CN114917345A - Application of compounds targeting SOAT1 protein in the preparation of drugs for preventing and/or treating liver cancer - Google Patents

Abstract

The invention discloses a group of drugs or compounds targeting SOAT1 protein, which are respectively Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595). Experiments show that the above drugs, like the positive control drug Avasimibe, can inhibit the conversion of cholesterol to cholesteryl ester by binding to SOAT1 and increase the level of intracellular cholesterol. Further, the above drugs were used for cell proliferation experiments on three types of liver cancer cell lines, HepG2, PLC and 97L, and it was found that they could significantly inhibit the growth of liver cancer cells, and obtained IC50 values better than the positive drug Avasimibe. From the perspective of targeting SOAT1 protein to affect cholesterol homeostasis, the present invention provides a new targeted drug for liver cancer treatment, and provides a new direction for clinical treatment of liver cancer.

Description

Application of compounds targeting SOAT1 protein in the preparation of drugs for preventing and/or treating liver cancer

This application is a divisional application with an application number of 202110371783.4, an application date of April 7, 2021, and an invention-creation titled "Application of a compound targeting SOAT1 protein in the preparation of a drug for the prevention and/or treatment of liver cancer".

technical field

The invention belongs to the technical field of medicine, and specifically relates to a group of small molecule drugs targeting the SOAT1 protein, a cholesterol homeostasis target, and has the prospect of treating liver cancer.

Background technique

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. Patients with different subtypes have different prognostic characteristics (S-I, S-II, S-III), and require different treatment options after surgery. Among them, the proteomic data study of the third type of hepatocellular carcinoma patients found that the cholesterol metabolism pathway was reprogrammed, and the high expression of the drug candidate target cholesterol esterase (SOAT1) had the worst prognosis risk, indicating that the SOAT1 protein It plays an important role in regulating cholesterol homeostasis and in the occurrence and development of liver cancer.

In recent years, the development of anti-tumor drugs targeting SOAT1 protein has also continued. For example, 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. However, 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.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a group of new uses of drugs/compounds targeting SOAT1 protein.

The 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.

The 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:

1) Prevention and/or treatment of liver cancer;

2) Inhibit the proliferation of hepatoma cells.

In the present invention, the liver cancer cells can specifically be HepG2, PLC/PRF/5 (PLC) and MHCC97L (97L).

The drug/compound targeting SOAT1 protein according to the present invention is specifically selected from at least one of the following: Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595).

Among them, Nilotinib (AMN-107), Cas No.: 641571-10-0, the structural formula is as follows:

Ramipril (Altace), Cas No.: 87333-19-5, the structural formula is as follows:

ABT-737, Cas No.: 852808-04-9, the structural formula is as follows:

Evacetrapib (LY2484595), CAS No.1186486-62-3, the structural formula is as follows:

The Nilotinib (AMN-107) has obvious inhibitory effect on the three kinds of hepatoma cells HepG2, PLC and 97L, with _IC50 of 448.5nM, 15.7μM and 2.44μM, respectively.

The Ramipril (Altace) has obvious inhibitory effect on three kinds of hepatoma cells, HepG2, PLC and 97L, with IC ₅₀ of 96.8nM, 25.1nM and 6.0μM, respectively.

The described ABT-737 has obvious inhibitory effect on three kinds of hepatoma cells, HepG2, PLC and 97L, with IC ₅₀ of 184.0 nM, 3.3 μM and 2.1 μM, respectively.

The described Evacetrapib (LY2484595) has obvious inhibitory effect on three kinds of hepatoma cells HepG2, PLC and 97L, with _IC50 of 9.2μM, 5.6μM and 2.0μM, respectively.

The present invention also provides a product, characterized in that the active ingredient of the product is selected from at least one of Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595).

The product has at least one of the following effects:

1) For the prevention and/or treatment of liver cancer;

2) Inhibit the proliferation of hepatoma cells.

Illustratively, the product may be a drug or a pharmaceutical formulation.

The product may contain, in addition to the active ingredient, suitable carriers or excipients. The carrier materials here 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.). Preferred among these are water-soluble carrier materials. 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. Examples of 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 inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils, etc.; absorption promotion agents, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets. For formulating the unit administration dosage form into a pill, a wide variety of carriers well known in the art can be used. Examples of 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. , ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc. For formulating the unit administration form as a suppository, 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. In order to make unit dosage forms into injection preparations, such as solutions, emulsions, lyophilized powders and suspensions, all 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. In addition, in order to prepare an isotonic injection, 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. In addition, colorants, preservatives, fragrances, flavors, sweeteners, or other materials can also be added to the pharmaceutical preparations, if desired. The above-mentioned 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.

In the present invention, the animal can be a mammal.

The four drugs/compounds described in the present invention can bind to SOAT1 protein and affect intracellular cholesterol homeostasis.

