Application of MAT2A inhibitor in preparation of medicine for preventing and / or treating inflammation

Abstract

The invention provides an application of a MAT2A inhibitor in preparation of a medicine for preventing and / or treating inflammation. A cell experiment result shows that the MAT2A inhibitor has a remarkable inhibition effect on an inflammatory cell model after administration, and mRNA expression levels of various inflammatory factors TNF-alpha, IFN-gamma, IL-1beta, IL-6, IL-12a and CXCL10 in cells are remarkably reduced; the invention provides a new direction for treating inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the use of a MAT2A inhibitor in preparing a drug for preventing and / or treating inflammation, and belongs to the field of medicine. Background Art

[0002] Methionine adenosyltransferase 2a (MAT2A) plays an important role in metabolism and epigenetics. The currently known functions and effects are:

[0003] 1. Participation in methionine metabolism: MAT2A is a member of the methionine adenosyltransferase family, which is mainly responsible for catalyzing the reaction of methionine with ATP to generate S-adenosylmethionine (SAM) and AMP. This reaction is the first step in methionine metabolism and is essential for maintaining normal methionine levels in the body.

[0004] 2. Influence on DNA methylation: SAM is an important methyl donor in the process of DNA methylation. Therefore, the activity of MAT2A directly affects the methylation state of DNA, which in turn may affect gene expression and regulation.

[0005] 3. Association with diseases: Studies have shown that abnormal expression or activity of MAT2A is associated with a variety of diseases, including tumors, cardiovascular diseases, and nervous system diseases. For example, in liver cancer, the expression level of MAT2A is often increased, which may be related to the proliferation and survival of tumor cells.

[0006] Since MAT2A is closely related to the occurrence and development of many diseases, it is expected to become a new diagnostic marker or therapeutic target for these diseases. For example, by detecting the expression level of MAT2A in blood or tissue, it can assist in the diagnosis of diseases such as liver cancer. At the same time, inhibitors or agonists targeting MAT2A may also provide new ideas and methods for the treatment of these diseases. Understanding the function and mechanism of action of MAT2A will help develop drugs or treatments targeting it. For example, reducing the amount of SAM produced by inhibiting the activity of MAT2A may help inhibit the proliferation and survival of tumor cells. In addition, drugs or compounds that can enhance the activity of MAT2A can also be studied to promote the proliferation and repair of normal cells.

[0007] 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one, i.e., the compound represented by formula (I), is a newly discovered MAT2A inhibitor. Mingzong Li et al. disclosed that the compound has MAT2A inhibitory activity (Leveraging Structure-Based Drug Design to Identify Next-Generation MAT2A Inhibitors, Including Brain-Penetrant and Peripherally Efficacious Leads, https: / / pubs.acs.org / doi / 10.1021 / acs.jmedchem.1c01595)

[0008]

[0009] Inflammation refers to the normal defense of the body or tissue against damaging factors. In general, inflammation activates macrophages to secrete a series of immune cytokines, which in turn kill pathogens, eliminate damaging factors, and clear and phagocytose necrotic cells. Therefore, as a defensive response of the body, a moderate inflammatory response is beneficial to the body. However, excessive inflammatory response can affect the body's normal metabolic process, cause septic shock, lead to multiple organ dysfunction, and even endanger life. Under normal conditions, a dynamic balance is maintained between pro-inflammatory factors and anti-inflammatory factors in cells or the body. After LPS stimulates macrophage activation, this balance is broken. On the one hand, it causes long-term high expression of pro-inflammatory factors, leading to tissue or organ damage. On the other hand, while a large amount of pro-inflammatory factors are released, the body will synthesize and secrete certain anti-inflammatory factors for defense in the short term. Among them, pro-inflammatory factors mainly include NO, iNOS, COX-2, TNF-α, IL-6, ROS, etc. The content of these inflammatory mediators involved in regulating the occurrence and development of inflammation in the body can indirectly reflect the degree of inflammatory response. Anti-inflammatory factors mainly include IL-4, IL-10, etc., whose large-scale secretion can inhibit the further development of inflammation.

