspend the cell pellet with 1 mL PBS;

[0155] Step 3: Fix the mouse: Fix the mouse in a fixator and wipe the mouse's tail with an alcohol cotton ball to make the blood vessels more obvious;

[0156] Step 4: Mouse transplantation: Use a 1 mL insulin syringe to take 100 μL of cell suspension and transplant it into the mouse via tail vein injection.

[0157] refer to Figure 3A and Figure 3B , Figure 3A .FASN knockdown and control MOLM-13 cells were respectively transplanted into NSGS mice by tail vein injection, and the survival time of the mice was observed. There were 10 mice in each group of shNC and shFASN#1, 9 mice in the shFASN#4 group, and 5 mice in each group of shFASN#5 and shFASN#6; Figure 3B FASN knockout and control MOLM-13 cells were transplanted into NSGS mice via tail vein injection, and the survival time of the mice was observed. There were 5 mice in each group. P value was calculated by Log-rank.

[0158] The results of the study found that knocking down FASN significantly prolonged the survival of mice and slowed down the progression of the disease.

[0159] 1.3 Study on the effect of FASN knockout on mouse hematopoietic function

[0160] FASN fl / fl Wild type, FASN fl / + -Mx1Cre heterozygous knockout and FASN fl / fl -Mx1Cre homozygous knockout mice were injected with pIpC to induce Cre enzyme to work, thus obtaining mice with FASN knockout specifically in the blood system, and the peripheral blood of FASN knockout mice was tested by a hemocytometer;

[0161] refer to FIG. 4A to FIG. 4D One month after pIpC-induced FASN deficiency, various indicators in peripheral blood were detected. Figure 4A .The number of white blood cells in the peripheral blood of mice; Figure 4B .The number of red blood cells in the peripheral blood of mice; Figure 4C .Neutrophil count in peripheral blood of mice; Figure 4D .Number of monocytes in peripheral blood of mice. Five mice in each group.

[0162] The test results showed that FASN did not affect the number of various blood cells in the peripheral blood of mice.

[0163] Flow antibody staining and flow cytometry were used to analyze the lineage cells and lineage progenitors in the bone marrow and spleen cells of wild-type, heterozygous knockout, and homozygous knockout mice;

[0164] The flow cytometry analysis method for mouse lineage cells is as follows:

[0165] Step 1: Prepare mouse spleen and bone marrow single cell suspension, filter through a 70μM filter, and adjust to the same volume;

[0166] Step 2: Take 20 μL of cells from the fixed volume of cell suspension and add them to a centrifuge tube; prepare CountingBeads (2.5 μL beads / sample) at 100 μL / sample (2.5 μL beads + 97.5 μL PBS); directly put it on the machine and collect 1000 Counting Beads.

[0167] Step 3: Flow cytometry for integration analysis of myeloid, lymphoid and erythroid cells:

[0168] Whole bone marrow: resuspend the whole bone marrow cells of one hind leg in 8 mL, and take 1 mL for erythroid and myeloid flow cytometry analysis; spleen (50 mg): resuspend in 5 mL PBS, and take 1 mL for erythroid and myeloid flow cytometry analysis;

[0169] Take 50 μL of cells in a 1.5 mL centrifuge tube, centrifuge at 5000 rpm for 5 minutes, and discard the supernatant;

[0170] Add the prepared antibody dilution (see Table 3) into the centrifuge tube and resuspend the cell pellet;

[0171] Stain on ice for 30 minutes in the dark;

[0172] After staining, add 1 mL of PBS to stop staining, centrifuge at 5000 rpm for 5 minutes, discard the supernatant and load onto the instrument.

[0173] Table 3 Antibody dilution formula for myeloid, lymphoid and erythroid cell integration analysis

[0174]

[0175]

[0176] Step 4: Purify the prepared bone marrow and spleen suspension to obtain Lin - Cells, purification steps are as follows:

[0177] After centrifuging the bone marrow and spleen cell suspensions, discard the supernatant and add RBC lysis buffer to lyse red blood cells, 1 mL / bone marrow cell and 1 mL / spleen cell, for 5 minutes, and add 10 mL PBS to terminate the lysis.

[0178] Centrifuge at 1800 rpm, 4°C for 8 min. Discard the supernatant, add 1 mL of PBS to resuspend the cells, add 25 μL of Biotin-Mouse Lineagent Depletion Cocktail per mouse, mix gently, and incubate on ice for 15 min.

