Linoleic acid compound as LPAR3 receptor antagonist and drug and application of linoleic acid compound as LPAR3 receptor antagonist and drug

Abstract

The invention relates to a natural product LPAR3 receptor antagonist and application of the natural product LPAR3 receptor antagonist as a medicine, and the compound is linoleic acid, has LPAR3 receptor antagonistic activity, is an antagonist of an LPAR3 receptor, and can be used for preparing a medicine. The traditional Chinese medicine composition can be used for preventing and treating various cancers (stellate cell tumor, large cell medulloblastoma, malignant pancreatic tumor, papillary mucus cancer in pancreatic ductus, malignant ventricular tubuloma and osteosarcoma), papillary mucus tumor in pancreatic ductus with low-grade dysplasia and other diseases; therefore, a novel LPAR3 receptor antagonist head compound with a clear action target can be provided for related diseases.

Description

Technical Field

[0001] The invention belongs to the field of LPAR3 (Lysophospholipid (LPA) receptors 3, LPAR3) receptor antagonists, and relates to the discovery of action targets of fatty acid compounds in safflower, specifically the discovery of action targets of linoleic acid in fatty acid compounds. Background Art

[0002] Safflower Carthamus tinctorius L. belongs to the Asteraceae family. The medicinal part is the dried flower of safflower, which is also known as red and blue flower, grass safflower, and thorny safflower. It was first recorded in the "Kaibao Materia Medica". Safflower seeds were brought back from the Western Regions by Zhang Qian in the Han Dynasty. It has a history of cultivation and use for more than 2,100 years. Safflower is in the heart and liver meridians and has the effect of promoting blood circulation and removing blood stasis. Safflower is also used as tea in the folks and is a crop with medicinal and edible properties. Globally, safflower is mainly produced in Iran, India, the United States, Australia and other places, and is widely planted in Xinjiang, Henan, Zhejiang, Yunnan and other places in my country. Safflower has multiple pharmacological activities such as anti-tumor, antioxidant, anti-inflammatory and analgesic. In addition, safflower seed oil also contains a large amount of vitamin E, which has a health-care effect. However, most of the current research focuses on the cellular level, and there is little research on the target sites of pure compounds (Hong Jing. Study on the quality of commercially available Chinese medicinal materials safflower. Strait Pharmacy, 2006, 18(5):106-107; Nanjing University of Chinese Medicine. Chinese Materia Medica Dictionary. 2nd edition. Shanghai: Shanghai Science and Technology Press, 2006; Zheng Zhiyong, Song Xiefeng, Fan Xuepeng, et al. Precious medicinal plants - safflower. Seed Science and Technology, 2010, 28(10):44; TAMOOR HUSSAIN, MUHAMMAD AZEEM TARIQ, ISHFAQ AHMAD, et al. Characters association analysis in safflower (carthamustinctorius l.). Journal of Biology, Agriculture and Healthcare, 2014, 4(6):2224-3208; Shi Zhu. Flora of China, Volume 1. Beijing: Science Press, 1987).

[0003] LPA receptors (LPARs) are known as heteromeric G protein-coupled receptors that specifically engage G(i) / G(o) / G(q). So far, six G protein-coupled receptors have been identified for LPARs. The expression pattern and signaling properties of LPARs are complex, exerting multiple effects on physiological, pathological, and developmental processes, which in turn act through G protein-coupled receptors (GPCRs). LPARs mediate a variety of cellular responses, such as calcium influx, cell proliferation, and cell survival. LPAR3 was discovered and cloned through homology searches and is located at locus 22.3 of human chromosome 1p and 71.03cM of mouse chromosome 3, encoding a 353-amino acid GPCR sequence with a molecular weight of approximately 40kDa. LPAR3 binds to Gαi / o and Gαq / 11 and promotes LPA-induced processes such as calcium mobilization, adenylate cyclase activation, MAPK activation, and PLC activation. LPAR3 has been shown to be involved in various cellular and physiological processes in the body, and is particularly closely related to a variety of cancers (astrocytic tumors, large cell medulloblastomas, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymomas, osteosarcomas), and intraductal papillary mucinous neoplasms of the pancreas with low-grade dysplasia. Therefore, discovering more LPAR3 receptor selective ligands with high activity, high selectivity and excellent drugability is of great significance for clarifying its pharmacological and biological functions, which will provide a new direction for targeted treatment of diseases (Blaho, VA; Chun, J. 'Crystal' Clear? Lysohphospholipid Receptor Structure Insights and Controversies. Trends Pharmacol. Sci. 2018, 39, 953-966; Yung, YC; Stoddard, NC; Chun, J. LPA receptor signaling: Pharmacology, physiology, and pathophysiology. J. Lipid Res. 2014, 55, 1192-1214; Bandoh, K.; Aoki, J.; Hosono, H.; Kobayashi, S.; Kobayashi, T.; Murakami-Murofushi, K.; Tsujimoto, M.; Arai, H.; Inoue, K. Molecular cloning and characterization of a novel human G-protein-coupledreceptor, EDG7, for lysophosphatidic acid. J. Biol. Chem. 1999, 274, 27776-27785).

