WO2022213878A1 - 靶向SOAT1蛋白的化合物Ramipril在制备预防和/或治疗肝癌药物中的应用 - Google Patents

靶向SOAT1蛋白的化合物Ramipril在制备预防和/或治疗肝癌药物中的应用 Download PDF

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WO2022213878A1
WO2022213878A1 PCT/CN2022/084430 CN2022084430W WO2022213878A1 WO 2022213878 A1 WO2022213878 A1 WO 2022213878A1 CN 2022084430 W CN2022084430 W CN 2022084430W WO 2022213878 A1 WO2022213878 A1 WO 2022213878A1
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liver cancer
soat1
drug
protein
ramipril
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French (fr)
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贺福初
孙爱华
汪志华
王苗苗
王磊
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北京蛋白质组研究中心
军事科学院军事医学研究院生命组学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to the application of a compound Ramipril targeting SOAT1 protein in the preparation of a medicine for preventing and/or treating liver cancer.
  • Cholesterol is an essential lipid component for maintaining cellular homeostasis. In addition to being a precursor for the synthesis of bile acids, steroid hormones and vitamin D, it is a major component of cell membranes, is enriched in lipid rafts and plays an important role in cell signaling. Studies have found that there is cholesterol metabolism reprogramming during the occurrence and development of tumor cells, the intracellular cholesterol level is significantly up-regulated, and its metabolites are abnormally accumulated. In the early stage, our research group found through proteomic and phosphorylated proteomic studies of 101 early-stage hepatocellular carcinoma and paired adjacent tissue samples that clinically considered early-stage hepatocellular carcinoma patients can be divided into three proteomic subtypes.
  • Avasimibe was first used as a lipid-lowering drug for the treatment of atherosclerosis. Recently, it has also been found to have therapeutic effects on pancreatic cancer and liver cancer. , such as Nevanimibe, CL-976 and other small molecules have been reported and SOAT1 protein co-crystal three-dimensional structure and has the effect of inhibiting protein activity.
  • these studies are still in preclinical research. Therefore, drug screening around SOAT1, a target that affects cholesterol homeostasis, combined with the analysis and verification of tumor cells and cholesterol homeostasis, found new small molecules with anti-tumor effects, which is a potential cause for liver cancer. targeted therapy provides a new direction.
  • Ramipril was originally a prescription drug for the treatment of essential hypertension, mainly for the prevention and treatment of cardiovascular and cerebrovascular diseases. There are few reports about the preventive and therapeutic effects of Ramipril on cancer.
  • the purpose of the present invention is to provide a group of new uses of drugs/compounds targeting SOAT1 protein.
  • novel use of the medicine/compound targeting SOAT1 protein provided by the present invention is its application in the preparation of a medicine for preventing and/or treating liver cancer.
  • novel use of the medicine/compound targeting SOAT1 protein provided by the present invention is its application in the preparation of a liver cancer cell proliferation inhibitor.
  • the drugs/compounds targeting SOAT1 protein of the present invention are combined with SOAT1 protein to inhibit the growth of liver cancer cells.
  • the present invention also includes the application of drugs/compounds targeting SOAT1 protein in the following aspects:
  • the liver cancer cells can specifically be HepG2, PLC/PRF/5 (PLC), MHCC97L (97L), Hep3B and Huh7.
  • the drug/compound targeting SOAT1 protein of the present invention is selected from at least one of the following: Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595).
  • the Ramipril (Altace) has obvious inhibitory effect on five kinds of hepatoma cells HepG2, PLC, 97L, Hep3B and Huh7, with IC50 of 96.8nM, 25.1nM, 6.0 ⁇ M, 3.0 ⁇ M and 9.8 ⁇ M, respectively.
  • the present invention also provides a product, characterized in that: the active ingredient of the product
  • the active ingredient of the product is selected from at least one of Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595).
  • liver cancer 1) For the prevention and/or treatment of liver cancer
  • the product may be a drug or a pharmaceutical formulation.
  • the product may contain, in addition to the active ingredient, suitable carriers or excipients.
  • suitable carriers or excipients include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers, etc. Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Of these, water-soluble carrier materials are preferred.
  • a variety of dosage forms can be prepared using these materials, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Buccal tablets, suppositories, freeze-dried powder injections, etc. It can be general formulation, sustained-release formulation, controlled-release formulation and various microparticle delivery systems. For tableting the unit administration dosage form, a wide variety of carriers well known in the art can be used.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin pulp, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, agar powder, alginate, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid esters, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration
  • the tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets.
  • a wide variety of carriers well known in the art can be used.
  • carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, etc.
  • disintegrating agents such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc.
  • a wide variety of carriers well known in the art can be used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.
  • diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like.
  • an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation, and in addition, conventional cosolvents, buffers, pH adjusters and the like can be added.
  • dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal cavity; mucosal administration.
  • the present invention also provides a method for preventing and/or treating liver cancer, comprising the steps of: administering the drug/compound targeting SOAT1 protein to a recipient animal or human to prevent and/or treat liver cancer.
  • the animal can be a mammal.
  • the drugs/compounds of the present invention can bind to SOAT1 protein and affect intracellular cholesterol homeostasis.
  • the drug or compound of the present invention can obviously inhibit the growth of liver cancer cells, and compared with the positive controls Avasimibe and Nevanimibe, the effect of Ramipril (Altace) is stronger than the positive drugs.
  • Cholesterol homeostasis experiments also showed that Ramipril (Altace) inhibits the conversion of cholesterol to cholesteryl ester by binding to SOAT1 and increases the level of intracellular cholesterol; and can achieve the effect of increasing intracellular cholesterol at lower drug concentrations.
