CN113456631B

CN113456631B - Small molecule drug targeting ACSL1 and application thereof in treatment of endometrial cancer

Info

Publication number: CN113456631B
Application number: CN202110902537.7A
Authority: CN
Inventors: 周雪妍; 郭晓莉; 高剑; 印晓星; 张蓓; 徐吟雪
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-08-06
Filing date: 2021-08-06
Publication date: 2022-06-21
Anticipated expiration: 2041-08-06
Also published as: CN113456631A

Abstract

The invention discloses an ACSL 1-targeted small molecule drug and application thereof in treatment of endometrial cancer, and the invention discovers that a small molecule compound EC02 has a treatment effect on the endometrial cancer for the first time, and in vivo and in vitro verification experiments show that the small molecule compound EC02 has a remarkable inhibition effect on the growth of endometrial cancer cells, so that a novel method and a novel thought are provided for clinical treatment of the endometrial cancer, and meanwhile, the small molecule compound EC02 has a remarkable significance in adjuvant treatment and prevention of the endometrial cancer, and therefore, the small molecule compound EC02 has a very good clinical application prospect.

Description

Small molecule drug targeting ACSL1 and application thereof in treatment of endometrial cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an ACSL 1-targeted small molecule medicine, more particularly to an ACSL 1-targeted small molecule medicine and application thereof in treatment of endometrial cancer.

Background

Endometrial Cancer (EC), which is one of the three most common malignancies of the female reproductive tract, accounts for 7% and 20% -30% of all malignancies in women, occurs on the endometrium, with adenocarcinoma being the most common, and accounts for the first of the gynecological malignancies in European and American countries (Jemal A, Bray F, Center MM, et al. Global Cancer statistics [ J ]. CA: a Cancer journer Cancer for clinics, 2011,61(2):69-90.), and in recent years with the widespread popularization of aging, obesity and hormone replacement, the incidence of Endometrial Cancer has a tendency to increase year by year and the incidence of renal Cancer tends to age (Szwarc M, Kommagi R, Putli V, et al. Steward Cancer Cooperation surgery-2 control [ RPJ. ], 2018,8(1): 13134.); in China, due to life style and metabolic diseases and other reasons, the incidence rate of endometrial cancer is also on a rising trend in recent years, and the health and the life quality of women are seriously threatened.

The etiology of endometrial cancer has not been clarified to date, and endometrial cancer has two major subtypes, which are classified into types I and II, estrogen-dependent and non-estrogen-dependent, depending on the etiology and prognosis. Endometrial cancer type I is estrogen-dependent and may occur because estrogen which lacks progestogen antagonism for a long time continuously acts on the endometrium, so that the endometrium is subjected to proliferative changes, and finally the endometrium is subjected to canceration, and the patients are relatively younger, but the tumor differentiation is better, the prognosis is better, mainly endometrioid adenocarcinoma accounts for 80-90 percent of the endometrial cancer, and the representative cell strain is Ishikawa; the II type endometrial cancer is non-estrogen dependent type, the pathogenesis is still unclear, a certain relation possibly exists with gene variation, the II type endometrial cancer mostly occurs in the old with a thin body after menopause, the estrogen level is not high, the endometrium distributed and shrunk around a cancer focus is high in tumor malignancy degree, poor differentiation and poor prognosis, common special pathological types are endometrial adenosquamous carcinoma, serous papillary carcinoma, clear cell carcinoma and mucinous adenocarcinoma, and the representative cell strain is HEC-1-B.

Early stage endometrial cancer can be obviously improved by operation treatment, but patients with late stage, recurrent metastasis or fertility requirements are limited in treatment options, and even the combined chemotherapy and/or hormone treatment cannot obviously improve the prognosis of the patients. Although large doses of medroxyprogesterone acetate (MPA), an artificially synthesized western progestogen, have 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 progestogen therapy (Chaudhry P, Asselin E.resistance to chemotherapy and hormone therapy in endometeric Cancer [ J ]. Endocrine Related Cancer,2009,16(2):363-380.), and in addition, the recurrence rate after progestogen therapy is high, so that there is an urgent need in the art to provide effective, safe and patient-compliant small molecule drugs for endometrial Cancer therapy to alleviate the pain of patients and improve the quality of life of patients.

