CN114558014A

CN114558014A - Novel PRMT5 inhibitor and application thereof in treating breast cancer

Info

Publication number: CN114558014A
Application number: CN202210334932.4A
Authority: CN
Inventors: 陈苏宁; 张健
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-05-31
Anticipated expiration: 2042-03-31
Also published as: CN114558014B

Abstract

The invention discloses a novel PRMT5 inhibitor and application thereof in treating breast cancer, belonging to the technical field of research on antitumor drugs. The invention carries out reconstruction on Tadalafil by computer-aided design, obtains 3 Tadalafil derivative compounds by screening, and names ZJ-781, ZJ-782 and ZJ-783. The ZJ-78 series compounds can bind with PRMT5 and inhibit PRMT5 function. In vivo and in vitro experiments further show that the ZJ-78 series compounds can inhibit the proliferation of breast cancer cells, promote apoptosis and promote the anti-tumor effect of chemotherapeutic drugs such as adriamycin, cisplatin and olaparib.

Description

Novel PRMT5 inhibitor and application thereof in treating breast cancer

Technical Field

The invention belongs to the technical field of research on antitumor drugs, relates to design synthesis of a new compound and research on antitumor effect, and particularly relates to a novel PRMT5 inhibitor and application thereof in treatment of breast cancer.

Background

In recent years, with the frequent occurrence of disease diversification and resistance problems, the demand for drugs has been increasing. The development of new drugs often has the risks of long development period, high cost and low success rate. The computer-aided drug design can greatly shorten the drug research and development time and improve the drug research and development efficiency in multiple aspects of drug target identification, active compound screening, compound property prediction, compound molecule reconstruction and the like. The molecular docking technology is a core technology of computer-aided drug design, and is characterized in that the technology is used for screening based on the structure of a biological macromolecule, simulating the three-dimensional structure of the combination of a small molecule and the biological macromolecule, calculating the combination strength of the small molecule and the biological macromolecule, comprehensively evaluating the combination activity of the small molecule and the biological macromolecule, and having good accuracy and high efficiency.

Protein arginine methyltransferase (PRMTs) is an important class of enzymes that catalyze the methylation of arginine in chromatin histones, and can catalyze the transfer of a methyl group on S-adenosyl-L-methionine (SAM) to arginine. In eukaryotes, methylated arginine can be divided into three main forms: monomethyl Arginine (MMA), asymmetric dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA). The family of PRMTs has 9 members in total, and is classified into three types according to its type of catalyzing the methylation of arginine. Type I PRMT includes 1, 2, 3, 4, 6, catalyzing monomethylation and asymmetric dimethylation of arginine residues, type II PRMT includes 5 and 9, catalyzing monomethylation and symmetric dimethylation, and type III PRMT includes 7, catalyzing only monomethylation. PRMT5 is the major type II PRMT, the N-terminus is the phosphoglycerate isomerase barrel structure, binds to the methylated-small-body protein 50(WD repeat-contacting protein methyl protein 50, MEP50), and the C-terminus is the methyltransferase catalytic domain. PRMT5 usually exists as a tetramer and forms a heterocomplex with its partner MEP 50.

The PRMT5 is highly expressed in various tumors, and the high expression of PRMT5 is closely related to the reduction of the survival rate of patients. Inhibition of PRMT5 in tumors causes DNA damage, functional defects associated with homologous recombination repair, and reduced genomic stability, and may also promote activation of the P53 signaling pathway, leading to cell cycle arrest and cell death.