  • the pharmacodynamic evaluation of human tumor transplanted into mouse model (PDX) also showed that Ramipril has a good inhibitory effect on the growth of PDX in patients with high SOAT1 expression, but has no obvious effect on the PDX model of patients with low SOAT1 expression, indicating that Ramipril has no obvious effect. It can be used for precise treatment of patients with high expression of SOAT1 in liver cancer.
  • the present invention provides a new targeted drug for liver cancer treatment from the perspective of targeting SOAT1 protein to affect cholesterol homeostasis, and provides a new direction for the clinical treatment of liver cancer.
  • Figure 1 is a high-throughput screening based on SOAT1 target proteins. Diagram showing the binding pockets and potential binding sites of several drugs or compounds to the SOAT1 protein.
  • Figure 2 shows the binding model and affinity determination of the drug and the target protein SOAT1.
  • the left of the figure shows the binding model of Ramipril and the positive control Nevanimibe and the target protein SOAT1; the right of the figure shows the results of the affinity determination of Ramipril and the positive control Nevanimibe and the target protein SOAT1.
  • Fig. 3 is the determination of the viability of liver cancer cells by screening small molecules.
  • the figure shows the cell viability measured by CCK-8 after 24h after four drugs were administered in HepG2 cells at two concentrations of 2 ⁇ M and 20 ⁇ M, Avasimibe was the positive control.
  • Figure 4 is the IC 50 determination of Nilotinib on the viability of three kinds of liver cancer cells HepG2, PLC and 97L.
  • Figure 5 is the IC 50 determination of the viability of Ramipril on three kinds of hepatoma cells HepG2, PLC and 97L.
  • Figure 6 is the IC 50 determination of the viability of ABT-737 on three kinds of hepatoma cells HepG2, PLC and 97L.
  • Figure 7 is the IC 50 determination of the viability of Evacetrapib on three kinds of liver cancer cells HepG2, PLC and 97L.
  • FIG. 8 is the assay of the viability of screening small molecules for liver cancer cells.
  • the figure shows the IC 50 of Ramipril on three kinds of hepatoma cells in HepG2, Hep3B and Huh7 cells, respectively.
  • Figure 9 is a drug-to-cell cholesterol homeostasis assay.
  • the figure shows the intracellular cholesterol staining with philipin at 2 times the IC 50 administration concentration of the above four drugs, and Avasimibe as the positive control.
  • Figure 10 is a comparison of the activities of the four drugs and the positive control Avasimibe in HepG2 cells.
  • Figure 11 is a drug-to-cell cholesterol homeostasis assay. Shown is intracellular cholesterol staining with Philippine at twice the IC50 of Ramipril and Nevanimibe as a positive control.
  • Figure 12 is a pharmacodynamic evaluation of Ramipril in a mouse tumor model derived from a liver cancer patient.
  • the left side of the figure shows the tumor tissues of two cases of liver cancer patients with different levels of SOAT1 expression; the right side of the figure shows the pharmacodynamic evaluation of Ramipril on two mouse tumor models of liver cancer patients with different origins.
  • the methods described in the following examples are conventional methods unless otherwise specified.
  • the raw materials can be obtained from open commercial sources unless otherwise specified.
  • Nilotinib (AMN-107) used in the following examples was purchased from Selleck, catalog number S1033; Ramipril (Altace) was purchased from Selleck, catalog number S1793, ABT-737 was purchased from Selleck, catalog number S1002; Evacetrapib (LY2484595) ) from Selleck, catalog number S2825; Avasimibe from Selleck, catalog number S2187, and Nevanimibe from Selleck, catalog number S1742.
  • liver cancer cells HepG2 used in the following examples were purchased from ATCC, product number ATCC HB-8065; Hep3B was purchased from ATCC, product number ATCC HB-8064; Huh7 was purchased from ATCC (agent of Wuhan Proceeds Biological Co., Ltd.), product number iCell-h107; PLC was purchased from ATCC, product number ATCC CRL-8024; 97L was purchased from ATCC (agent of Wuhan Proceeds Biological Co., Ltd.), product number CL-0497.
  • the three-dimensional crystal structure of the SOAT1 protein described in this example was derived from the RCSB PDB database.
  • the SOAT1 protein PDB code was selected as two models, 6L47 and 6VUM, for high-throughput ligand screening.
  • the small molecule database was derived from the FDA and selleck compounds library.
  • the docking software includes four docking methods of AutoDock 4.2, sybyl 2.0, and glide three softwares (glide has two docking methods, SP and XP) to conduct preliminary screening of all compounds to be screened.
  • the docking pocket was first defined.
  • the hydrophobic pocket of SOAT1 protein, C channel, T channel and NTD terminal binding pocket were used as the main active docking pockets.
  • lattices were created around the above four active pockets (distance sizes were set to about 15 ⁇ ) and were added to SOAT1 and the compounds to be docked by adding polar hydrogens and Gasteiger-Huckel partial charges.
  • the conformational sampling of SOAT1 and compounds were set to rigid and flexible, respectively. Genetic algorithms and empirical free energy functions were used to generate and account for docking poses, respectively.
  • the protein and compound pretreatment is the same as above, the docking results are refined by post-docking energy minimization, and the docking results are evaluated by the scoring function glideScore. After completing all these docking steps, the docking results will be merged, and the compounds ranked in the top 20% of all docking tools and aggregation greater than 80% are considered as potentially active compounds. Then, high-precision docking was further performed by glide docking, and the results were visualized and plotted in pymol software.
  • the docking results of Nilotinib (AMN-107), Ramipril (Altace), ABT-737, Evacetrapib (LY2484595) and the positive control Avasimibe are shown in Figure 1.
  • the docking scores of the four drugs are all lower than those of the positive control, and show good binding to SOAT1 .