Based on the current situation of the prior art, the inventor of the application researches the regulation and control effect of ACSL1 in endometrial cancer through experiments, further screens 40 small molecular compounds by taking ACSL1 as a target through computer-aided design, and proves that the small molecular compound EC02 has a treatment effect on the endometrial cancer through in vivo and in vitro experiments.

Disclosure of Invention

In view of this, in order to make up for the technical defects existing in the prior art, the present invention aims to provide a small molecule drug targeting ACSL1 and an application thereof in the treatment of endometrial cancer, wherein the small molecule drug inhibits the ACSL1 target, so as to inhibit the fatty acid β oxidation and ATP production of endometrial cancer cells, thereby significantly inhibiting the proliferation and migration of the endometrial cancer cells, and achieving the effect of inhibiting the progression of endometrial cancer.

The above object of the present invention is achieved by the following technical solutions:

the invention provides application of a small molecule drug targeting ACSL1 in preparation of a drug for treating and/or preventing endometrial cancer.

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

"Small molecule Compound E" according to the present inventionC02, similar to small molecule drugs EC02 and EC02, and has molecular formula of C₂₄H₂₁NO₃The structural formula is shown as a 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, and in the specific embodiment of the invention, the small molecular compound EC02 or the small molecular drug EC02 or EC02 corresponds to drug2, so that the small molecular compound is one of small molecular compounds screened by computer-aided design by taking ACSL1 as a target point.

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

Further, the small molecule drug is used at a concentration of 1 μ M to 500 μ M;

preferably, the small molecule drug is used at a concentration of 1 μ M to 100 μ M.

In a specific embodiment of the present invention, the concentration of the small molecule drug is preferably 1 μ M to 10 μ M, and the small molecule drug has a significant ability to inhibit proliferation and migration of endometrial cancer cells when the concentration of the small molecule drug is 1 μ M, so it should be clear to those skilled in the art that the concentration of the small molecule drug used in the present invention is not limited to 1 μ M to 10 μ M.

Furthermore, the small molecule drug can inhibit fatty acid beta oxidation and ATP generation of the endometrial cancer cells by inhibiting the ACSL1 target, so that proliferation, migration and invasion of the endometrial cancer cells are obviously inhibited, and apoptosis of the endometrial cancer cells is promoted.

Further, the medicine consists of a small molecular medicine with a therapeutically effective amount shown in the formula (I) and a pharmaceutically acceptable carrier and/or auxiliary material;

preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant;

preferably, the small molecule drug is used at a concentration of 1 μ M to 500 μ M;

more preferably, the small molecule drug is used at a concentration of 1 μ M to 100 μ M.

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, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid, alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.

Further, the surfactant includes, but is not limited to, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, and the like.

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

Further, the adsorption carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, bentonite, and the like.

Further, the lubricant includes, but is not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, and the like.

Further, the filler includes, but is not limited to, mannitol (granular or powder), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, and the like.

Further, the disintegrant includes, but is not limited to, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, croscarmellose sodium, soybean polysaccharide, and the like.

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

further, the agent is used in any one or more of the following aspects:

(1) inhibiting proliferation of endometrial cancer cells in vitro;

(2) inhibiting migration of endometrial cancer cells in vitro;

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

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

Further, 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, SK-UT-1;

preferably, the endometrial cancer cells are Ishikawa.

In a third aspect of the invention, there is provided a pharmaceutical composition for use in the prevention and/or treatment of endometrial cancer.

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

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

Further, said pharmaceutically acceptable carriers and/or adjuvants are well described in Remington's Pharmaceutical Sciences (19th ed.,1995) as needed to aid in the stability of the formulation or to aid in the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, and the formulations which may be used in such Pharmaceutical compositions may be in the form of their original compounds per se, or optionally in the form of their pharmaceutically acceptable salts, and the Pharmaceutical compositions so formulated may be selected as necessary for administration of the drug in any suitable manner known to those skilled in the art.

