CN114984007B - PRADX-EZH2 small molecule inhibitor and application thereof in preparation of tumor treatment medicines - Google Patents

PRADX-EZH2 small molecule inhibitor and application thereof in preparation of tumor treatment medicines Download PDF

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CN114984007B
CN114984007B CN202210753634.9A CN202210753634A CN114984007B CN 114984007 B CN114984007 B CN 114984007B CN 202210753634 A CN202210753634 A CN 202210753634A CN 114984007 B CN114984007 B CN 114984007B
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epic0307
pradx
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CN114984007A (en
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方川
康春生
檀艳丽
辛磊
肖梦麟
王琦雪
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Abstract

The invention discloses a PRADX-EZH2 small molecule inhibitor and application thereof in preparing a tumor treatment medicament, wherein the structural formula of the small molecule inhibitor is shown as a formula (I), the molecular weight is 416.54, and the small molecule inhibitor shows anti-tumor benefits in-vivo and in-vitro researches by blocking the combination of PRADX and EZH2 and can enhance the curative effect of temozolomide. Has wide application prospect in clinical application for resisting tumor, especially in the aspect of treating brain glioma.

Description

PRADX-EZH2 small molecule inhibitor and application thereof in preparation of tumor treatment medicines
Technical Field
The invention belongs to the technical field of biological medicines, relates to a small molecule inhibitor, and particularly relates to a small molecule inhibitor aiming at a PRADX-EZH2 compound and application thereof in preparing a tumor treatment medicine.
Background
Glioblastoma (GBM) is the most malignant of the primary brain tumors of the central nervous system. The tumor is mostly located under the cortex of supratentorial cerebral hemisphere and grows infiltratively, the deep structure is affected, the glioblastoma rapidly progresses, the course of disease of 70-80% of patients is 3-6 months, and the course of disease is only 10% for more than 1 year. The treatment method mainly comprises surgery, radiotherapy and chemotherapy. Although the median survival time of glioblastoma has been extended to 12-15 months in recent years, mortality remains high, with patient survival rates of only 30% and 13% for 1 and 5 years. Glioblastoma accounts for 52 percent of primary brain tumors, has a 5-year mortality rate second to pancreatic cancer and lung cancer, is located at the 3 rd position of a whole-body malignant tumor, and seriously threatens human health. Therefore, the research on the pathogenesis and the treatment strategy of the traditional Chinese medicine is of great significance.
Temozolomide (TMZ) is an alkylating agent, glioma is protected by blood-brain barrier (BBB), and Temozolomide is the only chemotherapeutic drug that can pass through the blood-brain barrier so far, and is widely used for treating primary and recurrent high-grade glioma, and the action mechanism of the Temozolomide is to enable DNA to be cross-linked at the position of guanine O6 through alkylation, block DNA replication, induce cell cycle arrest at the G2/M phase and finally cause cell apoptosis. However, since glioblastoma is susceptible to temozolomide, only 50% of patients can benefit from temozolomide treatment. The low potency and drug resistance of temozolomide are important causes of glioblastoma recurrence and death. Therefore, the development of glioblastoma and the molecular mechanism of drug resistance are deeply discussed, and the development of novel precise therapeutic methods and novel strategies are imperative on the basis.
The subject group of the present invention found that PRADX is highly expressed in tumor tissues of glioblastoma and colon adenocarcinoma. Further research shows that PRADX can inhibit gene transcription by interacting with two hairpin structures at the 5' end of PRADX and a core protein EZH2 of Polycomb regenerative Complex 2 (PRC 2), and catalyzing 27 th lysine of histone H3 to carry out trimethylation modification (H3K 27 methylation, H3K27me 3) by being recruited in a gene promoter region. Through a series of binding experiments, PRADX and EZH2 can be directly interacted, and the action site is between 200-500bp of PRADX. However, no small molecule drug and clinical treatment protocol for PRADX-EZH2 has been developed to date. The application of the small-molecule inhibitor aiming at the PRADX-EZH2 compound and shown in the formula (I) in treating glioblastoma is reported for the first time.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a small molecule inhibitor aiming at a PRADX-EZH2 complex and application thereof in preparing a tumor treatment drug.
The above object of the present invention is achieved by the following technical solutions:
the invention provides application of a small molecule inhibitor with a structural formula shown in a formula (I) in preparing a medicament for treating and/or preventing tumors;
Figure BDA0003719090950000021
further, the tumor is a tumor with high expression of PRADX and/or EZH 2;
preferably, the tumor comprises glioblastoma, oligodendroglioma, anaplastic glioma, colon adenocarcinoma, colon mucinous carcinoma, colon undifferentiated carcinoma, rectal cancer, small cell lung cancer, lung squamous carcinoma, lung adenocarcinoma, large cell lung cancer, breast cancer;
more preferably, the tumor is a glioblastoma.
Further, the small molecule inhibitor is effective in blocking the binding of PRADX and EZH 2.
Further, the small molecule inhibitor can interfere with the recruitment of PRADX to PRC 2;
preferably, the small molecule inhibitor can significantly reduce the levels of the target genes CDKN1A and BBC3 promoter region H3K27me3 of PRADX;
preferably, the small molecule inhibitor can block the cell cycle in the G1/S phase and induce apoptosis;
preferably, the small molecule inhibitor is capable of inhibiting DNA damage repair;
preferably, the small molecule inhibitor can inhibit STAT3 pathway, inhibit MGMT expression;
preferably, the small molecule inhibitor can enhance the therapeutic effect of temozolomide.
In one embodiment of the invention, the micromolecule inhibitor with the structural formula shown as the formula (I) is used as an effective component of a medicine for treating tumors.
The invention discovers for the first time that the small molecule inhibitor with the structural formula shown as the formula (I) can treat tumors and enhance the curative effect of temozolomide.
In one embodiment of the invention, the effective component of the drug for treating tumor (the micromolecule inhibitor with the structural formula shown in formula (I)) can inhibit DNA repair and enhance the curative effect of temozolomide.
In one embodiment of the invention, the effective component of the tumor treatment drug (the micromolecule inhibitor with the structural formula shown in formula (I)) can inhibit the STAT3 pathway, inhibit the transcription level and expression level of MGMT and enhance the curative effect of temozolomide.
Temozolomide is named Temozolomide in English and has a molecular formula of C 6 H 6 N 6 O 2 Molecular weight 194.15, CAS number 85622-93-1.
In the invention, the small molecule inhibitor with the structural formula shown in the formula (I) is also called compound 0307 or EPIC0307, and is the small molecule inhibitor which is screened and proved for the first time and can treat tumors and enhance the curative effect of temozolomide.
In a second aspect of the invention, a pharmaceutical composition for the treatment and/or prevention of a tumor is provided.
Further, the pharmaceutical composition comprises the small molecule inhibitor described in the first aspect of the invention;
preferably, the pharmaceutical composition consists of a therapeutically effective amount of the small molecule inhibitor described in the first aspect of the present invention and a pharmaceutically acceptable carrier and/or adjuvant;
more preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant;
preferably, the tumor is a tumor with high expression of PRADX and/or EZH 2;
more preferably, the tumor comprises glioblastoma, oligodendroglioma, anaplastic glioblastoma, colon adenocarcinoma, colon mucinous carcinoma, colon undifferentiated carcinoma, rectal cancer, small cell lung cancer, lung squamous carcinoma, lung adenocarcinoma, large cell lung cancer, breast cancer;
most preferably, the tumor is a glioblastoma.
Further, the pharmaceutical composition also comprises temozolomide.