[0179] Add 7 mL of PBS to terminate the reaction and centrifuge at 1800 rpm at 4°C for 8 min;

[0180] Discard the supernatant, add 1 mL PBS to resuspend the cells, add 25 μL Streptavidin particles plus-DM (shake well before use), and gently mix. Incubate on ice for 15 min;

[0181] Transfer 1 mL of the incubated cell suspension to the flow tube, wash the original tube with 1 mL of sterile PBS, merge the cells into the flow tube, and place them on a magnetic stand for 10 min.

[0182] Step 5: Lin-cell counts in bone marrow and spleen: Counting beads (take cells directly for counting)

[0183] From the fixed volume of cell suspension, take 100 μL of cells for counting;

[0184] Prepare counting beads (2.5μL beads / sample) and dispense them into 100μL / sample (2.5μL beads / sample + 97.5μL PBS); load directly onto the machine and collect 1000 beads.

[0185] Step 6: Lin-cell staining of bone marrow and spleen:

[0186] The leg bones of one hind leg and the Lin-cells of the spleen were resuspended in 1 mL, of which 450 μL was used for HSC staining, 450 μL was used for HPC staining, and 100 μL was used for Counting Beads counting (to facilitate the calculation of the difference in MEP / GMP / CMP in the spleen later)

[0187] The purified bone marrow and spleen Lin- cells were centrifuged, the supernatant was discarded, 50 μL of the prepared antibody diluent was added for staining, and the cells were resuspended by pipetting. The staining was carried out on ice for 30 minutes;

[0188] Stain on ice for 30 min, antibody dilution ratio 1:200;

[0189] Add 1 mL PBS to stop staining, 1800 rpm for 8 min;

[0190] The supernatant was discarded after centrifugation, and the precipitate was the cells. 200 μL PI was added to resuspend the cells and perform flow cytometry analysis.

[0191] The antibody dilution formula for HPC cell flow cytometry analysis and the antibody dilution formula for HSC cell flow cytometry analysis refer to Table 4 and Table 5;

[0192] Table 4 Antibody dilution formula for HPC cell flow cytometry analysis

[0193]

[0194] Table 5 Antibody dilution formula for HSC cell flow cytometry analysis

[0195]

[0196]

[0197] refer to FIG. 5A to FIG. 5J , Figure 5A .The ratio between spleen weight and body weight of mice; Figure 5B .The percentage of mature myeloid cells and erythrocytes in the bone marrow and spleen of mice; Figure 5C-5D The total number of mature myeloid cells and erythrocytes in the bone marrow and spleen of mice; Figure 5E .The percentage of HSC cells in the bone marrow and spleen of mice; Fig. 5F .The number of HSCs in the bone marrow and spleen of mice; Figure 5G .The cell percentages of LT-HSC, ST-HSC and MPP in the bone marrow and spleen of mice; Figure 5H .The number of LT-HSC, ST-HSC and MPP cells in the bone marrow and spleen of mice; Fig.5I .The cell percentages of CMP, GMP and MEP in the bone marrow and spleen of mice; Figure 5J .The number of CMP, GMP and MEP cells in the bone marrow and spleen of mice. 5 mice in each group.

[0198] The analysis results showed that FASN knockout had no effect on the number of cells of various lineages and progenitor cells in mice.

[0199] In order to study whether FASN knockout would damage the hematopoietic stem cells of mice, competitive bone marrow transplantation was performed on wild-type, heterozygous and homozygous knockout mice, and the ratio of CD45.1 to CD45.2 in the peripheral blood of mice was detected every month.

[0200] refer to Figure 6 , Figure 6 Results of competitive transplantation assay of CD45.1 / CD45.2 in FASN knockout mice.

[0201] The results showed that the ratio of CD45.1 to CD45.2 did not differ among the three groups of mice.

[0202] 1.4 Study on the signaling pathway of FASN affecting the pathogenesis of leukemia

[0203] Previous studies have found that knocking down or knocking out FASN in AML cells can induce apoptosis of tumor cells. Therefore, RNA sequencing was performed on MOLM-13 cells with FASN knockdown and MOLM-13 cells treated with MS-C19 (compound MS-C19 shown in the second part). By comparing the sequencing data of the control group and the knockdown group and the data of the DMSO group and the C-19 treatment group, 849 significantly differentially expressed genes were found in MOLM-13 cells after FASN knockdown, including 356 significantly upregulated genes and 492 significantly downregulated genes ( Fig. 7A ), there were 2971 differentially expressed genes in cells treated with MS-C19, including 2022 significantly up-regulated genes and 949 significantly down-regulated genes ( Figure 7B ).