Further, the appropriate dose of the pharmaceutical composition may be prescribed in various ways depending on factors such as the formulation method, the administration mode, the age, body weight, sex, disease state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose prescribed to be effective for the desired treatment or prevention in general.

Further, the pharmaceutical composition may further comprise conventional cosolvents, buffers, pH regulators, and the like, and if necessary, other materials may also be added to the pharmaceutical composition, and the pharmaceutical composition may be made into various dosage forms, including (but not limited to): the preparation comprises tablets, subcutaneous implants, vaginal or uterine cavity administration preparations, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like, wherein the pharmaceutical compositions of the various dosage forms can be prepared according to the conventional method in the pharmaceutical field, and can be used for injection administration, and the injection administration comprises subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; intraluminal, such as through the uterine cavity and vagina; respiratory administration, such as nasal administration; administration to the mucosa.

In a fourth aspect, the invention provides a method of screening for a candidate agent for the treatment and/or prevention of endometrial cancer.

Further, the method comprises the steps of:

(1) screening small molecular compounds by using ACSL1 as a drug target through computer-aided design;

(2) carrying out a verification experiment on the small molecule compound obtained by screening in the step (1), and screening the small molecule compound which can inhibit proliferation and/or migration and/or invasion of endometrial cancer cells, and/or promote apoptosis of the endometrial cancer cells, and/or inhibit growth of the endometrial cancer cells, and/or reduce volume of the endometrial cancer as a candidate drug;

preferably, the validation experiment comprises a cell proliferation experiment, a cell migration experiment, a cell invasion experiment, an apoptosis experiment and a xenograft tumor animal model experiment.

Further, the three-dimensional crystal structure of the humanized ACSL1 is not reported in the prior art at present, in the specific embodiment of the invention, SWISSMODEL online homologous modeling software is used for modeling the three-dimensional crystal structure of the humanized ACSL1, and the selected template protein is Oxidoreductase (PDB number is 6oz 1);

preferably, based on a template protein Oxidoreductase, a Surflex molecule docking module of a Sybyl-X2.1 drug design platform is utilized for virtual screening, a Chemdiv small molecule database (up to 150 ten thousand compounds) is selected, and a Compound Filtering module is utilized for carrying out first round screening of 'class five rules' on the compounds in Chemdiv;

preferably, a catalytic domain in a three-dimensional structure of the ACSL1 is selected as an inhibitor binding cavity in virtual screening, partial parameters are modified, and the speed of the second round of virtual screening is accelerated;

more preferably, the content of the modification part parameter is specifically as follows: reduce "Max constraints per Fragment" from default 20 to 10; reducing the 'Max number of rotatable blocks per mobile' from the default 100 to 50; the default options of ' Per-Dock Minimization ' and ' Post-Dock Minimization ' are cancelled, and the ' Maximum number of places Per ligand is reduced to 3 from the default 20, namely, only the molecular conformation 3 before each ligand molecule is ranked is reserved, and the docking speed is accelerated;

most preferably, small molecule compounds with a molecular score of 1% top (i.e., 1.5 ten thousand) are screened; recovering the Surflex molecular docking parameter as a default value based on the second round of screening results; compounds in the target of top300 are selected for cluster analysis and manual selection, and finally, 40 compounds in the target are confirmed and subsequent experiments are carried out for confirmation.

Further, the candidate drug for treating and/or preventing 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 invention has the advantages and beneficial effects that:

(1) the invention discovers that the small molecular compound EC02 has the effect of treating the endometrial cancer for the first time, and in-vivo and in-vitro verification experiments show that the small molecular compound EC02 has a remarkable inhibiting effect on the growth of endometrial cancer cells;

(2) the invention researches the molecular mechanism of a small molecular compound EC02 in the anti-tumor process, and the research result shows that EC02 can inhibit fatty acid beta oxidation and ATP generation of endometrial cancer cells by inhibiting the ACSL1 target spot for the first time, so that the proliferation and migration of the endometrial cancer cells are obviously inhibited, and the effect of inhibiting the progress of the endometrial cancer is achieved;