  • the first is molecular docking.
  • the three-dimensional crystal structure of the SOAT1 protein described in this example comes from the RCSB PDB database.
  • the SOAT1 protein PDB code is selected as 6L47 and 6VUM for model prediction and evaluation.
  • the docking software includes AutoDock 4.2, sybyl 2.0, Four docking methods of the three glide software (glide has two docking methods SP and XP) for molecular docking of all compounds to be screened. Specifically, before molecular docking, the docking pocket was first defined. In this experiment, the hydrophobic pocket of SOAT1 protein, C channel, T channel and NTD terminal binding pocket were used as the main active docking pockets.
  • the kd values of Ramipril, positive control and SOAT1 were further determined using surface plasmon resonance technology. Specifically, a CM5 chip was used for coupling the target protein, and the running buffer was PBS containing 5% DMSO. Before coupling, the chip channel was activated with EDC and NHS at a flow rate of 10uL/min for 60s. Then, the recombinantly expressed and purified SOAT1 protein was diluted to about 50 mg/mL with 10 mmol/L sodium acetate buffer (pH 5.0), the flow rate was 10 uL/min, the injection time lasted 120 s, and the coupling was repeated until the protein of each channel.
  • the coupling amount is about 8000RU, and finally the channel is blocked with ethanolamine, and the flow rate is 10uL/min for 120s.
  • each compound was diluted 11 times from 20 to 0.0195nmol/L, and the small molecules were passed through the chip coupled to the target protein from low concentration to high concentration, and then recorded in real time. and save the data. Simultaneous use of molecular weight adjustment and solvent correction to remove molecular effects of nonspecific binding and signal drift. Finally, all data processing was performed in Biacore T200 analysis software.
  • Example 3 Screening of small molecules to determine the viability of liver cancer cells
  • the cells used in this example were HepG2 cells, and the test drugs were Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595), and blank control and positive control group (Avasimibe). specifically:
  • Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595) can significantly inhibit the growth of HepG2 cells at the concentrations of 2 ⁇ M and 20 ⁇ M.
  • Example 3 IC 50 determination of four screening drugs/compounds on three liver cancer cells
  • liver cancer cells were seeded in a 96-well plate, and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 12 to 24 hours. When the cells grew to about 70%, the diluted drug was added.
  • Example 4 IC50 determination of Ramipril on three types of liver cancer cells
  • the IC50s of several drugs were measured in the three cells as shown in Figure 8, and all showed good cytostatic effects at lower drug concentrations.
  • This example is mainly used to investigate the effect of four compounds on intracellular cholesterol of liver cancer cells, and the method used is Philippine III intracellular cholesterol staining (Cholesterol cell-based detection assay kit, No. 10009779).
  • the principle is that philiptin III can combine with cholesterol on the membrane and emit blue light to develop color.
  • SOAT1 is an important cholesterol homeostasis regulator protein in cells. It can convert cholesterol to cholesterol ester. When SOAT1 is inhibited, the conversion of intracellular cholesterol to cholesterol ester is restricted, and intracellular cholesterol will increase.
  • This experiment investigated the effect of drugs on cellular cholesterol. specifically:
  • Nilotinib AN-107
  • Ramipril Altace
  • ABT-737 ABT-737
  • Evacetrapib LY2484595
  • Avasimibe Avasimibe
  • our disclosed four drugs can inhibit the conversion of cholesterol to cholesteryl ester and increase the level of intracellular cholesterol by binding to SOAT1. . They could significantly inhibit the growth of hepatoma cells, and obtained IC50 values better than the positive drug Avasimibe ( Figure 10).
  • the present invention provides a new targeted drug for liver cancer treatment from the perspective of targeting SOAT1 protein to affect cholesterol homeostasis, and provides a new direction for the clinical treatment of liver cancer.
  • This example is mainly used to explore the effect of Ramipril on intracellular cholesterol of liver cancer cells, and the method used is Philippine III intracellular cholesterol staining (Cholesterol cell-based detection assay kit, No. 10009779).
  • the principle is that philiptin III can combine with cholesterol on the membrane and emit blue light to develop color.
  • SOAT1 is an important cholesterol homeostasis regulator protein in cells. It can convert cholesterol to cholesterol ester. When SOAT1 is inhibited, the conversion of intracellular cholesterol to cholesterol ester is restricted, and intracellular cholesterol will increase.
  • This experiment investigated the effect of drugs on cellular cholesterol. specifically:
  • Ramipril can significantly affect intracellular cholesterol content. This suggests that drugs targeting the SOAT1 protein can cause changes in intracellular cholesterol.
  • Example 4 Evaluation of the efficacy of Ramipril on the mouse tumor model derived from tumor tissue of patients with liver cancer
  • This example is mainly used to explore the effect of Ramipril on tumor growth in vivo. specifically:
  • a tumor tissue from a patient with liver cancer with high SOAT1 expression and a tumor tissue from a patient with liver cancer with low SOAT1 expression were selected for mouse tumor transplantation models.
  • mice When the tumor volume of mice reaches 80-180 mm ⁇ 3, the mice will be randomly divided into groups, with at least 6 mice in each group. Cohort and first dose day were set as day 0, labeled D0.
  • mice were killed by cervical dislocation method, and the tumor was removed to take pictures, and the tumor tissue was preserved for other subsequent studies.
  • Ramipril has a significant inhibitory effect on the tumor model transplanted from the tumor tissue of the liver cancer patient with high SOAT1 expression to the mouse. This suggests that Ramipril targets SOAT1 and has potential therapeutic prospects for such liver cancer patients.
