CN114558014B

CN114558014B - PRMT5 inhibitor and application thereof in treating breast cancer

Info

Publication number: CN114558014B
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: 2024-01-30
Anticipated expiration: 2042-03-31
Also published as: CN114558014A

Abstract

The invention discloses a PRMT5 inhibitor and application thereof in treating breast cancer, belonging to the technical field of anti-tumor drug research. According to the invention, the Tadalafil is modified through computer aided design, 3 Tadalafil derivative compounds are obtained through screening, and the compounds are named as ZJ-781, ZJ-782 and ZJ-783.ZJ-78 compounds bind PRMT5 and inhibit PRMT5 function. In-vitro and in-vivo experiments further show that the ZJ-78 series compounds can inhibit proliferation of breast cancer cells, promote apoptosis and promote anti-tumor effects of chemotherapeutic drugs such as doxorubicin, cisplatin and olapanib.

Description

PRMT5 inhibitor and application thereof in treating breast cancer

Technical Field

The invention belongs to the technical field of anti-tumor drug research, relates to design synthesis of new compounds and anti-tumor effect research, and in particular relates to a PRMT5 inhibitor and application thereof in treating breast cancer.

Background

In recent years, with the frequent occurrence of disease diversification and drug 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 development time and improve the drug development efficiency in the aspects of drug target spot recognition, active compound screening, compound physical property prediction, compound molecular reconstruction and the like. The molecular docking technology is a core technology of computer-aided drug design, and is based on the structure of biological macromolecules for screening, simulating the three-dimensional structure of the combination of small molecules and biological macromolecules, calculating the combination strength of the small molecules and the biological macromolecules, comprehensively evaluating the combination activity of the small molecules and the biological macromolecules, and has the characteristics of good accuracy and high efficiency.

Protein arginine methyltransferases (protein arginine methyltransferase, PRMTs) are an important class of enzymes that catalyze the methylation of arginine in chromatin proteins, and can catalyze the transfer of methyl groups on S-adenosylmethionine (SAM) to arginine. In eukaryotes, methylated arginine can be divided into three main forms: monomethylarginine (monomethylated arginine, MMA), asymmetric dimethylarginine (asymmetric dimethylarginine, ADMA) and symmetric dimethylarginine (symmetric dimethylarginine, SDMA). The PRMTs family has a total of 9 members and is classified into three types according to the type of methylation that it catalyzes for arginine. Type I PRMTs include 1, 2, 3, 4, 6, catalyze both monomethylation and asymmetric dimethylation of arginine residues, type II PRMTs include 5 and 9, catalyze both monomethylation and symmetric dimethylation, and type III PRMTs include 7, catalyze only monomethylation. PRMT5 is the predominant type II PRMT, having a phosphoglycerate isomerase barrel at the N-terminus, binding to the methylated small protein 50 (WD repeat-containing protein methylosome protein, MEP 50), and a methyltransferase catalytic domain at the C-terminus. PRMT5 is typically present in tetrameric form and forms a heterogeneous complex with its chaperone MEP 50.

PRMT5 is highly expressed in a variety of tumors, and PRMT5 high expression is closely related to patient survival decline. Inhibition of PRMT5 in tumors causes DNA damage, homologous recombination repair-related functional defects, and reduced genomic stability, and may also contribute to activation of P53 signaling pathways, leading to cell cycle arrest and cell death.

Thus, due to the important role of PRMT5 in tumors, its inhibitor research has become a hotspot. Currently, although PRMT5 inhibitors such as GSK3326595, JNJ-64619178 and PF-06939999 have been reported to date to enter the clinical trial phase, no inhibitors are marketed, which is a significant challenge for PRMT5 inhibitor development. 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 arterial hypertension, prostatic hyperplasia, cardiac hypertrophy and the like. Among the marketed drugs, tadalafil is a novel PRMT5 inhibitor, which can effectively improve the sensitivity of tumor cells to chemotherapeutic drugs such as doxorubicin in breast cancer, but the binding ability with PRMT5 and the antitumor activity thereof are still to be further improved.