(3) the application of the small molecule compound EC02 targeting ACSL1 in preparation of the medicine for preventing and/or treating endometrial cancer is disclosed for the first time, a new method and a new thought are provided for clinical treatment of endometrial cancer, and the small molecule compound EC02 provided by the invention has a significant meaning in adjuvant treatment and prevention of endometrial cancer, so that the small molecule compound EC02 provided by the invention has a very good clinical application prospect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a statistical plot of the results of Ishikawa cell proliferation after ACSL1 knockdown and overexpression, # P <0.01 compared to SI-NC group, # P <0.01 compared to OE-NC group;

FIG. 2 is a graph showing the results of Ishikawa cell migration after ACSL1 knockdown and overexpression;

FIG. 3 shows a statistical plot of the results of Ishikawa cell migration after ACSL1 knockdown and overexpression, # P <0.01 compared to SI-NC group, # P <0.01 compared to OE-NC group;

FIG. 4 shows a statistical plot of the results of the relative expression levels of ACSL1mRNA in ACSL1 knockdown and over-expressed Ishikawa cells;

FIG. 5 shows representative images of tumor tissues from mice with ACSL1 knockdown and overexpression;

FIG. 6 shows the results of growth curves of tumors from mice with knockdown and over-expression of ACSL 1;

fig. 7 is a graph showing the results of observation of the appearance and structure of uterine tissue with hematoxylin and eosin staining, in which, panel a: SI-NC, B diagram: SI, C diagram: OE-NC, Panel D: OE;

figure 8 is a statistical plot of the results showing the effect of 40 compounds on endometrial cancer cell proliferation, wherein panel a: compounds 1-10, panel B: compounds 11-20, panel C: compounds 21-30, panel D: compounds 31-40, n-3, P <0.05, P <0.01, P <0.001, compared to Control group;

FIG. 9 is a statistical graph showing the results of proliferation of Ishikawa cells treated with EC02 at low (1. mu.M), medium (5. mu.M) and high (10. mu.M) concentrations for 48h, where n is 3,^**P<0.01, compared to group N;

FIG. 10 is a graph showing the results of Ishikawa cells migrating after 48h of EC02 treatment at low (1. mu.M), medium (5. mu.M) and high (10. mu.M) concentrations;

FIG. 11 is a statistical graph showing the results of the migration of Ishikawa cells after 48h of EC02 treatment at low (1. mu.M), medium (5. mu.M) and high (10. mu.M), where n is 3,^**P<0.01, compared to group N;

figure 12 shows a statistical plot of the results of fatty acid beta oxidation of Ishikawa cells after 48h of EC02 treatment at low (1 μ M), medium (5 μ M) and high (10 μ M), n-3,^*P<0.05，^**P<0.01, compared to group N;

FIG. 13 is a statistical plot showing the results of ATP production in Ishikawa cells treated with EC02 at low (1. mu.M), medium (5. mu.M) and high (10. mu.M) concentrations for 48h, where n is 3,^**P<0.01, compared to group N;

FIG. 14 shows a schematic structural diagram of the small molecule drug EC02 in the invention.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods in which specific conditions are not specified, and the detection is usually carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Example 1 Regulation of proliferation and migration Capacity of ACSL1 on endometrial cancer cells

To investigate the effect of ACSL1 on the proliferation and migration ability of endometrial cancer cells, the present example performed CCK8 cell proliferation and cell scratch experiments, respectively.

1. Cell culture and processing

The human endometrial cancer cell line Ishikawa is provided by a cell bank of the chinese academy of sciences (china, shanghai); ishikawa cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Clark, USA), 1% penicillin, 1% streptomycin, at 5% CO₂And culturing at 37 deg.C in a constant temperature incubator.