Further, the diluents include (but are not limited to): lactose, sodium chloride, glucose, urea, starch, water, powdered sugar, dextrin, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, calcium sulfate dihydrate, mannitol, etc. The adhesive includes (but is not limited to): starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, 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. The surfactants include (but are not limited to): polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, beeswax, lecithin, hydroxymethyl cellulose, polyethylene glycol caprylic acid, glyceryl decanoate, polyethylene glycol lauric glyceride, polyethylene glycol stearic glyceride, alkyl polyglucoside, etc. The humectants include, but are not limited to: distilled water, glycerin, starch, ethanol, etc. The adsorbent carrier includes (but is not limited to): starch, lactose, bentonite, silica gel, kaolin, bentonite, etc. The lubricants include (but are not limited to): zinc stearate, glyceryl monostearate, polyethylene glycol, pulvis Talci, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc. Such fillers include (but are not limited to): mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, pregelatinized starch, and the like. Such disintegrants include (but are not limited to): crospolyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose sodium, croscarmellose sodium, soybean polysaccharide, crospovidone, hydroxypropyl cellulose, etc.
Further, the pharmaceutically acceptable carrier and/or adjuvant added to the pharmaceutical composition is used to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or odor in case of oral administration, as required.
Furthermore, the pharmaceutical composition can be prepared into any pharmaceutically conventional dosage form.
The pharmaceutical composition can be prepared into injections or oral preparations, including injections, tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, sustained-release agents, nano preparations, syrups, medicated liquors, tinctures and lotions. The pharmaceutical composition is generally prepared into injection, and can also be developed into oral preparation, thereby improving the medication compliance of patients.
The pharmaceutical composition of the present invention can be prepared by conventional methods known to those skilled in the art, such as mixing the effective components, or mixing the effective components with corresponding adjuvants according to conventional methods for preparing various dosage forms. The pharmaceutical compositions of the present invention may also be used with other therapeutic agents for the treatment of tumors.
The therapeutically effective dose of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, administration time, administration route, excretion rate and reaction 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.
The invention also provides a method for treating tumors.
Further, the method comprises the steps of: administering an effective amount of a small molecule inhibitor having a structural formula shown in formula (I) and temozolomide to a subject.
Further, the subject may be a mammal or a mammalian tumor cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The tumor cell may be an ex vivo tumor cell.
Further, the subject may be a patient suffering from a tumor or an individual desiring treatment for a tumor. Or the subject is an isolated tumor cell of a tumor patient or an individual in whom treatment of a tumor is desired.
Further, the treatment methods can be administered to a subject before, during, or after receiving treatment for the tumor.
Further, the tumor is a tumor with high expression of PRADX and/or EZH 2;
preferably, the tumor comprises glioblastoma, oligodendroglioma, anaplastic glioblastoma, colon adenocarcinoma, colon mucinous carcinoma, colon undifferentiated carcinoma, rectal cancer, small cell lung cancer, lung squamous cancer, lung adenocarcinoma, large cell lung cancer, breast cancer;
more preferably, the tumor is a glioblastoma.
In a third aspect, the invention provides the use of a small molecule inhibitor as described in the first aspect of the invention in the preparation of a sensitizer for a chemotherapeutic drug for treating a tumor.
Further, the chemotherapeutic drug for treating the tumor is temozolomide;
preferably, the tumor comprises glioblastoma, oligodendroglioma, anaplastic glioma, colon adenocarcinoma, colon mucinous carcinoma, colon undifferentiated carcinoma, rectal cancer, small cell lung cancer, lung squamous carcinoma, lung adenocarcinoma, large cell lung cancer, breast cancer;
most preferably, the tumor is a glioblastoma.
Further, the sensitizer is in the form of any one or more of injection, tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, sustained-release agents, nano preparations, syrups, medicated liquors, tinctures and lotions, and is preferably injection or oral preparation.
The fourth aspect of the invention provides a sensitizer for tumor chemotherapy drugs.
Further, the sensitizer comprises the small molecule inhibitor described in the first aspect of the present invention, and the tumor chemotherapeutic drug is temozolomide.
In the specific embodiment of the invention, the invention discovers for the first time that the micromolecule inhibitor with the structural formula shown as the formula (I) can enhance the curative effect of temozolomide.
A fifth aspect of the invention provides the use of a small molecule inhibitor as described in the first aspect of the invention in combination with temozolomide for the preparation of a medicament for the treatment and/or prevention of a tumour;
preferably, the tumor comprises glioblastoma, oligodendroglioma, anaplastic glioma, colon adenocarcinoma, colon mucinous carcinoma, colon undifferentiated carcinoma, rectal cancer, small cell lung cancer, lung squamous carcinoma, lung adenocarcinoma, large cell lung cancer, breast cancer;
more preferably, the tumor is a glioblastoma.
A sixth aspect of the invention provides the use of any one of the following:
(1) Use of a small molecule inhibitor as described in the first aspect of the invention in the manufacture of an interfering agent for interfering with the binding of PRADX and EZH 2;
(2) Use of a small molecule inhibitor as described in the first aspect of the invention in the preparation of an interfering agent for interfering with the recruitment of PRADX to PRC 2;
(3) Use of a small molecule inhibitor as described in the first aspect of the invention in the manufacture of a promoter for increasing the level of a target gene CDKN1A, BBC3 of PRADX;
(4) Use of a small molecule inhibitor as described in the first aspect of the invention in the preparation of an inhibitor for reducing the level of the target gene CDKN1A, BBC3 promoter region H3K27me3 of PRADX;
(5) Use of a small molecule inhibitor as described in the first aspect of the invention in the preparation of a blocker for arresting the cell cycle in the G1/S phase, inducing apoptosis;
(6) Use of a small molecule inhibitor as described in the first aspect of the invention in the preparation of an inhibitor for inhibiting DNA damage repair;
(7) The application of the small molecule inhibitor in the first aspect of the invention in preparing the inhibitor for inhibiting STAT3 pathway and MGMT expression;
(8) The sensitizer of the fourth aspect of the invention is applied to the preparation of medicines capable of sensitizing temozolomide curative effect.
Compared with the prior art, the invention has the following advantages and beneficial effects:
according to the invention, by a computer simulation technology, aiming at the characteristic that a PRADX 5' functional structural domain is combined with PRC2, a small molecular compound which can effectively interfere the combination of PRADX and EZH2 is screened out for the first time, and a compound EPIC0307 which is simple in structure and low in molecular weight and IC50 is selected. In vivo and in vitro experiments of glioma, breast cancer and other tumors, EPIC0307 can effectively interfere the combination of PRADX and EZH2, influence the recruitment of PRADX to target gene, activate the transcription of target gene, and increase the expression, thereby inhibiting the tumor proliferation and DNA damage repair, inhibiting STAT3 pathway, inhibiting the transcription and expression of MGMT, and enhancing the curative effect of temozolomide, and EPIC0307 and temozolomide have synergistic effect. Has wide application prospect in clinical application for resisting tumor, especially in the aspect of treating brain glioma.