[0010] In summary, methionine adenosyltransferase 2a (MAT2A) is an enzyme that plays a key role in methionine metabolism, and its abnormal expression or activity is closely related to the occurrence and development of a variety of diseases. Therefore, in-depth research on the function and mechanism of action of MAT2A is of great significance for the diagnosis and treatment of diseases, and can also find new solutions for the treatment of inflammatory diseases. Summary of the invention

[0011] In view of the problems existing in the prior art, one of the purposes of the present invention is to provide a MAT2A inhibitor for use in preparing a drug for preventing and / or treating inflammation. The structural formula of the compound is shown in Formula I:

[0012]

[0013] Accordingly, the compound of formula I has the chemical formula C 22 H 16 F 3 N 4 O 2 , the chemical name is 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one, 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-((2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one.

[0014] Preferably, the above-mentioned drugs for preventing and / or treating inflammation also include pharmaceutically acceptable salts of the above-mentioned compounds, their crystal forms, their solvates, their stereoisomers or their isotope-substituted compounds.

[0015] Preferably, the above-mentioned drug for preventing and / or treating inflammation is an agent that inhibits the expression of inflammatory factors.

[0016] Preferably, the above-mentioned drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that inhibits inflammation caused by excessive release of inflammatory factors by macrophages.

[0017] More preferably, the inflammatory factors are one or more of TNF-α, IFN-γ, IL-1β, IL-6, IL-12a or CXCL10.

[0018] More preferably, the above-mentioned drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that reduces the mRNA expression level of inflammatory factors.

[0019] More preferably, the above-mentioned drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that reduces the mRNA expression level of inflammatory factors TNF-α, IFN-γ, IL-1β, IL-6, IL-12a or CXCL10.

[0020] More preferably, the above-mentioned drug for preventing and / or treating inflammation is an inhibitor of overexpression of inflammatory cytokines.

[0021] Preferably, the cell administration dosage of the above-mentioned drug for preventing and / or treating inflammation is 10-100 μM, preferably 20-50 μM.

[0022] Compared with the prior art, the MAT2A inhibitor is a new target. From the results of cell experiments, it can be seen that the MAT2A inhibitor has a significant inhibitory effect on the inflammatory cell model after administration, and the mRNA expression levels of multiple inflammatory factors TNF-α, IFN-γ, IL-1β, IL-6, IL-12a and CXCL10 in the cells are significantly reduced; the present invention provides a new direction for the treatment of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of TNF-α in LPS-induced primary mouse macrophages;

[0024] Figure 2 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of IFN-γ in LPS-induced primary mouse macrophages;

[0025] Figure 3 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of IL-1β in LPS-induced primary mouse macrophages;

[0026] Figure 4 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of IL-6 in LPS-induced primary mouse macrophages;

[0027] Figure 5 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of IL-12a in LPS-induced primary mouse macrophages;

[0028] Figure 6 This is a comparison chart of the effects of the experimental groups of the present invention on the mRNA expression level of CXCL10 in LPS-induced primary mouse macrophages. DETAILED DESCRIPTION

[0029] The present invention is further described in detail and completely below in conjunction with the embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] The experimental methods in the following examples, unless otherwise specified, are generally carried out under conventional conditions such as those described in Pharmacology Experimental Manual, 3rd edition, Science Press, 2002 or under conditions recommended by the manufacturer. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be purchased through commercial channels.

[0031] This embodiment provides the use of a MAT2A inhibitor as a drug for the prevention and / or treatment of inflammation. The MAT2A inhibitor is 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one (hereinafter referred to as AGI-41998), cas is 2377492-26-5, purchased from MedChemExpress.

[0032] In order to further verify the beneficial effects of the present invention, the following experimental examples are provided.

[0033] 1. Experimental materials:

[0034] BALb / c mice were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. and kept in the Experimental Animal Center of Central South University. This study has passed the ethical review of experimental animal welfare of Central South University. The "3R" principle was strictly followed during the experiment to minimize the pain and discomfort of the animals.