2. Ishikawa cell ACSL1 knockdown and over-expression cell model establishment

The ACSL1 lentivirus adopted in the embodiment is purchased from Shanghai Kjeka Gene chemistry Co., Ltd;

when Ishikawa cells grow to a density of about 50%, infection with the ACSL1 lentiviral plasmid was performed using a titer of 1X 10⁸Adding virus amount into TU/mL virus according to MOI of 50, changing into fresh culture medium after complete culture medium infection for 12h, continuously culturing for 1-2 days, changing primary liquid, infecting for 3-4 days, observing fluorescence intensity under a fluorescence microscope, detecting knocking-down efficiency by PCR and Western blot, screening the cells infected with lentivirus by puromycin to obtain an Ishikawa cell ACSL1 gene knocking-down and over-expression cell model, wherein the sequence information of ACSL1 knocking-down group is shown as SEQ ID NO:1 and SEQ ID NO:2, and QPCR detects the expression level of ACSL1 in cells of SI-NC, SI and OE-NC groups;

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

ACSL1-RNAi(83133-21)-b：5’-AATTCAAAAACGCAGATAGATGACCTCTATTCTCGAGAATAGAGGTCATCTATCTGCG-3’(SEQ ID NO:2)。

3. real-time fluorescent quantitative PCR

(1) Adding a corresponding amount of reactants into a 96-well plate according to a reaction system described in Table 1, sealing a membrane, and centrifuging (4000g, 2min), wherein the reaction primers are designed and synthesized by Shanghai biological engineering Co., Ltd, the mRNA expression level of beta-actin is used as a reference, the primer sequences of ACSL1 are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the primer sequences of beta-actin are shown as SEQ ID NO. 5 and SEQ ID NO. 6;

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

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

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

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

TABLE 1 qRT-PCR reaction System (10. mu.L)

(2) Putting the 96-well plate into an LC480 real-time fluorescent quantitative PCR instrument, and setting a 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 finished, calculating the Cp value of each target gene in the sample by using Roche LightCycle 480 software (the Cp value reflects the level of gene expression, and the larger the Cp value is, the lower the gene expression level is); data were derived and the relative mRNA expression level of each gene of interest was calculated by the Δ Δ CP method.

4. CCK8 cell proliferation assay

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

5. Cell scratch test

The Ishikawa cells of the OE group, the OE-NC group, the SI group and the SI-NC group are respectively inoculated in a 6-well plate, and after the cells of each group are attached to the wall and the cells grow to be 100 percent fused, 3 lines are drawn on the attached cells in parallel by using a 200 mu L gun head. The remaining cells in the well plate were washed twice with warm 1 × PBS to remove cell debris. Photographs were taken at this time point for 0 h. Putting the 6-hole plate back into the incubator, culturing for 24h, taking out the 6-hole plate, observing and photographing under a microscope, measuring the cell-cell area of each group of cells by using Imaje J software, and calculating the cell migration rate;

wherein, the calculation formula of the cell migration rate is as follows: cell mobility ═ 0h intercellular area-24 h intercellular area)/0 h intercellular area.

6. Results of the experiment

The results show that proliferation and migration are significantly increased in cells with over-expression of ACSL1 compared to the control group, and significantly decreased in cells with knockdown of ACSL1 compared to the control group (see FIGS. 1-3). In cells overexpressing ACSL1, the relative expression level of ACSL1mRNA was significantly increased compared to the control group; in contrast, in the cells with the knockdown of ACSL1, the relative expression level of ACSL1mRNA was significantly reduced compared to the control group (see fig. 4), indicating the successful establishment of a cell model with the knockdown and overexpression of ACSL 1.

Example 2 study of the relationship between ACSL1 and the occurrence and development of endometrial cancer

To investigate the relationship between ACSL1 and endometrial cancer malignancy characteristics, this example established ACSL1 knockout and overexpressing xenograft tumor model mice, and the experimental animals used in this example were purchased from Shanghai Witongliwa laboratory animals, Inc., female nude mice of 4-5 weeks of age (SPF grade) and weighing 10-12 g.