  • Ramipril can inhibit the conversion of cholesterol to cholesteryl ester by binding to SOAT1 and increase the intracellular cholesterol level. They can significantly inhibit the growth of hepatoma cells and inhibit tumor growth in human PDX mice.
  • the present invention provides a new targeted drug for liver cancer treatment from the perspective of targeting SOAT1 protein to affect cholesterol homeostasis, and provides a new direction for the clinical treatment of liver cancer.
  • the present invention provides a new targeted drug for liver cancer treatment from the perspective of targeting SOAT1 protein to affect cholesterol homeostasis, and provides a new direction for the clinical treatment of liver cancer.

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Abstract

本发明公开了靶向SOAT1蛋白的药物Ramipril(Altace)。实验表明，上述药物和阳性对照药nevanimibe一样，可通过与SOAT1结合从而抑制胆固醇向胆固醇酯转化，升高胞内胆固醇水平。进一步将上述药物对肝癌细胞系HepG2、Hep3B和Huh7三种细胞进行细胞增殖实验发现，他们可明显抑制肝癌细胞生长，并且得到了优于阳性药Avasimibe的IC 50值。进一步动物体内药效评价表明Ramipril可明显抑制SOAT1高表达的肝癌患者PDX模型小鼠肿瘤生长。本发明从靶向SOAT1蛋白影响胆固醇稳态的角度，提供了新的肝癌治疗靶向药物，为肝癌临床治疗提供了新方向。

Description

靶向SOAT1蛋白的化合物Ramipril在制备预防和/或治疗肝癌药物中的应用 技术领域
本发明属于医药技术领域，具体涉及靶向SOAT1蛋白的化合物Ramipril在制备预防和/或治疗肝癌药物中的应用。
背景技术
胆固醇是维持细胞稳态的必要脂质成分。它除了作为胆汁酸、类固醇激素和维生素D的合成前体外，还是细胞膜的主要组成成分，富含于脂筏并在细胞信号导中起重要作用。研究发现肿瘤细胞发生、发展过程中存在胆固醇代谢重编程，胞内胆固醇水平显著上调，其代谢产物异常堆积。前期，本课题组通过对101例早期肝细胞癌及配对癌旁组织样本的蛋白质组和磷酸化蛋白质组研究发现，目前临床上认为的早期肝细胞癌患者，可分成三种蛋白质组亚型，而不同亚型的患者具有不同的预后特征(S-I，S-II，S-III)，术后需要对应不同的治疗方案。其中，对第三类肝细胞癌患者的蛋白质组数据研究发现，胆固醇代谢通路发生了重编程，其中候选药靶胆固醇酯化酶(SOAT1)的高表达具有最差的预后风险，这表明SOAT1蛋白在调节胆固醇稳态及在肝癌发生发展中发挥重要作用。
近年来，靶向SOAT1蛋白的抗肿瘤药物开发也在不断进行，如Avasimibe最先被用于治疗动脉粥样硬化的调血脂药，最近不断被发现对于胰腺癌，肝癌等也具有治疗效果；另外，如Nevanimibe,CL-976等小分子先后被报道出和SOAT1蛋白的共晶三维结构并具有抑制蛋白活性的作用。但是这些研究都还处于临床前研究，因此，围绕影响胆固醇稳态的靶点SOAT1进行药物筛选，并结合肿瘤细胞和胆固醇稳态分析验证，发现具有抗肿瘤效应的新的小分子，这为肝癌的靶向治疗提供了新方向。
Ramipril原用于治疗原发性高血压类处方药，主要用于心脑血管疾病的预防和治疗，几乎没有关于Ramipril对癌症有预防和治疗作用的相关报道。
发明公开
本发明的目的是提供一组靶向SOAT1蛋白的药物/化合物的新用途。
本发明所提供的靶向SOAT1蛋白的药物/化合物的新用途是其在制备预防和/或治疗肝癌的药物中的应用。
本发明所提供的靶向SOAT1蛋白的药物/化合物的新用途是其在制备肝癌细胞增殖抑制剂中的应用。
本发明所述的靶向SOAT1蛋白的药物/化合物是通过与SOAT1蛋白结合并抑制肝癌细胞生长。
本发明还包括靶向SOAT1蛋白的药物/化合物在下述方面的应用：
1)预防和/或治疗肝癌；
2)抑制肝癌细胞的增殖。
本发明中所述肝癌细胞具体可为HepG2、PLC/PRF/5(PLC)、MHCC97L(97L)、Hep3B和Huh7。
本发明所述靶向SOAT1蛋白的药物/化合物选自下述至少一种：Ramipril(Altace)，ABT-737和Evacetrapib(LY2484595)。
其中，Ramipril(Altace)，Cas No.：87333-19-5，结构式如下所示：
Figure PCTCN2022084430-appb-000001
ABT-737，Cas No.：852808-04-9，结构式如下所示：
Figure PCTCN2022084430-appb-000002
Evacetrapib(LY2484595)，CAS No.1186486-62-3，结构式如下所示：