Disclosure of Invention

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

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

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

or a pharmaceutically acceptable salt thereof.

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

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

The invention discloses application of the PRMT5 inhibitor in preparing a chemotherapeutic drug sensitizer.

Preferably, the PRMT5 inhibitor is capable of enhancing the anti-tumor effect of the chemotherapeutic agents doxorubicin, cisplatin and olaparib.

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

The medicine for treating breast cancer is characterized by comprising the 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 invention carries out reconstruction on Tadalafil through computer aided design, firstly, on three parts (piperazine, indole and benzene ring connected with a mother ring) of Tadalafil molecules which are relatively independent, groups containing hetero atoms such as nitrogen, oxygen and sulfur are arranged, so that the Tadalafil molecules and proteins generate more hydrogen bonds; secondly, different alkyl substituents are introduced, so that the Tadalafil molecule and the protein generate more hydrogen bonds and simultaneously enhance the combination of hydrophobic interaction or aromatic groups and the protein; thirdly, changing the chirality of the chiral center, and exploring the influence of the chiral change on the binding process of the Tadalafil molecules and the proteins; fourth, the above modifications were introduced into Tadalafil molecules simultaneously, and the comprehensive effect of these modifications on the binding of Tadalafil molecules to proteins was explored, and 3 Tadalafl derivative compounds were obtained by screening through molecular docking analysis, and named ZJ-781, ZJ-782 and ZJ-783.ZJ-78 compounds bind PRMT5 and inhibit PRMT5 function. In-vitro and in-vivo experiments further show that the ZJ-78 series compounds can inhibit proliferation of breast cancer cells, promote apoptosis and promote anti-tumor effects of chemotherapeutic drugs such as doxorubicin, cisplatin and olapanib.

Drawings

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

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

FIG. 2b is a nuclear magnetic pattern of ZJ-782;

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

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

FIG. 4 is a graph showing that the ZJ-78 series compounds of the invention inhibit 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 compounds at various concentration gradients;

FIG. 6 is a graph showing apoptosis results of 150. Mu.M concentration of ZJ-78 series compounds and Tadalafil treatment of MDA-MB-231 cells;

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

FIG. 8 is a graph showing the results of cisplatin testing cell viability by treating MDA-MB-231 and MCF-7 cells with ZJ-78 series compounds and Tadalafil with the addition of corresponding concentration gradients;

FIG. 9 shows the results of the Olaparib test on MDA-MB-231 and MCF-7 cells treated with ZJ-78 series compounds and Tadalafil with the addition of corresponding concentration gradients;

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

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

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

FIG. 13 is a statistical analysis of volume and mass of MDA-MB-231 tumor material; wherein, (a) is tumor volume; (b) is tumor mass;

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

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise 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 attached drawing figures:

1. ZJ-78 series compound designed and synthesized by utilizing molecular docking technology

First, the binding capacity of the PRMT5 inhibitor to PRMT5 that has been reported was analyzed using Sybyl-X2.0 software. Minimizing energy of all compounds in Sybyl-X2.0 software by using a Tripos force field, taking distance as a dielectric function, taking Gasteiger-Hockel atomic charge as charge, optimizing by using a Powell energy gradient method to obtain the lowest energy conformation, and taking the maximum energy optimization frequency as 10000 and the energy convergence standard as 0.001 kcal.mol ^-1 Imitate the stable conformation of the molecule in natural systems. The PRMT5: MEP50 complex (PDB: 4 gqb) crystal structure was downloaded from the PDB database, and Sybyl-x 2.0 was introduced for molecular docking. The docking adopts a semi-flexible docking method, and is performed by using a Surflex-Dock module. The total score (Surflex-Dock) takes into account the combination of polarity, hydrophobicity, entropy and solubilityFactors such as the formulation of the agent can reflect the binding affinity of the ligand to the receptor. The greater the score, the more stable the ligand binding to the receptor and the more likely there is an interaction. Butt joint>7 minutes, which indicates that the molecules may have strong binding to the target,>a score of 5 indicates that there may be better binding of the molecule to the target.