[0204] At the same time, after GO analysis of the differentially expressed genes in the two groups of sequencing, the up-regulated genes were mainly enriched in signal pathways related to inflammation, lysosomes, and phagosomes, while the down-regulated genes were mainly enriched in signal pathways related to cell division, spindles, centrosomes, and DNA helicase activity ( Figure 8A-8D )

[0205] Fig. 8A .Pathway enrichment of significantly upregulated genes between KD group and control group; Figure 8B .Pathway enrichment of significantly downregulated genes between KD group and control group; Figure 8C .Pathway enrichment of genes significantly upregulated between DMSO groups after MS-C19 treatment; Fig.8D .Pathway enrichment of significantly downregulated genes between MS-C19 and DMSO groups.

[0206] After overlapping the two sets of sequencing data, the differentially expressed genes were found in both groups. The differentially expressed genes were also enriched, and the enriched pathways were mainly concentrated in signal pathways related to inflammation, lysosomes, and cell division ( Figure 9A-9C ).

[0207] Fig.9A .Overlap of significantly up-regulated genes between KD group and MS-C19 group; Fig. 9B .Overlap of significantly down-regulated genes between KD group and MS-C19 group; Fig. 9C .Pathway enrichment of significantly upregulated genes after Overlap; Fig.9D .Pathway enrichment of significantly upregulated genes after Overlap.

[0208] According to the analysis of RNA-seq data, this example believes that there is an inseparable connection between FASN knockdown and MS-C19 treatment to inhibit the pathogenesis of AML and the lysosome and inflammatory signaling pathways. Therefore, the expression of genes related to inflammation and lysosomes, such as PLD3, GRN, CTSD and LYZ, was detected in MOLM-13 cells with FASN knockdown and MOLM-13 cells after MS-C19 treatment;

[0209] refer to Fig. 10A and Fig. 10B , Fig. 10A and Fig. 10B Real-time fluorescence quantitative PCR was used to verify the expression of lysosome- and inflammation-related genes in the knockdown group and MS-C19 group. Fig. 10A . qPCR verification of the expression levels of differentially expressed genes related to lysosomes and inflammation in knockdown group cells; Fig. 10B . qPCR validation of differentially expressed genes related to lysosomes and inflammation in cells of the MS-C19-treated group; three data points per group.

[0210] The results showed that the expression of the above genes at the mRNA level increased significantly, and the results obtained in cells treated with MS-C19 were the same ( Fig. 10A -B).

[0211] In the screening and validation of target genes, the expression of GRN in the knockdown group was significantly upregulated compared with the control group. In addition, combined with the survival curve of AML patients in the public database, it was found that the survival time of AML patients with high expression of GRN was significantly prolonged (p = 0.033) ( Fig.11 ), which is exactly opposite to the relationship between FASN expression and the survival time of AML patients, and is in line with the expectation of the present invention.

[0212] In MOLM-13 cells with FASN knockdown and GRN knockdown, the proliferation, apoptosis and clone formation of the cells were detected. FIG. 12A to FIG. 12C , Fig. 12A Annexin V / PI staining was used to detect cell apoptosis in MOLM-13 cells with FASN and GRN knockdown on the fourth and sixth days; Fig. 12B . In methylcellulose semi-solid medium, nine groups of cells were subjected to clone formation experiments. After 7 days of culture, the clone formation was detected; Fig. 12C The survival curve of cells was recorded by hemocytometer, with three data points in each group.

[0213] The test results showed that knocking down GRN could restore the function of MOLM-13 cells.

[0214] The above research found that:

[0215] 1. FASN can be used as a target for anti-leukemia therapy;

[0216] 2. FASN-specific knockout does not affect hematopoiesis in mice;

[0217] 3. Knockdown of FASN and MS-C19 treatment further promoted leukemia apoptosis through lysosomal and inflammatory pathways.

[0218] Part II Synthesis and performance study of MS-C19

[0219] 2.1 Synthesis and screening of MS-C19

[0220] Based on the structure of the natural fatty acid synthase inhibitor tabacumycin, tabacumycin was modified using click chemistry to synthesize 70 tabacumycin derivatives.

