CN114984007A - PRADX-EZH2 small molecule inhibitor and application thereof in preparing tumor treatment medicine - Google Patents

Abstract

The invention discloses a PRADX-EZH2 small molecule inhibitor and application thereof in preparing tumor treatment medicines, wherein the structural formula of the small molecule inhibitor is shown as 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 preparing tumor treatment medicine

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 operation, 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 fatality rate only after pancreatic cancer and lung cancer, is located at the 3 rd position of a systemic 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 apoptosis. However, because glioblastoma is prone to developing resistance 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 has found that PRADX is highly expressed in glioblastoma and colon adenocarcinoma tumor tissues. Further research shows that PRADX can inhibit gene transcription by interacting with two hairpin structures at the 5' end of PRADX and the core protein EZH2 of Polycomb regenerative Complex 2 (PRC 2), and catalyzing the 27 th lysine of histone H3 to carry out trimethylation modification (H3K27 methylation, H3K27me3) by recruiting in a gene promoter region. Through a series of binding experiments, PRADX and EZH2 can directly interact, 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 complex shown in the formula (I) in the treatment of 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 first aspect of the invention provides an application of a small molecule inhibitor with a structural formula shown as a formula (I) in preparing a medicament for treating and/or preventing tumors;

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 and 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 drug for treating tumors (the micromolecule inhibitor with the structural formula shown in formula (I)) can inhibit STAT3 pathway, inhibit 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 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 pharmaceutical composition also comprises temozolomide.

Further, the diluents include (but are not limited to): lactose, sodium chloride, glucose, urea, starch, water, sugar powder, 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, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid, alginate, 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 octanoic acid, glyceryl decanoate, polyethylene glycol lauric acid glyceride, polyethylene glycol stearic acid 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, and the like.

Further, the pharmaceutically acceptable carriers and/or excipients added to the pharmaceutical composition are used as needed to aid the stability of the formulation or to aid the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

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 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.

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 manufacture 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 of PRADX, CDKN1A, BBC 3;

(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 blocking 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 a target gene from PRC2, lead the transcription of the target gene to be activated, increase the expression, inhibit the tumor proliferation and DNA damage repair, inhibit STAT3 pathway, inhibit the transcription and expression of MGMT, and enhance 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 representation 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 by the CCK8 test in a number of glioblastoma cell lines;

FIG. 4 is a graph of EPIC0307 versus PRADX expression, which is inversely correlated and statistically significant to IC 50;

FIG. 5 is a graph showing the results of RIP-qPCR assay, in which 15. mu.M EPIC0307 blocked PRADX-EZH2 binding after 48 h;

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 is a graph showing the results of detection in CHIRP co-immunoprecipitation assay, EPIC0307 was effective in blocking the binding of PRADX-EZH 2;

FIG. 8 is a graph showing the results of changes in RNA levels of the target genes CDKN1A, BBC3 after 48 hours of 15 μ M EPIC0307 treatment in TBD0220, U87-MG cell lines;

FIG. 9 is a graph showing the results of the RNA levels of the target genes CDKN1A, BBC3 as a function of the 0307 drug concentration 48 hours after EPIC0307 treatment in 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 the RNA levels of the target genes CDKN1A, BBC3 as a function of the 0307 drug treatment time after 15. mu.M EPIC0307 treatment in TBD0220, U87-MG cell lines;

FIG. 12 is a graph of the results of the change in protein levels of the target genes CDKN1A, BBC3 with EPIC0307 treatment time after 15 μ M EPIC0307 treatment in TBD0220, U87-MG cell lines;

FIG. 13 is a graph showing the results of confirming the decrease in enrichment of the promoter regions H3K27me3 of the target genes CDKN1A, BBC3 after 48 hours in EPIC 030715. 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 levels of CDKN1A and BBC3 after over-expression of PRADX were more significantly increased after 48 hours of EPIC 030715. mu.M treatment;

FIG. 15 is a graph showing that the expression of E2F1 is reduced by inhibiting the Rb pathway due to the change of cyclin after EPIC 030715. 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 to verify the change of apoptosis protein and apoptosis induction of EPIC 030715. mu.M after 48 hours of 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 change of apoptosis-related proteins Caspase 7and cleared Caspase3 after 48 hours of EPIC 030715. mu.M treatment by immunofluorescence assay;

