CN113456631B - A small molecule drug targeting ACSL1 and its application in the treatment of endometrial cancer - Google Patents

Abstract

The invention discloses a small molecule drug targeting ACSL1 and its application in the treatment of endometrial cancer. The invention discovers for the first time that the small molecule compound EC02 has a therapeutic effect on endometrial cancer, and the verification experiments in vivo and in vitro It is shown that the small molecule compound EC02 has a significant inhibitory effect on the growth of endometrial cancer cells, providing a new method and new idea for the clinical treatment of endometrial cancer, and has significant effects in the adjuvant therapy and prevention of endometrial cancer. Therefore, the small molecule compound EC02 has a very promising clinical application prospect.

Description

A small molecule drug targeting ACSL1 and its application in the treatment of endometrial cancer

technical field

The present invention belongs to the technical field of biomedicine, in particular, the present invention relates to a small molecule drug targeting ACSL1, more particularly, the present invention relates to a small molecule drug targeting ACSL1 and its use in the treatment of endometrial cancer Applications.

Background technique

Endometrial cancer (EC) is a group of epithelial malignant tumors that occur in the endometrium, among which adenocarcinoma is the most common one, and it is one of the three major malignant tumors of the female reproductive tract, accounting for 7% of all malignant tumors in women. %, accounting for 20%-30% of female reproductive tract malignant tumors. In European and American countries, the incidence of endometrial cancer ranks first in gynecological malignant tumors (Jemal A, Bray F, Center MM, et al.Global cancer statistics [J].CA:a cancerjournal for clinicians,2011,61(2):69-90.), in recent years, with the aging of the population, obesity and the widespread promotion of hormone replacement, the incidence of endometrial cancer is increasing year by year and the age of onset tends to be younger (Szwarc MM, Kommagani R, Putluri V, et al. Steroid Receptor Coactivator-2 Controls the Pentose Phosphate Pathway through RPIA in Human Endometrial Cancer Cells [J]. Sci Rep, 2018, 8 (1 ): 13134.); In my country, due to lifestyle and metabolic diseases, the incidence of endometrial cancer has also been on the rise in recent years, seriously threatening women's health and quality of life.

The etiology of endometrial cancer has not been clarified so far. There are two subtypes of endometrial cancer, which are divided into type I and type II according to different etiologies and prognosis, namely estrogen-dependent and estrogen-independent. Type I endometrial cancer is estrogen-dependent, and its occurrence may be due to the long-term lack of progesterone-antagonized estrogen that continues to act on the endometrium, resulting in proliferative changes and eventually cancerous changes. These patients are relatively Younger, but with better tumor differentiation and better prognosis, mainly endometrioid adenocarcinoma, accounting for 80%-90% of endometrial cancer, the representative cell line is Ishikawa; type II endometrial cancer is a non-female Hormone-dependent type, the pathogenesis is still unclear, and it may be related to gene variation. It mostly occurs in the postmenopausal and thin elderly people. The estrogen level is not high. High, poorly differentiated, poor prognosis, the common special pathological types are endometrial adenosquamous carcinoma, serous papillary carcinoma, clear cell carcinoma, mucinous adenocarcinoma, and the representative cell line is HEC-1-B.

Early-stage endometrial cancer can be significantly improved by surgical treatment, but patients with advanced stage, recurrence and metastasis, or patients with fertility requirements are still limited in treatment options. Improve patient outcomes. Although high-dose medroxyprogesterone acetate (MPA), a synthetic progestin-type western drug, has been approved for the treatment of type I endometrial cancer. However, up to 30% of patients with endometrial hyperplasia and endometrioid carcinoma are resistant to progesterone therapy (Chaudhry P, Asselin E. Resistance to chemotherapy and hormone therapy inendometrial cancer [J]. Endocrine Related Cancer, 2009, 16 (2): 363-380.), in addition, the recurrence rate after progesterone treatment is relatively high, therefore, there is an urgent need in this field to provide effective, safe, and patient-compliant small-molecule drugs for use in the endometrium Cancer treatment, thereby reducing the pain of patients and improving the quality of life of patients.

Based on the current state of the art, the inventors of the present application have experimentally studied the regulatory role of ACSL1 in endometrial cancer, and further screened 40 small molecule compounds by computer-aided design using ACSL1 as the target, and tested them in vivo and in vitro. Experiments have proved that the small molecule compound EC02 has a therapeutic effect on endometrial cancer. In addition, the inventors of the present application have also studied the molecular mechanism of this anti-tumor process. It inhibits the fatty acid beta oxidation and ATP generation of endometrial cancer cells, thereby significantly inhibiting the proliferation and migration of endometrial cancer cells, and achieving the effect of inhibiting the process of endometrial cancer, which has a very good clinical application prospect.