Therefore, due to the important role of PRMT5 in tumors, the study of its inhibitors has become a hotspot. Currently, although the PRMT5 inhibitors such as GSK3326595, JNJ-64619178 and PF-06939999 which have been reported so far have entered clinical trials, no inhibitors are yet on the market, which is a significant challenge in the development of PRMT5 inhibitors. Tadalafil (Tadalafil) is a phosphodiesterase 5 (PDE 5) inhibitor, has high efficiency, selectivity and reversibility, and has good effects in improving male erectile dysfunction, treating pulmonary hypertension, prostatic hyperplasia, myocardial hypertrophy and the like. In the marketed drugs, Tadalafil is a novel PRMT5 inhibitor, which can effectively improve the sensitivity of tumor cells to therapeutic drugs such as adriamycin and the like in breast cancer, but the binding capacity and the anti-tumor activity of Tadalafil and PRMT5 are still to be further improved.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a novel PRMT5 inhibitor and application thereof in treating breast cancer, and can solve the technical problems of low binding capacity of the conventional PRMT5 inhibitor and PRMT5 and low antitumor activity.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a novel PRMT5 inhibitor, which comprises a compound with the following structural formula:

or, a pharmaceutically acceptable salt thereof.

The invention discloses application of the novel PRMT5 inhibitor in preparing a medicament for treating breast cancer.

Preferably, the drug is a drug that inhibits the proliferation of MDA-MB-231 cells, MCF-7 cells and HCC1937 cells.

The invention discloses application of the novel PRMT5 inhibitor in preparing a chemosensitizer.

Preferably, the novel PRMT5 inhibitor is capable of potentiating the anti-tumor effect of the chemotherapeutic drugs doxorubicin, cisplatin and olaparib.

A pharmaceutical composition comprising the novel PRMT5 inhibitor described above and a pharmaceutically acceptable diluent or excipient.

The medicine for treating breast cancer is characterized by consisting of the novel PRMT5 inhibitor and a chemotherapeutic medicine.

Preferably, the chemotherapeutic agent is doxorubicin, cisplatin or olaparib.

Compared with the prior art, the invention has the following beneficial effects:

the method comprises the steps of reconstructing Tadalafil through computer-aided design, wherein firstly, groups containing heteroatoms such as nitrogen, oxygen, sulfur and the like are arranged on three parts (piperazine, indole and benzene rings connected with a mother ring) of the Tadalafil molecule which are relatively independent, so that more hydrogen bonds are generated between the Tadalafil molecule and protein; secondly, different alkyl substituents are introduced, so that the Tadalafil molecule and the protein generate more hydrogen bonds, and the combination of the Tadalafil molecule and the protein through hydrophobic interaction or aromatic groups is enhanced; thirdly, changing the chirality of the chiral center, and exploring the influence of the chiral change on the binding process of the Tadalafil molecule and the protein; fourthly, the modifications are introduced into Tadalafil molecules at the same time, the comprehensive influence of the modifications on the combination of the Tadalafil molecules and the proteins is explored, and 3 Tadalafil derivative compounds are obtained by molecular docking analysis and screening and named as ZJ-781, ZJ-782 and ZJ-783. The ZJ-78 series compounds can bind with PRMT5 and inhibit PRMT5 function. In vivo and in vitro experiments further show that the ZJ-78 series compounds can inhibit the proliferation of breast cancer cells, promote apoptosis and promote the anti-tumor effect of chemotherapeutic drugs such as adriamycin, cisplatin and olaparib.

Drawings

FIG. 1 is a schematic synthesis of a ZJ-78 series of compounds of the present invention;

FIG. 2a is a nuclear magnetic spectrum of ZJ-781;

FIG. 2b is a NMR spectrum of ZJ-782;

FIG. 2c is a nuclear magnetic spectrum of ZJ-783;

FIG. 3 shows the SPR analysis results of ZJ-78 series compounds and PRMT5 protein; wherein, (a) and (b) are respectively an influence result graph of ZJ-781 time and concentration; (c) and (d) graphs the effect of ZJ-782 time and concentration, respectively; (e) and (f) graphs of the effect of ZJ-783 time and concentration, respectively;

FIG. 4 is a graph of the ZJ-78 series of compounds of the present invention inhibiting the enzymatic function of PRMT 5;

