WO2019119275A1 - Gpr1 antagonizing polypeptide, derivative and application thereof - Google Patents

Abstract

The present invention provides a GPR1 antagonizing polypeptide having an amino acid sequence shown in any one of SEQ ID No. 1-3, and further provides a derivative of the polypeptide. The derivative is a product obtained by conventionally modifying the polypeptide on an amino acid side chain group, an amino terminal, or a carboxyl terminal or connecting the polypeptide to a tag for detection or purification. The polypeptide and the derivative thereof can combine with GRP1 in vitro, promote increasing of cAMP concentration by blocking a combination of chemerin and GRP1, inhibit chemerin-induced Ca2+ influx, inhibit cell proliferation of breast cancer, block the cell cycle thereof, and promote apoptosis of breast cancer so as to provide an effective treatment small molecule drug for female reproductive diseases such as breast cancer.

Description

GPR1 antagonist polypeptide and its derivative and application Technical field

The present invention relates to the fields of biotechnology and biomedicine, and in particular, the present invention is a target of the female reproductive disease GPR1 receptor antagonist polypeptide LRH7-G5 and its derivatives and applications.

Background technique

Breast cancer is the world's second-highest mortality fatal cancer after lung cancer, which continues to destroy the lives and health of millions of women and their families around the world. According to the latest statistics of the World Health Organization International Cancer Research Center, the number of new cases of breast cancer in women worldwide reached 1.67 million in 2012, accounting for 22.9% of all female malignant tumors; 460,000 women died of breast cancer, accounting for 13.7 of all female malignant tumor deaths. %, accounting for 1.7% of all female deaths, and about 1 in 4 women with cancer have breast cancer. The age-standardized incidence rate of breast cancer in Chinese women is 21.6/100,000, ranking first in female cancer; the mortality rate is 5.7/100,000, ranking sixth in female cancer death. 4% to 6% of breast cancers are metastatic breast cancer at the time of diagnosis, and 30% to 40% of early patients receiving adjuvant therapy can develop metastatic breast cancer, and the 5-year survival rate of patients is about 20%. Traditional treatments such as surgery, radiotherapy and chemotherapy have poor specificity and specificity, which inevitably have a killing effect on normal cells and tissues, and bring great side effects to patients. Therefore, finding highly effective and specific targeted drugs will greatly improve the therapeutic effect of breast cancer!

Clinical studies have found that early breast cancer often does not have typical symptoms and signs, and it is not easy to attract attention. It is often found through physical examination or breast cancer screening. Although early diagnosis of breast cancer can gain valuable time for the treatment of breast cancer, it can greatly improve the therapeutic effect of breast cancer, but the proportion of people who are currently diagnosed with early breast cancer in China is very small.

Metastasis to lymphatics after breast cancer surgery is a common metastatic symptom of breast cancer. Generally speaking, the chance of lateral breast lesions shifting to the axillary lymph nodes is 2/3, and the chance of metastasis to the sternum is 1/3. In the tumors on the medial and central parts of the breast, the chance of lymphatic metastasis to the medial and lateral breast cancer is 1/2, and breast cancer is prone to lung metastasis and lymphatic metastasis. The tumor microenvironment is crucial for the formation of tumors, which determines the occurrence and development of tumors. The first normal breast stem cells undergo carcinogenic mutations into breast cancer stem cells, resulting in poor prognosis of the tumor, and then these metastatic poor prognosis of the tumor, under the influence of stromal fibroblasts, part of the breast cancer stem cells metastasize, transferred to Brain, lungs, liver and bone marrow.

Studies have shown that the chemokine receptor CXCR4 is abnormally highly expressed in breast cancer tissues, and its ligand stromal cell-derived factor-1 (CXCL12) is also highly expressed in breast cancer metastasis sites such as bone marrow, lymph nodes, lungs and liver. The CXCR4/CXCL12 signaling system plays an important role in the homing of breast cancer to other distant organs, and thus has become a potential therapeutic target for breast cancer bone metastasis. Among them, CXCR4 antagonist polypeptide-CTCE-9908 has entered In the preclinical trial phase, bone metastasis of breast cancer is well inhibited in animal models of breast cancer. Therefore, chemokines play an important role in the development and metastasis of breast cancer. The targeted drugs for the treatment of breast cancer with chemokines as targets have broad market prospects and application value.

Breast cancer originates from various grades of ductal and acinar epithelium in the breast, and gradually develops from carcinoma of the gland to dysplasia to carcinoma in situ, early invasive carcinoma to invasive carcinoma. Carcinogenesis of different grades of catheters often varies in tissue type. More than 95% of breast cancers are malignant epithelial tumors, and breast sarcoma is very rare. The two most common invasive breast cancers in histopathology are: invasive ductal carcinoma (also known as "ordinary, NOS") and lobular carcinoma. These two types of breast cancer are formed by pre-invasive ductal and lobular tumors. The difference between invasive ductal carcinoma and lobular carcinoma as well as other types of breast cancer is mainly caused by morphological differences between cells.

Molecular targeted therapy for breast cancer is a treatment for the signaling pathways involved in the development and progression of breast cancer and their oncogene-related expression products. Affected by the high incidence of malignant tumors, the global anti-cancer drug market continues to grow. In 2010, the sales of anti-tumor drugs reached 45.97 billion US dollars, accounting for 5.8% of the world's drug sales, ranking first in the world. It is 8% more than in 2009. Among them, the sales of molecular targeted anti-tumor drugs was 31.19 billion US dollars, with a growth rate of 23%. In 1997, the US Food and Drug Administration (FDA) approved the application of a monoclonal antibody against trastomalin, a molecular targeted therapy against human epidermal growth factor receptor 2 (HER-2), and began breast cancer. A new era of molecular targeted therapy. According to global best-selling drug statistics, the global breast cancer drug market in 2015 has exceeded 10 billion US dollars, the highest sales of which is trastuzumab (Trastuzumab), sales of 6.79 billion US dollars, an increase of 4.2% over the same period. Many studies have found that epidermal growth factor receptor (EGFR) is a key molecule regulating the development and progression of breast cancer. By activating downstream Ras/Raf/MEK/ERK and PI3K/AKT signaling pathways, EGFR promotes breast cancer proliferation, migration and Invasion; in addition, epidermal growth factor receptor 2 (HER-2) is highly expressed in breast cancer cells, and promotes breast cancer by activating homologous dimers or heterodimers, activating downstream PI3K/Akt and Ras/MAPK signaling pathways. Proliferation, angiogenesis, migration and invasion. Currently, molecular targeted drugs for breast cancer treatment are: trastuzumab for epidermal growth factor receptor-2 treatment; against everolimus, an inhibitor in the PI3K/AKT/mTOR pathway; against anti-angiogenesis Drug bevacizumab; iniparib against BRCA1/2 mutation; and palbociclib against CDK4/6 inhibitor. The advent and application of these targeted drugs have brought new dawn to breast cancer patients, making breast cancer treatment more precise and greatly reducing the side effects of traditional treatment!