Drawings
FIG. 1 is a schematic diagram of three-dimensional structure simulation and drug target selection for PRADX and EZH 2;
FIG. 2 is a schematic representation of EPIC0307 interfering with the binding of the PRADX 5' domain to EZH 2;
FIG. 3 is a graph showing the results of toxicity tests of EPIC0307 in the CCK8 test in a plurality of glioblastoma cell lines;
FIG. 4 is a graph of EPIC0307 versus PRADX expression, which is statistically significant and inversely correlated with IC 50;
FIG. 5 is a graph showing the results of RIP-qPCR assay, in which 15. Mu.M EPIC0307 was treated for 48 hours to block the binding of PRADX-EZH 2;
FIG. 6 is a graph showing the effect of overexpression of PRADX on the sensitivity of EPIC0307, the sensitivity of EPIC0307 increased by overexpression of PRADX;
FIG. 7 shows the results of detection in the CHIRP co-immunoprecipitation assay, in which EPIC0307 can effectively block the binding of PRADX-EZH 2;
FIG. 8 is a graph showing the results of changes in RNA levels of target genes CDKN1A, BBC3 after 48 hours of treatment with 15. Mu.M EPIC0307 in TBD0220, U87-MG cell line;
FIG. 9 is a graph showing the results of the RNA levels of the target genes CDKN1A, BBC3 as a function of the drug concentration in 0307 after EPIC0307 treatment for 48 hours in the TBD0220, U87-MG cell line;
FIG. 10 is a graph showing the results of the changes in the protein levels of the target genes CDKN1A, BBC3 with 0307 drug concentrations after EPIC0307 treatment for 48 hours in TBD0220, U87-MG cell line;
FIG. 11 is a graph showing the results of RNA levels of target genes CDKN1A, BBC3 as a function of 0307 drug treatment time after 15. Mu.M EPIC0307 treatment in TBD0220, U87-MG cell line;
FIG. 12 is a graph of the results of the change in the protein levels of the target genes CDKN1A, BBC3 following treatment with 15 μ M EPIC0307 in the TBD0220, U87-MG cell line with the time of EPIC0307 treatment;
FIG. 13 is a graph showing the results of confirmation of decrease in enrichment of promoter regions H3K27me3 of target genes CDKN1A, BBC3 after 48 hours for EPIC0307. Mu.M by CHIP experiment;
FIG. 14 is a graph showing the results of the transcriptional expression changes of the target genes CDKN1A and BBC3 after over-expression of PRADX, and the transcriptional level of CDKN1A and BBC3 after over-expression of PRADX is more significantly increased after EPIC0307. Mu.M for 48 hours;
FIG. 15 is a graph showing that the expression of E2F1 is decreased by inhibiting the Rb pathway due to the change of cyclin after EPIC0307. Mu.M treatment for 48 hours by Western blot assay;
FIG. 16 is a graph showing the results of cell cycle arrest at G1/S phase by flow cytometry after EPIC0307 was treated for 48 hours at different concentrations;
FIG. 17 is a graph showing the result of Western blot analysis demonstrating the change of apoptotic proteins and induction of apoptosis after EPIC0307. Mu.M 48-hour treatment;
FIG. 18 is a graph showing the results of the induction of apoptosis by flow apoptosis assay 48 hours after different concentrations of EPIC0307 treatment;
FIG. 19 is a graph showing the results of the immunofluorescence assay demonstrating the changes in apoptosis-related proteins Caspase 7and cleared Caspase3 after 48 hours of EPIC0307. Mu.M treatment;
FIG. 20 is a graph showing the results of the inhibition rate of cells in TBD0220 and U87-MG cell lines, EPIC0307 and temozolomide in combination;
FIG. 21 is a graph showing the results of EPIC0307 in enhancing temozolomide effect by calculating the combination index CI through BISS model;
FIG. 22 is a graph showing the results of a plate clone cell formation assay, in which EPIC0307 at 10. Mu.M and temozolomide at 200. Mu.M were exposed for 2 weeks, EPIC0307 inhibited cell clone formation and enhanced temozolomide sensitivity;
FIG. 23 is a graph showing the statistical results of the numbers of clones formed in the plate clone cell formation test, the difference being statistically significant;
FIG. 24 is a graph showing the results of detecting the occurrence of EPIC 0307-induced and sensitized temozolomide-induced apoptosis by flow apoptosis after 48 hours of treatment with EPIC0307, temozolomide at corresponding concentrations;
FIG. 25 is a graph showing the results of detecting a decrease in the transcript level of a DNA repair-related indicator by qPCR after EPIC0307. Mu.M 48 hours treatment;
FIG. 26 is a graph showing the results of Western blot analysis showing that DNA damage protein is increased and repair protein is decreased after EPIC0307 and temozolomide at corresponding concentrations are treated for 48 hours;
FIG. 27 is a graph of the results of detecting changes in the transcript levels of PRADX and EZH2 by qPCR of temozolomide 50 μ M cells stimulated for 2 consecutive weeks;
FIG. 28 is a flow chart of intracranial in situ PDX model creation and drug treatment;
FIG. 29 is a graph showing the results of EPIC0307 and temozolomide combinations demonstrating that EPIC0307 can inhibit tumor growth by bioluminescence imaging;
FIG. 30 is a graph of the results of statistical analysis of bioluminescent data for statistical significance of differences;
fig. 31 is a result chart of survival analysis, EPIC0307 can prolong survival time properly, and the survival time of mice in drug combination group is longer;
FIG. 32 is a graph of the results of mouse brain sections with smaller tumor volumes in the combination and HE showing smoother tumor margins;
FIG. 33 is a graph showing the results of immunohistochemistry and immunofluorescence of mouse tissue sections, changes in Ki67, a protein involved in DNA damage repair;
FIG. 34 is a graph of the results of quantitative qPCR detection of background transcription levels of multiple tumor cell lines MGMT, with T98G cells as a normalization reference;
FIG. 35 is a graph showing the results of the Western blot assay demonstrating the protein expression levels of MGMT from a plurality of tumor cell lines;
FIG. 36 is a graph showing the results of the cell inhibition rate in the T98G cell line using EPIC0307 in combination with temozolomide;
FIG. 37 is a graph of the results of BISS model calculation of combination index CI showing that EPIC0307 enhances the effect of temozolomide;
FIG. 38 is a graph showing the results of a plate clone cell formation assay, in which EPIC0307 at 10. Mu.M and temozolomide at 400. Mu.M were used for 2 weeks, and EPIC0307 inhibits cell clone formation and sensitizes temozolomide;
FIG. 39 is a graph showing that the expression of E2F1 is decreased by inhibiting the Rb pathway due to the change of cyclin after EPIC0307. Mu.M treatment for 48 hours, as shown in Western blot;
FIG. 40 is a graph showing the results of detecting a decrease in the transcript level of a DNA repair-related indicator by qPCR after EPIC0307. Mu.M 48 hours treatment;
FIG. 41 is a result graph showing that the Western blot test shows that after EPIC0307. Mu.M and temozolomide 800. Mu.M are treated for 48 hours, MGMT expression is reduced, DNA damage protein is increased, and repair protein is reduced;
FIG. 42 is a graph showing the results of confocal immunoassay, showing the changes in γ -H2AX after 48 hours of EPIC0307. Mu.M and temozolomide 800. Mu.M treatment;
FIG. 43 is a graph showing the results of the RNA levels of MGMT, ATF3 after 48 hours of EPIC0307 as a function of the EPIC0307 drug concentration gradient;
FIG. 44 is a graph showing the results of the variation of RNA levels of MGMT, ATF3 after EPIC0307. Mu.M with the time gradient of EPIC0307 drug;
FIG. 45 is a graph showing the results of Western blot analysis demonstrating the changes of MGMT, ATF3, BLCAP, STAT3, P-STAT3 with concentration gradient and time gradient after EPIC0307 hours or EPIC0307. Mu.M treatment;
FIG. 46 is a graph showing the results of the decrease in the enrichment of the promoter region H3K27me3 of ATF3 after 48 hours of EPIC0307. Mu.M by CHIP experiment;
FIG. 47 is a graph demonstrating by CHIP experiments that enrichment of MGMT promoter region H3K27ac is decreased after EPIC0307. Mu.M for 48 hours, and that the decrease in enrichment of H3K27ac is restored after ATF3 is down-regulated by transfection;
FIG. 48 is a graph showing the results of detection of the change in O6-metG after 48 hours of treatment with EPIC0307. Mu.M and temozolomide 800. Mu.M by an immunoprophoresis experiment.
Detailed Description
An increasing number of long non-coding RNAs (lncrnas) have been reported to play a key role in the development of tumors, and therefore, the search for tumor treatment against lncrnas is urgent. The early stage of the subject group discovers that LncRNA PRADX is remarkably highly expressed in brain glioma tissues, the tumor promotion action mechanism is determined by combining a key member EZH2 of a PRC2 complex and further regulating and controlling the expression of a silent target gene through epigenetics, so that the tumor process is promoted, and researches show that PRADX has a structural domain combined with EZH2 within the range of 200-500 bp. The inventor utilizes a computer structure to simulate the 3D structure of PRADX and the 3D structure of EZH2, and screens important targets through molecular simulation of structural prediction and interaction of a PRADX and EZH2 compound. The ChemDiv and Specs small molecule databases were selected as screening databases, and the optimal compound "0307" (also known as EPIC 0307) was selected in combination with in vitro toxicity assay (IC 50) for analysis of binding patterns. Compound 0307 (also called compound EPIC 0307) can specifically interfere the combination of PRADX and EZH2, thus affecting the recruitment of PRC2 to target gene by PRADX, so that the important cancer suppressor gene in target gene can be released from transcription inhibition and expressed, and the proliferation of various tumors can be successfully inhibited and apoptosis can be induced.