SUMMARY OF THE INVENTION

In view of this, in order to make up for the technical defects existing in the current state of the art, the purpose of the present invention is to provide a small molecule drug targeting ACSL1 and its application in the treatment of endometrial cancer. ACSL1 targets, and then inhibits fatty acid β-oxidation and ATP production in endometrial cancer cells, thereby significantly inhibiting the proliferation and migration of endometrial cancer cells, and inhibiting the process of endometrial cancer.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

A first aspect of the present invention provides the use of a small molecule drug targeting ACSL1 in the preparation of a drug for the treatment and/or prevention of endometrial cancer.

Further, the small molecule drug is EC02, and the structural formula of the small molecule drug is shown in formula (I):

The "small molecule compound EC02" described in the present invention is the same as "small molecule drug EC02" and "EC02", its molecular formula is C ₂₄ H ₂₁ NO ₃ , the structural formula is shown in formula (I), the relative molecular weight is 371.4284, and the name is 3-hydroxy-3-(2-oxo-2-phenylethyl)-1-(2-phenylethyl)-1,3-dihydro-2H-indol-2-one, in a specific embodiment of the present invention, the The small-molecule compound EC02 or the small-molecule drug EC02 or EC02 corresponds to drug2, and is one of the small-molecule compounds screened by the inventors of the present application using ACSL1 as the target through computer-aided design.

Further, the small molecule drug inhibits the proliferation of endometrial cancer cells, inhibits the migration of endometrial cancer cells, inhibits the invasion of endometrial cancer cells, and promotes the apoptosis of endometrial cancer cells.

Further, the use concentration of the small molecule drug is 1 μM-500 μM;

Preferably, the use concentration of the small molecule drug is 1 μM-100 μM.

In a specific embodiment of the present invention, the use concentration of the small molecule drug is preferably 1 μM-10 μM, and when the use concentration of the small molecule drug is 1 μM, it has already exerted a significant inhibitory effect on the proliferation and migration of endometrial cancer cells. Therefore, it should be clear to those skilled in the art that the use concentration of the small molecule drugs described in the present invention is not limited to 1 μM-10 μM.

Further, the way that the small molecule drug exerts the above effect is that the small molecule drug inhibits the ACSL1 target, thereby inhibiting the fatty acid beta oxidation and ATP generation of endometrial cancer cells, thereby significantly inhibiting the proliferation and migration of endometrial cancer cells. , invasion, and promote the apoptosis of endometrial cancer cells.

Further, the medicine is composed of a therapeutically effective amount of the small molecule medicine shown in formula (I) and a pharmaceutically acceptable carrier and/or adjuvant;

Preferably, the pharmaceutically acceptable carriers and/or adjuvants include diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrating agents;

Preferably, the use concentration of the small molecule drug is 1 μM-500 μM;

More preferably, the use concentration of the small molecule drug is 1 μM-100 μM.

Further, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water, and the like.

Further, the binder includes (but is not limited to) starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, Polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid, alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc.

Further, the surfactant includes (but is not limited to) polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, cetyl alcohol and the like.

Further, the humectant includes (but is not limited to) glycerin, starch and the like.

Further, the adsorption carrier includes (but is not limited to) starch, lactose, bentonite, silica gel, kaolin, bentonite and the like.

Further, the lubricants include, but are not limited to, zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oils, Sodium stearyl fumarate, polyoxyethylene monostearate, monolaurin sucrose, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.

Further, the fillers include (but are not limited to) mannitol (granulated or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupled sugar, glucose, lactose, sucrose , dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

Further, the disintegrant includes (but is not limited to) cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide and the like.

The second aspect of the present invention provides the application of a small molecule drug targeting ACSL1 in the preparation of a reagent.

Further, the small molecule drug is EC02, and the structural formula of the small molecule drug is shown in formula (I):

Further, the reagent is used in any one or more of the following aspects:

(1) Inhibit the proliferation of endometrial cancer cells in vitro;

(2) Inhibit the migration of endometrial cancer cells in vitro;

(3) Inhibit the invasion of endometrial cancer cells in vitro;

(4) Promote the apoptosis of endometrial cancer cells in vitro.

Further, the endometrial cancer cells include Ishikawa, HEC-1-B, RL95-2, HEC-1-A, MFE-280, MFE-296, SNG-M, HECCL-1, KLE, ECC-1, ECC-10, ECC-12, HEC-151, HEC-251, SK-UT-1;

Preferably, the endometrial cancer cells are Ishikawa.