[0035] FBS was purchased from Beijing Sai Aomei Cell Technology Co., Ltd., DMEM medium was purchased from Gbico, USA; LPS was purchased from Sigma; DMSO was purchased from Changsha Dingguo Biotechnology Co., Ltd.; MagzoL lysis buffer was purchased from Magen; zirconium oxide beads were purchased from Wuhan Servicebio; chloroform, isopropanol and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; enzyme-free water was purchased from Sigma; 5xg DNA digester Mix was purchased from Shanghai Sangon Biotechnology Co., Ltd.; III SuperMix plus was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; primers for various inflammatory factors were purchased from Thermo Fisher Scientific; cDNA was purchased from Beijing Biolab Technology Co., Ltd.; and 2xSYBR Green qPCR Master Mix was purchased from Shanghai Yize Biotechnology Co., Ltd.

[0036] 2. Extraction of Primary Macrophages

[0037] Mouse peritoneal macrophages were isolated from Balb / c mice (20-25 g). The isolation method was described in (J VisExp. 2015Apr 29; (98): e52749.). After obtaining the cells, they were immediately centrifuged at 1000 rpm / 3 min, the supernatant was discarded, and the cells were cultured in DMEM medium containing 10% FBS. The cells were then seeded in a 6-well plate and incubated at 37°C with 5% CO. 2 , cultured for 2 h, and stimulated with LPS (100 ng / mL).

[0038] 3. Cell Model Processing

[0039] The cells used in the in vitro experiment were primary mouse macrophages, and a series of experimental studies on the in vitro LPS-stimulated macrophage inflammatory response model were established. LPS was prepared into a 100μg / ml stock solution using PBS in advance, and AGI-41998 was prepared into a 50mM stock solution using DMSO. The experimental groups were divided into four groups: blank group, lipopolysaccharide-induced group (LPS group), and treatment group (LPS-induced group incubated with 20μM AGI-41998 and LPS-induced group incubated with 50μM AGI-41998).

[0040] Blank group: No treatment was performed and the cells were directly placed in a 5% CO 2 The cells were cultured in a 37°C constant temperature incubator until they were sampled together with the other groups;

[0041] LPS group: stimulated with 100 ng / ml LPS for 6 h, and 1 μL DMSO was added at the same time, and the samples were collected immediately after the time was up;

[0042] Treatment Group:

[0043] 20μM AGI-41998+LPS group: cells were stimulated with 100ng / ml LPS and 0.4μL AGI-41998 stock solution was added, incubated for 6h, and then samples were collected;

[0044] 50μM AGI-41998+LPS group: cells were stimulated with 100ng / ml LPS and 1μL AGI-41998 stock solution was added, incubated for 6h, and then samples were collected.

[0045] 4. mRNA expression level detection

[0046] 4.1 RNA extraction

[0047] (1) Precool the grinder in advance, take out the cells from the -80℃ freezer, transfer the cells from the cryotube to a 1.5mL EP tube, add 1mL of MagzoL lysis buffer, and add 3 5mm zirconium oxide beads to each EP tube. Place the cells in the grinder and repeat 10 cycles at 60HZ, -10℃, grinding for 30s with a 10s interval.

[0048] (2) Add 200 μL of chloroform to the ground cells, vortex until fully mixed, let stand until the liquid separates, place the EP tube in a pre-cooled low-temperature ultracentrifuge, and centrifuge at 12,000 rpm and 4°C for 10 min.

[0049] (3) Transfer the supernatant to a new enzyme-free EP tube and add an equal amount of isopropanol. Mix thoroughly by shaking. Let stand at room temperature for 10 min and then centrifuge at 4°C and 12,000 rpm for 10 min. The white precipitate at the bottom of the EP tube is RNA.

[0050] (4) Carefully and slowly pour out the liquid in the EP tube and add 1 mL of 75% ethanol to the EP tube. Turn the EP tube upside down and place it in a low-temperature ultracentrifuge and centrifuge it at 12,000 rpm and 4°C for 10 min.

[0051] (5) Carefully and slowly remove the liquid in the EP tube, aspirate the liquid in the tube after separation, and add an appropriate amount of enzyme-free water to dissolve the RNA according to the amount of RNA precipitation.

[0052] RNA Reverse Transcription

[0053] (1) Detect RNA concentration using an enzyme-labeled instrument and gradually add the reaction substances to an 8-tube strip without enzyme according to the following system:

[0054] Table 1 Reverse transcription system

[0055]

[0056] (2) Remove the bubbles in the tube and place the 8-tube strip into the Veriti TM In the 96-Well Fast Thermal Cycler reverse transcription instrument, the program was set to react at 42°C for 2 minutes.