In the development of breast cancer, chemokines and their receptors are not only closely related to the establishment of tumor microenvironment and tumor metastasis, but also mediate the deterioration of tumor cells and promote the growth and proliferation of tumor cells. Studies have shown that CXCL8, CXCL12, CCL2, CCL5, CCL18 and other chemokines have a role in promoting breast cancer, while other chemokines such as XCL1, CXCL9, CXCL10, CXCL14, CCL16, CCL19, etc. may have anti-breast cancer effects. Tumor-associated fibroblasts (CAFs) and cancer cells can activate and secrete large amounts of CCL2, promote the self-renewal of cancer stem cells (CSC), thereby enhancing the resistance of tumor cells to chemotherapy and radiotherapy and promoting the metastasis of lesions. The positive rate of CXCR4 in breast cancer is as high as 60%. The CXCL12/CXCR4 network plays an important role in the growth and metastasis of various solid tumors including breast cancer. Blocking this biological axis can inhibit angiogenesis and tumors. Diffusion increases the sensitivity of tumor cells to chemotherapy and radiotherapy. In the 4T1 mouse breast cancer model, treatment with an oncolytic virus carrying a CXCR4 antagonist inhibits angiogenesis and tumor metastasis. The expression of CXCR4 in TNBC (71%) was higher than that of HER-2 positive breast cancer (44%) and Luminal type breast cancer (37%). The tumor of CXCR4+TNBC was large, prone to liver, lung and brain metastasis, and the prognosis was poor. Chemokines and their receptor antagonists or inhibitors have broad market prospects. Statistics show that about 30% of small molecule drugs are targeted by G-protein coupled receptors. The CXCR4 polypeptide antagonist CTCE-9908 has been shown to inhibit lung metastasis in mouse osteosarcoma and melanoma. CXCR1/CXCR2 Antagonist - Repertaxin inhibits tumor growth and metastasis in animal models and has been used in clinical trials of patients with advanced breast cancer. Therefore, studying the role of chemokines in tumor development, looking for new targets for chemokines in the treatment of breast cancer, is not only important for the interpretation of tumor metastasis, but also has broad drug market prospects.

The study of the relationship between obesity and breast cancer was first seen in the 1960s. Case-control studies first proposed that women with breast cancer tend to have postmenopausal obesity and hypertension. It has also been found in animal experiments that the tumor growth rate of obese mice is significantly increased compared with the control group. The World Cancer Research Foundation and the American Cancer Research conducted retrospective analysis of epidemiological evidence. Animal studies have shown that overweight and obesity can significantly increase the risk of breast cancer. Meta-analysis showed that the risk of breast cancer increased with the increase of BMI. The risk of breast cancer in patients with BMI>30kg/m2 was 1.3~2 times higher than that of normal people. Obesity can significantly increase the risk of breast cancer in women.

The possible mechanisms by which obesity promotes the development and progression of breast cancer include the following aspects: (1) Estrogen. Obesity increases estrogen synthesis, lowers serum estradiol and sex hormone binding globulin (SHBG) levels, serum estrogen excess, competes with estrogen receptors on target organs, causing hypothalamic-pituitary-ovarian gonadal axis dysfunction, Break the balance between breast tissue proliferation and rejuvenation, promote abnormal proliferation and transformation of breast cancer cells; (2) chronic inflammation. Activated macrophages in adipose tissue of obese patients can secrete a large number of pro-inflammatory mediators, causing chronic inflammatory reactions. A large number of inflammatory factors and various cells in the body form a tumor microenvironment for tumors, thereby promoting the occurrence and development of breast cancer cells. When the concentration of TG in the blood is too high, breast vascular lipid deposition leads to chronic inflammation, and inflammatory factors secreted by inflammatory cells are formed by interstitial fibroblasts, lymphocytes, macrophages, blood vessels and lymphatic networks, and extracellular matrix. The tumor microenvironment, which interferes with DNA replication and transcription, leads to malignant tumors; (3) insulin and insulin-like growth factor-1 (IGF-1). Obesity is prone to insulin resistance, which increases the levels of insulin and IGF-1, and contributes to cancer cell proliferation and angiogenesis. A study from the Swiss database collected data from 115,000 patients who received insulin therapy in 2005 and found that patients who were treated with insulin glargine for the next two years had a significantly higher risk of breast cancer than other insulin analogues. High (RR=1.99). (4) Adipocytokines. Obesity affects the production of adipocytokines, lowers adiponectin, raises the levels of leptin, PAI-1, etc., promotes cell proliferation, anti-apoptosis and accelerates angiogenesis, thereby promoting the proliferation and growth of cancer cells. Adiponectin can not only inhibit the mitotic effect of insulin and estrogen, but also promote the metastasis and infiltration of cancer cells by promoting the expression of Bax and p53 proapoptotic genes. Leptin can promote breast cancer growth by activating JAK/STAT3, MAPK-ERK1/2 or PI3K pathways; in addition, leptin can promote tumor-associated angiogenesis by inducing angiopoietin expression, and leptin can also Induces transcription of human epidermal growth factor receptor 2ErbB-2) and participates in the reaction of insulin-like growth receptor 1 (IGF-1) in triple-negative breast cancer cells, activating epidermal growth factor receptor (EGFR) to promote cell invasion With transfer. Obesity is a risk factor for postmenopausal breast cancer. Obesity is closely related to the occurrence and development of breast cancer. A large number of studies have shown that obesity can negatively regulate the occurrence, development, diagnosis, tumor characteristics and prognosis of breast cancer. Therefore, through the further exploration and research on the relationship between obesity, adipose tissue and its secreted adipokines and breast cancer, it can provide a very important reference for the future development of corresponding measures to reduce the incidence of breast cancer and improve the prognosis of the disease.

Fatty factor Chemerin

The adipokines Chemerin is also known as tazarotene-inducible gene 2 (TIG2) or retinol receptor-reactive protein 2. After being discovered by Nagpal and other clones in 1997, in 2003, Wittamer et al. isolated active proteins in reverse blood from ovarian cancer by reversed-phase high pressure liquid chromatography. The Chemerin protein precursor is 163 amino acids in length, has 6 cysteine residues, forms 3 disulfide bonds, removes the N-terminal signal peptide and several amino acids at the C-terminus, and has biological activity, and chemerin activation in the blood. The form contains 134 amino acids (about 16 kDa) and is structurally a family of bactericidal peptides/cysteine protease inhibitors. When an inflammatory reaction occurs, chemerin can be rapidly converted into an activated form by several proteases. Chemerin is widely expressed in various tissues of human body, mainly expressed in white adipose tissue, placenta and liver, and is combined with three receptors: 1G protein-coupled receptor CMKLR1; 2G protein-coupled receptor GPR1; 3CCRL-2. After binding to GPR1, Chemerin can release calcium ions in GPR1-positive cells, inhibit aggregation of cAMP, and phosphorylate MAP kinase. On the other hand, it can promote intracellular calcium influx. Chemerin acts as a chemokine, chemotaxis of dendritic cells and macrophages, and acts as a bridge between immune and adaptive immunity. Chemotactic natural killer cells reach the site of inflammation to participate in inflammatory reactions; on the other hand, as new fats The factor, produced by adipose tissue secretion, regulates the differentiation and lipolysis of fat cells, and promotes biological effects such as insulin signaling pathways in adipocytes. Chemerin plays an important role in the pathophysiological mechanisms of obesity and metabolic syndrome.