FIG. 5 is a graph showing the results of cell viability of MDA-MB-231, MCF-7 and HCC1937 cells treated with ZJ-78 series of compounds at different concentration gradients;

FIG. 6 is a graph showing the results of apoptosis detection of ZJ-78 series compounds at a concentration of 150. mu.M and Tadalafil-treated MDA-MB-231 cells;

FIG. 7 shows the results of testing cell viability for ZJ-78 series of compounds and Tadalafil by treating MDA-MB-231 and MCF-7 cells with doxorubicin added at corresponding concentration gradients;

FIG. 8 shows the results of cell viability of the ZJ-78 series of compounds and Tadalafil treated MDA-MB-231 and MCF-7 cells with the addition of a corresponding concentration gradient of cisplatin;

FIG. 9 shows the results of Olaparib cell viability test in which ZJ-78 series compounds and Tadalafil treated MDA-MB-231 and MCF-7 cells were added with corresponding concentration gradients;

FIG. 10 shows the results of the apoptosis assay of MDA-MB-231 cells treated with ZJ-781 and Tadalafil, respectively, in combination with doxorubicin; wherein (a) is Annexin V/PI double dyeing result; (b) the data are statistical data;

FIG. 11 is a typical photograph of MDA-MB-231 taken from subcutaneous tumor;

FIG. 12 is a tumor growth curve of an MDA-MB-231 subcutaneous tumor;

FIG. 13 is a statistical analysis of the volume and mass of MDA-MB-231 tumors when they were drawn; wherein, (a) is tumor volume; (b) is the tumor mass;

FIG. 14 shows Ki67 staining to detect tumor tissue proliferation; wherein, (a) a photograph of the proliferation; (b) is statistical result data.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

1. synthesis of ZJ-78 series compounds by molecular docking

First, the binding ability of the reported PRMT5 inhibitor to PRMT5 was analyzed using the Sybyl-X2.0 software. Minimizing energy of all compounds in Sybyl-X2.0 software by using a Tripos force field, wherein the distance is taken as a dielectric function, the charge is Gasteiger-Hockel atomic charge, the lowest energy conformation is obtained by optimizing the charge by a Powell energy gradient method, the maximum energy optimization frequency is 10000, and the energy convergence standard is 0.001 kcal.mol^-1Mimicking the stable conformation of molecules in the natural system. The PRMT5: MEP50 complex (PDB:4gqb) crystal structure was downloaded from the PDB database, and Sybyl-x 2.0 was introduced for molecular docking. The butt joint adopts a semi-flexible butt joint method and is carried out by using a Surflex-Dock moduleAnd (6) butting. The total score in Surflex-Dock takes into account factors such as polarity, hydrophobicity, entropy and solvation, which reflect the binding affinity of the ligand to the receptor. The larger the score, the more stable the ligand binding to the receptor and the more likely there is interaction. Butt joint branch>And 7 points, which indicates that strong binding between the molecule and the target point possibly exists,>and 5, the molecule and the target point can be better combined.

The results are shown in table 1 below, and the binding capacity of Tadalafil to PRMT5 is still to be further improved compared to EPZ015666 and other inhibitors:

TABLE 1

Name (R)	Score of	Energy of butt joint	Polarity
				CMP-5	6.2627	-0.763	0.1182
PJ-68	6.5977	-1.5616	0
				EPZ015666	6.931	-1.8667	1.003
HLCL-61	6.5104	-2.9024	0
				HLCL-65	6.3567	-1.2981	0.9458
Tadalafil	6.0896	-0.8536	0.0012

Thus, Tadalafil was modified as follows: firstly, groups containing heteroatoms such as nitrogen, oxygen, sulfur and the like are arranged on three parts (piperazine, indole and benzene rings connected with a mother ring) of the Tadalafil molecule which are relatively independent, so that the Tadalafil molecule and the protein generate more hydrogen bonds; secondly, different alkyl substituents are introduced, so that more hydrogen bonds are generated between the Tadalafil molecule and the protein, and the combination of the Tadalafil molecule and the protein through hydrophobic interaction or aromatic groups is enhanced; thirdly, changing the chirality of a chiral center, and exploring the influence of the chiral change on the binding process of the Tadalafil molecule and the protein; fourthly, the above modifications were introduced into the Tadalafil molecule simultaneously, exploring the combined effect of these modifications on the binding of the Tadalafil molecule to the protein.