1. Establishment of endometrial cancer nude mouse model

The nude mice were divided into 4 groups in total, which were: ACSL1 knockdown group (SI group), which mice inoculated with the ashikawa cell strain that was ACSL1 knocked down; ACSL1 knockdown negative control group (SI-NC group), which mice were inoculated with Ishikawa cell strain, ACSL1 knockdown negative control; an ACSL1 overexpression group (OE group), which mice were inoculated with an Ishikawa cell strain overexpressing ACSL 1; an ACSL1 overexpression negative control group (OE-NC group), which mice were inoculated with an Ishikawa cell strain in which ACSL1 overexpresses the negative control;

establishing an endometrial cancer nude mouse model: an Ishikawa cell strain with reduced ACSL1, an Ishikawa cell strain with reduced ACSL1 negative control, an Ishikawa cell strain with over-expressed ACSL1 and an Ishikawa cell strain with over-expressed ACSL1 negative control are respectively inoculated to the axillary part of the right side of a nude mouse to construct an endometrial cancer nude mouse model. Each mouse was injected with a 0.2mL volumetric amount of cells and inoculated with 3X 10 cells per mouse⁵And (4) one cell. After completion of the inoculation of the cells, the size of the tumor tissue was measured with a ruler at intervals of 4 days, and the major axis (a) and the minor axis (b) of the tumor tissue were recorded, and the time for 4 weeks was continuously recorded in this manner. After 4 weeks, the nude mice were sacrificed, tumor tissues were taken, the volume of the tumor tissues was calculated, and a tumor growth curve was drawn according to the time and the tumor volume;

wherein, the calculation formula of the tumor volume is as follows: tumor volume is a × b²/2；

2. HE staining of tumor tissue of nude mice

The tumor tissues of nude mice of the SI-NC group, SI group, OE-NC group and OE group were stained with Hematoxylin-Eosin (HE), and the specific HE staining experiment of each group was performed according to the experimental procedures in the specification.

3. Results of the experiment

The established ACSL1 knockout and overexpression xenograft tumor model mice are shown in a figure 5, the result shows that the ACSL1 can obviously increase the size of a tumor and promote the growth of the tumor, the knocking-down ACSL1 can obviously inhibit the growth of the tumor and reduce the volume of the tumor, the growth curve of the tumor is shown in a figure 6, the information of the specific tumor is shown in a table 2, and the result further shows that the overexpression of the ACSL1 can obviously promote the growth of the tumor, and the knocking-down ACSL1 can obviously inhibit the growth of the tumor; the staining results of the tumor tissues of the nude mice with hematoxylin and eosin showed that the OE group had a certain degree of disorder in arrangement, enlarged nuclei, deep staining, clearly visible pathological nuclear division and obvious cell heterogeneity compared to the other groups (see FIGS. 7A-D).

TABLE 2 tumor information of each experimental group and control group

Grouping	Tumor volume (mm)³)	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 having inhibitory effect on endometrial cancer cells targeting ACSL1

This example demonstrates the effect of 40 compounds on the proliferation of endometrial cancer cells by a CCK8 cell proliferation assay, which was based on the ACSL1 target and was determined by computer-aided design of 40 compounds that are theoretically capable of inhibiting the activity of ACSL 1.

1. Virtual screening based on human ACSL1 protein structure

At present, the three-dimensional crystal structure of the humanized ACSL1 is not reported, the three-dimensional crystal structure of the humanized ACSL1 is modeled by using SWISSMODEL online homologous modeling software, and the selected template protein is Oxidoreductase (PDB number is 6oz 1); the method comprises the steps of utilizing a Surflex molecule docking module of a Sybyl-X2.1 drug design platform to carry out virtual screening, selecting a Chemdiv small molecule database (up to 150 ten thousand compounds), and utilizing a 'Compound filtration' module to carry out first round screening of 'class five rules' on the compounds in Chemdiv; then selecting a catalytic structure domain in a three-dimensional structure of ACSL1 as an inhibitor binding cavity in virtual screening, modifying partial parameters and accelerating the speed of the second round of virtual screening; specifically, "Max constraints per Fragment" is reduced from 20 to 10 by default, and "Max number of rotatable blocks per module" is reduced from 100 to 50 by default. The options of "Per-Dock Minimization" and "Post-Dock Minimization" are cancelled by default; reducing the 'Maximum number of places per ligand' from default 20 to 3, namely only keeping the molecular conformation 3 before each ligand molecule rank, and accelerating the docking speed; screening out small molecule compounds with a molecular score of 1% at top (i.e. 1.5 ten thousand); finally, based on the second round of screening results, the Surflex molecular docking parameters are recovered as default values; selecting compounds in the target of top300 for cluster analysis and manual selection, finally confirming that 40 compounds in the target are commercially purchased, and carrying out subsequent experimental confirmation.