GPR1 receptor

GPR1 is the closest homolog of CMKLR1, sharing more than 40% sequence identity with CMKLR1. It is a G-protein coupled receptor that plays a key role in eukaryotic cells, usually for glucose stimulation, lipid accumulation, and signaling of nutrients to the cAMP pathway. Many signal transductions are mediated through G protein-switched receptors (GPCRs) in eukaryotes. G protein beta coupled receptors are found in some eukaryotes, including yeast, diflagellates and animals. GPR1 was originally a G protein-coupled receptor (GPCR) found in humans and was identified as a receptor for chemerin by in vitro experiments. Although the physiological function of Chemerin binding to GPR1 was not reported, GPR1 was reported to be highly expressed in brown adipose tissue, white adipose tissue and skeletal muscle of murine animals, and GPR1 was mainly expressed in vascular cells of white adipose tissue. In GPR1 knockout mice fed a high-fat diet, glucose intolerance was found to be more severe than in WT mice. Furthermore, in the pyruvate tolerance test, GPR1 knockout mice were able to inhibit glucose-stimulated insulin levels and cause elevated blood glucose. These results indicate that GPR1 is an active receptor for Chemerin that regulates glucose homeostasis during obesity.

The activity of CMKLR1 and GPR1 in HTLA cells after increasing Chemerin dose treatment was measured using a Tango bioassay. Chemerin was found to activate CMKLR1 and GPR1 with similar efficacy, achieving a maximum response (Emax) of 265.3 ± 182.2- and 185.6 ± 26.5-fold changes. Furthermore, Chemerin activation of GPR1 was significantly more potent than activation of CMKLR1 (EC50, 54.6 ± 30.7 nM) (EC50, 18.2 ± 2.9 nM), demonstrating that Chemerin activates GPR1 and that GPR1 is also a highly sensitive Chemerin receptor.

The GPR1 expressing cell line responds to chemerin by weak Ca ²⁺ mobilization and ERK1/2 activation, but it is efficiently internalized due to agonist binding. Therefore, GPR1 may act as a decoy receptor for chemerin, and to date, there has been no description of GPR1-mediated activity in or on primary cells. And GPR1 is expressed in the central nervous system, skeletal muscle, skin and adipose tissue, possibly regulating chemerin activity.

G-protein coupled receptor superfamilies (GPCRs) play an important role in extracellular signaling to intracellular transduction, regulating cell movement, growth, and gene transcription—a critical factor in these three cancer biology . In the past decade or so, the mediated signaling pathway has been shown to be a key regulator of protooncogene signaling and is a good drug target. Currently, 60% of the drugs sold on the market are designed for this receptor. . While GPR1 is a member of GPCRs, there is still no drug that directly targets GPR1 to treat clinical cancer. Therefore, targeting GPR1, screening new anti-cancer peptide drugs and humanized antibodies has important social significance and broad economic market.

Phage Display Technology is a specific polypeptide or protein screening technology. The technology can display the polypeptide encoded by the target gene as a fusion protein on the surface of the phage. The displayed polypeptide or protein can remain relatively independent. Spatial structure and biological activity, which establish a direct link between a large number of random polypeptides and their DNA coding sequences, enabling peptide ligands of various target molecules (such as antibodies, enzymes and cell surface receptors) to be obtained by in vitro affinity panning procedures. Quick identification. Since the introduction of phage display technology, phage antibody library technology has been progressing rapidly in recent decades due to its high storage capacity, high efficiency and convenience, and flexible screening. It has been widely used in many fields of life science, especially in tumor diagnosis and tumor. The field of antibody drug preparation and other fields is receiving more and more attention. At present, the main targets of peptide drugs are G-protein coupled receptors (GPCRs), and 39% of polypeptide drugs target GPCRs in clinical research. Peptide drugs have many advantages: (1) higher than general chemical drugs, high biological activity and specificity; (2) relatively weak toxicity, not easy to accumulate in the body; (3) less interaction with other drugs, and in vivo The affinity of the body is better. Humanized monoclonal antibodies have the advantages of high sensitivity, high specificity, high efficiency, little or no serum cross-reaction, and low preparation cost. The use of phage display technology to screen peptide drugs and humanized antibodies can achieve the unification of genotype and phenotype. It is a highly efficient and practical technical method for preparing antibodies. This technology utilizes the affinity of the expressed protein to the ligand to make it needed. Proteins or peptides are screened and widely used in the identification and screening of disease markers and in the screening and preparation of antibody drugs.

Summary of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the primary object of the present invention is to provide a GPR1 antagonist polypeptide which is screened by a phage display library, which has a specific high affinity with the Chemerin receptor GPR1 and can inhibit Chemerin and GPR1. The combination of these blocks to block the signaling pathway of Chemerin/GPR1, which proves that the peptide plays an important role in targeting inhibition of breast cancer cell proliferation and promoting apoptosis of breast cancer cells, and has great application in breast cancer targeted therapy. value.

Another object of the present invention is to provide a derivative of the above GPR1 receptor antagonist polypeptide which is also capable of specifically having high affinity with the GPR1 receptor and which specifically competes with the binding site of Chemerin and GPR1, and is capable of inhibiting Chemerin binding to GPR1. .

A further object of the present invention is to provide the use of the above GPR1 antagonist polypeptide and derivatives thereof.

In order to achieve the above tasks, the present invention adopts the following technical solutions:

A GPR1 receptor antagonist polypeptide characterized in that its amino acid sequence is: Met-(D)Pro-Arg-Leu-Pro-Pro-Ala-NH ₂ SEQ ID No. 1, LRH7-G5.

The screening method of the above GPR1 receptor antagonist polypeptide adopts a phage random peptide library, firstly transfects 293T cells with GPR1 plasmid to obtain a stable cell line with permanent high expression of GPR1, and adsorbs cells with wild type 293T cells as control, for 5 rounds. Whole cell subtractive screening, random selection of 50 positive phage amplification, extraction of single-stranded DNA sequencing. The basic characteristics of the amino acid sequence of the polypeptide are analyzed, the homology of the polypeptide is compared, and the polypeptide motif with high frequency of occurrence is searched. BLAST searches a protein database to detect proteins with high homology of polypeptide motifs, and discovers biological species containing a large amount of the polypeptide, and possibly cell surface receptors and ligands, which facilitate subsequent large-scale extraction and purification of polypeptides.