[0004] Currently, there are no reports on the effects of linoleic acid in fatty acid compounds on LPAR3 receptors (Lysophospholipid (LPA) receptors 3, LPAR3). Summary of the invention

[0005] The present invention relates to the discovery of the action target of linoleic acid in fatty acid compounds and the application of such compounds. One purpose is to provide a compound linoleic acid whose action target is LPAR3 receptor; the second purpose is to provide the clinical application scope of such compounds in diseases such as various cancers (astrocytic tumors, large cell medulloblastomas, malignant pancreatic tumors, pancreatic intraductal papillary mucinous carcinomas, malignant ependymomas, osteosarcomas), pancreatic intraductal papillary mucinous tumors with low-grade dysplasia, etc.

[0006] The technical solution of the present invention is:

[0007] The LPAR3 receptor antagonist is the compound linoleic acid.

[0008] The chemical structure of linoleic acid is as follows:

[0009]

[0010] The fatty acid compound of the present invention is used in the preparation of drugs for preventing and / or treating various cancers (astrocytic tumors, large cell medulloblastomas, malignant pancreatic tumors, intraductal papillary mucinous carcinomas of the pancreas, malignant ependymomas, osteosarcomas), intraductal papillary mucinous tumors of the pancreas with low-grade dysplasia, and the like, wherein the fatty acid compound is linoleic acid.

[0011] Beneficial effects of the present invention:

[0012] In vitro cell experiments show that the compound of the present invention is linoleic acid that acts on the LPAR3 receptor, which is a G protein-coupled receptor. The LPAR3 receptor is associated with a variety of cancers (astrocytic tumors, large cell medulloblastomas, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymomas, osteosarcomas), intraductal papillary mucinous tumors of the pancreas with low-grade dysplasia, and the like. Based on the correlation between the target and the disease, the clinical application scope of such compounds can be broadened.

[0013] The present invention finds that the natural product linoleic acid can be used as an LPAR3 (Lysophospholipid (LPA) receptors 3, LPAR3) receptor antagonist and its application, specifically the discovery of the target of fatty acid compounds, the target is the LPAR3 receptor, the LPAR3 receptor antagonist is linoleic acid or its derivatives, and one or more of the corresponding pharmaceutically acceptable salts thereof as active ingredients. In vitro cell experiments show that the compound of the present invention is an LPAR3 receptor antagonist. Current studies have shown that LPAR3 receptors are associated with a variety of cancers (astrocytic tumors, large cell medulloblastoma, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymoma, osteosarcoma), intraductal papillary mucinous tumor of the pancreas with low-grade dysplasia, and other diseases. LPAR3 receptor antagonists can prevent and treat a variety of cancers (astrocytic tumors, large cell medulloblastoma, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymoma, osteosarcoma), intraductal papillary mucinous tumor of the pancreas with low-grade dysplasia, and other diseases. Based on this, novel LPAR3 receptor antagonist lead compounds with clear targets can be provided for the above-mentioned related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The maximum DMR response (%) within 60 min of the negative control group (HBSS buffer containing 0.1% DMSO and 20 mM HEPES), CP 55940 (1.25 μM), linoleic acid (100 μM), and linoleic acid_CP 55940 on CHO-LPAR3 cells;

[0015] Figure 2 The maximum DMR response dose-effect curve of stimulation and desensitization of different concentrations of linoleic acid on CHO-LPAR3 cells within 60 minutes. DETAILED DESCRIPTION

[0016] The present invention is further described with reference to examples. The examples are only intended to illustrate the present invention, but not to limit the present invention.

[0017] Linoleic acid (Brand: Chengdu Pusi; Product No.: PS1853-0200) was purchased from Chengdu Pusi Biotechnology Co., Ltd. CHO cells (Product No.: R75807, containing Flp-In TM-CHO cell line, hereinafter referred to as CHO cells; pFRT / lacZeo and pOG44 tool plasmids) were purchased from Thermo Fisher Scientific; CHO-LPAR3 cells, a stable cell line with high expression of human LPAR3 receptors, were constructed in the laboratory (construction method: 1) pOG44 and LPAR3-pFRT / lacZeo plasmids were transfected into CHO cells: pOG44 and LPAR3-pFRT / lacZeo plasmids (188 μ and 2 μ, respectively) and transfection reagent Lipofectamine3000 (Thermo) (21.7.7); CHO-LPAR3, a cell line with high expression of LPAR3 receptors, was obtained. 2) After 8 hours of transfection, 10 mL of fresh F12 complete medium (10% FBS) was replaced. 3) After 24 hours of transfection, 12 mL of fresh F12 complete medium containing 600% aG418 antibiotics was replaced. 4) The cells were cultured for 7-10 days, and 12 mL of F12 complete medium containing 600 μL of amine30 was replaced every 2-3 days to obtain a stable cell line CHO-LPAR3 with high expression of LPAR3 receptor. L-9418 (brand: Echelon) was purchased from Beijing Bailingwei Technology Co., Ltd. F12 culture medium (brand: Gibco; item number: C11765500BT) was purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd. Dimethyl sulfoxide (DMSO, item number: A503039-0250). HBSS (brand: Gibco; item number: 14065056; specification: (10×) 500 mL) and HEPES (brand: Gibco; item number: 15630080; specification: (100×) 500 mL) buffer were purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd. Each compound was dissolved in DMSO to prepare a 100mM stock solution. The buffer volume ratio was: HEPES: HBSS: ultrapure water = 1:5:44, which was used for compound dilution during the experiment. The detection platform was Corning third generation The imager detects the wavelength shift caused by the dynamic mass resetting (DMR) of the cell.