Among them, CDKN1A is an important target gene of PRADX and can be significantly up-regulated by compound 0307, thereby preventing the progress of cell cycle, inhibiting the phosphorylation of Rb protein, reducing Rb release nuclear transcription factor E2F1, and inhibiting the transcriptional expression of E2F1 downstream genes. In addition, compound 0307 can significantly up-regulate the expression of another important target gene, PUMA, a Bcl-2 family member, of PRADX, which induces apoptosis. In addition, in vivo and in vitro experiments, the inventor finds a novel combined treatment scheme that the compound 0307 can enhance the treatment effect of temozolomide, inhibit tumor proliferation and induce apoptosis by inhibiting a DNA damage repair pathway. The inventor finds that the method for influencing PRADX to PRC2 recruitment in the form of small molecule compounds by interfering LncRNA PRADX and EZH2 combination for the first time changes the malignancy apparent regulation of tumors, and provides a new drug treatment way and a new combined treatment scheme for tumor treatment.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any number between the two endpoints are optional unless otherwise specified in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) METHODS IN ENZYMOLOGY, vol.304, chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.
The invention will now be further illustrated with reference to specific examples, which are provided for illustration 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 experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 EPIC0307 is a simple, easily synthesized small molecule compound that is more sensitive in tumors with high PRADX and EZH2 expression
1. Experimental methods
In the embodiment, a computer structure is used for simulating a 3D structure of PRADX and a 3D structure of EZH2, and important targets are screened by molecular simulation of structure prediction and interaction of a PRADX and EZH2 compound. The ChemDiv and Specs small molecule databases were selected as screening databases for analysis of binding patterns and screening for optimal compounds in combination with in vitro toxicity assays (IC 50).
The experimental procedure for in vitro toxicity experiments (IC 50) is as follows:
selected GBM cell lines include: U87-MG, LN229, U251-MG, T98G, DMEM medium; GBM primary cell lines include: TBD0220, TBD0118, N33, N9, DMEM/F-12 medium was used. The medium is prepared into a complete medium containing 10% FBS and 1% penicillin-streptomycin (P/S), the cells are placed at 37 ℃ and the content of 5% CO 2 The constant temperature cell incubator of (1) for culture.
In vitro toxicity test (IC 50) adopting CCK-8 method, a blank control group, a DMSO control treatment group and an EPIC0307 drug treatment group are respectively arranged, wherein, the concentration gradient of EPIC0307 is set as 1, 5, 10, 15, 20, 25, 30, 35 and 40 μ M. According to experimental groups, 3-5 duplicate wells are set per group, and 100. Mu.L of cell suspension is added to each well, containing 2000-3000 cells in logarithmic growth phase. Observing the cell state, diluting the drug to be detected to different concentrations by using a culture medium in advance when the confluence degree is 50-60%, absorbing and removing the culture medium, adding 100 mu L of culture medium containing different drug concentrations into each hole, adding 10 mu L of CCK-8 solution into each hole for 48h of drug treatment, incubating for 1-4 hours (color change in the hole plate can be observed), detecting the absorbance at 450nm by using an enzyme labeling instrument, and recording data. According to the calculation formula: cell survival (%) = (As-Ab)/(Ac-Ab), as is the OD value of drug-treated group, ab is the average OD value of blank control group, ac is the average OD value of DMSO control-treated group. Drug IC50 curves were plotted using GraphPad software.
2. Results of the experiment
In 4 compounds screened out, the compound EPIC0307 is selected to aim at the target G187-LYS639, as shown in figure 1, and the structural formula of EPIC0307 is shown as the formula (I); EPIC0307 has simple structure, small molecular weight and easy synthesis, and structural simulation shows that EPIC0307 can be combined in a combining pocket consisting of hydrophobic amino acids at two sides to form hydrogen bonds with the side chain of alanine 736 (A736) and lysine 574 (K574), as shown in figure 2. In addition, LYS574, LYS703 in the protein form a p-pi interaction with EPIC0307. The benzene rings of TYR701 and EPIC0307 also form a π - π stacking effect. Then, toxicity tests were performed on cell lines of a plurality of tumors of the glioma, as shown in fig. 3, EPIC0307 was found to have obvious tumor inhibition effect on cell lines of the glioblastoma U87-MG, LN229, T98G, U251 and primary cell lines TBD0220, N33, N9, and the IC50 was lower in cell lines with high PRADX expression, as shown in fig. 4, correlation analysis was performed on the IC50 of each cell line and the expression level of PRADX, and a statistically significant negative correlation was found. As shown in figure 6, EPIC0307 treated cells after transfection with PRADX had a lower IC50 compared to TBD0220 cell lines transfected with empty virus, and therefore EPIC0307 was more sensitive to tumor cell lines with high expression of PRADX and EZH 2.
Figure BDA0003719090950000101
Example 2 EPIC0307 can affect PRC2 recruitment by specifically blocking PRADX binding to EZH2, reduce H3K27me3 level at target gene locus, and make target gene transcriptionally expressed
1. Experimental methods
In the embodiment, RIP and ChIRP experiments are used for verifying that EPIC0307 can affect the recruitment of PRC2 by specifically blocking the combination of PRADX and EZH2, the concentration dependence and time dependence of the transcription level and the translation level of target genes CDKN1A and BBC3 are obviously increased by RT-PCR and Western Blot detection, and further ChIP experiments prove that EPIC0307 can reduce the H3K27me3 level of a target gene locus, the transcription level of the target gene is reduced by over-expressing PRADX, and interestingly, the transcription level of the target gene is increased obviously after over-expressing PRADX.
The cell lines selected in this experiment were TBD0220 and U87-MG (culture conditions were the same as those described in example 1), the cell state was observed, and EPIC0307 drug treatment was performed for 48 hours at EPIC0307 concentration gradients of 10, 15, 20, and 25. Mu.M. The time gradient is 0, 12, 24 and 48h, and the treatment concentration is 15 mu M. Control group was DMSO. CHIRP, RIP, CHIP experiments EPIC0307 treatment concentration was 15. Mu.M, treatment time was 48h.