Then, molecular docking analysis is carried out again on the simulated molecular structure after modification, 3 compounds with the highest scores are screened out from the 90 obtained compounds and are respectively named as ZJ-781, ZJ-782 and ZJ-783, and the docking scores and the chemical structures of the 3 compounds are as follows:

2. chemical synthesis process of ZJ-78 series compound

The synthesis method is shown in figure 1, and comprises the following specific synthesis steps:

(1) treating D-tryptophan methyl ester hydrochloride with sodium bicarbonate to obtain D-tryptophan methyl ester, dissolving the D-tryptophan methyl ester hydrochloride serving as a raw material with Dichloromethane (Dichloromethane, DCM, 10mL/g of the raw material), adding 1.08 equivalents of aldehyde compound into a reaction bottle, fully replacing the aldehyde compound with nitrogen, cooling a reaction system to about 0 ℃, slowly dropwise adding 2.05 equivalents of trifluoroacetic acid, and raising the temperature to room temperature for reacting completely after the addition is finished.

And (3) post-treatment: after the reaction is completed, adjusting the pH of the reaction solution to about 9, extracting with DCM, combining organic layers, spin-drying through a column (200-mesh 300-mesh silica gel column chromatography: petroleum ether/ethyl acetate 150/1-80/1), and obtaining ZJ-781-1/ZJ-782-1/ZJ-783-1 (if the reaction raw material is not pretreated by sodium bicarbonate, the reaction time is longer, the reaction is more complicated, the product has isomers, the low-polarity product is the target compound, and the yield is about 35 percent basically).

(2) Placing ZJ-781-1/ZJ-782-1/ZJ-783-1 and 1.2 equivalents of sodium bicarbonate into a reaction bottle, adding chloroform (20mL/g of raw material) for dissolving, fully replacing with nitrogen, cooling the reaction system to 0 ℃, slowly adding 2.55 equivalents of chloroacetyl chloride, heating to room temperature after the addition is finished, and stirring until the reaction is complete (about 1 h);

and (3) post-treatment: dilute with DCM, wash with sodium bicarbonate, wash with water, combine the organic layers, dry over anhydrous sodium sulfate, and spin dry. ZJ-781-2/ZJ-782-2/ZJ-783-2 can be obtained by the same synthesis method (the post-treatment of the reaction is relatively simple, and the purer target product can be obtained by adding ether for ultrasonic treatment and filtering, ZJ-782-2 is not suitable for the treatment of the method, and the direct rotary drying is adopted for the next reaction, so that the yield is basically about 80 percent).

(3) Putting ZJ-781-2/ZJ-782-2/ZJ-783-2 into a reaction bottle, adding methanol for dissolving (35mL/g of raw material), fully replacing with nitrogen, adding 2 equivalents of methylamine alcohol solution, heating to reflux after the addition is finished, and stirring until the reaction is complete (about 16 h);

and (3) post-treatment: after the reaction is finished, directly spin-drying ZJ-781, and pulping by petroleum ether/ethyl acetate (3/1) to obtain a relatively pure target product, wherein ZJ-782/ZJ-783 is purified by a column through the synthetic method, and the yield of (200-mesh 300-mesh silica gel column chromatography: 150/1-80/1) is basically about 75%.

The nuclear magnetic spectra of the three synthesized compounds ZJ-781, ZJ-782 and ZJ-783 are shown in FIG. 2a, FIG. 2b and FIG. 2c, respectively.