2. CCK8 cell proliferation assay

After treating the endometrial cancer cells Ishikawa with different reagents (drug 1-drug40 at 10. mu.M) for 48h, the effect of 40 compounds on the proliferation of the endometrial cancer cells was tested using Cell Counting Kit-8(CCK-8) (Dojindo, Japan) according to the manufacturer's instructions;

wherein, the control group is a negative control group; the drug1-drug40 group was 40 experimental groups obtained by treating endometrial cancer cells with 40 compounds at a concentration of 10 μ M.

3. Results of the experiment

The results show that only EC02(drug2) has the most obvious inhibition effect on endometrial cancer cells in 40 compounds (see figures 8A-D), and the molecular formula of the compound EC02 is C₂₄H₂₁NO₃The compound has the relative molecular weight of 371.4284 and the structural formula shown in figure 14, and is named as 3-hydroxy-3- (2-oxo-2-phenylethyl) -1- (2-phenylethyl) -1, 3-dihydro-2H-indol-2-one.

Example 4 EC02 inhibition of energy metabolism by endometrial cancer cells

To evaluate the effect of EC02 on endometrial cancer cell proliferation, CCK8 cell proliferation experiments were performed 48h after endometrial cancer cells were treated with EC02 and triacin C, respectively, and the effect of EC02 on endometrial cancer migration capacity was also examined by cell scratch experiments;

endometrial cancer cells Ishikawa 48h were treated with EC02 at different concentrations: high concentration 10. mu.M, medium concentration 5. mu.M and low concentration 1. mu.M;

wherein, N groups: a negative control group; 1 μ M group: cells were treated at EC02 concentration of 1 μ M; 5 μ M group: cells were treated at EC02 concentration of 5 μ M; 10 μ M group: cells were treated at an EC02 concentration of 10. mu.M.

1. CCK8 cell proliferation assay

After treating different groups of endometrial cancer cells Ishikawa with different reagents for 48h, the effect of the different reagents on the proliferation of endometrial cancer cells was tested using Cell Counting Kit-8(CCK-8) (Dojindo, Japan) according to the manufacturer's instructions.

2. Cell scratch test

The endometrial cancer cells of different treatment groups are inoculated in a 6-well plate, after the cells are attached to the wall, the cells grow to be 100% fused, and 3 lines are drawn on the attached cells in parallel by using a 200-microliter gun head. The remaining cells in the well plate were washed twice with warm 1 × PBS to remove cell debris. Photographs were taken at this time point for 0 h. Putting the 6-hole plate back into the incubator, taking out the 6-hole plate after 24 hours, observing and photographing under a microscope, measuring the area between cells by using Imaje J software, and calculating the cell migration rate;

3. Results of the experiment

The results of CCK8 cell proliferation experiments show that the EC02 treatment concentration of 1 μ M has obvious inhibition on the proliferation of endometrial cancer cells, and the inhibition effect is gradually enhanced along with the increasing of the EC02 concentration (see figure 9);

the results of the cell scratching experiments show that the EC02 can obviously inhibit the migration of endometrial cancer cells, and the inhibition capability is gradually enhanced along with the increase of the concentration of EC02 (see fig. 10 and 11);

the results show that EC02 has obvious inhibition effect on endometrial cancer cells and has good clinical application value.

Example 5 EC02 inhibition of lipid metabolism in endometrial cancer cells

To further investigate the effect of EC02 on lipid metabolism of endometrial cancer cells, MDA experiments were performed in this example, and ATP production by endometrial cancer cells was also tested using the ATP Lite kit.