Hydrophilic analysis indicated that LRH7-G5 is a hydrophilic polypeptide;

The purity of LRH7-G5 synthesis was 99.85% by high performance liquid chromatography (HPLC) and mass spectrometry (MS).

According to another aspect of the present invention, the derivative of the GPR1 receptor antagonist polypeptide of the present invention is obtained by conventionally modifying the amino terminus or the carboxy terminus of the GPR1 receptor antagonist polypeptide fragment on the amino acid side chain group of the GPR1 receptor antagonist polypeptide. a product, or a product obtained by ligating a tag for detection or purification of a polypeptide or protein on a GPR1 receptor antagonist polypeptide;

Preferably, the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotinylation , fluorophore modification, polyethylene glycol PEG modification or immobilization modification;

Preferably, the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.

In a further aspect of the invention there is provided a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide of the invention, which is set forth in SEQ ID No. 2-3:

MPRLPPA SEQ ID No. 2

MPRLPPA-NH ₂ SEQ ID No. 3.

The present invention also provides a derivative of a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide which antagonizes an amino acid side chain group, an amino terminus or a carboxyl group of a derivative of a biologically active fragment or analog of the polypeptide a product obtained by conventional modification, or a product obtained by linking a tag for detection or purification of a polypeptide or protein to a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide;

Preferably, the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotinylation , fluorophore modification, polyethylene glycol PEG modification or immobilization modification;

Preferably, the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.

In the technical solution of the present invention, the GPR1 receptor antagonist polypeptide and derivatives thereof can be applied to the preparation of a medicament for preventing and/or treating female reproductive diseases in the form of 7 amino acids. The GPR1 antagonist polypeptide derivative is a product obtained by conventional modification of the amino acid side chain group of the GPR1 antagonist polypeptide, the amino terminus or the carboxy terminus of the GPR1 antagonist polypeptide fragment, or the GPR1 antagonist polypeptide is ligated for polypeptide or protein detection. Or a purified tag resulting in a product; the conventional modification is preferably amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization , biotinylation, fluorophore modification, polyethylene glycol PEG modification or immobilization modification, etc.; the label is preferably His ₆ , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact Wait;

The GPR1 antagonist polypeptide and its derivatives may be derived from insects, humans, monkeys, birds, fungi, bacteria, fish, bats, mammals or birds, such as primates (humans); rodents, including Mouse, rat, hamster, rabbit, horse, cow, dog, cat, etc.

Preferably, the derivative of the GPR1 antagonist polypeptide is: the second amino acid residue of the above GPR1 antagonist polypeptide is a D-configuration proline, and the terminal is amidated, that is, Met-(D)Pro-Arg-Leu-Pro -Pro-Ala-NH ₂ SEQ ID No. 1.

The GPR1 antagonist polypeptide and the derivative thereof are obtained by a known method in the prior art, and can be chemically synthesized by an automatic peptide synthesizer; the nucleotide sequence is deduced from the short peptide sequence, and then cloned into a vector. Biosynthesis is carried out; it can also be extracted and purified in large quantities from existing organisms.

In particular, the GPR1 antagonist polypeptide LRH7-G5 and its derivatives include the following polypeptides naturally present in the organism:

SEQ ID No. 2 MPRLPPA (insect, human, monkey, bird, fungus, bacteria, fish, bat)

SEQ ID No. 3 MPRLPPA-NH ₂ (insect, human, monkey, bird, fungus, bacteria, fish, bat).

A further aspect of the invention provides a polynucleotide encoding the polypeptide of any one of SEQ ID No. 1-3.

In a further aspect of the invention there is provided a vector comprising a nucleotide of the invention which can be linked to a promoter sequence by genetic means.

In a further aspect of the invention there is provided a host cell transfected with a vector of the invention.

According to still another aspect of the present invention, a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a bioactive fragment or the like of the GPR1 receptor antagonist polypeptide, and a derivative thereof are provided in the preparation of a therapeutic chemerin-GPR1 Use in drugs that cause disease.

According to still another aspect of the present invention, a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a biologically active fragment or the like of the GPR1 receptor antagonist polypeptide, and a derivative thereof are provided in the treatment of chemerin-GPR1. Use in disease.

In the technical solution of the present invention, the chemerin-GPR1 mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response, and polycystic ovary syndrome.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide and the derivative thereof of the present invention provide cAMP for inhibiting chemerin production. The use of reduced concentrations of the drug.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide and the derivative thereof of the present invention provide a cAMP concentration caused by inhibition of chemerin. The use of the lowering.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide, and the derivative thereof are provided for inhibiting calcium induced by chemerin. Use in drugs with (Ca ²⁺ ) influx.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide, and the derivative thereof of the present invention provide calcium inhibiting by chemerin ( Use in Ca ²⁺ ) influx.

According to still another aspect of the present invention, a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a biologically active fragment or the like of the GPR1 receptor antagonist polypeptide, and a derivative thereof according to the present invention provide a cell which inhibits chemerin production. Use in chemotactic drugs.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide and the derivative thereof of the present invention provide a cell line induced by chemerin inhibition. Use in the role of chemical.

According to still another aspect of the present invention, a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a bioactive fragment or the like of the GPR1 receptor antagonist polypeptide, and a derivative thereof are provided for treating breast cancer and fat. Use in liver, diabetes, inflammatory response, and drugs for polycystic ovary syndrome.

According to still another aspect of the present invention, the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the bioactive fragment or the like of the GPR1 receptor antagonist polypeptide and the derivative thereof are provided for treating breast cancer and fatty liver. , diabetes, inflammatory response, use in polycystic ovary syndrome.

According to still another aspect of the present invention, a pharmaceutical composition comprising the GPR1 receptor antagonist polypeptide of the present invention, a derivative of a GPR1 receptor antagonist polypeptide, a biologically active fragment or analog of the GPR1 receptor antagonist polypeptide, and the like thereof are provided. One or more of the substances are used as active ingredients.

The pharmaceutical composition may contain one or a plurality of pharmaceutically acceptable carriers;

The pharmaceutically acceptable carrier is preferably a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant;

The pharmaceutical composition may be further prepared into various forms such as tablets, granules, capsules, oral liquids or injections, and the medicaments of various dosage forms may be prepared according to conventional methods in the pharmaceutical field;

A medicament for preventing and/or treating a female reproductive disease or a tumor, comprising the GPR1 receptor antagonist polypeptide of the present invention, a derivative of a GPR1 receptor antagonist polypeptide, a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide, and At least one of its derivatives.

A medicament for preventing and/or treating a disease which highly expresses a GPR1 receptor, comprising a GPR1 receptor antagonist polypeptide, a derivative of a GPR1 receptor antagonist polypeptide, a biologically active fragment of GPR1 receptor antagonist polypeptide or the like and a derivative thereof At least one of them.