The four drugs or compounds described in the present invention can obviously inhibit the growth of liver cancer cells. Compared with Avasimibe, Nilotinib (AMN-107), Ramipril (Altace) and ABT-737 are more effective than the positive drug Avasimibe. Basically, the effect of positive drugs is equivalent. Cholesterol homeostasis experiments also showed that these drugs inhibited the conversion of cholesterol to cholesteryl esters by binding to SOAT1 and increased intracellular cholesterol levels; and the effect of increasing intracellular cholesterol could be achieved at lower drug concentrations. 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.

Description of drawings

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.

Fig. 2 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 μM and 20 μM, Avasimibe was the positive control.

Figure 3 is the IC ₅₀ determination of Nilotinib on the viability of three kinds of hepatoma cells HepG2, PLC and 97L.

Figure 4 is the IC ₅₀ determination of the viability of Ramipril on three kinds of liver cancer cells HepG2, PLC and 97L.

Figure 5 is the IC ₅₀ determination of the viability of ABT-737 on three kinds of hepatoma cells HepG2, PLC and 97L.

Figure 6 is the IC ₅₀ determination of the viability of Evacetrapib on three kinds of liver cancer cells HepG2, PLC and 97L.

Figure 7 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 8 is a comparison of the activities of the four drugs and the positive control Avasimibe in HepG2 cells.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. The methods 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.

The liver cancer cell HepG2 used in the following examples was purchased from ATCC, product number ATCC HB-8065; PLC was purchased from ATCC, product number ATCC CRL-8024; 97L was purchased from ATCC (the agent of Wuhan Proceeds Biological Co., Ltd.), product number CL-0497.

Example 1: High-throughput screening based on SOAT1 target protein

The three-dimensional crystal structure of the SOAT1 protein described in this example was derived from the RCSB PDB database. Two models of the SOAT1 protein PDB code, 6L47 and 6VUM, were selected 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.

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. For AutoDock 4.2 and sybyl2.0, lattices were created around the above four active pockets (distance sizes were set to about 15 Å) and were added to SOAT1 and the compounds to be docked by adding polar hydrogen 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. For glide SP and XP modes, protein and compound pretreatments were the same as above, docking results were refined by post-docking energy minimization, and docking results were 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 .

Example 2: Screening small molecules to determine the viability of liver cancer cells

To explore the activity of the four screened compounds on hepatoma cells, we proceeded to perform cell viability assays.

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:

1) Seed 8000/well HepG2 cells in a 96-well plate, culture in DMEM medium containing 10% fetal bovine serum, 37°C, 5% CO ₂ culture environment for 12-24 hours, when the cells grow to about 70%, Start adding the diluted drug.

2) The above four drugs and positive control were diluted to 2 μM and 20 μM with cell culture medium respectively, and DMSO under the same conditions was used for blank control.

3) Remove the old culture medium, add the diluted drug (prepared with cell culture medium) into the 96-well plate containing cells and make a label, and repeat in three groups for each concentration.

4) Continue culturing for 24-48 hours in a 37°C, 5% CO ₂ culture environment, add 10 μL/well of CCK-8 reagent, and continue culturing for 1 hour.

5) Measure the absorbance value of the incubated sample at 450nM of the microplate reader, collect and organize the data, and perform analysis and graphing in graphpadprism 9.0.

The experimental results are shown in Figure 2. Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595) can significantly inhibit the growth of HepG2 cells at the concentrations of 2 μM and 20 μM.

Example 3: IC ₅₀ determination of four screening drugs/compounds on three liver cancer cells

To further explore the activity of the four compounds on hepatoma cells, we performed cellular _IC50 assays. specifically:

1) 8000/well HepG2, PLC and 97L 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.

2) Dilute the above four drugs from 10mM to ten concentration points of 100μM, 50μM, 25μM, 10μM, 5μM, 1μM, 100nM, 10nM, 1nM, 0 respectively.

3) Remove the old culture medium, respectively add the diluted drugs (prepared in the medium) into the 96-well plate containing HepG2, PLC and 97L liver cancer cells and label them. Each concentration is repeated in three groups.

4) Continue culturing for 24-48 hours in a 37°C, 5% CO ₂ culture environment, then add 10 μL/well of CCK-8 reagent, and continue culturing for 1 hour.

5) Measure the absorbance value of the incubated sample at 450nM of the microplate reader, collect and organize the data, and perform analysis and graphing in graphpadprism 9.0.

The _IC50s of several drugs measured in the three cells are shown in Figures 3 to 6, and they all showed a good inhibitory effect on cell growth at lower drug concentrations.

Example 4: Drug-to-Cell Cholesterol Homeostasis Assay

This example is mainly used to explore the effects 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:

1) Seed 8000/well HepG2 in a 96-well plate, culture in DMEM medium containing 10% fetal bovine serum, 37°C, 5% CO ₂ culture environment for 12-24 hours, when the cells grow to about 70%, start Add the diluted drug.

2) Nilotinib (AMN-107), Ramipril (Altace), ABT-737, Evacetrapib (LY2484595) and Avasimibe were diluted to 1μM, 200nM, 400nM, 20μM and 10μM respectively ( _IC50 double dose of each drug to HepG2) .