Affinity analysis of ZJ-78 series Compounds with PRMT5 protein

Based on Biacore T200 analysis platform and Surface Plasmon Resonance (SPR) technology, the ZJ-78 series compound designed and synthesized is subjected to binding affinity analysis with target protein PRMT 5. The medicine Tadalafil is a product of MCE company, and has the catalog number HY-90009A. The PRMT5/MEP50 purified protein is a product of Active Motif company, catalog number 31521. The dilution concentrations of Tadalafil were in order: 31.75, 62.5, 125, 250, 500. mu.M. Referring to fig. 3, the results show that the compounds of the ZJ-78 series have better affinity with PRMT5 protein.

Functional analysis of ZJ-78 series of compounds for inhibiting PRMT5 enzyme

MDA-MB-231 breast cancer cell lines were treated with ZJ-78 series compounds at different concentrations (50. mu.M, 100. mu.M and 150. mu.M) to detect the expression level of the downstream substrate H3R8me2s of PRMT 5. Western blotting results are shown in FIG. 4, and the results show that the level of total intracellular H3R8me2s is remarkably reduced, and the inhibition effect is dose-dependent. The results indicate that the ZJ-78 series compounds can inhibit the enzymatic activity of PRMT 5.

ZJ-78 series compounds for promoting the inhibition of breast cancer cell proliferation and promoting apoptosis

In order to further compare the antitumor abilities of ZJ-78 series compounds and Tadalafil, different types of breast cancer cell lines were treated with the 3 compounds and Tadalafil, and CCK8 detection revealed that cell viability was significantly inhibited in MDA-MB-231 cells, MCF-7 cells and HCC1937 cells, wherein ZJ-781 had the most significant effect on inhibiting cell proliferation (FIG. 5).

In addition, the effect of ZJ-78 series compounds on apoptosis was also analyzed by Annexin V/PI double staining in MDA-MB-231 cell line. The results show that the ZJ-78 series compounds can promote MDA-MB-231 apoptosis to different degrees, and the promotion effect of ZJ-781 on apoptosis is most obvious (figure 6)

In FIG. 5, MDA-MB-231, MCF-7 and HCC1937 cells were treated with ZJ-78 series compounds and Tadalafil at different concentration gradients, and cell viability was measured by CCK8 method after 48 h. In FIG. 6, MDA-MB-231 cells were treated with ZJ-78 series compounds at a concentration of 150. mu.M and Tadalafil, and apoptosis was detected by Annexin V/PI method after 48 h. P <0.05, P <0.01, P <0.001, P <0.0001, n.s., not significan. Bars ═ means ± SEM.

The ZJ-78 series compounds can improve the sensitivity of breast cancer cells to chemotherapeutic drugs in vitro

Because the difference between the drugs is most obvious when the ZJ-78 series compounds and Tadalafil are used for treating the cells at the concentration of 150 mu M, the concentration of the ZJ-78 series compounds and Tadalafil is set as the concentration of 150 mu M, the concentration gradient of adriamycin treatment is established on the basis, and the CCK8 method is used for detecting the influence of the compounds and the adriamycin on the cell viability. The results show that the ZJ-78 series and Tadalafil can further enhance the inhibition of the cell viability by the adriamycin at the adriamycin treatment concentration of 0.2 mug/ml, but the inhibition of the cell viability by the ZJ-781 and the adriamycin is more obvious (FIG. 7). Similarly, the ZJ-78 series of compounds also promoted the inhibitory effect of cisplatin and Olaparib on cell viability (FIGS. 8 and 9).