In a specific experiment of the present invention, the present invention utilizes the obtained GPR1 antagonist polypeptide LRH7-G5 to effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway. Among them, the GPR1 antagonist polypeptide LRH7-G5 derivatives (SEQ ID Nos. 1 to 3) have the same effects.

In another specific experiment of the present invention, the GPR1 antagonist polypeptide LRH7-G5 can effectively inhibit the influx of calcium (Ca ²⁺ ) induced by chemerin. Among them, the GPR1 antagonist polypeptide LRH7-G5 derivatives (SEQ ID Nos. 1 to 3) have the same effects.

In further experiments, the inventors demonstrated the function of the GPR1 antagonist polypeptide LRH7-G5 using the breast cancer cell MDA-MB-231, which highly expresses GPR1, as a cell model. Compared with the control group, LRH7-G5 was found to significantly inhibit breast cancer cells. Proliferation of MDA-MB-231. Among them, the GPR1 antagonist polypeptide LRH7-G5 derivatives (SEQ ID Nos. 1 to 3) have the same effects.

In another specific experiment of the present invention, the inventors used flow cytometry to detect that the GPR1 antagonist polypeptide LRH7-G5 can block the MDA-MB-231 cycle of breast cancer cells and promote the apoptosis of MDA-MB-231. Among them, the GPR1 antagonist polypeptide LRH7-G5 derivatives (SEQ ID Nos. 1 to 3) have the same effects.

The present invention has the following advantages and effects over the prior art:

(1) The present invention provides a GPR1 antagonist polypeptide LRH7-G5 and a derivative thereof (SEQ ID No. 1 to 3), which are capable of specifically binding to GPR1 and specifically competing for Chemerin Binding to the GPR1 site inhibits the Chemerin/GPR1 signaling pathway.

(2) The GPR1 antagonist polypeptides and the derivatives thereof (SEQ ID No. 1 to 3) screened by the present invention can inhibit the proliferation of breast cancer cells by blocking the binding of Chemerin/GPR1, and promote apoptosis of breast cancer cells, and can be used as The bio-peptide drug of the Chemerin/GPR1 binding site can be used for the preparation of drugs for preventing and/or treating female reproductive diseases, such as breast cancer, fatty liver, diabetes, and inflammatory reactions. It can be widely used in the fields of medicine and biology, and it has enormous social and economic benefits.

DRAWINGS

Figure 1: LRH7-G5 high performance liquid chromatography (HPLC) detection map.

Figure 2: LRH7-G5 mass spectrometry (MS) detection map.

Figure 3: Comparative analysis of hydrophobic profiles of GPR1, chemerin and LRH7-G5 polypeptides. Among them: A: GPR1 hydrophobic profile; B: chemerin hydrophobic profile; C: LRH7-G5 polypeptide hydrophobic profile; D: GPR1, chemerin and LRH7-G5 polypeptide hydrophobic profile comparison. The GPR1 outline is red, the chemerin outline is blue, and the LRH7-G5 outline is green;

Figure 4: LRH7-G5 attenuates the inhibitory effect of chemerin on the cAMP signaling pathway. Among them, A, LRH7-G5 and Forsklin and Chemerin in 293T wild-type cells; B, LRH7-G5 alone in 293T GPR1 ^{+ / +} ; C, LRH7-G5 and Forsklin and Chemerin at 293TGPR1 ^{+ / +} Role in

Figure 5: LRH7-G5 inhibits the influx of calcium (Ca ²⁺ ) induced by chemerin.

Figure 6: LRH7-G5 inhibits the proliferation of breast cancer cell MDA-MB-231.

Figure 7: LRH7-G5 blocks the cell cycle progression of MDA-MB-231 in breast cancer cells. A: flow cytometry to detect cell cycle; B: cell cycle data statistics.

Figure 8: LRH7-G5 promotes apoptosis of breast cancer cell MDA-MB-231. A: Flow cytometry detects apoptosis; B: Apoptosis data statistics.

Detailed ways

In order to more clearly understand the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings. The examples are for illustrative purposes only and are not intended to limit the invention in any way. In the examples, each of the original reagent materials is commercially available, and the experimental methods not specifying the specific conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.

Example 1: Panning, amplification, purification, sequencing and synthesis of the GPR1 antagonist polypeptide LRH7-G5.

This example is mainly for screening for positive phage which specifically binds to GPR1, and then by amplifying and purifying the positive phage, extracting phage single-stranded DNA (ssDNA) for sequencing, comparing the obtained sequence analysis, and finally synthesizing high purity. Antagonistic polypeptide LRH7-G5.

details as follows:

1. Establish a 293T cell line that permanently expresses GPR1: 293T-GPR1 ^+/+ /LRH

1 The luminescent human 293T cells were selected and inoculated into 6-well plates at 5×10 ⁵ cells/well one day before transfection, and the cell fusion degree was 60% after the second day of culture;

2 Transfection was performed on the second day, and 3 μg of the plasmid was diluted with 200 μL of opti-MEM medium in a culture well of a 6-well plate, and 6 μL of liposome Lipofectamine 2000 was diluted with 200 μL of opti-MEM medium, respectively. After mixing, leave it at room temperature for 5 minutes;

3 gently mix the two tube dilutions, let stand for 20 minutes at room temperature, gently add 600 μL of opti-MEM medium to the mixed dilution;

4 The cells to be transfected were gently rinsed once with PBS, and then the mixed dilutions were gently added to the culture wells and placed in a carbon dioxide incubator for cultivation;

5 After 4 to 6 hours of culture, discard the medium used for transfection, and add 3 mL of complete medium to the well;

After 648 hours, a medium containing 1 μg/mL puromycin was selected for screening; a 293T cell line stably expressing GPR1 was obtained after the cells no longer died.

7 Total RNA was extracted with TRIzol, 2 μg of RNA was quantified for reverse transcription (reverse transcription kit, purchased from Promega), and qPCR was performed using specific primer sequences.

The specific primer sequence used is the Hu-GPR1 primer sequence:

Fw 5'-AATGCCATCGTCATTTGGTT-3' (SEQ ID No. 4)

Rv 5'-CAACTGGGCAGTGAAGGAAT-3' (SEQ ID No. 5)

8 Compared with transfected pSM2c-Hu-scramble RNA, the high expression level of GPR1 was detected and named as: 293T-GPR1 ^+/+ /LRH, which can be used for positive phage screening.