A third aspect of the present invention provides a pharmaceutical composition for preventing and/or treating endometrial cancer.

Further, the pharmaceutical composition comprises the small molecule drug as described in the first aspect of the present invention;

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.

Further, the pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's PharmaceuticalSciences (19th ed., 1995), and these substances are used to help the stability of the formulation or to improve the activity or its effects as needed. Bioavailability or producing an acceptable mouthfeel or odor in the case of oral administration, the formulations that can be used in such pharmaceutical compositions can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. Form, the pharmaceutical composition so formulated can be administered in any suitable manner known to those skilled in the art as desired.

Further, the suitable dosage of the pharmaceutical composition depends on the formulation method, the administration method, the patient's age, body weight, sex, morbidity, diet, administration time, administration route, excretion rate, response sensitivity, etc. A variety of prescriptions can be made depending on the factors, and the skilled physician can usually easily determine the prescription and the dosage that will be effective for the desired treatment or prophylaxis.

Further, conventional cosolvents, buffers, pH adjusters, etc. can also be added to the pharmaceutical composition, and if necessary, other materials can also be added to the pharmaceutical composition preparation, and the above-mentioned pharmaceutical composition can be made into a variety of dosage forms, Including (but not limited to): tablets, subcutaneous implants, vaginal or intrauterine formulations, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, lipids Plasmids, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc., the pharmaceutical compositions of the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field, and the above-mentioned dosage forms can be administered by injection, and the described Injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection, etc.; cavity administration, such as intrauterine cavity and vaginal administration; respiratory tract administration, such as nasal cavity administration; mucosal administration.

A fourth aspect of the present invention provides a method of screening drug candidates for the treatment and/or prevention of endometrial cancer.

Further, the method includes the following steps:

(1) Taking ACSL1 as the drug target, screening small molecule compounds by computer-aided design;

(2) Carry out a verification experiment on the small molecule compounds screened in step (1), and screen again to inhibit the proliferation and/or migration and/or invasion of endometrial cancer cells, and/or promote the apoptosis of endometrial cancer cells, And/or small molecule compounds that inhibit the growth of endometrial cancer and/or reduce the volume of endometrial cancer are drug candidates;

Preferably, the verification experiments include cell proliferation experiments, cell migration experiments, cell invasion experiments, cell apoptosis experiments, and xenograft tumor animal model experiments.

Further, the three-dimensional crystal structure of human ACSL1 has not yet been reported in the prior art. In the specific embodiment of the present invention, the SWISSMODEL online homology modeling software is used to model the three-dimensional crystal structure of human ACSL1, and the selected template protein is Oxidoreductase (PDB No. 6oz1);

Preferably, based on the template protein Oxidoreductase, use the Surflex molecular docking module of the Sybyl-X2.1 drug design platform to perform virtual screening, select the Chemdiv small molecule database (up to 1.5 million compounds), and use the "Compoud Filtering" module. Compounds are screened in the first round of the "Five Rules for Similar Drugs";

Preferably, the catalytic domain in the three-dimensional structure of ACSL1 is selected as the inhibitor-binding cavity in the virtual screening, and some parameters are modified to speed up the second round of virtual screening;

More preferably, the modified content of the parameters of the modification part is as follows: reduce "Max conformations perFragment" from the default 20 to 10; reduce "Max number of rotatable bonds per molecule" from the default 100 to 50; cancel the default The options of "Per-Dock Minimization" and "Post-Dock Minimization" reduce the "Maximum number of poses per ligand" from the default 20 to 3, that is, only the top 3 molecular conformations of each ligand molecule are retained to speed up docking speed;

Most preferably, small molecule compounds with molecular scores in the top 1% (ie, 15,000) are screened; based on the results of the second round of screening, the Surflex molecular docking parameters are restored to the default values; the top 300 target compounds are selected for cluster analysis and analysis. After manual selection, 40 target compounds were finally confirmed and confirmed by subsequent experiments.