FIG. 20 is a graph showing the results of the inhibition of cells using 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 cell formation experiment for plate clones, in which EPIC0307 at 10. mu.M and temozolomide at 200. mu.M were allowed to act for 2 weeks, and EPIC0307 inhibited cell colony 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 EPIC 030710. mu.M treatment for 48 hours;

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 are treated for 48 hours at corresponding concentrations;

FIG. 27 is a graph of the results of detecting changes in the transcription levels of PRADX and EZH2 by qPCR in 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, with changes in Ki67 and proteins involved in DNA damage repair;

FIG. 34 is a graph of the results of quantitative qPCR detection of background transcript levels of multiple tumor cell lines MGMT, with T98G cells as a normalization reference;

FIG. 35 is a graph showing the results of confirming the protein expression levels of MGMT of a plurality of tumor cell lines by Western blot assay;

FIG. 36 is a graph showing the results of the inhibition of cells by EPIC0307 in combination with temozolomide in the T98G cell line;

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 cell formation experiment with plate clones, in which EPIC0307 at 10. mu.M and temozolomide at 400. mu.M were allowed to act for 2 weeks, and EPIC0307 was able to inhibit cell clone formation and sensitize temozolomide;

FIG. 39 is a graph showing that the expression of E2F1 is decreased by inhibiting the Rb pathway due to the cyclin change and Rb pathway after 48 hours of EPIC 030710. mu.M treatment using Western blot;

FIG. 40 is a graph showing the results of detecting a decrease in the transcription level of DNA repair-related indicators by qPCR after EPIC 030710. mu.M treatment for 48 hours;

FIG. 41 is a result graph showing that the Western blot test shows that after EPIC 030710. mu.M and temozolomide 800. mu.M are treated for 48 hours, the 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 change in γ -H2AX after 48 hours of EPIC 030710. mu.M and temozolomide 800. mu.M treatment;

FIG. 43 is a graph showing the results of the RNA levels of MGMT and ATF3 after EPIC 030748 hours as a function of the EPIC0307 drug concentration gradient;

FIG. 44 is a graph showing the results of the RNA levels of MGMT, ATF3 after EPIC 030710. mu.M as a function of the EPIC0307 drug time gradient;

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 EPIC 030748 hours or EPIC 030710. mu.M treatment;

FIG. 46 is a graph showing the result of the decrease in the enrichment of the promoter region H3K27me3 of ATF3 after 48 hours of EPIC 030710. mu.M by CHIP experiment;

FIG. 47 is a graph showing that the decrease in enrichment of MGMT promoter region H3K27ac after 48 hours of EPIC 030710. mu.M and the decrease in enrichment of H3K27ac after transfection to lower ATF3 are restored by CHIP experiments;

FIG. 48 is a graph showing the results of detecting the changes of O6-metG in EPIC 030710. mu.M and temozolomide 800. mu.M after 48 hours of treatment by an immunopropfocusing 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 thus, the search for tumor treatment modalities for LncRNA 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 silencing target gene through epigenetics, so that the tumor progress 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 the structure 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 EPIC0307) was selected in combination with in vitro toxicity assay (IC50) for analysis of binding patterns. Compound 0307 (also called compound EPIC0307) is confirmed to be capable of specifically interfering the binding of PRADX and EZH2, thereby affecting the recruitment of PRC2 to target genes by PRADX, so that the important cancer suppressor genes in the target genes are released from transcriptional inhibition and expressed, thereby successfully inhibiting the proliferation of various tumors and inducing apoptosis.

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 gene. 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 interfering the combination of LncRNA PRADX and EZH2 to influence the recruitment of PRADX to PRC2 in the form of small molecule compound for the first time, changes the malignant appearance regulation of tumors, and provides a new drug treatment path and a new combined treatment scheme for treating the tumors.

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 value therebetween can be selected unless the invention otherwise indicated. 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 expression of PRADX and EZH2

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 interaction and structure prediction 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 conjunction with in vitro toxicity experiments (IC 50).