2. Purification, amplification, purification, sequencing and synthesis of GPR1 antagonist polypeptides.

Preparation of 1ER2738 host bacterial solution: Aseptic technique, first take 200μl LB-Tet liquid medium in 1.5ml sterile centrifuge tube, then mix 0.2μl bacterial solution from E.coli ER2738 glycerol frozen solution and mix well with it. All the tubes were blotted onto LB-Tet plates, and the plates were labeled, placed at room temperature for 3 min, and then placed in a 37 ° C incubator for overnight incubation. The next day of observation, the clone was grown and sealed with a parafilm, and stored at 4 ° C in the dark. A single colony was picked by a sterile technique using a sterile tip, placed in a 10 ml sterilized centrifuge tube to which 3 ml of LB-Tet liquid medium was previously added, labeled, and shake-cultured at 37 ° C, 300 rpm/min overnight at a constant temperature shaker. The next day, the bacterial amplification solution was stored at 4 ° C until use. Take 10ml sterile centrifuge tube, add 3ml LB-Tet liquid medium aseptically, take 30μl of overnight cultured bacteria to inoculate it, shake the incubator at 37°C, shake at 300rpm/min for 2~3h, the bacteria are in exponential growth phase, naked eye Observed as a mist (OD ₆₀₀ ~ 0.5).

Panning of 2GPR1 antagonist peptide: high-expression GPR1 cells were inoculated into a 60×15 mm ² culture dish pre-coated with polylysine in 10 ⁵ / culture dishes, and cultured until the cell density was 80% to 90%. For panning (while using a cell line that does not express GPR1 as a blank control), take 1μl of titer for each round of eluate, and add the remaining to 20ml of LB medium for amplification, purification, and finally measurement. After the increased titer, the amplification was stored at 4 ° C for a short period of time and taken for an equal number of stages for the next round of panning, and the remaining amplifications were stored at 50 ° C with glycerol at -20 ° C.

3 Determination of phage titer: Take 4 sterile 10 ml centrifuge tubes, prepare 1 sterile centrifuge tube for each phage dilution, melt the top agar (Agarose Top) in a microwave oven, add 3 ml top agar to each tube, and use a 45 ° C water bath. . One LB/IPTG/Xgal plate was prepared for each phage dilution, and preheated in a 37 °C incubator. E. coli ER2738 E. coli having an OD ₆₀₀ to 0.5 was dispensed according to a phage dilution of 200 μl/tube, and stored at 4 ° C until use. Take 4 sterile 1.5ml centrifuge tubes, respectively, containing 100μl, 90μl, 90μl, 90μl LB-Tet medium, and take 1μl of the phage to be tested into 100μl LB-Tet medium, and dilute it by 10-fold gradient. For 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , gently mix and shake each dilution and centrifuge instantaneously. 10 μl of each dilution of the phage to be titrated was mixed with 200 μl of E. coli ER2738, gently shaken and mixed, centrifuged instantaneously, and incubated at room temperature for 5 min. The mixed bacterial solution was quickly added to the top agar, mixed rapidly by shaking, and immediately poured into a preheated LB/IPTG/Xgal plate, which was uniformly flattened, cooled at room temperature for 5 min, and cultured in a 37 ° C incubator at room temperature overnight.

4 Amplification and purification of eluted phage: Take a 250 ml Erlenmeyer flask, add the ER2738 host broth cultured overnight to 20 ml LB liquid medium at a ratio of 1:100, and incubate vigorously at 37 ° C, 250 rpm for 2 h; The phage liquid to be amplified was added to an Erlenmeyer flask, and cultured vigorously at 37 ° C, 250 rpm for 4.5 h; the culture was transferred to a 50 ml centrifuge tube and centrifuged at 10,000 rpm for 10 min at 4 ° C. The supernatant was transferred to another clean centrifuge tube, centrifuged again at 10,000 rpm for 10 min at 4 ° C; 80% of the supernatant was transferred to another clean centrifuge tube, 1/4 volume of PEG/NaCl was added, and the mixture was inverted and mixed. The pellet was precipitated overnight at 4 ° C; the next day, the pellet was centrifuged at 12,000 rpm for 20 min at 4 ° C. The supernatant was carefully aspirated with a clean tip and centrifuged at 12,000 rpm for 1 min at 4 ° C to remove the residual supernatant; then the pellet was resuspended in 1 ml of TBS and gently pipetted 100 times. The suspension was then transferred to a 2 ml centrifuge tube and centrifuged at 10,000 rpm for 5 min at 4 ° C for 5 min to remove residual cells; the supernatant was added to 1/4 volume of PEG/NaCl, and then incubated on ice for 60 min to precipitate again; the centrifuge tube was removed, 12,000 °C at 4 °C After centrifugation at rpm for 20 min, the supernatant was removed; the pellet was resuspended in 200 μl of TBS and centrifuged at 10,000 rpm for 1 min at 4 °C. The supernatant is transferred to another centrifuge tube. Short-term storage at 4 ° C, can also be stored with 50% glycerol at -20 ° C for a long time. Amplification of monoclonal phage, including (1) adding the ER2738 host broth cultured overnight to 2 mL LB liquid medium at a ratio of 1:100, vigorously shaking at 37 ° C, 250 rpm for 2 h; using a sterile toothpick, from the fourth round The plate with less than 100 plaques was selected from the titer plate, and the well-separated blue plaques were picked and added to the culture tube. The culture was vigorously shaken at 37 ° C for 250 h/min for 4.5 h; then the culture was transferred to Centrifuge at 10,000 rpm for 30 sec at 4 ° C in a fresh centrifuge tube. The supernatant was transferred to a fresh tube and centrifuged again; 80% of the supernatant was transferred to a fresh centrifuge tube and stored at 4 ° C. It can also be stored with 50% glycerol at -20 ° C for a long time.

5 Agarose gel electrophoresis identification of M13 phage ssDNA: Place the gel forming mold horizontally, place the selected comb, leave 1mm space between the bottom of the comb and the mold; weigh 1g of DNA electrophoresis with agarose into 250ml triangle Add 100 ml of 1×TAE buffer to the flask, mix well, place the flask in a microwave oven, heat and boil until the agarose is completely dissolved; close the induction cooker, take out the Erlenmeyer flask, and let it cool to room temperature at room temperature (hand holding the flask) Can be tolerated), and then add 5μl of ethidium bromide, after mixing, the gel solution is poured into the rubber sheet. The rubber plate used in this experiment requires about 100ml of glue; the gel is completely solidified at room temperature, it takes about 30 minutes, the comb is pulled out, and the rubber plate is placed in the electrophoresis tank; 1×TAE buffer is added to the electrophoresis tank. It is better to raise the surface of the gel by 2mm; the sample is diluted with the Loading buffer and added to the rubber plate. Note that the applicator tip should be placed in the gel sample hole, the gel should not be pierced, and the sample should be prevented. Outside the overflow hole; turn on the power, adjust the voltage to 50 volts, after electrophoresis for 90 minutes, remove the gel plate and observe the result under UV light.

6ssDNA sequencing and sequence analysis: The extracted M13 phage ssDNA was sent to Shanghai Yingji Jieji Biotechnology Co., Ltd. for DNA sequencing. Sequence analysis was performed using Bioedit software after sequencing. According to the analysis results, the sample sequence is Met-(D)Pro-Arg-Leu-Pro-Pro-Ala-NH ₂ , wherein the second proline is in the D configuration, represented by LRH7-G5, and the short peptide is Shanghai Qiang Yao Biological Company Synthetic.