Further, the candidate drug for the treatment and/or prevention of endometrial cancer screened in step (2) is EC02, and the structural formula of the small molecule drug is shown in formula (I):

Compared with the prior art, the present invention has the advantages and beneficial effects:

(1) The present invention finds for the first time that the small molecule compound EC02 has a therapeutic effect on endometrial cancer, and the verification experiments in vivo and in vitro show that the small molecule compound EC02 has a significant inhibitory effect on the growth of endometrial cancer cells;

(2) The present invention studies the molecular mechanism of the small molecule compound EC02 in the anti-tumor process. The research results show for the first time that EC02 inhibits the β-oxidation of fatty acids and the generation of ATP in endometrial cancer cells by inhibiting the ACSL1 target, thereby significantly inhibiting the production of ATP. Proliferation and migration of endometrial cancer cells to inhibit the process of endometrial cancer;

(3) The use of the small molecule compound EC02 targeting ACSL1 provided by the present invention in the preparation of a drug for preventing and/or treating endometrial cancer is disclosed for the first time, and provides a new method and new idea for the clinical treatment of endometrial cancer, At the same time, it has significant significance in the adjuvant therapy and prevention of endometrial cancer. Therefore, the small molecule compound EC02 provided by the present invention has a very good clinical application prospect.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows the statistical chart of the proliferation of Ishikawa cells after ACSL1 knockdown and overexpression, **P<0.01, compared with SI-NC group, ##P<0.01, compared with OE-NC group;

Figure 2 shows the results of the migration of Ishikawa cells after ACSL1 knockdown and overexpression;

Figure 3 shows the result statistics of the migration of Ishikawa cells after ACSL1 knockdown and overexpression, **P<0.01, compared with SI-NC group, ##P<0.01, compared with OE-NC group;

Figure 4 shows a statistical graph of the results of the relative expression levels of ACSL1 mRNA in ACSL1 knockdown and overexpressed Ishikawa cells;

Figure 5 shows representative images of tumor tissue from mice with ACSL1 knockdown and overexpression;

Figure 6 shows the results of the growth curve of mouse tumors with ACSL1 knockdown and overexpression;

Figure 7 shows the results of observing the appearance and structure of uterine tissue by hematoxylin and eosin staining, wherein, picture A: SI-NC, picture B: SI, picture C: OE-NC, picture D: OE;

Figure 8 shows the statistical chart of the results of the effects of 40 compounds on the proliferation of endometrial cancer cells, wherein, Panel A: Compounds 1-10, Panel B: Compounds 11-20, Panel C: Compounds 21-30, Panel D: Compound 31 -40, n=3, *P<0.05, **P<0.01, ***P<0.001, compared with the Control group;

Figure 9 shows the results of the proliferation of Ishikawa cells treated with low concentration (1 μM), medium concentration (5 μM) and high concentration (10 μM) of EC02 for 48 h, n=3, ^** P<0.01, compared with the N group ;

Figure 10 shows the results of migration of Ishikawa cells treated with low concentration (1 μM), medium concentration (5 μM) and high concentration (10 μM) of EC02 for 48 h;

Figure 11 shows the results of the migration of Ishikawa cells treated with low concentration (1 μM), medium concentration (5 μM) and high concentration (10 μM) of EC02 for 48 h, n=3, ^** P<0.01, compared with the N group ;

Figure 12 shows the results of β-oxidation of fatty acids in Ishikawa cells treated with low concentration (1 μM), medium concentration (5 μM) and high concentration (10 μM) of EC02 for 48 h, n=3, ^* P<0.05, ^** P< 0.01, compared with N group;

Figure 13 shows the results of ATP production in Ishikawa cells treated with low concentration (1 μM), medium concentration (5 μM) and high concentration (10 μM) of EC02 for 48 h, n=3, ^** P<0.01, compared with the N group Compare;

Figure 14 shows a schematic diagram of the structural formula of the small molecule drug EC02 in the present invention.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention, and should not be construed as a limitation of the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents . In the following examples, the experimental methods without specific conditions are usually detected according to conventional conditions or according to the conditions suggested by the manufacturer.

Example 1 The regulatory effect of ACSL1 on the proliferation and migration of endometrial cancer cells

In order to study the effect of ACSL1 on the proliferation and migration ability of endometrial cancer cells, CCK8 cell proliferation experiments and cell scratch experiments were carried out in this example.

1. Cell culture and processing

The human endometrial cancer cell line Ishikawa was provided by the cell bank of the Chinese Academy of Sciences (Shanghai, China); Ishikawa cells were cultured in RPMI-10% fetal bovine serum (Clark, USA), 1% penicillin, 1% streptomycin 1640 medium, and cultured in a constant temperature incubator at 5% CO ₂ and 37°C.