The experimental procedure for the in vitro toxicity test (IC50) was 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 culture medium is prepared into complete culture medium containing 10% FBS and 1% penicillin-streptomycin (P/S), and the cells are placed at 37 deg.C and contain 5% CO ₂ The constant temperature cell culture box is used for culturing.

In vitro toxicity test (IC50) adopts CCK-8 method, and blank control group, DMSO control treatment group, and EPIC0307 drug treatment group are respectively set, wherein, concentration gradient of EPIC0307 is set as 1, 5, 10, 15, 20, 25, 30, 35, 40 μ M. According to experimental groups, 3-5 duplicate wells were set per group, and 100. mu.L of cell suspension was 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 being the OD value of the drug-treated group, Ab being the average OD value of the blank control group, Ac being the average OD value of the 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 and EPIC0307 in the protein form p-pi interactions. The benzene rings of TYR701 and EPIC0307 also form a π - π stacking effect. Then, toxicity tests were performed on cell lines of multiple tumors of glioma, as shown in fig. 3, EPIC0307 was found to have obvious tumor inhibition effect on glioblastoma cell lines U87-MG, LN229, T98G, U251 and primary cell lines TBD0220, N33, N9, and IC50 was lower in cell lines with high PRADX expression, as shown in fig. 4, correlation analysis was performed on the expression levels of IC50 and PRADX of each cell line, and a statistically significant negative correlation was found. As shown in figure 6, EPIC0307 treated cells after transfection with PRADX had 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.

Example 2 EPIC0307 can affect PRADX recruitment to PRC2 by specifically blocking PRADX binding to EZH2, and reduce H3K27me3 level at target gene locus, so that target gene can be 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 PRADX from being combined with EZH2, the concentration-dependent and time-dependent significant increase is shown in the transcription level and translation level of target genes CDKN1A and BBC3 detected by RT-PCR and Western Blot, 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 target gene is given after over-expressing PRADX, and the transcription level of the target gene is increased more obviously.

The cell lines selected in this experiment were TBD0220 and U87-MG (culture conditions were the same as those described in example 1), and the cell state was observed, and EPIC0307 drug treatment was performed with EPIC0307 concentration gradients of 10, 15, 20, 25. mu.M for 48 h. The time gradient is 0, 12, 24 and 48h, and the treatment concentration is 15 mu M. Control group was DMSO. CHIRP, RIP, and CHIP experiments EPIC0307 was given at a treatment concentration of 15. mu.M for a treatment time of 48 h.

(1) RIP experiments are used to identify the interaction of specific RNA molecules with binding proteins within cells. The specific antibody against EZH2 was used to capture the endogenous RNA binding protein EZH2 in the nucleus or cytoplasm, preventing non-specific RNA binding. Immunoprecipitation isolated EZH2 along with its associated RNA, 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 1 mL; remove 10. mu.L from the lysate as 10% input; mixing the centrifuge tube 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 by 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 performing shake incubation 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 each tube of supernatant; to each tube was added 400 μ L phenol: chloroform: isoamyl alcohol (125:24:1pH 4.3), vortexing and centrifuging, absorbing 350 μ L of upper aqueous phase and transferring to a new EP tube, adding 400 μ L of chloroform, vortexing and centrifuging, absorbing 300 μ L of upper aqueous phase and transferring to a new EP tube; 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

(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 biotin probe set is enriched, the PRDAX enriched protein EZH2 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: firstly, designing 1 probe for every 100 nucleotides of RNA molecules, secondly, controlling the GC content to be about 45 percent, thirdly, controlling the length of the probe to be 20 nucleotide molecules, and fourthly, arranging adjacent probes at intervals of 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

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 minus 20 ℃.

Cell preparation and cross-linking: digesting the cells by using trypsin, and resuspending the cells by using a PBS buffer solution; 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, 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 rest 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 1 min; 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 is carried out. The sequences of ChIP primers designed for the target genes CDKN1A and BBC3 promoter region are shown in table 3 below.