In addition, the effect of ZJ-781 and Tadalafil, respectively, in combination with doxorubicin on MDA-MB-231 apoptosis was examined. The results show that, when Tadalafil and the compound ZJ-781 are added, compared with the simple addition of adriamycin, the proportion of living cells is obviously reduced, the proportion of cells undergoing early apoptosis and late apoptosis is obviously increased, and the necrotic cell population with single positive PI is not obviously changed after the compound is added. In general, ZJ-781 combined with doxorubicin promotes apoptosis, and the effect is stronger than that of doxorubicin taken alone and combined effect of Tadalafil and doxorubicin. As a result, referring to FIG. 10, MDA-MB-231 cells were treated with 150. mu.M ZJ-781 and Tadalafil in combination with 0.2. mu.g/ml doxorubicin, respectively, and apoptosis was detected by Annexin V/PI double staining after 48 hours. P <0.05, P <0.01, P <0.001, P <0.0001, n.s., not significan. Bars ═ means ± SEM.

7. The compound ZJ-781 can improve the sensitivity of breast cancer cells to chemotherapeutic drugs in vivo

MDA-MB-231 cells are inoculated under the skin of a nude mouse, ZJ-781 with the best traditional Chinese medicine effect of ZJ-78 series compounds is selected to be combined with chemotherapy drugs of adriamycin, cisplatin and olaparib, and the anti-tumor effect of the novel PRMT5 inhibitor in vivo is analyzed. On day 10 post-inoculation, mice were given 8 different intervention regimens, respectively: control, ZJ-781, doxorubicin, ZJ-781+ doxorubicin, cisplatin, ZJ-781+ cisplatin, Olaparib, ZJ-781+ Olaparib. Wherein, the compound ZJ-781 is administrated by intragastric administration once a day at the dose of 2mg/kg, the adriamycin is administrated by caudal vein injection once a week at the dose of 2mg/kg, the olaparib is administrated by intraperitoneal injection once a day at the dose of 50mg/kg, the cisplatin is administrated by intraperitoneal injection once every 3 days at the dose of 2.5mg/kg, and the material drawing analysis is carried out on the 31 th day after inoculation. Similar to the results of in vitro experiments, the growth of tumors was already slowed by ZJ-781 alone, but when ZJ-781 was used in combination with chemotherapeutic agents, the efficacy of the chemotherapeutic agents was further enhanced and tumor growth was slowed (FIGS. 11, 12 and 13). Also, Ki67 immunohistochemistry showed that the proliferation potency of tumor cells was further inhibited when ZJ-781 was used in combination with chemotherapeutic agents (FIG. 14).

In conclusion, the invention designs and synthesizes 3 novel PRMT5 inhibitors based on Tadalafil, namely ZJ-781, ZJ-782 and ZJ-783. The 3 novel PRMT5 inhibitors can effectively combine with PRMT5, inhibit the enzyme activity of PRMT5, inhibit the proliferation of breast cancer cells, promote apoptosis and enhance the anti-tumor effect of chemotherapy drugs of adriamycin, cisplatin and olaparib. Among them, ZJ-781 has the most significant anti-tumor effect and can play a role in sensitizing chemotherapeutic drugs in vivo.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A novel class of PRMT5 inhibitors, comprising compounds of the formula:

or, a pharmaceutically acceptable salt thereof.

2. Use of a novel PRMT5 inhibitor according to claim 1 in the manufacture of a medicament for the treatment of breast cancer.

3. The use of claim 2, wherein the agent is an agent that inhibits the proliferation of MDA-MB-231 cells, MCF-7 cells, and HCC1937 cells.

4. The use of a novel PRMT5 inhibitor according to claim 1 in the preparation of a chemotherapeutic drug sensitizer.

5. The use according to claim 4, wherein the novel PRMT5 inhibitor is capable of potentiating the anti-tumor effect of the chemotherapeutic agents doxorubicin, cisplatin and olaparib.

6. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable diluent or excipient.

7. A medicament for the treatment of breast cancer, consisting of the novel PRMT5 inhibitor of claim 1 and a chemotherapeutic agent.

8. The medicament of claim 7, wherein the chemotherapeutic agent is doxorubicin, cisplatin, or olaparib.