2. Establishment of Ishikawa cell ACSL1 knockdown and overexpression cell model

The ACSL1 lentivirus used in this example was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.;

When the Ishikawa cells grow to a density of about 50%, the ACSL1 lentiviral plasmid is used for infection. The titer of 1×10 ⁸ TU/mL virus is used, and the amount of virus is added according to MOI=50. After 12h of infection in the complete medium, it is replaced with Fresh medium, continue to culture for 1-2 days, change the medium, observe the fluorescence intensity under a fluorescence microscope for 3-4 days after infection, and detect the knockdown efficiency by PCR and Western blot. The cells infected with lentivirus can be screened by puromycin to obtain Ishikawa cells ACSL1 Gene knockdown and overexpression cell models, in which the sequence information of the ACSL1 knockdown group is shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the expression of ACSL1 in the cells of the SI-NC, SI, and OE-NC groups was detected by QPCR Level;

ACSL1-RNAi(83133-21)-a: 5'-CCGGCGCAGATAGATGACCTCTATTCTCGAGAATAGAGGTCATCTATCTGCGTTTTTG-3' (SEQ ID NO: 1);

ACSL1-RNAi(83133-21)-b: 5&apos;-AATTCAAAAACGCAGATAGATGACCTCTATTCTCGAGAATAGAGGTCATCTATCTGCG-3&apos; (SEQ ID NO: 2).

3. Real-time fluorescent quantitative PCR

(1) according to the reaction system described in table 1, add the reactant of corresponding amount in the 96-well plate, seal film, centrifuge (4000g, 2min), described reaction primer is designed and synthesized by Shanghai Shenggong Bioengineering Co., Ltd., Taking the mRNA expression level of β-actin as a reference, the primer sequences of ACSL1 are as shown in SEQ ID NO:3 and SEQ ID NO:4, and the primer sequences of β-actin are as shown in SEQ ID NO:5 and SEQ ID NO:6. Show;

ACSL1 upstream primer: 5'-ATGCCAGAGCTGATTGACATT-3' (SEQ ID NO:3);

ACSL1 downstream primer: 5'-CAAGGACTGCTGATCTTCGG-3' (SEQ ID NO:4);

β-actin upstream primer: 5'-TCCACCACCAGGCAGAAGAC-3' (SEQ ID NO:5);

β-actin downstream primer: 5'-TTTAATGTCACGCACGATTTC-3' (SEQ ID NO: 6).

Table 1 qRT-PCR reaction system (10μL)

(2) Put the 96-well plate into the LC480 real-time fluorescence quantitative PCR instrument, and set the reaction program according to the following reaction conditions:

Activation: 95℃, 30s;

PCR: 95℃, 5s; 60℃, 30s; 40cycles, Quantification;

Melting curves: 95℃, 5s; 60℃, 60s; 95℃, Continuous;

Cooling: 50℃, 30s.

(3) After the reaction is completed, use Roche LightCycle 480 software to calculate the Cp value of each target gene in the sample (Cp value reflects the level of gene expression level, the larger the Cp value, the lower the gene expression level); export the data, through ΔΔCP The relative mRNA expression levels of each target gene were calculated by the method.

4. CCK8 cell proliferation assay

The Ishikawa cells of the OE group, OE-NC group, SI group, and SI-NC group, respectively, were tested for ACSL1 overexpression or knockout by Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) according to the manufacturer's instructions. Effects on proliferation of endometrial cancer cells Ishikawa.

5. Cell scratch test

The Ishikawa cells of the OE group, the OE-NC group, the SI group, and the SI-NC group were seeded in 6-well plates. Draw 3 lines in parallel on the cells. Cells remaining in the plate were washed twice with warm 1×PBS to remove cell debris. Take this time point as 0h to take a picture. The 6-well plate was put back into the incubator, and after culturing for 24 hours, the 6-well plate was taken out and observed and photographed under a microscope, and the intercellular area of each group of cells was measured by Imaje J software, and the cell migration rate was calculated;

The calculation formula of cell mobility is as follows: cell mobility=(0h intercellular area-24h intercellular area)/0h intercellular area.

6. Experimental results

The results showed that in ACSL1-overexpressing cells, both proliferation and migration were significantly increased compared with their control group, while in ACSL1-knockdown cells, their proliferation and migration were significantly decreased compared with their control group (see Figure 1-3). In ACSL1-overexpressing cells, the relative expression level of ACSL1 mRNA was significantly increased compared with the control group; while in ACSL1-knockdown cells, the relative expression level of ACSL1 mRNA was significantly decreased compared with the control group (see Figure 4). , indicating the successful establishment of a cell model of ACSL1 knockdown and overexpression.

Example 2 Study on the correlation between ACSL1 and the occurrence and development of endometrial cancer

In order to study the relationship between ACSL1 and the malignant characteristics of endometrial cancer, ACSL1 knockout and overexpression xenograft tumor model mice were established in this example. The experimental animals used in the study in this example were purchased from Shanghai Weitong Lihua Experimental Animal Co., Ltd. is a 4-5 week old female nude mouse (SPF grade) with a body weight of 10-12 g.