Figure PCTCN2022084430-appb-000003
所述的Ramipril(Altace)对五种肝癌细胞HepG2、PLC、97L、Hep3B和Huh7均具有明显抑制作用，IC 50分别为96.8nM、25.1nM、6.0μM、3.0μM和9.8μM。
本发明还提供了一种产品，其特征在于：所述产品的活性成分
所述产品的活性成分选自Ramipril(Altace)，ABT-737和Evacetrapib(LY2484595)中的至少一种。
包括Ramipril(Altace)。
所述产品具有下述至少一种功效：
1)用于预防和/或治疗肝癌；
2)抑制肝癌细胞的增殖。
示例性的，所述产品可为药物或药物制剂。
所述产品除含有活性成分外，还可含有适宜的载体或赋形剂。这里的载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。其中优选的是水溶性载体材料。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、 乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注射和腔内注射等；腔道给药，如经直肠和阴道；呼吸道给药，如经鼻腔；粘膜给药。
本发明还提供了预防和/或治疗肝癌的方法，包括如下步骤：给受体动物或人施用所述靶向SOAT1蛋白的药物/化合物以预防和/或治疗肝癌。
本发明中，所述动物可为哺乳动物。
本发明所述的药物/化合物可与SOAT1蛋白结合并影响胞内胆固醇稳态。
本发明所述的药物或化合物可明显抑制肝癌细胞生长，与阳性对照Avasimibe和Nevanimibe相比，Ramipril(Altace)效果强于上述阳性药。胆固醇稳态实验也表明，Ramipril(Altace)通过与SOAT1结合从而抑制胆固醇向胆固醇酯转化，升高胞内胆固醇水平；且可以在更低的药物浓度下达到增高胞内胆固醇效果。人源肿瘤移植到小鼠模型(PDX)药效学评价也表明，Ramipril对于SOAT1高表达的病人PDX具有很好的抑制生长作用，而对于SOAT1低表达的病人PDX模型则无明显效果，表明Ramipril可用于肝癌SOAT1高表达这类病人的精准治疗。本发明从靶向SOAT1蛋白影响胆固醇稳态的角度，提供了新的肝癌治疗靶向药物，为肝癌的临床治疗提供了新方向。
附图说明
图1是基于SOAT1靶蛋白的高通量筛选。图示为几种药物或化合物与SOAT1蛋白结合口袋和潜在结合位点。
图2是药物与靶蛋白SOAT1结合模型及亲和力测定。图左示为Ramipril及阳性对照Nevanimibe和靶蛋白SOAT1结合模型；图右示为Ramipril及阳性对照Nevanimibe和靶蛋白SOAT1亲和力测定结果。
图3是筛选小分子对肝癌细胞活力测定。图示是在HepG2细胞中分别给予四种药2μM和20μM两个浓度，24h后用CCK-8测得的细胞活力，Avasimibe为阳性对照。
图4是Nilotinib对三种肝癌细胞HepG2、PLC、97L细胞活力IC 50测定。
图5是Ramipril对三种肝癌细胞HepG2、PLC、97L细胞活力IC 50测定。
图6是ABT-737对三种肝癌细胞HepG2、PLC、97L细胞活力IC 50测定。
图7是Evacetrapib对三种肝癌细胞HepG2、PLC、97L细胞活力IC 50测定。
图8是筛选小分子对肝癌细胞活力测定。图示是在HepG2，Hep3B和Huh7三种细胞中分别测定Ramipril对三种肝癌细细胞的IC 50。
图9是药物对细胞胆固醇稳态测定。图示分别是在上述四种药物2倍IC 50给药浓度下，利用菲利宾菌素进行胞内胆固醇染色，Avasimibe为阳性对照。
图10是四种药物与阳性对照Avasimibe在HepG2细胞中活性比较。
图11是药物对细胞胆固醇稳态测定。图示是在Ramipril的2倍IC 50给药浓度下，利用菲利宾菌素进行胞内胆固醇染色，Nevanimibe为阳性对照。
图12是Ramipril对与肝癌病人来源的小鼠肿瘤模型药效学评价。图左示为SOAT1表达高低不同的二例肝癌患者肿瘤组织；图右示为Ramipril对二种不同来源的肝癌患者小鼠肿瘤模型的药效学评价。
实施发明的最佳方式
下述实施例中的实验方法，如无特别说明，均为常规方法
下面结合实施例对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所做的任何变更或改进，均属于本发明的保护范围。
以下实施例中所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
下述实施例中使用的Nilotinib(AMN-107)购自Selleck，商品目录号S1033；Ramipril(Altace)购自Selleck，商品目录号S1793，ABT-737购自Selleck，商品目录号S1002；Evacetrapib(LY2484595)购自Selleck，商品目录号S2825；Avasimibe购自Selleck，商品目录号S2187，Nevanimibe购自Selleck，商品目录号S1742。
下述实施例中使用的肝癌细胞HepG2购自ATCC，产品编号ATCC HB-8065；Hep3B购自ATCC，产品编号ATCC HB-8064；Huh7购自ATCC(武汉普诺赛生物有限公司代理)，产品编号iCell-h107；PLC购自ATCC，产品编号ATCC CRL-8024；97L购自ATCC(武汉普诺赛生物有限公司代理)，产品编号CL-0497。
实施例1：基于SOAT1靶蛋白的高通量筛选