Figure 1 High performance liquid chromatography (HPLC) and Figure 2 mass spectrometry (MS) showed that the purity of LRH7-G5 polypeptide was 99.85%, and the molecular weight was consistent with the predicted value. The hydrophobic profile analysis of Figure 3 demonstrates that the LRH7-G5 polypeptide has some similarity to chemerin with a similarity of 0.0245399 (PAM250).

Example 2 GPR1 antagonist polypeptide LRH7-G5 can effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway.

(1) Cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay:

1 cell plating: wild type 293T cells and 293T cells (293TGPR1 ^+/+ ) with high expression of GPR1 were seeded at 5×10 ⁵ cells/well in 6-well cell culture plates at a volume of 1 mL per well. After incubation for 24 h in the incubator, starvation overnight, adding different concentration gradients (3 μM, 0.3 μM, 0.03 μM) of LRH7-G5 polypeptide, Fosklin (25 μM) and chemerin (30 nM) for 6 h;

2 Sample preparation: 300 μL of cell lysate was added to each well, and placed at 4 ° C for 20 minutes. The cells were collected by scraping with a cell scraper, mixed upside down, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected;

3 sample concentration determination: sample concentration is determined by BCA method;

4 cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay:

a, prepare the required reagents, prepare 3 duplicate wells for each sample;

b, 50 μL of the sample or standard is added to the antibody-coated 96-well plate; then 25 μL of cAMP peroxidase tracer conjugate is added to each well;

c, 50 μL of Anti-cAMP antibody was added to each well, and incubated at room temperature for 30 minutes on a shaker;

d, wash 5 times with eluent, add 100 μL of chemical reflective agent per well, incubate for 5 minutes at room temperature;

e, the microplate reader reads the plate and records the luminescence value.

Figure 4 shows that chemerin can reduce cellular cAMP concentration at a concentration of 30 nM, but different concentrations of LRH7-G5 (3 μM, 0.3 μM, 0.03 μM) are added to 293T cells (293T GPR1 ^+/+ ) with high expression of GPR1. After that, the cAMP concentration can be significantly increased. In wild-type 293T cells, GPR1 receptor was not expressed, so LRH7-G5 had no significant inhibitory effect on chemerin's effect on reducing cAMP concentration. Similarly, LRH7-G5 did not increase cAMP concentration in the LRH7-G5 short peptide alone group. In summary, it can be inferred that LRH7-G5 can specifically inhibit the decrease of cAMP concentration by chemerin by acting with GPR1. *P<0.05, **P<0.01, ***P<0.001 compared with the Forskolin+chemerin group.

Example 3 The GPR1 antagonist polypeptide LRH7-G5 can effectively inhibit the influx of calcium (Ca ²⁺ ) induced by chemerin.

1 cell plating: wild type 293T cells and 293T cells (293TGPR1 ^+/+ ) with high expression of GPR1 were seeded at ⁵ ×10 ³ cells/well in 96-well cell culture plates at a volume of 200 μL per well. After culturing for 24 hours in the box, starve overnight;

2 Reagent configuration: Mix probenecid into 1mL buffer solution, configure to a concentration of 250nM probenecid, shake well, add fluorescent reagents to prepare for use;

3 Remove the cell culture medium, add LRH7-G5 polypeptide with different concentration gradient (30μM, 3μM, 0.3μM, 0.03μM, 0.003μM) and chemerin (0.3nM) for 30 minutes, then add 100μL of the above fluorescent reagent per well;

Leave at 437 ° C for 30 minutes, then leave at room temperature for 30 minutes;

5 Fluorescence absorbance was measured at excitation light 494 nm and emission light 516 nm.

Figure 5 shows that in 293T cells (293T GPR1 ^+/+ ) with high expression of GPR1, chemerin can promote calcium (Ca ²⁺ ) flow signaling pathway and increase calcium ion (Ca ²⁺ ) concentration at a concentration of 0.3 nM. . In LRH7-G5 After addition of various concentrations of a short peptide, can significantly reduce the calcium ion (Ca ²⁺⁾ concentrations, inhibition chemerin activation of calcium (Ca ²⁺⁾ signaling pathway stream. However, in wild-type 293T cells that do not express GPR1, chemerin has no activation effect on calcium (Ca ²⁺ ) flow signaling pathway, and LRH7-G5 has no effect on calcium (Ca ²⁺ ) flow signaling pathway. Chemerin can activate the calcium (Ca ²⁺ ) flow signaling pathway by binding to the receptor GPR1, while the LRH7-G5 short peptide can specifically inhibit the chemerin/GPR1 signaling pathway to reduce calcium (Ca ²⁺ ) concentration. *P<0.05, **P<0.01, ***P<0.001 compared with the chemerin group.

Example 4 LRH7-G5 significantly inhibited the proliferation of human breast cancer cell MDA-MB-231.

1 Human breast cancer cells MDA-MB-231 were seeded at ⁵ ×10 ³ cells/well in 96-well cell culture plates at a volume of 200 μL per well, cultured for 24 hours, and then starved overnight;

2 LRH7-G5 polypeptides with different concentration gradients (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM) were added for 24 hours, 48 hours, and 72 hours, respectively;

3 Add 20 μL of MTT working solution per well and continue to incubate for 4 hours in a carbon dioxide incubator;

4 Discard the supernatant in the culture plate, add 150 μL of DMSO (dimethyl sulfoxide), shake for 10 minutes, select the wavelength of 490 nm on the microplate reader for detection, and plot the growth curve of the cells.

In the preliminary work of the inventor's laboratory, it was found that breast cancer cell MDA-MB-231 highly expressed GPR1 by q-PCR assay. Figure 6 shows that different concentrations of LRH7-G5 short peptide can significantly inhibit the proliferation of breast cancer cell MDA-MB-231 cells, and with the increase of the action time, the effect of short peptide on the proliferation of breast cancer cell MDA-MB-231 cells is also More significant.

Example 6 LRH7-G5 blocked the cell cycle progression of human breast cancer cell MDA-MB-231.