1. Establishment of a nude mouse model of endometrial cancer

Nude mice were divided into 4 groups in total, namely: ACSL1 knockdown group (SI group), the mice in this group were inoculated with ACSL1 knockdown Ishikawa cell line; ACSL1 knockdown negative control group (SI-NC group), this group was small. The mice were inoculated with the Ishikawa cell line with the negative control of ACSL1 knockdown; the ACSL1 overexpression group (OE group), the mice in this group were inoculated with the Ishikawa cell line with the overexpression of ACSL1; the ACSL1 overexpression negative control group (OE-NC group), the mice in this group were small. Mice were inoculated with the Ishikawa cell line that overexpressed the negative control of ACSL1;

Establishment of endometrial cancer nude mouse model: ACSL1 knockdown Ishikawa cell line, ACSL1 knockdown negative control Ishikawa cell line, ACSL1 overexpression Ishikawa cell line, and ACSL1 overexpression negative control Ishikawa cell line were inoculated in nude mice. A nude mouse model of endometrial carcinoma was constructed subcutaneously in the right armpit of mice. Cells were injected in a volume of 0.2 mL per mouse, with 3 x 10 ⁵ cells per mouse. After the inoculation of cells is completed, every 4 days, measure the size of the tumor tissue with a ruler, record the long diameter (a) and short diameter (b) of the tumor tissue, and record the time continuously for 4 weeks according to this method. . After 4 weeks, the nude mice were sacrificed, the tumor tissue was taken, the volume of the tumor tissue was calculated, and the tumor growth curve was drawn according to the time and tumor volume;

The formula for calculating tumor volume is as follows: tumor volume=a×b ² /2;

2. HE staining of tumor tissue in nude mice

Hematoxylin-Eosin (HE) staining was performed on the tumor tissues of nude mice in SI-NC group, SI group, OE-NC group and OE group. The experimental steps are performed.

3. Experimental results

The established ACSL1 knockout and overexpression xenograft tumor model mice are shown in Figure 5. The results show that ACSL1 significantly increases tumor size and promotes tumor growth, while knockdown of ACSL1 can significantly inhibit tumor growth and reduce tumor volume. The growth curve is shown in Figure 6, and the specific tumor information is shown in Table 2. The results further show that the overexpression of ACSL1 significantly promotes the growth of the tumor, and the knockdown of ACSL1 can significantly inhibit the growth of the tumor. The staining results of the tissue showed that, compared with other groups, the arrangement of the OE group had a certain degree of disorder, the nuclei were enlarged, the staining was deep, the pathological mitosis was clearly visible, and the cell atypia was also obvious (see Figure 7A-D).

Table 2 Tumor information of each experimental group and control group

grouping Tumor volume (mm³) Tumor weight (g) Mouse weight (g) SI-NC 1209.58±266.74 0.72±0.08 18.65±0.71 SI 546.83±104.35 0.4±0.06 19.32±0.87 OE-NC 1221±434.32 0.72±0.13 19.28±1.05 OE 2500.75±569.57 1.45±0.08 19±1.33

Example 3 Screening of compounds with inhibitory effect on endometrial cancer cells using ACSL1 as a target

In this example, according to the ACSL1 target, 40 compounds that can theoretically inhibit the activity of ACSL1 were designed by computer aided, and the effect of the 40 compounds on the proliferation of endometrial cancer cells was further detected by CCK8 cell proliferation assay.

1. Virtual screening based on the structure of human ACSL1 protein

The three-dimensional crystal structure of human ACSL1 has not been reported yet. The SWISSMODEL online homology modeling software was used to model the three-dimensional crystal structure of human ACSL1. The selected template protein was Oxidoreductase (PDB No. 6oz1); Sybyl-X2 .1 The Surflex molecular docking module of the drug design platform conducts virtual screening, selects the Chemdiv small molecule database (up to 1.5 million compounds), and uses the "Compoud Filtering" module to conduct the first round of the "Five Rules of Drug-like" for the compounds in Chemdiv Screening; then select the catalytic domain in the three-dimensional structure of ACSL1 as the inhibitor-binding cavity in the virtual screening, modify some parameters to speed up the second round of virtual screening; specifically, reduce the "Maxconformations per Fragment" from the default 20. To 10, reduce the "Max number of rotatable bondsper molecule" from the default of 100 to 50. Cancel the default "Per-Dock Minimization" and "Post-DockMinimization" options; reduce the "Maximum number of poses per ligand" from the default 20 to 3, that is, only keep the top 3 molecular conformations of each ligand molecule, Speed up the docking speed; screen out the small molecule compounds with molecular scores in the top 1% (ie 15,000); finally, based on the results of the second round of screening, restore the Surflex molecular docking parameters to the default values; select the top 300 target compounds for clustering Through analysis and manual selection, 40 target compounds were finally confirmed for commercial purchase, and follow-up experimental confirmation was carried out.