本实施例所述的SOAT1蛋白三维晶体结构来源于RCSB PDB数据库，选取了SOAT1蛋白PDB code为6L47和6VUM二个模型进行高通量配体筛选，小分子数据库来源于FDA及selleck compounds library。对接软件包括AutoDock 4.2、sybyl 2.0、glide三种软件的四种对接方法(glide分SP和XP二种对接方法)对所有待筛选化合物进行初步筛选。
具体地，在进行分子对接之前，首先定义对接口袋，本实验主要以SOAT1蛋白疏水口袋，C通道，T通道及NTD端结合口袋作为主要活性对接口袋。对于AutoDock4.2和sybyl 2.0，在上述四个活性口袋周围创建了晶格(距离大小设置约15埃)，并通过将极性氢和Gasteiger-Huckel部分电荷添加到SOAT1和待对接化合物中。SOAT1和化合物的构象采样分别设置为刚性和柔性。遗传算法和经验自由能函数分别用于生成和计入对接姿势。对于glide SP和XP模式，蛋白和化合物前处理同上，对接结果通过对接后能量最小化完善，并通过评分功能glideScore评估对接结果。在完成所有这些对接步骤之后，对接结果将被合并，在所有对接工具中排名前20％的化合物及聚集度大于80％都被视为潜在的活性化合物。而后，进一步通过glidedocking进行高精度对接，并将结果在pymol软件中进行可视化展示及作图。Nilotinib(AMN-107)，Ramipril(Altace)，ABT-737，Evacetrapib(LY2484595)及阳性对照Avasimibe对接结果如图1所示，四种药对接得分均小于阳性对照，并显示着和SOAT1有良好结合。
实施例2：化合物Ramipril与靶蛋白SOAT1的结合测定
首先是分子对接，本实施例所述的SOAT1蛋白三维晶体结构来源于RCSB PDB数据库，选取了SOAT1蛋白PDB code为6L47和6VUM二个模型进行模型预测和评价，对接软件包括AutoDock 4.2、sybyl 2.0、glide三种软件的四种对接方法(glide分SP和XP二种对接方法)对所有待筛选化合物进行分子对接。具体地，在进行分子对接之前，首先定义对接口袋，本实验主要以SOAT1蛋白疏水口袋，C通道，T通道及NTD端结合口袋作为主要活性对接口袋。对于AutoDock 4.2和sybyl 2.0，在上述四个活性口袋周围创建了晶格(距离大小设置约15埃)，并通过将极性氢和Gasteiger-Huckel部分电荷添加到SOAT1和待对接化合物中。SOAT1和化合物的构象采样分别设置为刚性和柔性。遗传算法和经验自由能函数分别用于生成和计入对接姿势。对于glide SP和XP模式，蛋白和化合物前处理同上，对接结果通过对接后能量最小化完善，并通过评分功能glideScore评估对接结果。在完成所有这些对接步骤之后，进一步通过glidedocking进行高精度对接，并将结果在pymol软件中进行可视 化展示及作图。Ramipril(Altace)及阳性对照Nevanimibe对接结果如图2所示，Ramipril显示着和SOAT1有良好结合,并且得分小于阳性对照。
而后，使用表面等离子共振技术又进一步测定了Ramipril及阳性对照和SOAT1的kd值。具体地，使用CM5芯片用于偶联目标蛋白，运行缓冲液为含5％DMSO的PBS。偶联前先用EDC和NHS活化芯片通道，流速10uL/min，持续60s。后将重组表达纯化好的SOAT1蛋白用10mmol/L醋酸钠缓冲液(pH 5.0)稀释至约50mg/mL，流速10uL/min，一次进样时间持续120s，多次偶联直至每个通道的蛋白偶联量约为8000RU，最后用乙醇胺封闭好通道，流速10uL/min，持续120s。进行蛋白-小分子相互作用亲和力kd值测定时，将每种化合物从20至0.0195nmol/L稀释11倍，依次从低浓度到高浓度将小分子通过偶联好目标蛋白的芯片，然后实时记录并保存数据。同步使用分子量调整和溶剂校正去除非特异性结合和信号漂移的分子效应。最后所有数据处理均在Biacore T200分析软件中进行。实验结果显示，Ramipril与SOAT1蛋白亲和力kd值为1.02E -8M，远低于阳性对照药Nevanimibe的KD＝5.79E -7M，这表明Ramipril与蛋白具有更好的亲和力。
实施例3：筛选小分子对肝癌细胞活力测定
为了探究筛选的四种化合物对肝癌细胞的活性，我们继续进行了细胞活力测定。
本实施例所用细胞为HepG2细胞，测试药物分别为Nilotinib(AMN-107)，Ramipril(Altace)，ABT-737和Evacetrapib(LY2484595)，另有空白对照和阳性对照组(Avasimibe)。具体地：
1)在96孔板中种入8000/孔HepG2细胞，在DMEM含10％胎牛血清培养基中，37℃，5％CO 2培养环境中培养12～24h，待细胞长到70％左右，开始加入稀释好的药物。
2)分别将上述四种药物及阳性对照用细胞培养基稀释至2μM和20μM，空白对照用等条件下DMSO。
3)移去旧的培养基，将稀释好的药物(用细胞培养基配的)加入含细胞的96孔板并做好标记，每个浓度三组重复。
4)37℃，5％CO 2培养环境中继续培养24-48h后加入10μL/孔CCK-8试剂，继续培养1h。
5)将孵育好的样品于酶标仪450nM处测吸光值，收集整理数据，并在graphpad prism 9.0中进行分析作图。
实验结果如图3所示，Nilotinib(AMN-107)，Ramipril(Altace)，ABT-737和Evacetrapib(LY2484595)在2μM和20μM浓度下均可明显抑制HepG2细胞生长。
实施例3：四种筛选药物/化合物对三种肝癌细胞IC 50测定
为了进一步探究四种化合物对肝癌细胞的活性，我们进行了细胞IC 50测定。具体地：