1 Human breast cancer cells MDA-MB-231 were seeded at ⁵ ×10 ⁵ cells/well in 6-well cell culture plates at a volume of 1 mL per well, cultured for 24 hours, and then starved overnight;

2 LRH7-G5 polypeptides with different concentration gradients (1 μM, 0.1 μM, 0.01 μM) were added for 24 hours, 48 hours, and 72 hours, respectively;

3 Carefully collect the cell culture medium into a centrifuge tube for later use. The cells were digested with trypsin, and when the cells were gently pipetted with a pipette or a pipette tip, the previously collected cell culture medium was added, all adherent cells were blown, and the cells were gently blown off. Collect again into the centrifuge tube. Centrifuge for about 3-5 minutes at 1000g to precipitate cells;

4 Carefully remove the supernatant and leave about 50 μl of the culture solution to avoid aspiration. Add about 1 ml of ice bath to pre-cooled PBS, resuspend the cells, and transfer to a 1.5 ml centrifuge tube. The cells were again pelleted by centrifugation, and the supernatant was carefully aspirated, leaving about 50 μl of PBS remaining to avoid aspiration of the cells. Gently bomb the bottom of the centrifuge tube to properly disperse the cells to avoid cell agglomeration;

5 Add 1 ml of ice bath to pre-cool 70% ethanol, mix gently by blowing, and fix at 12 °C for 12 h. Centrifuge the cells for about 3-5 minutes by centrifugation at 1000 g. Carefully aspirate the supernatant to leave approximately 50 microliters of 70% ethanol to avoid aspiration. Add about 1 ml of ice bath pre-chilled PBS and resuspend the cells. The cells were again pelleted by centrifugation, and the supernatant was carefully aspirated, leaving about 50 μl of PBS remaining to avoid aspiration of the cells. Gently bomb the bottom of the centrifuge tube to properly disperse the cells to avoid cell agglomeration;

6 Add 0.5 ml of propidium iodide staining solution to each cell sample, slowly and fully resuspend the cell pellet, and leave the bath at 37 ° C for 30 minutes. It can then be stored in the dark at 4 ° C or in an ice bath;

7 The red fluorescence was detected by a flow cytometer at an excitation wavelength of 488 nm, and the light scattering was detected. Analyze the cell results.

Figure 7 shows that by flow cytometry analysis, on the one hand, with the increase of the concentration of action, LRH7-G5 short peptide can significantly reduce the number of cells in MDA-MB-231G2/M phase, increase the number of G0/G1 and S phase, Blocking the normal progression of the cell cycle; on the other hand, with the prolongation of the action time, the LRH7-G5 short peptide also significantly decreased the number of cells in the G2/M phase, increased the number of G0/G1 and S phases, and blocked the normal progression of the cell cycle. In summary, it can be inferred that the LRH7-G5 short peptide inhibits the proliferation of MDA-MB-231 by blocking the cell cycle. * P <0.05, ** P < 0.01 control group G0 / G1 phase compared; ^# P <0.005 compared to the control group of S; ^& P <0.005 with the control group G2 / M phase compared.

Example 7 LRH7-G5 can promote apoptosis of human breast cancer cell MDA-MB-231 cells.

1 Human breast cancer cells MDA-MB-231 were seeded at ⁵ ×10 ⁵ cells/well in 6-well cell culture plates at a volume of 1 mL per well, cultured for 24 hours, and then starved overnight;

2 LRH7-G5 polypeptides with different concentration gradients (1 μM, 0.1 μM, 0.01 μM) were added for 24 hours, 48 hours, and 72 hours, respectively;

3 The supernatant was collected into a centrifuge tube, and the cell culture medium was carefully digested with trypsin without EDTA to the centrifuge tube. Centrifuge for 5 minutes at about 500g to precipitate the cells;

4 Wash the cells twice with pre-cooled PBS, and centrifuge the cells for about 5 minutes to collect the cells;

5 Resuspend the cells by adding 100 μL of pre-cooled 1xAnnexinV Binding Buffer;

6 Add 5 μL of Annexin V-FITC and 5 μL of PI, mix gently, and react at room temperature for 15 minutes in the dark;

7 Add 400 μL of pre-cooled 1xAnnexinV Binding Buffer, mix gently, place the sample on ice and protect it from light, and measure it by flow cytometry within 1 h. Analyze the test results.

Figure 8 shows that by flow cytometry analysis, on the one hand, with the increase of the concentration of action, LRH7-G5 short peptide can significantly promote the apoptosis of breast cancer cells MDA-MB-231, on the other hand with the action time Prolonged, LRH7-G5 short peptide also significantly increased the number of apoptosis of breast cancer cells MDA-MB-231. In summary, LRH7-G5 short peptide can significantly promote the apoptosis of human breast cancer cell MDA-MB-231. *P<0.05, **P<0.01 compared with the control group.

Claims (11)

1. A GPR1 antagonist polypeptide characterized in that the amino acid residue sequence is Met-(D)Pro-Arg-Leu-Pro-Pro-Ala-NH ₂ SEQ ID No. 1.
2. A biologically active fragment or analog of a GPR1 antagonist polypeptide, as set forth in SEQ ID No. 2-3:

   MPRLPPA SEQ ID No. 2

   MPRLPPA-NH2 SEQ ID No. 3.
3. A derivative of a biologically active fragment or analog of the GPR1 antagonist polypeptide of claim 1 or the GPR1 antagonist polypeptide of claim 2, wherein:

   The derivative is a product obtained by conventional modification of an amino acid side chain group, an amino terminal or a carboxy terminus of a GPR1 antagonist polypeptide or a biologically active fragment live analog thereof, or a label for ligation or purification of a polypeptide or protein. The product obtained;

   Preferably, the conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorophore Group modification, polyethylene glycol PEG modification or immobilization modification; the label is His ₆ , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.
4. A polynucleotide encoding the polypeptide of any one of SEQ ID No. 1-3.
5. A vector comprising any one of the polynucleotides of claim 4 and linked to a promoter sequence by genetic means.
6. A host cell transfected with the vector of claim 5.
7. Use of the GPR1 receptor antagonist polypeptide of claim 1, the biologically active fragment or analog of claim 2, and the derivative of claim 3 for the treatment of a chemerin-GPR1 mediated disease;

   Preferably, the chemerin-GPR1 mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response, and polycystic ovary syndrome.
8. The use of the GPR1 receptor antagonist polypeptide of claim 1, the biologically active fragment or analog of claim 2, and the derivative of claim 3 for the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin.
9. The GPR1 receptor antagonist polypeptide according to claim 1, the biologically active fragment or the analog of claim 2, and the derivative of claim 3, for preparing a drug for inhibiting calcium (Ca ²⁺ ) influx caused by chemerin Use in.
10. Use of the GPR1 receptor antagonist polypeptide of claim 1, the biologically active fragment or analog of claim 2, and the derivative of claim 3 for the preparation of a medicament for inhibiting chemotaxis caused by chemerin.
11. A pharmaceutical composition comprising one or more of the GPR1 receptor antagonist polypeptide of claim 1, the biologically active fragment or analog of claim 2, and the derivative of claim 3, Active ingredient

    Preferably, the pharmaceutical composition may contain one or a plurality of pharmaceutically acceptable carriers;

    The pharmaceutically acceptable carrier is preferably a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant;

    The pharmaceutical composition may be further prepared into various forms such as tablets, granules, capsules, oral solutions or injections, and the medicaments of various dosage forms may be prepared according to a conventional method in the pharmaceutical field.

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