2. CCK8 cell proliferation assay

After treatment of endometrial cancer cells Ishikawa with different reagents (drug1-drug40 at 10 μM) for 48 h, Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was used to detect the effect of 40 compounds on the uterus according to the manufacturer's instructions. Influence on the proliferation of endometrial cancer cells;

Among them, the control group was the negative control group; the drug1-drug40 group was the 40 experimental groups obtained by treating endometrial cancer cells with 40 compounds at a concentration of 10 μM.

3. Experimental results

_The results showed that among the 40 compounds, only _EC02 ( _drug2 ) had the most obvious inhibitory effect on endometrial cancer cells (see Figure 8A-D). The structural formula is shown in Figure 14, and the name of the compound is 3-hydroxy-3-(2-oxo-2-phenylethyl)-1-(2-phenylethyl)-1,3-dihydro-2H-indol-2-one.

Example 4 EC02 inhibits the energy metabolism of endometrial cancer cells

In order to evaluate the effect of EC02 on the proliferation of endometrial cancer cells, in this example, EC02 and TriacsinC were used to treat endometrial cancer cells for 48 h, respectively, and a CCK8 cell proliferation experiment was performed. In addition, this example also used a cell scratch test to detect EC02 Effects on the ability of endometrial cancer to migrate;

Endometrial cancer cells Ishikawa were treated with different concentrations of EC02 for 48h, the concentrations were: high concentration of 10 μM, medium concentration of 5 μM and low concentration of 1 μM;

Among them, N group: negative control group; 1 μM group: cells were treated with EC02 concentration of 1 μM; 5 μM group: cells were treated with EC02 concentration of 5 μM; 10 μM group: cells were treated with EC02 concentration of 10 μM.

1. CCK8 cell proliferation assay

After different groups of endometrial cancer cells Ishikawa were treated with different reagents for 48h, Cell Counting Kit-8 (CCK-8) (Japan, Dojindo) was used to detect the proliferation of endometrial cancer cells according to the manufacturer's instructions. impact of the situation.

2. Cell scratch test

Endometrial cancer cells of different treatment groups were seeded in 6-well plates. After the cells adhered, the cells grew to 100% confluence. Three lines were drawn in parallel on the adherent cells with a 200 μL pipette tip. Cells remaining in the plate were washed twice with warm 1×PBS to remove cell debris. Take this time point as 0h to take a picture. Put the 6-well plate back into the incubator, take out the 6-well plate after 24 hours, observe and take pictures under the microscope, measure the area between cells with Imaje J software, and calculate the cell migration rate;

The calculation formula of cell mobility is as follows: cell mobility=(0h intercellular area-24h intercellular area)/0h intercellular area.

3. Experimental results

The results of the CCK8 cell proliferation experiment showed that the proliferation of endometrial cancer cells was significantly inhibited by EC02 treatment at a concentration of 1 μM, and the inhibitory effect was gradually enhanced with the increasing concentration of EC02 (see Figure 9);

The results of cell scratch experiments showed that EC02 could significantly inhibit the migration of endometrial cancer cells, and this inhibitory ability gradually increased with the increase of EC02 concentration (see Figure 10 and Figure 11 );

All the above results indicated that EC02 had a significant inhibitory effect on endometrial cancer cells and had good clinical application value.

Example 5 EC02 inhibits lipid metabolism of endometrial cancer cells

In order to further study the effect of EC02 on lipid metabolism of endometrial cancer cells, MDA experiment was carried out in this example. In addition, ATP Lite kit was used in this example to detect the production of ATP in endometrial cancer cells.

Endometrial cancer cells Ishikawa were treated with different concentrations of EC02 for 48h, the concentrations were: high concentration of 10 μM, medium concentration of 5 μM and low concentration of 1 μM;

Among them, N group: negative control group; 1 μM group: cells were treated with EC02 concentration of 1 μM; 5 μM group: cells were treated with EC02 concentration of 5 μM; 10 μM group: cells were treated with EC02 concentration of 10 μM.