The results are shown in Table 1 below, where the binding capacity of Tadalafil to PRMT5 is still further improved compared to inhibitors such as EPZ 015666:

TABLE 1

Name of the name	Score of	Butt joint energy	Polarity of
				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, the Tadalafil was modified as follows: firstly, on three relatively independent parts (piperazine, indole and benzene ring connected with a mother ring) of the Tadalafil molecule, installing groups containing hetero atoms such as nitrogen, oxygen and sulfur, so that the Tadalafil molecule and the protein generate more hydrogen bonds; secondly, different alkyl substituents are introduced, so that the Tadalafil molecule and the protein generate more hydrogen bonds and simultaneously enhance the combination of hydrophobic interaction or aromatic groups and the protein; thirdly, changing the chirality of the chiral center, and exploring the influence of the chiral change on the binding process of the Tadalafil molecules and the proteins; fourth, the above modifications were introduced simultaneously into the Tadalafil molecule, and the combined effect of these modifications on the binding of the Tadalafil molecule to the protein was explored.

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

2. chemical synthesis process of ZJ-78 series compounds

The synthesis method is shown in fig. 1, and the specific synthesis steps are as follows:

(1) D-tryptophan methyl ester hydrochloride is treated by sodium bicarbonate to obtain D-tryptophan methyl ester, the D-tryptophan methyl ester hydrochloride is taken as a raw material, dichloromethane (DCM, 10mL/g raw material) is used for dissolving, 1.08 equivalent of aldehyde compound is added into a reaction bottle, nitrogen replacement is sufficient, the reaction system is cooled to about 0 ℃, 2.05 equivalent of trifluoroacetic acid is slowly added dropwise, and the reaction is completed after the addition, and the reaction is carried out at room temperature.

Post-treatment: after the reaction is completed, the reaction solution is adjusted to pH=9 or so, DCM is used for extraction, the organic layers are combined, the mixture is dried by spin-drying and subjected to column chromatography (200-300 meshes of silica gel column chromatography: petroleum ether/ethyl acetate=150/1-80/1), and ZJ-781-1/ZJ-783-1 is obtained by the same synthesis method (the reaction raw materials are not pretreated by sodium bicarbonate, the reaction time is longer, the reaction is more complicated, the isomer exists in the product, the small-polarity product is the target compound, and the yield is basically about 35%).

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

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

(3) Adding ZJ-781-2/ZJ-782-2/ZJ-783-2 into a reaction bottle, adding methanol for dissolution (35 mL/g raw material), fully replacing nitrogen, adding 2 equivalents of methylamine in the mixture, heating to reflux, and stirring until the reaction is complete (about 16 h);

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

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

Affinity assay of ZJ-78 series Compounds with PRMT5 protein

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

Functional analysis of PRMT5 enzyme inhibition by ZJ-78 series Compounds

MDA-MB-231 breast cancer cell lines were treated with various concentrations (50. Mu.M, 100. Mu.M, and 150. Mu.M) of ZJ-78 series compounds to detect the expression level of the substrate H3R8me2s downstream of PRMT 5. Western blotting results are shown in FIG. 4, and the results show that the level of total H3R8me2s in the cells is significantly reduced, and the inhibition is dose-dependent. The results indicate that ZJ-78 series compounds inhibit PRMT5 enzymatic activity.

ZJ-78 series compounds can promote inhibition of breast cancer cell proliferation and promote apoptosis

To further compare the antitumor ability of ZJ-78 series compounds with that of Tadalafil, treatment of different types of breast cancer cell lines with these 3 compounds and Tadalafil, CCK8 assays found significant inhibition of cell viability in MDA-MB-231 cells, MCF-7 cells, and HCC1937 cells, with the inhibition of cell proliferation by ZJ-781 being most pronounced (FIG. 5).