3) Remove the old medium, respectively add the diluted drugs (prepared with the medium) into the 96-well plate containing cells and mark them, and repeat in three groups for each concentration.

4) Continue to culture for 12h-24h in a 37°C, 5% CO ₂ culture environment (the best observation effect is when the cells grow to about 70%-80%) and then stain according to the instructions of the cholesterol philippin III alcohol staining kit.

5) After removing the culture medium from the cultured cells, at room temperature, shake at 100 rpm for 3 times with PBS, and shake for 5 minutes with 50 μL of PBS buffer each time.

6) Fix the washed HepG2 cells with a cell fixative solution (Item No. 10009866) at room temperature for 15 minutes. It was then placed on a shaker at 100 rpm, washed three times with 50 μL of PBS buffer each time, and shaken for 5 minutes.

7) Dilute the Philippinein III stock solution at 1:100 (Item No. 10009868), add 100 μL of the diluted solution to each well, and incubate in the dark for 30-60 min (note that the following steps should be avoided as much as possible). It was then placed on a shaker at 100 rpm, washed three times with 50 μL of PBS buffer each time, and shaken for 5 minutes.

8) The washed samples were observed under a 20 times microscope, the excitation wavelength was 340-380 nm, and the emission wavelength was 385-470 nm, and the data were collected and sorted.

As shown in Figure 7, several drugs can significantly affect the intracellular cholesterol content at corresponding concentrations. And with the SOAT1 protein knockdown group (gene knockdown experiment, using sh-RNA to interfere with SOAT1 protein expression, sh-RNA was purchased from the United States sigma-aldrich company (http://www.sigmaaldrich.com). The SOAT1 sh-RNA The sequences are as follows: 5'-CCGGTGGTCCATGACTGGCTATATTCTCGAGAATATAGCCAGTCATGGACCATTTTTTG-3') The results are consistent, which indicates that drug targeting SOAT1 protein can cause changes in intracellular cholesterol.

In summary, our disclosed four drugs, Nilotinib (AMN-107), Ramipril (Altace), ABT-737 and Evacetrapib (LY2484595), 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 (Fig. 8). 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.

Claims (10)

1. The application of the drug/compound targeting the SOAT1 protein in the preparation of the drug for preventing and/or treating liver cancer;

the drug/compound targeting the SOAT1 protein is selected from at least one of the following: ramipril (Altace), ABT-737 and Evactrapib (LY 2484595);

wherein, Ramipril (Altace), Cas No.: 87333-19-5, the structural formula is as follows:

ABT-737, Cas No.: 852808-04-9, the structural formula is shown as follows:

evacet rapib (LY2484595), CAS No.1186486-62-3, having the following structural formula:

2. the use of the drug/compound targeting SOAT1 protein according to claim 1 in the preparation of inhibitors of liver cancer cell proliferation.

3. Use according to claim 2, characterized in that: the liver cancer cell is selected from any one of the following: HepG2, PLC/PRF/5 and MHCC 97L.

4. Use according to claim 3, characterized in that:

the IC of Ramipril (Altace) for three liver cancer cells HepG2, PLC/PRF/5 and MHCC97L ₅₀ 96.8nM,25.1nM and 6.0. mu.M, respectively, in that order;

the IC of the ABT-737 to three hepatoma cells HepG2, PLC/PRF/5 and MHCC97L ₅₀ 184.0nM, 3.3. mu.M and 2.1. mu.M, respectively, in this order;

the IC of the Evacet rapib (LY2484595) on three liver cancer cells HepG2, PLC/PRF/5 and MHCC97L ₅₀ This was followed by 9.2. mu.M, 5.6. mu.M and 2.0. mu.M, respectively.

5. Use according to any one of claims 2-4, characterized in that: the drug/compound targeting the SOAT1 protein is combined with the SOAT1 protein and influences the intracellular cholesterol homeostasis, so that the liver cancer cell proliferation is inhibited.

6. A product characterized by: the active ingredient of the product is selected from at least one of Ramipril (Altace), ABT-737 and Evactrapib (LY 2484595).

7. The product of claim 6, wherein:

the product has at least one of the following effects:

1) for preventing and/or treating liver cancer;

2) inhibiting proliferation of hepatocarcinoma cell;

the product is a medicament or pharmaceutical formulation.

8. The use of the drug/compound targeting the SOAT1 protein of claim 1 in:

1) preventing and/or treating liver cancer;

2) inhibiting proliferation of hepatocarcinoma cell.

9. Use according to claim 8, characterized in that: the liver cancer cell is selected from any one of the following: HepG2, PLC/PRF/5 and MHCC 97L.

10. A method for preventing and/or treating liver cancer, comprising the steps of: administering a drug/compound targeting the SOAT1 protein according to claim 1 or a product according to claim 6 or 7 to a recipient animal or human for the prevention and/or treatment of liver cancer.

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