(1) RIP experiments are used to identify the interaction of specific RNA molecules with binding proteins within cells. The specific antibody of the EZH2 is used for capturing endogenous RNA binding protein EZH2 in nucleus or cytoplasm, and non-specific RNA binding is prevented. Immunoprecipitation isolates EZH2 and its associated RNA together, and the RNA sequence was identified by RT-PCR. Throughout the experimental protocol, standard precautions should be taken to reduce ribonuclease contamination. The detailed experimental procedure is as follows:
preparation of complete RIP lysates: taking 100 mu L of RIP lysate, and adding 0.5 mu L of protease inhibitor and 0.25 mu L of RNase inhibitor; the cells were removed and washed twice with 10mL PBS buffer; adding 10mL of PBS buffer solution, scraping the cells by using a cell scraper, and transferring the cells into a centrifuge tube; centrifuging to precipitate cells, and discarding the supernatant; resuspending and uniformly mixing the cell precipitate by using complete RIP lysate, placing the centrifuge tube on ice for standing for 5min, cracking the cells by using the hypotonic action of the RIP lysate, and storing the cell lysate for later use. Magnetic beads were prepared. And (3) performing immunoprecipitation: preparing RIP immunoprecipitation buffer: 900 μ L of RIP immunoprecipitation buffer was required for each RIP reaction, and 35 μ L of 0.5M EDTA solution and 5 μ L of RNA inhibitor were added to 860 μ L of RIP wash buffer; placing the centrifuge tube on a magnetic frame, standing, removing the supernatant after the magnetic beads are adsorbed on the tube wall, and adding 900 μ L RIP immunoprecipitation buffer solution into each tube; centrifuging the RIP cell lysate, taking the supernatant to a new centrifuge tube, and adding 100 mu L of lysate into each RIP reaction to ensure that the total volume of the RIP reaction is 1mL; 10% input as 10% by taking 10. Mu.L of the lysate; mixing the centrifuge tubes evenly, and incubating for 3 hours to overnight; after centrifugation, placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the magnetic beads are adsorbed on the tube wall; adding 500 mu L of RIP washing buffer solution into each tube, and uniformly mixing; placing the centrifugal tube on a magnetic frame for standing, and discarding the supernatant after the magnetic beads are adsorbed on the tube wall; washing the magnetic beads with RIP washing buffer solution repeatedly; the immunoprecipitation efficiency was detected by western blotting: in the last washing process, 12.5. Mu.L of 5 × loading buffer was added to the suspension of magnetic beads, and the mixture was placed in a homomixer, and incubated with shaking to elute the proteins bound to the magnetic beads. The magnetic beads were pelleted by centrifugation and the supernatant was used for SDS-PAGE detection. RNA extraction and detection: preparing a proteinase K buffer solution: each system corresponded to 150. Mu.L proteinase K buffer, containing 117. Mu.L RIP wash buffer, 15. Mu.L 10% SDS, and 18. Mu.L 10mg/mL proteinase K; after discarding the supernatant, 150. Mu.L of proteinase K buffer solution was added to resuspend; adding 107. Mu.L of RIP washing buffer, 15. Mu.L of 10% SDS and 18. Mu.L of 10M/mL proteinase K in a total volume of 150. Mu.L, and shake-incubating to digest the protein; after centrifugation, placing the centrifuge tube on a magnetic frame, and transferring supernatant to a new tube after magnetic beads are adsorbed on the tube wall; 250 mul of RIP washing buffer solution is added into the supernatant of each tube; to each tube was added 400 μ L phenol: chloroform: isoamyl alcohol (125; adding salt solution I (50 μ L), salt solution II (15 μ L), precipitation enhancing solution (5 μ L) and anhydrous ethanol (850 μ L) into each tube, mixing, placing in a refrigerator at-80 deg.C to precipitate RNA, and standing for 1 hr to overnight; centrifuging and discarding the supernatant; adding 1mL of 80% ethanol, washing once, centrifuging, removing supernatant, and air-drying at room temperature; the RNA was resuspended using 10-20. Mu.L of enzyme-free water and placed on ice, at which time concentration determination, reverse transcription, qPCR and the like can be performed. Specific primer sequences are shown in table 1 below.
TABLE 1 primer sequences
Figure BDA0003719090950000111
Figure BDA0003719090950000121
(2) The ChIRP assay is an assay for studying the interaction between RNA and DNA, RNA and protein. The biotin probe set of PRADX is designed, PRADX is specifically pulled down through base complementary pairing, and simultaneously DNA, RNA or protein (RBPs) interacting with the PRADX is enriched, the protein EZH2 enriched by PRDAX is researched in the experiment, and the verification is carried out through Western Blot. The detailed experimental procedure is as follows:
the design of ChIRP probe is carried out aiming at the determined PRADX sequence, and the principle is as follows: (1) the RNA molecule is designed with 1 probe at every 100 nucleotides, (2) the GC content is about 45 percent, (3) the length of the probe is 20 nucleotide molecules, and (4) the adjacent probes are spaced with 60-80 nucleotides. 10 PRADX probes are designed according to the principle, the specific sequence is shown in the following table 2, and the sequence information of the probes 1-10 is respectively shown in SEQ ID NO. 25-34.
TABLE 2 ChIRP Probe sequence information
Figure BDA0003719090950000122
The designed probe sequence is handed over to Tianjin Jinzhi biology company for synthesis, and biotin labeling is needed to be added at the 3' end of the probe. The probe is dissolved in water without enzyme to a concentration of 50. Mu.M. The odd probe and the even probe are measured according to the following ratio of 1:1, respectively mixing the components together, subpackaging and storing in a refrigerator at the temperature of-20 ℃.
Cell preparation and cross-linking: digesting the cells with trypsin, and resuspending the cells with PBS buffer; centrifuging the cell suspension at room temperature for 5 minutes, removing the supernatant, adding glutaraldehyde crosslinking solution, and reversing and uniformly mixing at room temperature for 10 minutes; adding glycine to terminate the crosslinking, reversing for several times, mixing, and incubating at room temperature for 5 min; centrifuging and removing supernatant, and washing cells by PBS buffer solution; the supernatant was discarded by centrifugation, PBS was resuspended in cells, and the supernatant was discarded by centrifugation.
And (3) cracking ultrasonic cells: add lysis solution (containing 5. Mu.L of 200 Xprotease inhibitor and 5. Mu.L of RNase inhibitor), resuspend the cell pellet, place on ice, prepare for sonication, and fragment DNA ultrasonically. Taking out the cell lysate for later use, carrying out ultrasonic treatment on the remaining lysate, and centrifuging after ultrasonic treatment to precipitate fragmented DNA; mixing the same sample, taking out 5 mu L of the mixture, performing crosslinking and agarose gel electrophoresis with the non-ultrasonic sample taken out in the first step, and detecting the DNA fragmentation effect; mixing the DNA samples to be detected with 1 mu L of 6 Xloading buffer solution, adding the mixture into agarose gel for electrophoretic separation, and after the electrophoresis is finished, placing the gel into a DNA imager for imaging.
RNA enrichment: this step uses probes for RNA enrichment for RNA and protein extraction. RNA extraction: after RNA extraction, reverse transcription, qPCR and other experimental operations can be carried out. Protein extraction: adding the magnetic beads into a 2.5 × loading buffer directly, and shaking for 10min at 95 ℃; centrifuging, placing on a magnetic frame, and standing for 1min; the supernatant is removed to a new tube, at which time the protein can be directly detected by Western Blot or the like.
(3) ChIP is used to study the interaction of proteins with DNA in vivo, and is currently the best method for determining the genomic region to which a particular protein binds, or for determining the proteins that bind to a particular genomic region. The protein and DNA in the cells are crosslinked in a living cell state, the proteins and the DNA are randomly cut into small chromatin fragments in a certain length range by ultrasonic waves, and then the protein-DNA complex is immunoprecipitated by using a specific antibody against H3K27me3, thereby specifically enriching the DNA fragments bound by the target protein. After separation and purification, qPCR detection. The sequences of ChIP primers designed for the target gene CDKN1A and BBC3 promoter regions are shown in table 3 below.
Table 3 design ChIP primer sequence information for target gene CDKN1A, BBC3 promoter region
Figure BDA0003719090950000131
(4) RNA extraction, reverse transcription and real-time quantitative PCR: standard precautions should be taken to reduce contamination with ribonucleases. The detailed experimental procedure is as follows:
extraction of RNA: the cells were removed, washed twice with PBS, 1mL of TRIol was added to each well, lysed, transferred to an EP tube, and lysed at room temperature for 5 minutes to completely separate the nucleoprotein and nucleic acid from the cells. Adding 0.2mL of chloroform, shaking, standing, centrifuging, and layering to obtain a colorless and transparent water phase as the upper layer. The upper aqueous phase was transferred to a new EP tube, 0.5mL of isopropanol was added, left to stand, centrifuged and the supernatant was discarded, 1mL of 75% ethanol was added, mixed and centrifuged for 5 minutes. Discard the supernatant, air dry RNA precipitate for 5-10 minutes, add 20-30 μ L DEPC water, vortex and mix well. Total RNA concentration and purity were determined using a Nanodrop 1000 microspectrophotometer, with OD260/OD280 ratios close to 2.00 being preferred.