Table 3 ChIP primer sequence information designed for target genes CDKN1A and BBC3 promoter region

(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 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

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

The Uni All-in-One Supermix for qPCR and gDNA Remover were mixed at a ratio of 4:1, and 5. mu.L was added to each PCR tube to give a final volume of 20. mu.L. 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 subjected to polymerase chain amplification using a ChamQ Universal SYBR qPCR Master Mix of Novowed, 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

TABLE 5 qPCR primer sequence information

(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 from the incubator, the cell culture solution was discarded, and then the cell culture dishes were washed 2 to 3 times with precooled PBS, followed by addition of an appropriate amount of cell lysis solution (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 EPIC0307 has a significant inhibitory effect on the binding of PRADX to EZH2 at 15. mu.M concentration, as shown in FIG. 7, and CHIRP experiment results further demonstrate that 15. mu.M EPIC0307 can effectively inhibit the binding of PRADX-EZH2 and interfere with the recruitment of PRADX to PRC2, as shown in FIG. 8, and that the transcriptional levels of target genes CDKN1A and BBC3 significantly increase after TBD0220 cells and U87-MG cells have been exposed to EPIC0307 at 15. mu.M concentration for 48 hours, as shown in FIGS. 9and 10, and the transcriptional and translational levels of target genes CDKN1, A and BBC3 have been significantly increased after EPIC0307 at different concentrations (0. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M) in TBD0220 cells and U87-MG cells, as shown in FIGS. 11 and 12, and after EPIC 03048 hours at different concentrations of EPIC 03048 hours, 72h) Then, the transcription and translation levels of the target genes CDKN1A and BBC3 are increased in a time-dependent manner. As shown in fig. 14, the transcript levels of the target genes CDKN1A and BBC3 were significantly reduced after overexpression of PRADX, and the increase of the target gene caused by EPIC0307 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 (culture conditions are the same as the previous conditions), EPIC 030715 mu M treatment is carried out for 48 hours, a control group DMSO is used, Western blotting is used for detecting P21, Rb signal channel, cycle related protein, PUMA and apoptosis related protein are detected; EPIC0307 gradient concentrations of TBD0220, U87-MG are treated for 48h at 10, 15, 20 and 25 mu M, control group DMSO, and 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 DMSO control group, EPIC 030710. mu.M and TMZ 200. mu.M, combined group EPIC 030710. 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, EPIC 030710 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. Centrifuge and wash 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 the 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 (IC50), 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, EPIC 030710. mu.M, TMZ 200. mu.M, combined EPIC 030710. mu.M and TMZ 200. mu.M were added to TBD0220 and U87-MG cell lines after 24 hours, respectively. 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 fixative was discarded, a small amount of crystal violet dye was added at room temperature for 30 minutes, the six well plates were rinsed and air dried. 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

(6) The experimental procedure for immunofluorescent staining was as follows:

the treated slides were placed in a 12-well plate, cells were plated on the 12-well plate at the appropriate density, after treatment, washed 2 times with PBS, fixed with 4% paraformaldehyde for 10min, washed 3 times with PBS, 1% triton for 15min, washed 3 times with PBS, blocked with 5% BSA for 30min, after which slides were incubated overnight at 4 ℃, washed 3 times with PBS for 5min each, sections were incubated at 37 ℃ for 1h with secondary antibody, washed 3 times with PBS for 5min each, all slides 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 μ M EPIC0307 for 48h, P21 was significantly increased, which caused changes in cyclin-related proteins, Rb pathway was inhibited, P-Rb was decreased, and E2F1 expression was decreased. As shown in fig. 16, the results of flow cell cycle assays showed that EPIC0307 blocked the cell cycle in the G1 phase in a concentration-dependent manner in glioblastoma TBD0220, U87-MG cell line. As shown in fig. 17 and 19, PUMA expression was significantly increased in TBD0220, U87-MG cell lines treated with 15 μ M EPIC0307 for 48h, causing changes 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, U87-MG cell lines in a concentration-dependent manner. As shown in fig. 20 and 21, in TBD0220, U87-MG cell lines, EPIC0307 and temozolomide have synergistic effect, and as shown in fig. 22 and 23, the results of plate cloning show that the number of cell clones treated by the combination of 10 μ M EPIC0307 and 200 μ M temozolomide is significantly reduced, i.e. EPIC0307 and temozolomide have better cytotoxicity in combination, and both have synergistic therapeutic effect. As shown in figure 24, EPIC0307 in combination with temozolomide was 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 fig. 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 after EPIC0307 and temozolomide were combined, and the expression of DNA damage protein γ -H2AX was increased, which indicates that EPIC0307 sensitizes the therapeutic effect of TMZ by inhibiting DNA damage repair.