1)在96孔板中分别种入8000/孔HepG2，PLC及97L三种肝癌细胞，在DMEM含10％胎牛血清培养基中，37℃，5％CO2培养环境中培养12～24h，待细胞长到70％左右，开始加入稀释好的药物。
2)分别将上述四种药物由10mM稀释至100μM，50μM，25μM，10μM，5μM，1μM，100nM，10nM，1nM，0十个浓度点。
3)移去旧的培养基，分别将稀释好的药物(培养基配的)加入含HepG2，PLC及97L三种肝癌细胞的96孔板并做好标记，每个浓度三组重复。
4)37℃，5％CO 2培养环境中继续培养24-48h后加入10μL/孔CCK-8试剂，继续培养1h。
5)将孵育好的样品于酶标仪450nM处测吸光值，收集整理数据，并在graphpad prism 9.0中进行分析作图。
几种药物在三种细胞中测得IC 50如图4～图7所示，在较低的药物浓度下均表现出良好的抑制细胞生长效果。
实施例4：Ramipril对三种肝癌细胞IC 50测定
为了进一步探究Ramipril对肝癌细胞的活性，我们进行了细胞IC 50测定。具体地：
1)在96孔板中分别种入8000/孔HepG2，Hep3B及Huh7三种肝癌细胞，在DMEM含10％胎牛血清培养基中，37℃，5％CO2培养环境中培养12～24h，待细胞长到70％左右，开始加入稀释好的药物。
2)分别将药物由10mM稀释至100μM，50μM，25μM，10μM，5μM，1μM，100nM，10nM，1nM，0十个浓度点。
3)移去旧的培养基，分别将稀释好的药物(培养基配的)加入含HepG2，Hep3B及Huh7三种肝癌细胞的96孔板并做好标记，每个浓度三组重复。
4)37℃，5％CO 2培养环境中继续培养24-48h后加入10μL/孔CCK-8试剂，继续培养1h。
5)将孵育好的样品于酶标仪450nM处测吸光值，收集整理数据，并在graphpad prism 9.0中进行分析作图。
几种药物在三种细胞中测得IC 50如图8所示，在较低的药物浓度下均表现出良好的抑制细胞生长效果。
实施例5：药物对细胞胆固醇稳态测定
本实施例主要用于探究四种化合物对肝癌细胞胞内胆固醇影响，所用方法为菲律宾菌素III胞内胆固醇染色(Cholesterol cell-based detection assay kit,No.10009779)。其原理是菲律宾菌素III可以与膜上胆固醇结合并发出蓝光而显色。而SOAT1是细胞内重要的胆固醇稳态调节蛋白，它可以将胆固醇转化为胆固醇酯，当SOAT1被抑制，胞内胆固醇向胆固醇酯转化被限制，胞内胆固醇将会升高。本实验探究药物对细胞胆固醇影响。具体地：
1)在96孔板中种入8000/孔HepG2或Huh7，在DMEM含10％胎牛血清培养基中，37℃，5％CO 2培养环境中培养12～24h，待细胞长到70％左右，开始加入稀释好的药物。
2)分别将上述Nilotinib(AMN-107)，Ramipril(Altace)，ABT-737，Evacetrapib(LY2484595)和Avasimibe稀释至1μM，200nM，400nM，20μM和10μM(各药物对HepG2的IC 50二倍剂量)。
3)移去旧的培养基，分别将稀释好的药物(培养基配的)加入含细胞的96孔板并做好标记，每个浓度三组重复。
4)37℃，5％CO 2培养环境中继续培养12h～24h(细胞长到约70％～80％观察效果最佳)后按照胆固醇菲律宾菌素III醇染色试剂盒说明书进行染色。
5)将上述养好的细胞去除培养基后，室温，100转/分钟的摇床，PBS清洗3次，每次50μL PBS缓冲液，震荡5分钟。
6)将清洗后的HepG2细胞用细胞固定液(Item No.10009866)，室温，固定15分钟。再置于100转/分钟的摇床，清洗3次，每次50μL PBS缓冲液，震荡5分钟。
7)将菲律宾菌素III储液按1：100稀释(Item No.10009868)，每孔加入100μL稀释好的溶液，避光孵育30-60min(注意此后步骤尽量避光操作)。再置于100转/分钟的摇床，清洗3次，每次50μL PBS缓冲液，震荡5分钟。
8)将洗好的样品置于20倍显微镜下观察，激发波长为340-380nm，发射波长为385-470nm，收集整理数据。
如图9所示，几种药物在对应浓度下均可明显影响胞内胆固醇含量。且与SOAT1蛋白敲低组(基因敲低实验，用sh-RNA干扰SOAT1蛋白表达，sh-RNA购自美国sigma-aldrich公司(http://www.sigmaaldrich.com).The SOAT1sh-RNA的序列如下所示：5’-CCGGTGGTCCATGACTGGCTATATTCTCGAGAATATAGCCAGTCATGGACCATTTTTTG-3’，如序列表中序列1所示)，结果一致，这表明，药物靶向SOAT1蛋白可引起了胞内胆固醇变化。
综上所述，我们公开的四种药物Nilotinib(AMN-107)，Ramipril(Altace)，ABT-737，Evacetrapib(LY2484595)可通过与SOAT1结合从而抑制胆固醇向胆固醇酯转化，升高胞内胆固醇水平。它们可明显抑制肝癌细胞生长，并且得到了优于阳性药Avasimibe的IC 50值(图10)。本发明从靶向SOAT1蛋白影响胆固醇稳态的角度，提供了新的肝癌治疗靶向药物，为肝癌的临床治疗提供了新方向。
实施例6：Ramipril对细胞胆固醇稳态测定
本实施例主要用于探究Ramipril对肝癌细胞胞内胆固醇影响，所用方法为菲律宾菌素III胞内胆固醇染色(Cholesterol cell-based detection assay kit,No.10009779)。其原理是菲律宾菌素III可以与膜上胆固醇结合并发出蓝光而显色。而SOAT1是细胞内重要的胆固醇稳态调节蛋白，它可以将胆固醇转化为胆固醇酯，当SOAT1被抑制，胞内胆固醇向胆固醇酯转化被限制，胞内胆固醇将会升高。本实验探究药物对细胞胆固醇影响。具体地：