Reverse transcription: using Beijing gold
Figure BDA0003719090950000132
Uni RT&The qPCR Kit carries out rapid reverse transcription to synthesize the first strand cDNA. The labeling was done using a high pressure nuclease-free PCR tube. According to the concentration of the RNA sample, taking a sample with a corresponding volume of 1 mu g, and adding DEPC water into the sample to enable the volume of the sample to be 15 mu L; will be provided with
Figure BDA0003719090950000133
The Uni All-in-One Supermix for qPCR and gDNA Remover were mixed at a ratio of 4. After centrifugation and mixing, the mixture was put into a Thermal Cycler S1000 PCR instrument, and the program was set up: incubation was carried out at 65 ℃ for 5 minutes and at 85 ℃ for 5 seconds. Reverse transcription is initiated. And storing the reverse transcribed product at the temperature of-20 ℃.
Fluorescent quantitative PCR: primers were designed, synthesized by Kingchi, and stored at-20 ℃ using DEPC water to dissolve the primers at 100. Mu.M. The DNA was polymerase chain amplified using a ChemQ Universal SYBR qPCR Master Mix of Novonoprazan, running an Applied Biosystems QuantStudio 3 real-time fluorescent quantitative PCR system. Three duplicate wells were set for each sample, and the procedure was completed by reading CT values, calculating Δ Δ CT values with GAPDH as an internal reference, and evaluating the relative expression of the relevant genes in each sample. The reaction system and procedure are shown in Table 4, and the primer sequence information is shown in Table 5.
TABLE 4 reaction System and procedure for qPCR
Figure BDA0003719090950000141
TABLE 5 qPCR primer sequence information
Figure BDA0003719090950000142
(5) The detailed experimental procedure for western blot analysis is as follows:
after the cell culture dishes of the different treatment groups were taken out of the incubator and the cell culture fluid was discarded, the cell culture dishes were washed 2-3 times with pre-cooled PBS, followed by addition of an appropriate amount of cell lysis fluid (RIPA: PMSF = 100: 1), lysis was performed on ice for 30min, and then transferred to a 1.5mL centrifuge tube. The cell lysate collected in the centrifuge tube was centrifuged at 12000rpm × 15min at low temperature (4 ℃), the supernatant containing the protein was aspirated and transferred to a new 1.5mL centrifuge tube, the concentration of the collected protein was detected using the BCA kit, and each treatment histone was diluted to the same concentration using a 5 × loading buffer according to the measured protein concentration. The diluted protein solution was then boiled in boiling water for 7 minutes to denature the protein. After the denaturation is finished, the protein is put into a refrigerator at minus 80 ℃ for long-term storage, and repeated freezing storage is avoided. Mixing glue, electrophoresis, film transfer and color development. And placing the PVDF film on an exposure plate, dropwise adding ECL luminous liquid on the PVDF film, and placing the PVDF film on a gel imager for exposure and photographing.
2. Results of the experiment
As shown in fig. 5, RIP experiment results show that at 15 μ M concentration, EPIC0307 has significant inhibitory effect on binding of PRADX to EZH2, as shown in fig. 7, and CHIRP experiment results further demonstrate that 15 μ M EPIC0307 can effectively inhibit binding of PRADX to EZH2 and interfere with PRADX recruitment to PRC2, as shown in fig. 8, and that TBD0220 cells and U87-MG cells have significantly increased transcription levels of target genes CDKN1A and BBC3 after 48 hours of action of EPIC0307 at 15 μ M concentration, as shown in fig. 9and 10, and that TBD0 cells and U87-MG cells have concentration-dependent increase of transcription and translation levels of target genes CDKN1A and BBC3 after 48 hours of action of EPIC0307 at different concentrations (0 μ M, 10 μ M, 15 μ M, 20 μ M, 25 μ M), as shown in fig. 11 and 12, and that TBD0 cells and U0220 cells have concentration-dependence of transcription and translation levels of target genes epkn 7 at 15 μ M, 72h, and 24 μ h of epkn 0307 at 15 μ M, and 24h, respectively. As shown in fig. 14, the transcription levels of the target genes CDKN1A and BBC3 were significantly reduced after overexpression of PRADX, and EPIC0307 caused an increase in the target gene after overexpression of PRADX was more significant. Finally, as shown in fig. 13, it is verified by CHIP experiments in the present invention that EPIC0307 affects PRADX recruitment to PRC2 after blocking PRADX-EZH2 binding, so that the level of the target gene promoter region H3K27me3 is significantly reduced, and the target gene is transcriptionally activated.
Example 3 EPIC0307 causes cell cycle arrest, induces apoptosis, inhibits the Rb Signaling pathway, sensitizes the therapeutic effects of TMZ by inhibiting DNA Damage repair
1. Experimental methods
The selected cell lines of the experiment are TBD0220 and U87-MG (the culture conditions are the same as the previous conditions), EPIC0307 mu M is applied for treatment for 48h, a control group DMSO is used, western blotting is used for detecting P21, rb signal channel, cycle related protein, PUMA and apoptosis related protein; EPIC0307 is treated for 48h by TBD0220, U87-MG at gradient concentrations of 10, 15, 20 and 25 mu M, and DMSO is used as a control group, and the cell cycle and apoptosis are detected by flow cytometry; in combination with IC50, EPIC0307 sets concentration gradients 0, 5, 10, 15, 20. Mu.M, corresponding to TBD0220, TMZ sets concentration gradients 0, 50, 100, 200, 400, 800. Mu.M, corresponding to U87-MG sets concentration gradients 0, 200, 400, 800, 1200, 1600. Mu.M; TBD0220 and U87-MG are respectively provided with a DMSO control group, EPIC0307 mu M and TMZ 200 mu M, a combination group of EPIC0307 mu M and TMZ 200 mu M, cell proliferation is detected by plate cloning, and the detection of proteins such as DNA damage repair and apoptosis is detected after respective treatment for 48 h; TBD0220 and U87-MG are respectively provided with a DMSO control group, EPIC0307 mu M and treated for 48h, and RT-PCR is carried out to detect the downstream DNA repair index of E2F 1.
(1) Western blot analysis experiments were as before.
(2) The experimental procedure for flow cytometry was as follows:
in direct immunofluorescence staining, cells are incubated with antibodies directly coupled to a fluorescent dye. Well-grown cells were taken, trypsinized and resuspended in PBS. PBS was aspirated, resuspended in 70% ethanol, blown into single cells, and the cells were fixed at room temperature. Centrifuged and washed twice with PBS. The PI staining solution was diluted with PBS and the cells were resuspended. When apoptosis is detected, FITC staining solution is added, and incubation is carried out in dark. Cycle and apoptosis were detected on demand using flow cytometry.
(3) The experimental procedure was as before in combination with in vitro toxicity experiments (IC 50), wherein EPIC0307 was set up concentration gradients 0, 5, 10, 15, 20. Mu.M for selected cell lines TBD0220, U87-MG, each gradient being in combination with TMZ, concentration gradients 0, 50, 100, 200, 400, 800. Mu.M for TBD0220 and 0, 200, 400, 800, 1200, 1600. Mu.M for U87-MG.
(4) The experimental procedure for plate cloning is as follows:
taking tumor cells in a good growth state, digesting and centrifuging, then resuspending the cells by using a culture medium and blowing the cells into a single cell suspension for later use. Each well was seeded with 2000 cells and contained 2mL of medium. DMSO, EPIC0307. Mu.M, TMZ 200. Mu.M, EPIC0307. Mu.M in combination with TMZ 200. Mu.M were added to TBD0220 and U87-MG cell lines after 24 hours. Then cultured in an incubator for 2 weeks. The culture was terminated when macroscopic clumps of cells appeared in the plates. Washed 2 times with PBS and then fixed for 30 minutes at room temperature by adding 4% paraformaldehyde. The stationary liquid is discarded, a small amount of crystal violet dye solution is added for 30 minutes at room temperature, the six-hole plate is washed, and air drying is carried out. The six-well plate was inverted and a piece of film with a grid was placed underneath, the number of clones counted and photographed.