Example 4 therapeutic effects of EPIC0307 capable of inhibiting proliferation of glioblastoma in situ model and sensitizing 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 for 48h by using a Luciferase negative control lentivirus to obtain a cell line stably expressing Luciferase; digesting cells conventionally to obtain single cell suspension, counting cells with a hemocytometer, centrifuging to remove supernatant, washing serum with precooled PBS, and adjusting the concentration to 3-5 × 10 ⁵ 3 mu L of the seed/seed; 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 (5mg/kg/d, 5d/w), EPIC0307 (7.5mg/kg/d), EPIC0307+ TMZ (7.5mg/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 status using bioluminescent imaging and survival was recorded.

(2) H & E staining was used to show tumor volume size in nude mice, and expression of Ki67, r-H2AX, etc. in immunohistochemical staining brain tumor samples.

2. Results of the experiment

To further demonstrate the in vivo tumor-inhibiting and TMZ-sensitizing effects of EPIC0307, a murine model of glioblastoma in situ was constructed using the TBD0220 cell line. As shown in fig. 28, the mice were gavaged at a dose of EPIC 03077.5 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 of the mice and the results of staining of HE tissue 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 with 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 EPIC0307 treated group was significantly prolonged compared to DMSO solvent group, and that of mice in EPIC0307 combined with TMZ treated group was significantly prolonged compared to TMZ treated group alone. As shown in fig. 33, immunohistochemical staining results show that changes in tumor cell proliferation index Ki-67 and DNA damage and repair-related indices are consistent with the results of in vitro experiments, i.e., EPIC0307 is capable of significantly inhibiting proliferation of tumor cells in mouse model of glioblastoma in situ and sensitizing the therapeutic effect of TMZ.

Example 5 EPIC0307 in MGMT-highly expressed cell line can simultaneously inhibit the expression of MGMT and enhance the therapeutic effect of TMZ

1. Experimental methods

The selected GBM cell line with high MGMT expression is T98G, and DMEM medium is used; the GBM primary cell line TBD0118, DMEM/F-12 medium was used. The culture medium is prepared into complete culture medium containing 10% FBS and 1% penicillin-streptomycin (P/S), and the cells are placed at 37 deg.C and contain 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 48 h. Time gradients of 0, 12, 24 and 48h, treatment concentration of 15 μ M, concentration-dependent and time-dependent changes of transcription level and translation level of ATF3 and MGMT detected by RT-PCR and Western Blot; T98G and TBD0118 are respectively provided with a DMSO control group, EPIC 030710 mu M and TMZ 800 mu M, a combination group of EPIC 030710 mu M and TMZ 800 mu M, the cell proliferation is detected by plate cloning, the treatment is respectively carried out 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, EPIC 030710 mu M and treated for 48h, and RT-PCR is carried out to detect the downstream DNA repair index of E2F 1.

(1) The experimental procedure was as before in conjunction with in vitro toxicity experiments (IC50), where selected cell lines T98G, TBD0118 (culture conditions as before), EPIC0307 was set at concentration gradients 0, 5, 10, 15, 20 μ M, each gradient in conjunction with TMZ, set at concentration gradients 0, 400, 800, 1200, 1600, 2000 μ 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

(3) Plate cloning was performed as before.

(4) The experimental procedure for lentivirus infection was as follows:

tumor cells in a good growth state are taken and paved into a six-well plate, shATF3#1 and shATF3#2 lentiviruses are added into T98G cells after 12 hours according to the instructions, 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 over-expression identification and subsequent experiments of the cells. The shRNA sequences are shown in Table 8 below.