1. Lipid oxidation (MDA) experiment

Cells in each group were lysed, and the supernatant was collected after centrifugation at 12,000 g. After adding the malondialdehyde detection working solution, mix well, heat in a boiling bath for 15 min, cool to room temperature in a water bath, and centrifuge at 1000 g for 10 min at room temperature. Take 200 μL of the supernatant, add it to a 96-well plate, and measure the absorbance at 532 nm with a microplate reader. The level of MDA production was analyzed by a lipid oxidation (MDA) assay kit (Beyotine, China) according to the manufacturer's instructions.

2. ATP generation detection experiment

Cells of each group were seeded in a 96-well plate, and 50 μL of mammalian cell lysis solution was added to each well, and the plate was shaken in a shaker at 700 rpm for 5 min to lyse cells and stabilize ATP. Add 50 μL of matrix solution to the well, and shake the well plate for 5 min. Protect from light for 10 min, and measure the chemiluminescence value. ATP production was analyzed by ATP-LITE assay kit (PerkinElmer, USA) according to the manufacturer's instructions.

3. Experimental results

The results showed that EC02 could significantly inhibit the β-oxidation of fatty acids in endometrial cancer cells in a concentration-dependent manner (see Figure 12), and EC02 could significantly inhibit the production of ATP in endometrial cancer cells in a concentration-dependent manner (see Figure 12). Figure 13), the above results show that EC02 can inhibit the β-oxidation of fatty acid and the generation of ATP in endometrial cancer cells by inhibiting the target of ACSL1, thereby significantly inhibiting the proliferation and migration of endometrial cancer cells, and inhibiting intrauterine cancer cells. The role of the process of endometrial cancer, EC02 (see Figure 14 for the structural formula) is expected to become a new compound for the treatment of endometrial cancer, and has a very good clinical application prospect.

The description of the above embodiment is only for understanding the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

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Claims (13)

1. the application of the small molecule medicine of targeting ACSL1 in the preparation of the medicine for the treatment and/or prevention of endometrial cancer, it is characterized in that, described small molecule medicine is EC02, and the structural formula of described small molecule medicine is such as formula ( I) shows:

Formula (I). 2 . The use according to claim 1 , wherein the small molecule drug inhibits the proliferation of endometrial cancer cells and the migration of endometrial cancer cells. 3 . 3. The application according to claim 1, wherein the use concentration of the small molecule drug is 1 μM-500 μM. 4. The application according to claim 3, wherein the use concentration of the small molecule drug is 1 μM-100 μM. 5. The application according to claim 1, wherein the medicine is composed of a therapeutically effective amount of the small molecule medicine shown in formula (I) and a pharmaceutically acceptable carrier and/or adjuvant. 6. application according to claim 5 is characterized in that, described pharmaceutically acceptable carrier and/or adjuvant comprises diluent, adhesive, surfactant, wetting agent, adsorption carrier, lubricant and/or or disintegrant. 7. The application according to claim 5, wherein the use concentration of the small molecule drug is 1 μM-500 μM. 8. The application according to claim 7, wherein the use concentration of the small molecule drug is 1 μM-100 μM. 9. the application of the small-molecule drug targeting ACSL1 in the preparation reagent, it is characterized in that, described small-molecule drug is EC02, and the structural formula of described small-molecule drug is as shown in formula (I):

Formula (I); The agent is used in any one or more of the following aspects: (1) Inhibit the proliferation of endometrial cancer cells in vitro; (2) Inhibit the migration of endometrial cancer cells in vitro. 10. The application according to claim 9, wherein the endometrial cancer cells comprise Ishikawa, HEC-1-B, RL95-2, HEC-1-A, MFE-280, MFE-296, SNG -M, HECCL-1, KLE, ECC-1, ECC-10, ECC-12, HEC-151, HEC-251 or SK-UT-1. 11. The use according to claim 10, wherein the endometrial cancer cells are Ishikawa. 12. A method for screening a candidate drug for the treatment and/or prevention of endometrial cancer, characterized in that the method comprises the steps of: (1) Taking ACSL1 as the drug target, screening small molecule compounds by computer-aided design; (2) Carry out a verification experiment on the small molecule compounds screened in step (1), and screen again to inhibit the proliferation and/or migration and/or invasion of endometrial cancer cells, and/or promote the apoptosis of endometrial cancer cells, and/or small molecule compounds that inhibit the growth of endometrial cancer and/or reduce the volume of endometrial cancer are candidate drugs; the candidate drugs screened in step (2) for treating and/or preventing endometrial cancer Be EC02, the structural formula of described EC02 is shown in formula (I):

Formula (I). 13 . The method according to claim 12 , wherein the verification experiments include cell proliferation experiments, cell migration experiments, cell invasion experiments, cell apoptosis experiments or xenograft tumor animal model experiments. 14 .

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