(5) The experimental processes of RNA extraction, reverse transcription and real-time quantitative PCR are the same as the previous ones. The qPCR primer sequences are shown in table 6 below.
TABLE 6 qPCR primer sequence information
Figure BDA0003719090950000161
(6) The experimental procedure for immunofluorescent staining was as follows:
the treated slides were placed in 12-well plates, the cells were plated in the 12-well plates at the appropriate density, after the treatment, washed 2 times with PBS, fixed for 10min with 4% paraformaldehyde, washed 3 times with PBS, 1 triton for membrane disruption 15min, washed 3 times with PBS, blocked for 30min with 5% BSA, after which the slide was incubated at 4 ℃ for one antibody overnight, washed 3 times with PBS, 5min each, the sections were incubated at 37 ℃ for two antibodies for 1h, washed 3 times with PBS, 5min each, all the sections were stained with DAPI for 10min, and finally photographed with a confocal microscope (TCS SP5, leica).
2. Results of the experiment
As shown in FIG. 15, in glioblastoma TBD0220 and U87-MG cell lines, after treatment with 15. Mu.M EPIC0307 for 48h, P21 is significantly increased, which causes changes in cyclin-related proteins, rb pathway is inhibited, P-Rb is reduced, and E2F1 expression is reduced. As shown in FIG. 16, the results of flow cell cycle assays showed that EPIC0307 concentration-dependently arrests cell cycle in the G1 phase in glioblastoma TBD0220, U87-MG cell line. As shown in FIGS. 17 and 19, PUMA expression was significantly increased in TBD0220 and U87-MG cell lines treated with 15. Mu.M EPIC0307 for 48h, causing the change in expression of the relevant apoptotic proteins, and as shown in FIG. 18, the results of flow cell cycle assays showed that EPIC0307 induced apoptosis in glioblastoma TBD0220 and U87-MG cell lines in a concentration-dependent manner. As shown in fig. 20 and 21, in TBD0220 and U87-MG cell lines, EPIC0307 and temozolomide have synergistic effect, as shown in fig. 22 and 23, the results of plate cloning show that the number of cell clones after combined treatment with 10 μ M EPIC0307 and 200 μ M temozolomide is significantly reduced, i.e. EPIC0307 and temozolomide have better cytotoxicity, and both have synergistic therapeutic effect. As shown in fig. 24, EPIC0307 in combination with temozolomide is able to induce apoptosis in more tumor cells. Further studies found that the transcription level of the downstream associated DNA repair target of E2F1 was significantly reduced in TBD0220, U87-MG cell lines after 48h treatment with 10 μ M EPIC0307, as shown in figure 25. As shown in fig. 26, the expression of DNA damage repair-related protein was significantly increased in TBD0220, U87-MG cell lines after TMZ treatment, the expression of DNA damage repair protein was significantly decreased and the expression of DNA damage protein γ -H2AX was increased after the combined use of EPIC0307 and temozolomide, which indicates that EPIC0307 sensitizes the therapeutic effect of TMZ by inhibiting DNA damage repair.
Example 4 therapeutic effects of EPIC0307 on inhibition of glioblastoma in situ model proliferation and sensitization of TMZ and Experimental methods
(1) The experimental method for constructing the GBM orthotopic xenograft glioma mouse model is as follows:
female BALB/c nude mice at 4 weeks of age were purchased from Beijing Vihe laboratory animal science and technology Co. Infecting a TBD0200 cell line by using Luciferase negative control lentivirus for 48h to obtain a cell line stably expressing Luciferase; digesting the cells conventionally to form a single cell suspension, using a blood cell counting plateCounting cells, centrifuging, discarding supernatant, washing serum with precooled PBS, and adjusting concentration to 3-5 × 10 5 3 mu L of the seed/grain; the naked mouse is anesthetized by a conventional method, the exposed bone of the scalp is cut, the right side of the median connecting line of bregma and bregma is opened by 2mm, a hole is drilled, a 10 mu L micro-needle is used for sucking cell suspension, the naked mouse is fixed by using a stereotaxic apparatus, the needle insertion depth is 3mm, the needle withdrawal is 1mm, the micro-injection is 3 mu L, the needle is stopped for 1min after the fixation is finished, the naked mouse is taken out, the surface of the exposed bone is wiped by a sterilized cotton swab, and the skin is sutured; after 1 week, mice were randomized into DMSO groups, and mice were given TMZ (5 mg/kg/d,5 d/w), EPIC0307 (7.5 mg/kg/d), EPIC0307+ TMZ (7.5 mg/kg/d EPIC0307,5mg/kg/d TMZ) for 2 weeks of gavage treatment. On days 7, 14 and 21, mice were examined for intracranial tumor growth using bioluminescent imaging and survival recorded.
(2) H & E staining was used to show tumor volume size in nude mice, and expression of Ki67, r-H2AX, etc. in brain tumor samples were immunohistochemically stained.
2. Results of the experiment
To further demonstrate the tumor-inhibiting effect and the sensitizing effect of EPIC0307 on TMZ in vivo, this example constructed a mouse model of glioblastoma in situ using TBD0220 cell line. As shown in fig. 28, the mice were gavage at a dose of EPIC 0307.5 mg/kg for 2 weeks (1/day) and TMZ 5mg/kg for 2 weeks (1/day, 5 days/week), and as shown in fig. 29 and fig. 30 and fig. 32, the results of luminescence imaging and HE tissue staining of the mice showed a significant reduction in tumor volume in the EPIC0307 treated group compared to the DMSO solvent group and a significant reduction in tumor volume in the EPIC0307 combined TMZ treated group compared to the TMZ treated group alone. As shown in fig. 31, the survival analysis results showed that the survival of mice in the EPIC0307 treated group was significantly prolonged compared to the DMSO solvent group, and the survival of mice in the EPIC0307 combined TMZ treated group was significantly prolonged compared to the TMZ treated group alone. As shown in fig. 33, immunohistochemical staining results show that the changes of the tumor cell proliferation index Ki-67 and the DNA damage and repair related index are consistent with the results of in vitro experiments, i.e., EPIC0307 can significantly inhibit the proliferation of tumor cells in mouse model of glioblastoma in situ and enhance the therapeutic effect of TMZ.
Example 5 EPIC0307 in MGMT high expression cell line can inhibit MGMT expression and enhance TMZ therapeutic effect
1. Experimental method
In this example, the selected GBM cell line with high MGMT expression is T98G, and DMEM medium is used; the primary cell line of GBM TBD0118, DMEM/F-12 medium was used. The culture medium is prepared into a complete medium containing 10% FBS, 1% penicillin-streptomycin (P/S), the cells are placed at 37 deg.C, containing 5% CO 2 The constant temperature cell incubator. EPIC0307 drug treatments were given at T98G, TBD0118 with concentration gradients of 10, 15, 20, 25 μ M and treatment time of 48h. The time gradient is 0, 12, 24 and 48h, the treatment concentration is 15 mu M, and the transcriptional level and the translational level of ATF3 and MGMT detected by RT-PCR and Western Blot show concentration-dependent and time-dependent changes; T98G and TBD0118 are respectively provided with a DMSO control group, EPIC0307 mu M and TMZ 800 mu M, a combination group of EPIC0307 mu M and TMZ 800 mu M, the cell proliferation is detected by plate cloning, the cells are respectively treated for 48h, and protein detection such as DNA damage repair and apoptosis is detected; T98G and TBD0118 are respectively provided with a DMSO control group, EPIC0307 mu M and treated for 48h, and RT-PCR is carried out to detect E2F1 downstream DNA repair indexes.
(1) The experimental procedure was as before in combination with in vitro toxicity experiments (IC 50), where selected cell lines T98G, TBD0118 (culture conditions as before), EPIC0307 was set with concentration gradients 0, 5, 10, 15, 20. Mu.M, each gradient in combination with TMZ, set with concentration gradients 0, 400, 800, 1200, 1600, 2000. Mu.M.
(2) Western blot analysis experiments were as before.