TABLE 8 shRNA sequences

(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, EPIC 030710 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 isolation and purification, ATF3 was detected by qPCR. Selecting a T98G cell line, selecting lentivirus stable transformation shATF3#1 and shATF3#2, setting T98G DMSO, T98G EPIC 030710 mu M, shATF3#1+ EPIC 030710 mu M, shATF3#2+ EPIC 030710 mu M, treating for 48H, and immunoprecipitating a protein-DNA complex by using a specific antibody against H3K27ac, thereby specifically enriching the DNA fragment bound by the target protein. After separation and purification, MGMT was detected by qPCR. ChIP primers are designed aiming at ATF3 and MGMT promoter regions of genes, and the sequences of the primers are shown in the following table 9.

TABLE 9 primer sequences

(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 EPIC 030710 mu M treatment are respectively arranged for 48h, and specific antibodies of anti-P-STAT 3 are utilized to detect ATF3 and HDAC 1.

2. Results of the experiment

As shown in fig. 34 and 35, MGMT was significantly highly expressed in 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 the Western blot assay showed that the expression levels of P21 and cyclin were significantly reduced, Rb pathway was inhibited, and the expression of E2F1 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-associated protein was significantly reduced in tumor cells after 48 hours of treatment with 10 μ M EPIC 0307. 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 indicator 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 EPIC 0307. 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 and ATF3 is transcriptionally activated through CHIP experiment verification. The shATF3 virus transfected by the invention knocks down the expression of ATF3, as shown in FIG. 47, and the result shows that the level of MGMT promoter H3K27ac is obviously reduced after EPIC0307 treatment, however, the level of MGMT promoter H3K27ac is restored after ATF3 knockdown, as shown in FIG. 48, and the level of O6-metG also meets the 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

<110> subsidiary hospital of Hebei university

<120> PRADX-EZH2 small molecule inhibitor and application thereof in preparing tumor treatment medicine

<141> 2022-06-28

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tagtttgcca aatggcccgt a 21

Claims (10)

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 tumors;

2. the use according to claim 1, wherein the tumor is a tumor highly expressing 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.

3. The use of claim 1, wherein the small molecule inhibitor is effective to block the binding of PRADX and EZH 2.

4. The use of claim 1, wherein the small molecule inhibitor interferes with the recruitment of PRADX to PRC 2;

preferably, the small molecule inhibitor can significantly reduce the levels of the target gene CDKN1A, 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 and inhibit MGMT expression;

preferably, the small molecule inhibitor can enhance the therapeutic effect of temozolomide.

5. A pharmaceutical composition for treating and/or preventing tumors, comprising the small molecule inhibitor of claim 1;

preferably, the pharmaceutical composition consists of a therapeutically effective amount of the small molecule inhibitor described in claim 1 and a pharmaceutically acceptable carrier and/or adjuvant;

more preferably, the pharmaceutically acceptable carrier and/or adjuvant includes diluent, binder, surfactant, humectant, adsorbent 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 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.

6. The pharmaceutical composition of claim 5, further comprising temozolomide.

7. 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;

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;

most preferably, the tumor is a glioblastoma.

8. Sensitizer for tumor chemotherapy drugs, comprising the small molecule inhibitor of claim 1, wherein said tumor chemotherapy drugs are temozolomide.

9. 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;

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.

10. The use of any one of the following aspects, wherein said use comprises:

(1) use of a small molecule inhibitor as claimed in claim 1 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 claimed in claim 1 in the manufacture of an interfering agent for interfering with the recruitment of PRADX to PRC 2;

(3) use of a small molecule inhibitor as claimed in claim 1 in the manufacture of a promoter for increasing the level of a target gene of PRADX, CDKN1A, BBC 3;

(4) use of a small molecule inhibitor as claimed in claim 1 for 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 claimed in claim 1 in the preparation of a blocker for blocking the cell cycle in the G1/S phase, inducing apoptosis;

(6) use of a small molecule inhibitor as claimed in claim 1 in the preparation of an inhibitor for inhibiting DNA damage repair;

(7) use of a small molecule inhibitor as claimed in claim 1 in the preparation of an inhibitor for inhibiting the STAT3 pathway, inhibiting MGMT expression;

(8) use of the sensitiser of claim 8 in the manufacture of a medicament capable of sensitising the therapeutic effects of temozolomide.

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