1. Lipid Oxidation (MDA) experiments

Each group of cells was lysed and the supernatant was collected after 12000g centrifugation. Adding malondialdehyde detection working solution, mixing, heating in boiling bath for 15min, cooling to room temperature in water bath, and centrifuging at 1000g room temperature for 10 min. 200. mu.L of the supernatant was added to a 96-well plate, and the absorbance at 532nm was measured with a microplate reader. The level of MDA production was analyzed by lipid oxidation (MDA) detection kit (beyosine, china) according to the manufacturer's instructions.

2. ATP Generation test

Each group of cells was seeded in a 96-well plate, and 50. mu.L of a mammalian cell lysis solution was added to each well, and the well plate was shaken in a shaker at 700rpm for 5min, thereby lysing the cells and stabilizing ATP. Add 50. mu.L of matrix solution to the wells and shake the well plate on a shaker for 5 min. Protected from light for 10min and the chemiluminescence was measured. ATP production was analyzed by the ATP-LITE assay kit (PerkinElmer, USA) according to the manufacturer's instructions.

3. Results of the experiment

The results show that EC02 can obviously inhibit fatty acid beta oxidation of endometrial cancer cells and is concentration-dependent (see fig. 12), EC02 can obviously inhibit ATP production of endometrial cancer cells and is concentration-dependent (see fig. 13), and the results show that EC02 can obviously inhibit fatty acid beta oxidation and ATP production of endometrial cancer cells by inhibiting ACSL1 targets, so that proliferation and migration of endometrial cancer cells are obviously inhibited, and the effect of inhibiting endometrial cancer progression is achieved, and EC02 (structural formula shown in fig. 14) is expected to be a novel compound for treating endometrial cancer and has a very good clinical application prospect.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims

1. The application of the small molecule drug targeting ACSL1 in the preparation of the drug for treating and/or preventing endometrial cancer is characterized in that the small molecule drug is EC02, and the structural formula of the small molecule drug is shown in formula (I):

formula (I).

2. The use of claim 1, wherein the small molecule drug inhibits proliferation of endometrial cancer cells and inhibits migration of endometrial cancer cells.

3. The use of claim 1, wherein the small molecule drug is used at a concentration of 1 μ Μ -500 μ Μ.

4. The use of claim 3, wherein the small molecule drug is used at a concentration of 1 μ M to 100 μ M.

5. The use of claim 1, wherein the medicament consists of a therapeutically effective amount of a small molecule drug represented by formula (I) and a pharmaceutically acceptable carrier and/or adjuvant.

6. Use according to claim 5, wherein the pharmaceutically acceptable carrier and/or adjuvant comprises diluents, binders, surfactants, humectants, adsorbent carriers, lubricants and/or disintegrants.

7. The use of claim 5, wherein the small molecule drug is used at a concentration of 1 μ M to 500 μ M.

8. The use of claim 7, wherein the small molecule drug is used at a concentration of 1 μ M to 100 μ M.

9. The application of the small molecule drug targeting ACSL1 in the preparation of the reagent is characterized in that the small molecule drug is EC02, and the structural formula of the small molecule drug is shown in the formula (I):

formula (I);

the agent is used in any one or more of the following aspects:

(1) inhibiting proliferation of endometrial cancer cells in vitro;

(2) inhibiting migration of endometrial cancer cells in vitro.

10. The use of 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 of claim 10, wherein the endometrial cancer cells are Ishikawa.

12. A method of screening for a candidate agent for the treatment and/or prevention of endometrial cancer, said method comprising the steps of:

(2) carrying out a verification experiment on the small molecule compound obtained by screening in the step (1), and screening the small molecule compound capable of inhibiting proliferation and/or migration and/or invasion of endometrial cancer cells, and/or promoting apoptosis of the endometrial cancer cells, and/or inhibiting growth of the endometrial cancer cells, and/or reducing volume of the endometrial cancer as a candidate drug; the candidate drug screened in the step (2) for treating and/or preventing endometrial cancer is EC02, wherein the structural formula of EC02 is shown in the formula (I):

the formula (I).

13. The method of claim 12, wherein the validation experiment comprises a cell proliferation experiment, a cell migration experiment, a cell invasion experiment, an apoptosis experiment, or a xenograft tumor animal model experiment.