(3) The experimental processes of RNA extraction, reverse transcription and real-time quantitative PCR are the same as the previous ones. The qPCR primer sequences are shown in table 7 below.
TABLE 7 qPCR primer sequences
Figure BDA0003719090950000181
(3) Plate cloning was performed as before.
(4) The experimental procedure for lentivirus infection was as follows:
tumor cells in good growth state are taken and paved on a six-hole plate, shATF3#1 and shATF3#2 lentiviruses are added into T98G cells after 12 hours according to the instruction, puromycin is added according to the resistance of a lentivirus vector after 2-3 days of culture, and after 1 week of screening, RNA and protein are extracted for carrying out over-expression identification and subsequent experiments on the cells. The shRNA sequences are shown in Table 8 below.
TABLE 8 shRNA sequences
Figure BDA0003719090950000182
(5) The experimental process of immunofluorescence staining was the same as before.
(6) The experimental procedure for ChIP is as before: wherein, the selected cell lines T98G and TBD0118 are provided with a DMSO control group, EPIC0307 mu M is treated for 48H, and a protein-DNA complex is immunoprecipitated by using a specific antibody of anti-H3K 27me3, thereby specifically enriching the DNA fragment bound by the target protein. After separation and purification, the ATF3 was detected by qPCR. Selecting a T98G cell line, selecting lentivirus stable transformation shATF3#1 and shATF3#2, setting T98G DMSO, T98G EPIC0307 muM, shATF3#1+ EPIC0307 muM and shATF3#2+ EPIC0307 muM, treating for 48H, and immunoprecipitating a protein-DNA complex by using a specific antibody of anti-H3K 27ac, thereby specifically enriching the DNA fragment combined with the target protein. After separation and purification, MGMT was detected by qPCR. ChIP primers are designed aiming at the promoter regions of the ATF3 and MGMT genes, and the primer sequences are shown in the following table 9.
TABLE 9 primer sequences
Figure BDA0003719090950000183
Figure BDA0003719090950000191
(7) Co-immunoprecipitation (Co-IP) experiments
The Co-IP experiment is a classical experiment for detecting protein interaction, and the principle is that a specific antibody is utilized to hook protein A, and the specific antibody of anti-protein B is incubated after SDS-PAGE electrophoretic separation to determine whether a binding relationship exists between A and B. The cell lines of the experimental cell line are T98G wt, T98G shATF3#1 and T98G shATF3#2+, DMSO and EPIC0307 mu M treatment are respectively arranged for 48h, and specific antibodies for resisting P-STAT3 are utilized to detect ATF3 and HDAC1.
2. Results of the experiment
As shown in fig. 34 and 35, MGMT was significantly highly expressed in the glioblastoma cell line T98G. As shown in fig. 36 and 37, EPIC0307 and temozolomide have a synergistic effect. As shown in fig. 38, EPIC0307 and temozolomide combined treatment groups were more cytotoxic to tumor cells compared to DMSO solvent group, EPIC0307 treatment group alone, and temozolomide treatment group alone. As shown in fig. 39, the results of Western blot assay showed that the expression levels of P21 and cyclin were significantly reduced, rb pathway was inhibited, and E2F1 expression was reduced in tumor cells treated with 10 μ M EPIC0307 for 48 hours. As shown in fig. 40, the transcription level of DNA damage repair-related proteins was significantly reduced in tumor cells after 48 hours of treatment with 10 μ M EPIC0307. As shown in fig. 41, the Western blot test results show that the expression of MGMT is significantly reduced, DNA damage protein is increased, and repair protein is reduced in tumor cells treated with 10 μ M EPIC0307 and 800 μ M temozolomide for 48 hours. As shown in fig. 42, the results of the immune confocal analysis show that the DNA damage index γ -H2AX was significantly increased when EPIC0307 and TMZ were used together. Further studies found that, as shown in fig. 43 and 45, the expression level of MGMT decreased in a concentration-dependent manner and the expression level of ATF3 increased in a concentration-dependent manner in tumor cells after 48 hours of treatment with different concentrations of EPIC0307. As shown in fig. 44 and 45, the expression level of MGMT decreased in a time-dependent manner and the expression level of ATF3 increased in a time-dependent manner in tumor cells treated with 10 μ M EPIC0307 at different times. In addition, as shown in fig. 46, EPIC0307 shows that the level of ATF3 promoter region H3K27me3 is significantly reduced by CHIP experiment, and ATF3 is transcriptionally activated. The shATF3 virus of the invention was further transfected to knock down the expression of ATF3, as shown in fig. 47, and the results show that the level of MGMT promoter H3K27ac was significantly reduced after treatment with EPIC0307, however, the level of MGMT promoter H3K27ac was restored after the ATF3 knock-down, as shown in fig. 48, and the level of O6-metG also follows this trend. The invention further proves that EPIC0307 can simultaneously inhibit the expression of MGMT and enhance the therapeutic effect of TMZ in a cell line with high MGMT expression.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
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<120> PRADX-EZH2 small molecule inhibitor and application thereof in preparing tumor treatment medicine
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aggtggacct ggagactctc ag 22
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tcctcttgga gaagatcagc cg 22
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gtctcctctg acttcaacag cg 22
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<400> 53
gtgtcagagt ctcccagtgg at 22
<210> 54
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cttgtttcgg cactttgcag ctg 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
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<210> 61
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 63
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gatccggaga agacggagtg cctgcactcg agtgcaggca ctccgtcttc tcctttttt 59
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<400> 66
ccgagattcg agctgagacc 20
<210> 67
<211> 21
<212> DNA
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tagtttgcca aatggcccgt a 21

Claims (12)

1. The application of the micromolecule inhibitor with the structural formula shown as the formula (I) in preparing the medicine for treating and/or preventing PRADX and/or EZH2 high-expression tumors;
Figure 957791DEST_PATH_IMAGE002
formula (I);
the tumor is a glioblastoma.
2. The use of claim 1, wherein the small molecule inhibitor is effective to block the binding of PRADX and EZH 2.
3. The use of claim 1, wherein the small molecule inhibitor interferes with the recruitment of PRC2 by PRADX.
4. The use of claim 3, wherein the small molecule inhibitor significantly reduces the level of CDKN1A, BBC3 promoter region H3K27me3 of the target gene of PRADX.
5. The use of claim 4, wherein the small molecule inhibitor retards the cell cycle at the G1/S phase and induces apoptosis.
6. The use of claim 5, wherein the small molecule inhibitor inhibits DNA damage repair.
7. The use according to claim 6, wherein the small molecule inhibitor is capable of inhibiting the STAT3 pathway and inhibiting the expression of MGMT.
8. The use of claim 7, wherein the small molecule inhibitor sensitizes the therapeutic effects of temozolomide.
9. The use of a small molecule inhibitor as claimed in claim 1 for the preparation of a sensitizer for a chemotherapeutic agent for treating tumors, wherein said chemotherapeutic agent for treating tumors is temozolomide;
the tumor is a glioblastoma.
10. Use of a small molecule inhibitor as claimed in claim 1 in combination with temozolomide for the preparation of a medicament for the treatment and/or prevention of a tumour;
the tumor is a glioblastoma.
11. Use of a small molecule inhibitor as claimed in claim 1 in the preparation of a blocker for blocking the cell cycle in the G1/S phase, inducing apoptosis;
the cell is a TBD0220 cell line or a U87-MG cell line.
12. The use of a small molecule inhibitor according to claim 1 for the manufacture of a medicament capable of sensitizing temozolomide to the therapeutic effect of glioblastoma.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAS号894949-88-3;STN数据库;《STN数据库中REGISTRY》;20060720;894949-88-3 *
LncRNA PRADX-mediated recruitment of PRC2/DDX5 complex suppresses UBXN1 expression and activates NF-κB activity, promoting tumorigenesis;Yansheng Li等;《Theranostics》;20210304;第11卷(第9期);第4516-4530页 *
STN数据库.CAS号894949-88-3.《STN数据库中REGISTRY》.2006, *

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