1)在96孔板中种入8000/孔HepG2，在DMEM含10％胎牛血清培养基中，37℃，5％CO 2培养环境中培养12～24h，待细胞长到70％左右，开始加入稀释好的药物。
2)将Ramipril(Altace)和nevanimibe稀释至对上述细胞所测得的IC 50二倍剂量。
3)移去旧的培养基，分别将稀释好的药物(培养基配的)加入含细胞的96孔板并做好标记，每个浓度三组重复。
4)37℃，5％CO 2培养环境中继续培养12h～24h(细胞长到约70％～80％观察效果最佳)后按照胆固醇菲律宾菌素III醇染色试剂盒说明书进行染色。
5)将上述养好的细胞去除培养基后，室温，100转/分钟的摇床，PBS清洗3次，每次50μL PBS缓冲液，震荡5分钟。
6)将清洗后的HepG2细胞用细胞固定液(Item No.10009866)，室温，固定15分钟。再置于100转/分钟的摇床，清洗3次，每次50μL PBS缓冲液，震荡5分钟。
7)将菲律宾菌素III储液按1：100稀释(Item No.10009868)，每孔加入100μL 稀释好的溶液，避光孵育30-60min(注意此后步骤尽量避光操作)。再置于100转/分钟的摇床，清洗3次，每次50μL PBS缓冲液，震荡5分钟。
8)将洗好的样品置于20倍显微镜下观察，激发波长为340-380nm，发射波长为385-470nm，收集整理数据。
如图11所示，Ramipril可明显影响胞内胆固醇含量。这表明，药物靶向SOAT1蛋白可引起了胞内胆固醇变化。
实施例4：Ramipril对肝癌患者肿瘤组织来源小鼠肿瘤模型药效评价
本实施例主要用于探究Ramipril在体内对肿瘤生长的影响。具体地：
1)通过免疫组化分析，选择了一例SOAT1高表达的肝癌患者肿瘤组织和一例SOAT1低表达的肝癌患者肿瘤组织分别进行小鼠肿瘤移植模型。
2)将肿瘤按照3×3×3mm大小进行分割后，移植于NOD-SCID小鼠右后皮下位置。
3)当小鼠肿瘤体积达到80～180mm^3开始随机分组,每组至少6只小鼠。分组和首次给药日被设置为第0天，标记为D0。
4)将Ramipril用30％PEG400+0.5％Tween 80+5％propylene glycol溶液溶解稀释，并按20mg/kg的剂量腹腔注射到小鼠体内，每天给药，持续28天，每隔三天测量和记录肿瘤大小。
5)给药周期结束，使用颈部脱臼法处死小鼠，并取下肿瘤拍照，将肿瘤组织保存以备后续其他研究。
6)使用graphpad prism8.0处理数据并作图。
如图12所示，Ramipril对于SOAT1高表达的肝癌患者肿瘤组织移植到小鼠的肿瘤模型具有明显抑制作用。这表明，Ramipril靶向SOAT1并对此类肝癌病人具有潜在治疗前景。
综上所述Ramipril(Altace)可通过与SOAT1结合从而抑制胆固醇向胆固醇酯转化，升高胞内胆固醇水平。它们可明显抑制肝癌细胞生长，并且抑制人源PDX小鼠肿瘤生长。本发明从靶向SOAT1蛋白影响胆固醇稳态的角度，提供了新的肝癌治疗靶向药物，为肝癌的临床治疗提供了新方向。
工业应用
本发明从靶向SOAT1蛋白影响胆固醇稳态的角度，提供了新的肝癌治疗靶向药物，为肝癌的临床治疗提供了新方向。

Claims (10)

  1. 靶向SOAT1蛋白的药物/化合物在制备预防和/或治疗肝癌的药物中的应用；
    所述靶向SOAT1蛋白的药物/化合物为Ramipril；
    所述Ramipril，Cas No.：87333-19-5，结构式如下所示：
    Figure PCTCN2022084430-appb-100001
  2. 权利要求1所述的靶向SOAT1蛋白的药物/化合物在制备肝癌细胞增殖抑制剂中的应用。
  3. 根据权利要求2所述的应用，其特征在于：所述肝癌细胞选自下述任意一种：HepG2、PLC/PRF/5、MHCC97L、Hep3B和Huh7。
  4. 根据权利要求3所述的应用，其特征在于：所述的Ramipril对下述肝癌细胞HepG2、PLC/PRF/5、MHCC97L、Hep3B和Huh7的IC 50依次分别为96.8nM,25.1nM，6.0μM，3.0μM和9.8μM。
  5. 根据权利要求2-4中任一项所述的应用，其特征在于：所述的靶向SOAT1蛋白的药物/化合物是通过与SOAT1蛋白结合并影响胞内胆固醇稳态，从而抑制肝癌细胞增殖。
  6. 一种产品，其特征在于：所述产品的活性成分包括Ramipril。
  7. 根据权利要求6所述的产品，其特征在于：
    所述产品具有下述至少一种功效：
    1)用于预防和/或治疗肝癌；
    2)抑制肝癌细胞的增殖；
    所述产品为药物或药物制剂。
  8. 权利要求1所述的靶向SOAT1蛋白的药物/化合物在下述方面的应用：
    1)预防和/或治疗肝癌；
    2)抑制肝癌细胞的增殖。
  9. 根据权利要求6所述的应用，其特征在于：所述肝癌细胞选自下述任意一种：HepG2、PLC/PRF/5、MHCC97L、Hep3B和Huh7。
  10. 一种预防和/或治疗肝癌的方法，包括如下步骤：给受体动物或人施用权利要求1所述靶向SOAT1蛋白的药物/化合物或权利要求8或9所述的产品以预防和/或治疗肝癌。
PCT/CN2022/084430 2021-04-07 2022-03-31 靶向SOAT1蛋白的化合物Ramipril在制备预防和/或治疗肝癌药物中的应用 WO2022213878A1 (zh)

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