In addition, the effect of ZJ-78 series compounds on apoptosis was also analyzed by an Annexin V/PI double staining method in the MDA-MB-231 cell line. The results show that the ZJ-78 series compounds can promote the apoptosis of MDA-MB-231 cells to different degrees, and the promotion effect of the ZJ-781 on the 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 various concentration gradients, and after 48h, the cell viability was examined by CCK8 method. In FIG. 6, MDA-MB-231 cells were treated with ZJ-78 series compounds and Tadalafil at 150. Mu.M concentration, and apoptosis was detected by the Annexin V/PI method after 48 h. * P <0.05, < P <0.01, < P <0.001, < P <0.0001, n.s., not signature. Bars = means ± SEM.

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

Since the difference between the ZJ-78 series compound and the Tadalafil is most obvious when the cells are treated at the concentration of 150 mu M, the concentration of the ZJ-78 series compound and the Tadalafil is set to be 150 mu M, and a concentration gradient of doxorubicin treatment is established on the basis, and the influence on the cell viability after the compound is combined with the doxorubicin is detected by a CCK8 method. The results showed that at a doxorubicin treatment concentration of 0.2 μg/ml, both ZJ-78 series and Tadalafil further enhanced the inhibition of cell viability by doxorubicin, but that the inhibition of cell viability was more pronounced following the combination of ZJ-781 with doxorubicin (FIG. 7). Similarly, ZJ-78 series of compounds may also promote inhibition of cell viability by cisplatin and Olaparib (FIGS. 8 and 9).

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

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

MDA-MB-231 cells are inoculated subcutaneously in nude mice, ZJ-781 with the best efficacy in ZJ-78 series compounds is selected, and the combination of the ZJ-781 with chemotherapeutic drugs of doxorubicin, cisplatin and olapanib is used for analyzing the anti-tumor effect of PRMT5 inhibitors in vivo. On day 10 post-inoculation, mice were given 8 different intervention modes: control group, ZJ-781, doxorubicin, ZJ-781+doxorubicin, cisplatin, ZJ-781+cisplatin, olaparib, ZJ-781+Olaparib. Wherein, the compound ZJ-781 is infused once per day at a dose of 2mg/kg, doxorubicin is injected once per week at a dose of 2mg/kg for tail vein, olaparib is injected once per day at 50mg/kg for abdominal cavity, cisplatin is injected once per 3 days at 2.5mg/kg for abdominal cavity, and the materials are taken and analyzed on day 31 after inoculation. Similar to the results of in vitro experiments, ZJ-781 alone resulted in a reduction in tumor growth, but when ZJ-781 was used in combination with chemotherapeutic agents, the efficacy of the chemotherapeutic agents was further enhanced and tumor growth was reduced (FIGS. 11, 12 and 13). Meanwhile, ki67 immunohistochemical results showed that ZJ-781 was further inhibited in proliferation capacity of tumor cells when combined with chemotherapeutic drugs (fig. 14).

In summary, the present invention is based on Tadalafil design and synthesis of 3 PRMT5 inhibitors, namely ZJ-781, ZJ-782 and ZJ-783. The 3 PRMT5 inhibitors can be effectively combined with PRMT5, inhibit the enzyme activity of PRMT5, inhibit the proliferation of breast cancer cells, promote apoptosis, and enhance the anti-tumor effect of chemotherapeutic drugs such as doxorubicin, cisplatin and olaparib. Wherein, the ZJ-781 has the most obvious anti-tumor effect and can play a role in sensitization of chemotherapeutic drugs in vivo.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. The application of PRMT5 inhibitors in preparing medicaments for treating breast cancer is characterized in that the PRMT5 inhibitors are compounds shown in the following structural formula:

or a pharmaceutically acceptable salt thereof.

2. The use of claim 1, wherein the agent is an agent that inhibits proliferation of MDA-MB-231 cells, MCF-7 cells, and HCC1937 cells.

3. The application of PRMT5 inhibitors in preparing chemotherapeutic drug sensitizers is characterized in that the PRMT5 inhibitors are compounds shown in the following structural formula:

or a pharmaceutically acceptable salt thereof;

the chemotherapeutic drugs are doxorubicin, cisplatin and olapanib, and the PRMT5 inhibitor can enhance the antitumor effect of the chemotherapeutic drugs doxorubicin, cisplatin and olapanib.