[0057] (3) Add 10 μL to the 8-tube strip III SuperMix plus, mix well, put it into the reverse transcription instrument again, and set the program to react at 25°C for 5 minutes, 55°C for 15 minutes, and 85°C for 5 minutes. The reacted sample was diluted 10 times with enzyme-free water and then stored in a 4°C refrigerator.

[0058] RT-qPCR

[0059] (1) The primers (primers for various inflammatory factors are shown in Table 1) were centrifuged at 4000 rpm for 1 min, and then enzyme-free water was added to maintain the primer concentration at 100 μM. 10 μ of each 100 μM upstream and downstream primers were taken, and 980 μ of enzyme-free water was added to dilute the primers to 1 μM.

[0060] Table 1

[0061]

[0062] (2) Add 5 μL of diluted primers, 5 μL of diluted cDNA, and 10 μL of 2xSYBR Green qPCR Master Mix to an 8-tube strip, so that the final system is 20 μL.

[0063] (3) Remove the bubbles in the 8-tube strip and separate them into QuantStudio TM 3. Real-Time PCR System was installed. The reaction program was set as pre-denaturation at 95°C for 10 min, PCR reaction was denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, PCR reaction was repeated for 40 cycles, melting curve stage was reacted at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, and the program was completed.

[0064] (4) According to 2 -ΔΔCt The formula is used to calculate the CT value in the experiment to obtain the relative expression of the gene.

[0065] 5. Experimental results:

[0066] Figures 1 to 5 The experimental results of the blank control group, LPS-induced group, LPS-induced group incubated with 20μM AGI-41998, and LPS-induced group incubated with 50μM AGI-41998 were used as experimental subjects to show the effect of mRNA expression levels of various inflammatory factors. As can be seen from the figure, the data show that whether it is 20μM AGI-41998 or 50μM AGI-41998 incubated with LPS-induced mouse primary macrophages, the mRNA expression levels of TNF-α, IFN-γ, IL-1β, IL-6, IL-12a and CXCL10 in the cells are significantly reduced (*P<0.05, **P<0.01, ***P<0.001, ****p value <0.0001). The results show that the MAT2A inhibitor AGI-41998 can significantly inhibit the cellular inflammatory response at a lower concentration.

[0067] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Any technician familiar with this profession, without departing from the scope of the technical solution of this application, using the above-disclosed technical content to make slight changes, modifications, substitutions, combinations, and simplifications should all be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A use of a MAT2A inhibitor, characterized in that: Use of 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one in the preparation of a drug for the prevention and / or treatment of inflammation, wherein the structure of the MAT2A inhibitor is shown in formula (I):

2. The use according to claim 1, characterized in that The drug for preventing and / or treating inflammation contains a pharmaceutically acceptable salt, a crystal form, a solvate, a stereoisomer or an isotope-substituted compound of 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one.

3. The use according to claim 1, characterized in that The drug for preventing and / or treating inflammation is for inhibiting the expression of inflammatory factors.

4. The use according to claim 1, characterized in that The drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that inhibits inflammation caused by excessive release of inflammatory factors by macrophages.

5. The use according to claim 4, characterized in that The inflammatory factor is one or more of TNF-α, IFN-γ, IL-1β, IL-6, IL-12a or CXCL10.

6. The use according to claim 1, characterized in that: The drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that reduces the mRNA expression level of inflammatory factors.

7. The use according to claim 1, characterized in that The drug for preventing and / or treating inflammation is a drug for preventing and / or treating inflammation that reduces the mRNA expression level of inflammatory factors TNF-α, IFN-γ, IL-1β, IL-6, IL-12a or CXCL10.

8. The use according to claim 1, characterized in that The drug for preventing and / or treating inflammation is an inhibitor of overexpression of inflammatory cytokines.

9. The use according to claim 1, characterized in that The cell administration dosage of the drug for preventing and / or treating inflammation is 10 to 100 μM.

10. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is 8-(4-bromophenyl)-6-(4-methoxyphenyl)-2-(2,2,2-trifluoroethyl)amino)pyrido[4,3-d]pyrimidin-7(6H)-one.

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