[0221] The specific synthesis method is:

[0222] General Procedure: All ^1H and ^13C nuclear magnetic resonance (NMR) spectra were recorded on Brucker 600, 500 or 400 MHz instruments. Chemical shifts are reported with tetramethylsilane (δ = 0 ppm) as internal standard in ^1H NMR and with deuterated chloroform (δ = 77.00 ppm) as internal standard in ^13C NMR spectra. The following abbreviations are used to indicate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. High resolution mass spectra (HRMS) were recorded on a LTQ-ORBITRAP-ETD instrument. Samples were analyzed on a Waters e2695 high performance liquid chromatography (HPLC) system equipped with a PDA detector and a Waters Sunfire C18 column (150×4.6 mm). The mobile phase consisted of buffer A (ultrapure water containing 0.1% HCOOH and 0.1% CH3CN) and buffer B (containing 0.1%

[0223] The flow rate was 1 mL / min. All compounds used for minimum inhibitory concentration (MIC) determination and cell viability assays had a purity of at least 95% according to HPLC and ^1H NMR analysis.

[0224] Synthesis of 2a: In ethanol / water (8.8 mL / 0.88 mL), platensimycin epoxide solution was added, followed by NaN3 (866 mg, 0.66 mmol) and NH4Cl (70 mg, 1.32 mmol). The reaction mixture was stirred at 80°C for 12 hours under reflux conditions. Then, the reaction mixture was concentrated under reduced pressure and neutralized with 2M HCl (2.0 mL). The residue was dissolved in ethyl acetate and washed with water and brine in sequence. The organic phase was dried and concentrated; the crude product obtained was purified by column chromatography (eluent: PE / EA / AcOH = 100:20:0.25 to PE / EA / AcOH = 50:50:0.25) to obtain 2a (157.0 mg, yield 87%).

[0225] Synthesis of the compound library: 2a (70 mg, 145.25 μmol) was dissolved in 3.75 mL DMF and then transferred to 70 test tubes containing 50 μL of 2a solution. Next, 50 μL of TBTA (81.75 mg, 145.25 μmol, dissolved in 3.75 mL DMF) was added to the above reaction tubes similarly. Next, 70 alkyne substrates (311.2 μmol in total) were dissolved in 5 mL DMF, and 50 μL of each was added to each test tube. Finally, CuSO4 (10.38 μmol, 20 μL), sodium ascorbate (51.88 μmol, 110 μL) and deionized water (50 μL) were added. The entire reaction mixture was stirred at room temperature for 12 hours, and then the solvent was removed under reduced pressure. An appropriate amount of dimethyl sulfoxide (DMSO) was added to the crude product to make the concentration of the final product about 5 mg / mL.

[0226] General procedure for the synthesis of CuAAC reaction products (a): Dissolve 2a in tert-butyl alcohol / water (5.0 mL / 5.0 mL), add alkyne substrate (228.25 μmol), CuSO4 (1 mg, 4.15 μmol) and sodium ascorbate (4.1 mg, 20.5 μmol). The mixture is stirred at room temperature for 12 hours. The reaction mixture is then concentrated. The resulting residue is dissolved in ethyl acetate and washed with brine and fresh water in sequence. The organic phase is dried and concentrated; the resulting crude product is purified by column chromatography (eluent: PE / EA / AcOH = 100:20:0.25) to obtain the compound (yield greater than 95%).

[0227] General procedure for the synthesis of CuAAC reaction products (b): Dissolve 2a in DMF / H2O (5.0mL / 5.0mL), add alkyne substrate (228.25μmol), TBTA (109mg, 207.5μmol), CuSO4 (200μL, 100mg / mL aqueous solution), sodium ascorbate (1100μL, 100mg / mL aqueous solution). The mixture was stirred at room temperature for 12 hours. The reaction mixture was then concentrated, and the residue was dissolved in ethyl acetate and washed with brine and fresh water. The organic phase was dried and concentrated; the crude product was purified by column chromatography (eluent: PE / EA / AcOH = 100:20:0.25) to obtain the product (yield greater than 95%).

[0228] By the above method, 70 plate mycin derivatives were synthesized; Fig.13A Show the specific reaction equation; Fig. 13B The structures of the characteristic groups of 70 plate mycin derivatives synthesized by them are shown.

[0229] 70 plate mycin derivatives were screened in K562 cells to obtain MS-C19 ( Fig. 13C , Fig. 13C The results of screening 70 inhibitors in K562 cells are shown);

[0230] Thermal migration experiments confirmed the interaction between MS-C19 and FASN ( Fig.13D , Fig.13D The thermal shift experiment verifies the interaction between MS-C19 and FASN protein, and TVB-3166 is a positive control). MS-C19 exerts its effect by inhibiting the expression of FASN protein ( Fig.13E Figure 5 shows the protein expression of FASN detected by WB in MOLM-13 treated with MS-C19).