[0018] Example 1: Discovery that the fatty acid compound linoleic acid acts on the LPAR3 receptor

[0019] CHO-LPAR3 cells in the logarithmic growth phase were inoculated In the 384-well biosensor microplate, the volume of the cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 1.5×10 4 Then, the 384-well plate was placed in a cell culture incubator (5% CO2 volume concentration in air, 37°C) for 22-24 hours. When the cell confluence reached about 95%, the experiment was performed.

[0020] First, 10 μL of linoleic acid stock solution with a final concentration of 100 μM and a negative control group (0.1% DMSO (volume ratio) in 20 mM HEPES HBSS buffer) were added to different wells inoculated with CHO-LPAR3 cells and detected on the EPIC instrument for 1 hour. Then, L-9418 (0.5 μM, calculated based on the final volume of each well) was added and detected on the EPIC instrument for another 1 hour. The results are shown in FIG. Figure 1 As shown, linoleic acid has no DMR response signal in CHO-LPAR3 cells, but it can antagonize the DMR response signal of LPAR3 receptor agonist L-9418, indicating that it may have LPAR3 receptor antagonist activity.

[0021] Example 2: Verification of the effect of fatty acid compound linoleic acid on LPAR3 receptor

[0022] CHO-LPAR3 cells in the logarithmic growth phase were inoculated In the 384-well biosensor microplate, the volume of the cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 1.5×10 4 Then, the 384-well plate was placed in a cell culture incubator (5% CO2 volume concentration in air, 37°C) for 22-24 hours. When the cell confluence reached about 95%, the experiment was performed.

[0023] DMR antagonism detection: Linoleic acid with different final concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, HBSS Buffer containing 0.1% DMSO (volume ratio)) was pre-added to CHO-LPAR3 cells and its DMR response signal was detected in real time on the EPIC instrument for 1 hour. Then, LPAR3 receptor agonist L-9418 (0.5 μM, calculated according to the final volume of each well) was added and monitored in real time on the EPIC instrument for 1 hour. The results are shown in Figure 2. Figure 2 Linoleic acid has no DMR response signal on CHO-LPAR3 cells, but inhibits the DMR response signal of LPAR3 receptor agonist L-9418 in a concentration-dependent manner, indicating that it has LPAR3 receptor antagonist activity and is an LPAR3 receptor antagonist. Its antagonistic IC 50 The value was 23.06±4.56μM.

[0024] Pharmacological DMR experiments showed that LPAR3 receptor is the target of linoleic acid for the compound. Current studies have shown that LPAR3 receptor is associated with various cancers (astrocytic tumors, large cell medulloblastoma, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymoma, osteosarcoma), intraductal papillary mucinous tumor of the pancreas with low-grade dysplasia, etc. According to the correlation between the target and the disease, the clinical application range of this compound can be broadened.

Claims

1. A use of a linoleic acid compound as an LPAR3 receptor antagonist or for preparing a medicine, characterized in that: The LPAR3 receptor antagonist contains the following linoleic acid, or a derivative of the linoleic acid, and one or more of the pharmaceutically acceptable salts of the linoleic acid as active ingredients, and the compound structural formula is as follows:

2. The use according to claim 1, characterized in that: The linoleic acid compound described in claim 1 is used as an active ingredient in the preparation of therapeutic drugs for the following diseases, specifically including: one or more of astrocytic tumors, large cell medulloblastoma, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymoma, osteosarcoma, and intraductal papillary mucinous tumor of the pancreas with low-grade dysplasia.

3. The use according to claim 1 or 2, characterized in that: The LPAR3 receptor agonist or drug further comprises a pharmaceutically acceptable carrier and / or excipient.

4. A drug or LPAR3 receptor antagonist, characterized in that: The active ingredients are one or more of the following compounds, derivatives of the compounds, and pharmaceutically acceptable salts of the compounds; The chemical structure is shown below:

5. The drug or LPAR3 receptor antagonist according to claim 4, characterized in that: The medicine also includes a pharmaceutically acceptable carrier and / or excipient.

6. The drug or LPAR3 receptor antagonist according to claim 4 or 5, characterized in that: The medicine is a medicine for treating and / or preventing the following diseases, and the diseases include one or more of astrocytic tumors, large cell medulloblastoma, malignant pancreatic tumors, intraductal papillary mucinous carcinoma of the pancreas, malignant ependymoma, osteosarcoma, intraductal papillary mucinous tumor of the pancreas with low-grade dysplasia, and the like.

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