[0231] 2.2 Functional studies of FASN inhibitors on leukemia cell lines

[0232] Four FASN inhibitors, MS-C19, PTM, Orlistat and TVB-3166, were used to treat MOLM-13, K562 and NB-4 cells. For MOLM-13 and NB-4 cells, five concentration gradients of 0μM, 1μM, 2.5μM, 5μM and 10μM were designed, and for K562 cells, the following five concentration gradients were selected, namely 0μM, 2.5μM, 5μM, 10μM and 20μM.

[0233] Cell apoptosis was detected 48 hours after drug treatment. Flow cytometry showed that TVB-3166 had no effect on cell apoptosis in MOLM-13, high concentrations of Orlistat and PTM could induce cell apoptosis, and MS-C19 could induce cell apoptosis at low concentrations with increasing concentrations, and its effect was significantly better than the other three drugs.

[0234] In K562 cells, PTM had no effect on cell apoptosis. Orlistat and TVB-3166 could induce slight apoptosis at high concentrations, and MS-C19 could induce cell apoptosis at low concentrations with increasing concentrations.

[0235] In NB-4 cells, PTM had no effect on cell apoptosis. TVB-3166, Orlistat, and MS-C19 could induce cell apoptosis at low concentrations with increasing concentrations, and the effect of Orlistat was slightly better than that of MS-C19 ( Fig.14A -C).

[0236] In addition, the cell cycle of the cells treated with the drugs was analyzed. In the three cell lines, the effects of PTM, Orlistat and TVB-3166 on the cell cycle were not obvious. However, the cell cycle of cells treated with MS-C19 was blocked at different stages. The number of G1 phase cells of MOLM-13 cells increased significantly, while the number of S phase cells decreased significantly. The number of G2-M phase cells of K562 and NB-4 cells increased significantly, while the number of S phase cells decreased significantly ( Fig.14D -F).

[0237] At the same time, in MOLM-13 and NB-4 cells, by using a drug concentration of 5 μM, in K562 cells, using a drug concentration of 10 μM, these three cell lines were subjected to cloning experiments under different drug treatments. After 1-2 weeks of culture, the number of clones was counted. The statistical results showed that Orlistat had no effect on the cloning ability of MOLM-13 cells. PTM, TVB-3166 and MS-C19 could inhibit the cloning ability of cells to a certain extent, but MS-C19 had the best inhibitory effect. In the analysis results of K562 cells, Orlistat and PTM had no effect on the cloning ability of cells, and TVB-3166 and MS-C19 had the ability to inhibit the formation of cell clones. In NB-4 cells, the four FASN inhibitors could inhibit the cloning ability of cells, and the effects of Orlistat and MS-C19 were comparable ( Figure 14G -I).

[0238] in, FIG. 14A to FIG. 14IOrlistat, PTM, MS-C19 and TVB-3166 were used to treat MOLM-13 and NB-4 cells at 0 μM, 1 μM, 2.5 μM, 5 μM and 10 μM, respectively; K562 cells were treated with 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM concentrations.

[0239] Figure 14A-Figure 14C . Detect cell apoptosis after drug intervention for 48 hours, and collect three data points in each group A. MOLM-13 cells; Fig. 14B .K562 cells, Fig. 14C .NB-4 cells;

[0240] Figure 14D-14F . Detect the progression of the cell cycle after 48 hours of drug intervention, and collect three data points in each group; Fig.14D .MOLM-13 cells; Fig.14E .K562 cells; Fig.14F .NB-4 cells;

[0241] Figure 14G-14I . Detection of cell clone formation after 7 days of drug intervention, four data points in each group; Figure 14G .MOLM-13 cells; H.K562 cells; Fig.14I .NB-4 cells.

[0242] Through the above research, we found that: 1. Through the synthesis and screening process, it was accidental that MS-C19 could exist as a FASN candidate;

[0243] 2.MS-C19 is more effective than other FASN inhibitors and has broad application prospects.

Claims

1. Use of FASN inhibitors in the preparation of drugs for treating blood tumors; The blood tumor is leukemia; The FASN inhibitor is MS-C19; The structural formula of MS-C19 is shown in Formula 1; Formula 1.

2. A compound, characterized in that Its structural formula is shown in Formula 1; Formula 1.

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